- Email: [email protected]
A novel FGFR1-binding peptide attenuates the degeneration of articular cartilage in adult mice
Accepted Manuscript A novel FGFR1-binding peptide attenuates the degeneration of articular cartilage in adult mice Qiaoyan Tan, MD, Bo Chen, MS, Quan Wang, MD, Wei Xu, MD, Yuanqiang Wang, PhD, Zhihua Lin, PhD, Fengtao Luo, MD, Shuo Huang, PhD, Ying Zhu, MD, Nan Su, MD, Min Jin, MD, Can Li, BS, Liang Kuang, MD, Huabing Qi, PhD, Zhenghong Ni, MD, Zuqiang Wang, MD, Xiaoqing Luo, MS, Wanling Jiang, MS, Hangang Chen, BS, Shuai Chen, BS, Fangfang Li, BS, Bin Zhang, BS, Junlan Huang, BS, Ruobin Zhang, BS, Kexin Jin, BS, Xiaoling Xu, PhD, Chuxia Deng, PhD, Xiaolan Du, MS, Yangli Xie, MD, Lin Chen, MD. PhD PII:
S1063-4584(18)31434-1
DOI:
10.1016/j.joca.2018.08.012
Reference:
YJOCA 4300
To appear in:
Osteoarthritis and Cartilage
Received Date: 29 November 2017 Revised Date:
13 August 2018
Accepted Date: 28 August 2018
Please cite this article as: Tan Q, Chen B, Wang Q, Xu W, Wang Y, Lin Z, Luo F, Huang S, Zhu Y, Su N, Jin M, Li C, Kuang L, Qi H, Ni Z, Wang Z, Luo X, Jiang W, Chen H, Chen S, Li F, Zhang B, Huang J, Zhang R, Jin K, Xu X, Deng C, Du X, Xie Y, Chen L, A novel FGFR1-binding peptide attenuates the degeneration of articular cartilage in adult mice, Osteoarthritis and Cartilage (2018), doi: 10.1016/ j.joca.2018.08.012. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
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Running title: FGFR1-binding peptide and Osteoarthritis
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Title: A novel FGFR1-binding peptide attenuates the degeneration of articular
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cartilage in adult mice
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Authors: Qiaoyan Tan1†, MD, Bo Chen1†, MS, Quan Wang1, MD, Wei Xu1, MD,
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Yuanqiang Wang2, PhD, Zhihua Lin2, PhD, Fengtao Luo1, MD, Shuo Huang1, PhD,
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Ying Zhu1, MD, Nan Su1, MD, Min Jin1, MD, Can Li1, BS, Liang Kuang1, MD,
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Huabing Qi1, PhD, Zhenghong Ni1, MD, Zuqiang Wang1, MD, Xiaoqing Luo1, MS,
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Wanling Jiang1, MS, Hangang Chen1, BS, Shuai Chen, BS1, Fangfang Li1, BS, Bin
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Zhang1, BS, Junlan Huang1, BS, Ruobin Zhang1, BS, Kexin Jin1, BS, Xiaoling Xu3,
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PhD, Chuxia Deng3, PhD, Xiaolan Du1*, MS, Yangli Xie1**, MD, Lin Chen1***, MD,
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PhD
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Institution:
Department
of Rehabilitation
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Rehabilitation of Traumatic Injuries, State Key Laboratory of Trauma, Burns and
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Combined Injury, Trauma Center, Research Institute of Surgery, Daping Hospital,
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Third Military Medical University, Chongqing 400042, China.
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Bioengineering, Chongqing Institute of Technology, Chongqing 400050, China.
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Laboratory for the
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College of
Faculty of Health Sciences, University of Macau, Macau SAR 00853, China.
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Medicine,
†These authors contributed equally to this work.
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***
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Medicine, Laboratory for the Rehabilitation of Traumatic Injuries, State Key
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Laboratory of Trauma, Burns and Combined Injury, Trauma Center, Research
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Institute of Surgery, Daping Hospital, Third Military Medical University, Chongqing
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400042, China.
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Tel: 86-23-68757041. Fax: 86-23-68702991. E-mail: [email protected].
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**
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Laboratory for the Rehabilitation of Traumatic Injuries, State Key Laboratory of
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Trauma, Burns and Combined Injury, Trauma Center, Research Institute of Surgery,
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Daping Hospital, Third Military Medical University, Chongqing 400042, China.
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Tel: 86-23-68757042. Fax: 86-23-68702991.E-mail: [email protected].
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*
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Laboratory for the Rehabilitation of Traumatic Injuries, State Key Laboratory of
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Trauma, Burns and Combined Injury, Trauma Center, Research Institute of Surgery,
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Daping Hospital, Third Military Medical University, Chongqing 400042, China.
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Tel: 86-23-68757042. Fax: 86-23-68702991.E-mail: [email protected].
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Corresponding authors. Address: Y. Xie, Department of Rehabilitation Medicine,
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Corresponding authors. Address: X. Du, Department of Rehabilitation Medicine,
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Corresponding authors. Address: L. Chen, Department of Rehabilitation
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Abstract
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Objective: We previously reported that genetic ablation of Fgfr1 in knee cartilage
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attenuates the degeneration of articular cartilage in adult mice, which suggests that
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FGFR1 is a potential targeting molecule for osteoarthritis (OA). Here, we identified
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R1-P1, an inhibitory peptide for FGFR1 and investigated its effect on the
ACCEPTED MANUSCRIPT pathogenesis of OA in mice induced by destabilization of medial meniscus (DMM).
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Design: Binding ability between R1-P1 and FGFR1 protein was evaluated by ELISA
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and molecular docking. Alterations in cartilage were evaluated histologically. The
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expression levels of molecules associated with articular cartilage homeostasis and
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FGFR1
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immunohistochemistry. The chondrocyte apoptosis was detected by TUNEL assay.
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Results: R1-P1 had highly binding affinities to human FGFR1 protein, and efficiently
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inhibited ERK1/2 pathway in mouse primary chondrocytes. In addition, R1-P1
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attenuated the IL-1β induced significant loss of proteoglycan in full-thickness
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cartilage tissue from human femur head. Moreover, this peptide can significantly
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restore the IL-1β mediated loss of proteoglycan and type II collagen (Col II) and
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attenuate the expression of matrix metalloproteinase-13 (MMP13) in mouse primary
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chondrocytes. Finally, intra-articular injection of R1-P1 remarkably attenuated the
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loss of proteoglycan and the destruction of articular cartilage and decreased the
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expressions of ECM degrading enzymes and apoptosis in articular chondrocytes of
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mice underwent DMM surgery. Conclusions: R1-P1, a novel inhibitory peptide for
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FGFR1, attenuates the degeneration of articular cartilage in adult mice, which is a
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potential leading molecule for the treatment of OA.
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by
qRT-PCR,
Western
blotting
and
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analyzed
Key Words: FGFR1; Peptide; Osteoarthritis; Mice
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were
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signaling
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Introduction
ACCEPTED MANUSCRIPT Osteoarthritis (OA) is one of the most prevalent chronic joint diseases with
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progressive cartilage destruction and insufficient extracellular matrix synthesis.
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Currently, there are no effective biological therapies to prevent or treat OA, the most
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common final therapeutic option is the total joint replacement.
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In recent years, a variety of molecules and signaling pathways, such as mTOR, IHH
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and Wnt/β-catenin signaling have been found to be involved in cartilage homeostasis
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and OA development (1,2). Fibroblast growth factors (FGF) and their receptors
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(FGFRs) regulate the maintenance of cartilage and therefore, play a crucial role in
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joint homeostasis and OA development (3,4,5). FGF2 promotes cartilage catabolism
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via FGFR1-Ras-Raf-MEK1/2-ERK1/2 axis in human articular chondrocytes (6).
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FGFR1 and FGFR3 are highly expressed in human articular chondrocytes, and the
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expression ratio of FGFR1 to FGFR3 was significantly increased in OA chondrocytes
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compared to normal chondrocytes (7), which suggests the potential role of FGF
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signaling in cartilage homeostasis. FGF18 can promote chondrogenesis through
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FGFR3 and is a well-established anabolic growth factor for articular cartilage (8).
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Surgical DMM in mice is a well-established OA model (9,10), which is commonly
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used to screen biological and pharmacological agents for OA treatment (11,12).
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FGFR3 deficiency mice exhibited abnormal cartilage metabolism and early signs of
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OA, including increased cellular hypertrophy and loss of proteoglycan with
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upregulated expressions of MMP13, type X collagen (Col X) (13,14). Regarding to
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FGFR1, our previous study showed that conditional knockout (cKO) of Fgfr1 in
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cartilage attenuates articular cartilage degeneration, which is associated with
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ACCEPTED MANUSCRIPT down-regulation of MMP13 and increased proteoglycan synthesis. Thus, we expected
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that pharmacological down-regulation of the activity of FGFR1 or its downstream
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pathways may attenuate the development of OA. Since we previously found a short
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FGFR1-binding peptide (GPPDWHWKAMTH, named as R1-P1) using phage display
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and demonstrated that it can blunt the activity of ERK1/2 MAPK pathway. We thus
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tested whether it may exert a protective effect on OA in mouse model. Our results
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demonstrated that R1-P1 can attenuate the severity of OA induced by DMM in mice.
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Materials and Methods
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Sequence analysis and molecular docking
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Sequence analysis and molecular docking were carried out according to previous
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protocols (15,16), The peptide sequence was analyzed with Bioedit and ProtParam
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programs (http://au.expasy.org/tools/protparam.html). The crystal structure of FGFR1
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was accessed from RCSB database (ID: 5AM6) and prepared as receptor with
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biopolymer package of crystal. We determined the binding site based on the crystal
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structure of FGFR1. Then, the peptide was docked into receptor by surflex-dock
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method and the pose with highest score was used to generate the peptide/FGFR1
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complex. Molecular dynamics simulation was performed as following steps: (1)
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Minimization (1000 steps, restraint receptor); (2) Heating (NVE, 50 ps, 0K-300 K,
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restraint receptor); (3) Density adjustment (NPT, 50 ps, 300 K, constraint receptor); (4)
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Equilibrium (NPT, 500 ps, 300 K); (5) Production (NPT, 120 ns, 300 K). The
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molecular dynamics simulation was performed by amber.
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Peptide synthesis
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The peptide was synthesized at Shanghai China Peptides Co., Ltd. by solid-phase
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synthesis and purified by high-performance liquid chromatography (HPLC). Peptide
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with high purity grade (95.43%) was obtained after purification. The synthetic
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product was further confirmed by mass spectrometry analysis.
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ELISA assay for binding ability
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Binding affinities were checked by ELISA using previous protocol (17). Briefly,
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microtiter plates were coated with 20 mg/ml FGFR1 overnight at 4°C. After being
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blocked at 37°C for 1 h, the plates were washed six times. R1-P1 at different
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concentrations (0,1,5,10,15,25,50,100 mM) were added and incubated at room
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temperature for 1 h. After the plates were washed six times, anti-FGFR1 antibody
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were added and incubated at 37°C for 1 h. The plates were washed six times. Goat
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anti-rabbit immunoglobulin G (IgG)-HRP was then added and incubated at 37°C for
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30 min. The plates were washed, and color development was initiated with addition of
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3,3’,5,5’-tetramethylbenzidine (TMB; 100 ml/well of TMB). The reaction was
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terminated 20 min later by adding 20 µl stopping buffer, and the absorbance was
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measured at 450 nm.
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Cell culture
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Mouse embryonic carcinoma-derived cell line ATDC5 cells were cultured in 12-well
ACCEPTED MANUSCRIPT plates until 90% confluence, then cells were pre-incubated with FGF2 (10 ng/ml) for
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30 min, followed by treatment with peptide R1-P1 (0, 1, 10, 25 or 50mM) for 2 hours.
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PD173074 (Selleck), an inhibitor of FGFR1, was used as the positive control. Mouse
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primary chondrocytes were isolated as previously described (18). Chondrocytes were
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cultured in 12-well plates until 90% confluence and were pre-incubated with R1-P1
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(25 or 50 mM) alone for 2 hours, before treatment with IL-1β (10 ng/ml) for 24 hours.
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Culture of human articular cartilage explants
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This project received approval from the Ethical and Protocol Review Committee of
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Daping Hospital, Chongqing, and the culture were carried out according to the
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previous protocols (19). Briefly, human full-thickness femur head cartilage tissue was
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cut into 4 mm diameters, following 48 hours culture, explants were treated with IL-1β
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(20 ng/ml) and peptide R1-P1 (25 or 50 mM) every 3 days for 14 days under
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serum-free conditions (plus mini-ITS™ + Premix). Following 14 days of incubation,
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the medium was collected, and all explants were fixed in 4% paraformaldehyde
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overnight, followed by paraffin embedding.
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Dimethylmethylene Blue (DMMB) assay
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DMMB dye solution and sample digestion solution were prepared following previous
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protocol (20). Briefly, 250 µl of DMMB reagent was added to 40 µl of culture
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medium and the absorbance was measured at 525 nm immediately. Chondroitin
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sulfate (Sigma-Adrich) with several concentrations was used to plot a standard curve,
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which was used to measure the amount of aggrecan released into culture medium.
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Aggrecan released into the medium was normalized as mass of GAG per milliliter (ml)
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of culture medium.
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RNA extraction and quantitative real-time PCR (qPCR)
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Total RNA of treated mouse primary chondrocytes was isolated by TRIZOL reagent
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(Invitrogen). Real-time PCR was repeated at least three times using Mx3000P PCR
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machine (Stratagene) and SYBR Premix Ex TaqTM kit (Takara). The primers for
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genes measured were listed in Table 1. Expression values were normalized to
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Cyclophilin A.
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Western blotting
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Protein was extracted using ice-cold RIPA lysis buffer containing protease inhibitors
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(Roche). Equal amount of protein samples (30 ug) were dissolved by 10%
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SDS-PAGE gels and transferred onto a polyvinylidene difluoride membrane
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(Millipore). After being blocked with 8% nonfat milk in tris buffered saline tween
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buffer, the membrane was probed with antibody specific to ERK1/2 (1:1000 dilution;
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Cell Signaling Technology (CST)), p-ERK1/2 (1:1000 dilution; CST), MMP13
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(1:1000 dilution; Millipore), Aggrecan (1:1000 dilution; Abcam) and β-Actin
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(1:10000 dilution; Sigma) followed by chemiluminescent (Pierce) detection. All
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antibodies were solvated with antibody diluent (Beyotime). Intensity values were
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analyzed with the Image J software and were normalized to those of β-Actin. Each
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sample was analyzed three times and the mean gray values of immunoblot band were
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calculated (21).
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Surgical model of OA in mice
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All mice were maintained in the Animal Facility (specific pathogen free) of the
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Daping Hospital (Chongqing, China). All procedures were approved by the
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Institutional Animal Care and Use Committee of Daping Hospital (Chongqing, China).
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8-week-old male C57BL/6J mice (25-30 g) were purchased from the Beijing HFK
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Bioscience Co. Ltd.. According to previous protocol (22), DMM surgery was
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performed on the right knee joints, sham surgery was performed with medial
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capsulotomy only on the left knee joints of mice. Mice divided into groups at
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randomisation. After 1 week, mice in treatment group received R1-P1 (once a week,
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15µg/week) postoperatively via articular cavity injection for 4, 8 and 12 weeks. In
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control mice, their articular cavities were injected with physiological saline only.
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Histologic assessment
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Microscopic scoring of mouse cartilage degeneration was performed in accordance
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with the recommendations of the Osteoarthritis Research Society International
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(OARSI) (23). Briefly, the joints were fixed in 4% paraformaldehyde, then decalcified
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in 20% formic acid, and embedded in paraffin. Then serial sagittal sections were
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obtained across the entire knee joint. Histologic grading of cartilage degeneration was
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performed using the OARSI recommended subjective scoring system (on a scale of
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0-6) (23). In addition, cartilage aggrecan depletion was scored (on a scale of 0-5) as a
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complement measure of cartilage degeneration
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agreement between the two blinded observers (Quan Wang and Wei Xu).
(24). There was a high level of
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Synovitis score
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Mouse articular joint sections were stained with hematoxylin and eosin (H&E) to
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assess the grade of synovitis. Scoring was performed by two blinded observers (25).
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Three features of synovitis (enlargement of lining cell layer, cellular density of
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synovial stroma, leukocytic infiltrate) were semiquantitatively evaluated (from 0,
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absent to 3, strong) and each feature was graded separately. The sum provided the
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synovitis score, which is interpreted as follows: 0-1, no synovitis; 2-4, low-grade
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synovitis; 5-9, high grade synovitis.
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Immunohistochemistry (IHC)
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IHC were carried out using SP-9000 Histostain-Plus kits (Zsgb Bio) as previous
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protocols (14). Incubated with antibody specific to Aggrecan (1:200 dilution; Abcam),
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Col II (1:400 dilution; Chondrex), Adamts5 (1:200 dilution; Abcam), MMP13 (1:200
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dilution; Abcam), Col X (1:200 dilution; Millipore) and FGFR3 (1:200 dilution; Santa
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Cruz Biotechnology) followed by diaminobenzidine (DAB) kit detection, all
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antibodies were solvated with antibody diluent (Origene).
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TUNEL assay
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Apoptotic cells in articular cartilage were detected by TdT mediated dUTP-X nick
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end labeling (TUNEL) assay using in situ cell death detection kit (peroxidase (POD))
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of Roche according to the manual.
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Statistical analysis
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We used 5-10 animals in each group for in vivo studies and three independent
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biological replicates for in vitro studies. Individual cell experiments were also
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performed in triplicate as technical replicates. The sample sizes for the groups of
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interest were big enough based on a power of 80% and p < 0.05 using power and
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sample size calculation online software (http://powerandsamplesize.com/Calculators/)
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(Supplementary Table 1). All tested variables were tested for normality and
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homogeneity using Shapiro-Wilk test and Levine’s test, respectively. Quantitative
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data of histologic grading of cartilage degeneration, IHC analysis of MMP13, ColX,
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FGFR3 and TUNEL assay were compared by using the non-parametric
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Mann-Whitney U test. For the other data, all the tested variables did not violate the
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assumptions of normality and equal variance. Differences between two groups were
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evaluated using Student’s unpaired t-test, and comparisons of multiple groups were
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evaluated using analysis of variance (ANOVA) followed by Tukey’s test. All data
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were analyzed using GraphPad Prism v.6.01 software and depicted in univariate
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scatter plots as previously described (26). P < 0.05 was considered to be statistically
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significant.
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Results
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R1-P1 binds to human FGFR1 protein and inhibits MAPK/ERK signaling
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pathway
ACCEPTED MANUSCRIPT All 11 clones with high binding affinity to FGFR1 protein shared the
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“GPPDWHWKAMTH” amino acid sequences, which was named as peptide R1-P1.
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ProtParam programs (http://web.expasy.org/protparam/) were then applied to analyze
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the sequence and predict its properties. The molecular weight of the R1-P1 is 1462.65,
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theoretical isoelectric point of the peptide is 6.92, grand average of hydropathicity of
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the R1-P1 is -1.35 (Table 2).
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To evaluate the binding ability between peptide R1-P1 and the extracellular region of
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FGFR1 (5AM6) protein, we first determined the binding site based on the crystal
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structure of FGFR1. Results indicate that there was a large groove in the surface of
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FGFR1 (Figure 1A), which could accommodate the peptide. Then the peptide was
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docked into binding site by surface-dock. The best pose, total score and C score were
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7.87 and 5 respectively, was combined with FGFR1 to generate FGFR1/pep complex.
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To achieve a stable structure, a 20 nanoseconds MD simulation was performed.
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According to the RMSD plot (Figure 1B), the RMSD was fluctuated close to 2.5Å
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after 70 nanoseconds, which means the structure of complex was reasonable. The
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peptide could bind to FGFR1 stably (Figure 1C) relied on hydrogen bonds,
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electrostatic and hydrophobic interaction (Figure 1D). Figure 1D shows that there
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were 8 hydrogen bonds between peptide with residue Arg493, Arg512, Asp513, Leu484,
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Gly490, Asp527, Glu571, and Asn568.
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We examined the binding affinity of peptide R1-P1 at different concentrations
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(0,1,5,10,15,25,50,100 mM) to FGFR1, ELISA result showed that R1-P1 can bind to
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FGFR1 protein in a dose-response manner, and the binding affinities were not further
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ACCEPTED MANUSCRIPT increased from 50 to 100 mM (Figure 1E). In addition, ELISA results demonstrated
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that R1-P1 strongly bound to FGFR1, although it also slightly bound to FGFR2 and
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FGFR3 (Figure 1F). FGFs/FGFRs exert their diverse biological effects through
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multiple intracellular signaling pathways, among which the MAPK pathway has been
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found to inhibit chondrocyte differentiation and matrix synthesis (6). We examined
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whether R1-P1 could affect the protein level of total ERK1/2 and phosphorylated
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ERK1/2 induced by FGF2. Furthermore, western blotting result showed that
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FGF2-mediated ERK1/2 phosphorylation were partially blocked by R1-P1 at 25 and
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50mM concentration (Figure 1G), indicating that R1-P1 inhibited ERK1/2 MAPK
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signaling pathway, a classic downstream signaling pathway of FGFR1 in
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chondrocytes.
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R1-P1 attenuates IL-1β induced proteoglycan loss in adult human articular
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cartilage
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To determine the effect of R1-P1 on proteoglycan loss, we treated cultured healthy
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human femoral head cartilage explant with IL-1β (20 ng/ml) with or without the
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R1-P1 (25 or 50 mM) for 14 days. Safranin O staining showed that IL-1β induced
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significant proteoglycan loss, which was partially blunted by peptide R1-P1 treatment
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(Figure 2A).
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The GAG released into the culture medium was significantly increased after IL-1β
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treatment (P < 0.001), which were markedly decreased after being incubated with 25
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or 50 mM R1-P1 (all P < 0.001, Figure 2B), respectively. These data were consistent
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with the results of Safranin O staining. Together, these results indicate that R1-P1 can
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attenuate the catabolic effect of IL-1β on chondrocytes.
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R1-P1 inhibits the phosphorylation of ERK1/2 and rescues IL-1β induced
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dysregulated ECM metabolism in mouse primary chondrocytes
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To explore the mechanisms for the anti-catabolic effects of R1-P1 on chondrocytes,
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real-time qPCR and western blotting were performed to examine the effects of R1-P1
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on the synthesis and breakdown of extracellular matrix in mouse primary
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chondrocytes. The mRNA expressions of Adamts5 (P < 0.001) and MMP13 (P <
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0.001) were significantly increased in mouse primary chondrocytes after IL-1β
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treatment. R1-P1 treatment resulted in a marked reduction in the expressions of these
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two catabolic markers (P < 0.001 and P = 0.006, respectively). Meanwhile, the
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mRNA expressions of Aggrecan (P < 0.001) and Col II (P < 0.001) were decreased
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after IL-1β treatment, which were partially recovered (P < 0.001 and P < 0.001,
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respectively) by peptide R1-P1 (Figure 3A).
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Western blotting results showed that following the stimulation of IL-1β, ERK1/2
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MAPK signaling pathway was activated, the protein levels of Aggrecan was
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decreased and MMP13 was increased (all P < 0.001). Treatment with 25 or 50 mM
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R1-P1 significantly decreased the protein levels of MMP13 and phosphorylated
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ERK1/2, while up-regulated the protein levels of Aggrecan in mouse primary
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chondrocytes (all P < 0.001, Figure 3B and 3C). Taken together, these results
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demonstrate that R1-P1 may help to maintain the homeostasis of mouse primary
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chondrocytes.
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R1-P1 delays articular cartilage degradation in a mouse OA model
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We determined the effect of R1-P1 on the development of OA in mice induced by
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DMM. There is no remarkable toxicity observed in the R1-P1 treatment group, as
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evidenced by no signs of abnormal gross phenotypes including weight loss during
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observation period. Histologic examination at 4 weeks after DMM surgery revealed
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the loss of proteoglycan content (data not shown). Furthermore, 8 weeks after DMM
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mice showed an early OA-like manifestations including loss of proteoglycan content
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and cartilage tissue (Figure 4A-B). The OA-like phenotype became more profound in
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mice at 12 weeks after DMM surgery, which exhibited as more severe destruction of
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the articular cartilage associated with greater loss of proteoglycan content (Figure
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5A-B). However, intra-articular injection of R1-P1 significantly decreased the
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cartilage destruction and proteoglycan loss in mouse knee joint after DMM surgery
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(Figure 4A-B and 5A-B).
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Accordingly, the OARSI scores were significantly increased in mice at 8 and 12
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weeks after DMM surgery compared to those of sham operation mice (all P < 0.001,
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Figure 4C-F and 5C-F). R1-P1 treated mice had a significantly lower sum score and
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maximal score in medial femoral condyle and medial tibial plateau than those in the
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vehicle treated mice at 8 weeks (P = 0.001, P = 0.014, P = 0.002 and P = 0.008,
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respectively, Figure 4C-F) and 12 weeks (P < 0.001, P = 0.002, P = 0.021 and P =
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0.006, respectively, Figure 5C-F) following DMM surgery. In addition, H&E
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ACCEPTED MANUSCRIPT staining and scoring of synovitis showed that treatment with R1-P1 attenuated the
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severity of synovitis at 8 weeks (P = 0.002, Figure 4G-H) and 12 weeks (P < 0.001,
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Figure 5G-H) following DMM surgery compared with vehicle. Taken together, these
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results demonstrate that intra-articular injection of R1-P1 partially delayed OA
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development.
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R1-P1 attenuates ECM loss and chondrocyte hypertrophy and apoptosis in knee
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joints of DMM mice
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To explore the cellular mechanisms underlying the delayed progression of cartilage
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degradation in peptide R1-P1 treated mice with DMM, we performed IHC to examine
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the expressions of Aggrecan, Col II, Col X, Adamts5 and MMP13. The results
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showed that the expressions of Adamts5, MMP13 were significantly increased and the
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expressions of Aggrecan, Col II were decreased in the articular cartilage after DMM
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surgery (Figure 6A-D). In addition, after DMM surgery, the ratio of cells with
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positive Col X in the articular cartilage was significantly increased (Figure 6E).
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However, peptide R1-P1 largely lowered the ratio of Adamts5, MMP13, Col X
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positive cells and significantly increased the ratio of Aggrecan, Col II positive cells in
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the articular cartilage after DMM surgery compared to vehicle treatment (all P <
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0.001, Figure 6H). Furthermore, the ratio of FGFR3 positive cells was
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down-regulated in the articular cartilage when OA was induced by DMM surgery.
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However, inhibition of FGFR1 activity led to increased ratio of FGFR3 positive cells
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in the articular cartilage (P < 0.001, Figure 6F and Figure 6H).
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ACCEPTED MANUSCRIPT Since it has been reported that chondrocyte apoptosis is strongly related to the OA
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development (27,28). We performed TUNEL assay to analyze the apoptosis of
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chondrocytes in knee joints. Our results showed that peptide R1-P1 treatment largely
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decreased the apoptosis of chondrocytes in the articular cartilage 8 weeks after DMM
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surgery compared to vehicle treatment (as shown in red arrows, P < 0.001, Figure 6G
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and Figure 6H). Taken together, these results demonstrate that local intra-articular
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injection of R1-P1 reduced articular cartilage damage, at least in part, through its
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inhibition on the hypertrophy and apoptosis process of articular chondrocytes.
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Discussion
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OA is a degenerative disease characterized with cartilage degradation, synovial
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inflammation and dysregulated subchondral bone remodeling. The mechanisms
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underlying OA is not well understood. Currently, there is no effective biological
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treatment for OA. Finding targeting cells and/or molecules for effective therapies of
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OA is urgently needed.
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Decades of studies have demonstrated that FGFs/FGFRs play vital roles in cartilage
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homeostasis and OA development (11). We previously demonstrated that conditional
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deletion of Fgfr1 in mouse articular chondrocytes delays the progression of cartilage
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degeneration (29), suggesting that pharmacological down-regulation of FGFR1
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activity is a potential therapy for OA. Over the past decades, several FGFR inhibitors,
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such as PD173074, SU5402 and PD166866 have been developed as candidates for the
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treatment of FGF signaling related diseases (30). Because of the low-specificity and
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ACCEPTED MANUSCRIPT toxicity, majority of them failed to enter clinical application. Peptides can be easily
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synthesized and have low immunogenicity. Furthermore, peptide drugs have been
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successfully used in clinic such as a1-thymosin (31). Here, we, for the first time,
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provide evidence showing that a novel FGFR1 antagonist, peptide R1-P1, has high
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binding affinity to FGFR1 protein and can alleviate cartilage degradation in a mouse
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model of OA.
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The major characteristic of OA is an imbalance between anabolic effects mediated
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extracellular matrix synthesis and catabolic effects regulated matrix degradation (32).
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We demonstrated that R1-P1 inhibited the proteoglycan degradation induced by IL-1β
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in cultured human cartilage explants. Furthermore, we demonstrated that
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R1-P1attenuated the IL-1β induced up-regulation of the mRNA levels of Adamts5 and
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MMP13 and down-regulation of Aggrecan and Col II mRNA levels, in mouse articular
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chondrocytes. We speculate that the mechanism for the protective effect of R1-P1 on
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OA is mainly via its inhibition on the activities of ERK1/2 MAPK signaling pathway.
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Chondrocyte hypertrophy, which can result in increased metabolic activity of articular
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chondrocytes and trigger unbalanced cartilage homeostasis favoring degenerative
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changes (33). We found that R1-P1 treatment reduced the expressions of MMP13 and
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Col X, markers for hypertrophic chondrocytes, in mouse OA model induced by DMM
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(12,34). These findings revealed that intra-articular injection of R1-P1 prevented
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articular chondrocytes from hypertrophy, which may contribute to the inhibitory
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effect of R1-P1 on OA development. The inhibitory effect of R1-P1 on chondrocyte
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hypertrophy is consistent with our previous findings showing that Fgfr1 deficiency in
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ACCEPTED MANUSCRIPT chondrocytes decreased chondrocyte hypertrophy in articular cartilage (29). FGF
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signaling pathway is highly associated with chondrocyte apoptosis (35).
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Gain-of-function mutation of Fgfr3 promotes chondrocyte apoptosis in thanatophoric
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dysplasia (TD) mice (36). Fgf2 transgenic mice exhibit chondrodysplasic phenotype
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resulting from both reduced proliferation and increased apoptosis of growth plate
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chondrocytes (37). FGF18 markedly reduces chondrocyte apoptosis and enhances the
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repair response of cartilage following cartilage insult (38). In current study, we
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showed that pharmacologically inhibiting FGFR1 activity by R1-P1 decreased
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chondrocyte apoptosis in mouse OA model, indicating that R1-P1 positively
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maintains cartilage homeostasis partially through downregulating chondrocyte
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apoptosis.
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During OA development, the expression ratio of FGFR1 to FGFR3 was up-regulated
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in articular chondrocytes from patients with OA (7), suggesting that down-regulation
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of FGFR3 is at least partially responsible for the development of OA in FGFR1
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deficiency related OA. We found that intra-articular injection of R1-P1 can increase
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ratio of FGFR3 positive cells in mouse OA model.
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Whether R1-P1 have the same effects in other animal model need to be studied. In
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addition, inflammation also plays essential role in the pathogenesis of OA by inducing
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catabolic effects on ECM, etc. Inhibition of inflammation leads to attenuated OA
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severity and progression (39,40). As a classic signaling pathway in development and
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homeostasis of a variety of tissues/organs, FGF signaling appears to have crosstalk
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with inflammation related signaling pathways. For example, inhibition of FGF23
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ACCEPTED MANUSCRIPT might serve as a novel anti-inflammatory strategy in chronic obstructive pulmonary
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disease (COPD) (41). So, we speculate that R1-P1 and anti-inflammatory drug may
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have overlapping but different effects on attenuating OA progression.
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In summary, we found a novel inhibitory peptide, R1-P1, for FGFR1 that can
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maintain the ECM of chondrocytes through its down-regulation of matrix-degrading
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enzymes and prevent articular chondrocytes from hypertrophy and apoptosis, which
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will facilitate the development of effective therapeutic agents for OA.
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Author contributions
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All authors listed have read and approved all versions of the manuscript.
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Conception and design: Qiaoyan Tan, Xiaolan Du, Yangli Xie, Lin Chen.
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Analysis and interpretation of the data: Qiaoyan Tan, Quan Wang, Wei Xu, Yuanqiang
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Wang, Huabing Qi, Yangli Xie.
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Drafting of the article: Qiaoyan Tan, Yangli Xie, Lin Chen.
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Critical revision of the article for important intellectual content: Chuxia Deng, Yangli
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Xie, Lin Chen.
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Final approval of the article: Qiaoyan Tan, Xiaolan Du, Yangli Xie, Lin Chen.
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Provision of study materials or patients: Qiaoyan Tan, Shuo Huang, Zhenhong Ni,
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Liang Kuang, Nan Su, Xiaolan Du.
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Statistical expertise: Qiaoyan Tan, Liang Kuang, Wei Xu.
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ACCEPTED MANUSCRIPT Administrative, technical, or logistic support: Yin Zhu, Wanling Jiang, Hangang Chen,
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Shuai Chen, Fangfang Li, Bin Zhang, Junlan Huang, Ruobing Zhang, Kexin Jin, Can
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Li, Min Jin, Xiaolan Du.
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Collection and assembly of data: All authors.
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Fundings
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This work was supported by Special Funds for Major State Basic Research Program
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of China (973 program) (No.2014CB942904), National Natural Science Foundation
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of China (No. 81530071, No.81472074), State Key Laboratory of Trauma, Burn and
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Combined Injury, China (No.201601).
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Conflict of interest
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The authors declare that they have no competing interests.
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Figure Legends
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Figure 1. Specific binding of the R1-P1 to FGFR1. (A) The binding site base
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crystal structure of FGFR1. (B) R1-P1 was docked into binding site by surface-dock.
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(C) R1-P1 could bind to FGFR1 stably. (D) Showed there were 8 hydrogen bonds
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between peptide with residue. (E) Binding affinities of R1-P1 at different
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concentrations (0,1,5,10,15,25,50,100 mM) to FGFR1 revealed by ELISA (n = 3). (F)
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Binding affinities of R1-P1 to FGFRs measured by ELISA (n = 6). (G) Results
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showed peptide R1-P1 inhibited phosphorylation of ERK1/2 induced by FGF2 (n = 3).
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Data in were expressed as the mean ± 95% confidence intervals.
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Figure 2. Effects of R1-P1 on proteoglycan loss in adult human articular
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cartilage. Full thickness cartilage samples from human femur were cultured in the
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absence or presence of IL-1β (20 ng/ml) and in the presence or absence of R1-P1 (25
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or 50 mM) for 14 days. (A) Sections were stained with Safranin O to identify the
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cartilage proteoglycan loss (n = 3). (B) Culture medium were collected, and the
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amount of GAG released into the medium were quantified by DMMB assay. GAG
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released into the medium was normalized as mass of GAG per ml of culture medium
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(n = 9). Scale bar: 100 µm. Data were expressed as the mean ± 95% confidence
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intervals.
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Figure 3. Effects of R1-P1 on mRNA levels of MMP13, Adamts5, Col II and
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Aggrecan and on the protein levels of MMP13, Aggrecan and p-ERK1/2 in IL-1β
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treated mouse primary chondrocytes. (A) Total RNA was isolated, and levels of
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mRNA of the catabolic markers (MMP13 and Adamts5) and chondrocyte markers
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(Col II and Aggrecan) were detected by Real-time qPCR (n = 9). (B) Cell lysates
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were analyzed by western blotting using antibodies specific for MMP13, Aggrecan
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and p-ERK1/2 (n = 3). (C) The signal intensities of MMP13, Aggrecan and
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phospho-ERK1/2 were quantitatively analyzed (n = 3). Data were expressed as the
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mean ± 95% confidence intervals.
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Figure 4. Histologic features of mouse articular at 8 weeks following DMM
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surgery. (A) The articular cartilage was stained with Safranin O-fast green at 8 weeks
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after surgery to assess the extent of articular cartilage degeneration. (B) Expansion of
623
the region occupied by articular cartilage. (C-F) OARSI scoring system showed more
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severe articular cartilage destruction in the medial femur and tibia of mice at 8 weeks
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following DMM surgery compared with sham operation (n = 8 or 10 mice per group).
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(G-H) H&E staining and scoring of synovitis showed that treatment with R1-P1 can
627
attenuate the severity of synovitis after DMM surgery compared with vehicle (n = 8
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or 10 mice per group). MFC: medial femoral condyle; MTP: medial tibial plateau.
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Scale bar: 100 µm. Data were expressed as the mean ± 95% confidence intervals.
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Figure 5. Histologic features of mouse articular cartilage at 12 weeks following
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DMM surgery. (A) The articular cartilage was stained with Safranin O-fast green at
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12 weeks after surgery to assess the extent of articular cartilage degeneration. (B)
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Expansion of the region occupied by articular cartilage. (C-F) OARSI scoring system
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showed more severe articular cartilage destruction in the medial femur and tibia of
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mice at 12 weeks following DMM surgery compared with sham operation (n = 5 mice
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per group). (G-H) H&E staining and scoring of synovitis showed that treatment with
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peptide R1-P1 can attenuate the severity of synovitis after DMM surgery compared
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with vehicle (n = 5 mice per group). MFC: medial femoral condyle; MTP: medial
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tibial plateau. Scale bar: 100 µm. Data were expressed as the mean ± 95% confidence
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intervals.
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Figure 6. Effects of peptide R1-P1 on cartilage ECM levels and degradation
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enzymes and chondrocyte apoptosis in mice at 8 weeks after DMM. IHC analysis
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of Aggrecan (A), Col II (B), Adamts5 (C), MMP13 (D), Col X (E), FGFR3 (F)
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protein expression in articular cartilage of mice from either sham operation group or
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surgery group at 8 weeks after DMM surgery with intra-articular injection of peptide
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R1-P1 or vehicle (n = 3). (G) TUNEL assay was performed on knee joints to measure
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chondrocyte apoptosis either sham operation or at 8 weeks after DMM surgery with
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intra-articular injection of peptide R1-P1 or vehicle (as shown in red arrows) (n = 3).
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(H) The percentage of cells that are positive for MMP13, Col X, FGFR3 and TUNEL
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in the articular cartilage were calculated (n = 3). Scale bar: 100 µm. Data were
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expressed as the mean ± 95% confidence intervals.
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ACCEPTED MANUSCRIPT Table 1. Primers and sequences for real-time PCR analysis. Forward primer
5'-CTGTGGAGGTCACTGTAGACT-3'
Adamts5
5'-GGAGCGAGGCCATTTACAAC-3'
5'-CGTAGACAAGGTAGCCCACTTT-3'
Col II
5'-CTGGTGGAGCAGCAAGAGCAA-3'
5'-CAGTGGACAGTAGACGGAGGAAAG-3'
Aggrecan
5'-CCTGCTACTTCATCGACCCC-3'
5'-AGATGCTGTTGACTCGAACCT-3'
Cyclophilin A
5'-CGAGCTCTGAGCACTGGAGA-3'
5'-TGGCGTGTAAAGTCACCACC-3'
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5'-CTTCTTCTTGTTGAGCTGGACTC-3'
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MMP13
Reverse primer
Table 2. Properties of peptides displayed by positive phages
Clones
Sequence
Molecular weight
Theoretical pI a
GRAVY b
R1-P1
11
GPPDWHWKAMTH
1462.65
6.92
-1.35
PI, Isoelectric Point.
b
GRAVY, Grand Average of Hydropathicity.
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S1063-4584(18)31434-1
DOI:
10.1016/j.joca.2018.08.012
Reference:
YJOCA 4300
To appear in:
Osteoarthritis and Cartilage
Received Date: 29 November 2017 Revised Date:
13 August 2018
Accepted Date: 28 August 2018
Please cite this article as: Tan Q, Chen B, Wang Q, Xu W, Wang Y, Lin Z, Luo F, Huang S, Zhu Y, Su N, Jin M, Li C, Kuang L, Qi H, Ni Z, Wang Z, Luo X, Jiang W, Chen H, Chen S, Li F, Zhang B, Huang J, Zhang R, Jin K, Xu X, Deng C, Du X, Xie Y, Chen L, A novel FGFR1-binding peptide attenuates the degeneration of articular cartilage in adult mice, Osteoarthritis and Cartilage (2018), doi: 10.1016/ j.joca.2018.08.012. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
ACCEPTED MANUSCRIPT 1
Running title: FGFR1-binding peptide and Osteoarthritis
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Title: A novel FGFR1-binding peptide attenuates the degeneration of articular
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cartilage in adult mice
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Authors: Qiaoyan Tan1†, MD, Bo Chen1†, MS, Quan Wang1, MD, Wei Xu1, MD,
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Yuanqiang Wang2, PhD, Zhihua Lin2, PhD, Fengtao Luo1, MD, Shuo Huang1, PhD,
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Ying Zhu1, MD, Nan Su1, MD, Min Jin1, MD, Can Li1, BS, Liang Kuang1, MD,
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Huabing Qi1, PhD, Zhenghong Ni1, MD, Zuqiang Wang1, MD, Xiaoqing Luo1, MS,
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Wanling Jiang1, MS, Hangang Chen1, BS, Shuai Chen, BS1, Fangfang Li1, BS, Bin
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Zhang1, BS, Junlan Huang1, BS, Ruobin Zhang1, BS, Kexin Jin1, BS, Xiaoling Xu3,
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PhD, Chuxia Deng3, PhD, Xiaolan Du1*, MS, Yangli Xie1**, MD, Lin Chen1***, MD,
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PhD
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Institution:
Department
of Rehabilitation
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Rehabilitation of Traumatic Injuries, State Key Laboratory of Trauma, Burns and
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Combined Injury, Trauma Center, Research Institute of Surgery, Daping Hospital,
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Third Military Medical University, Chongqing 400042, China.
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Bioengineering, Chongqing Institute of Technology, Chongqing 400050, China.
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Laboratory for the
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College of
Faculty of Health Sciences, University of Macau, Macau SAR 00853, China.
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Medicine,
†These authors contributed equally to this work.
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***
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Medicine, Laboratory for the Rehabilitation of Traumatic Injuries, State Key
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Laboratory of Trauma, Burns and Combined Injury, Trauma Center, Research
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Institute of Surgery, Daping Hospital, Third Military Medical University, Chongqing
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400042, China.
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Tel: 86-23-68757041. Fax: 86-23-68702991. E-mail: [email protected].
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**
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Laboratory for the Rehabilitation of Traumatic Injuries, State Key Laboratory of
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Trauma, Burns and Combined Injury, Trauma Center, Research Institute of Surgery,
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Daping Hospital, Third Military Medical University, Chongqing 400042, China.
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Tel: 86-23-68757042. Fax: 86-23-68702991.E-mail: [email protected].
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*
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Laboratory for the Rehabilitation of Traumatic Injuries, State Key Laboratory of
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Trauma, Burns and Combined Injury, Trauma Center, Research Institute of Surgery,
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Daping Hospital, Third Military Medical University, Chongqing 400042, China.
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Tel: 86-23-68757042. Fax: 86-23-68702991.E-mail: [email protected].
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Corresponding authors. Address: Y. Xie, Department of Rehabilitation Medicine,
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Corresponding authors. Address: X. Du, Department of Rehabilitation Medicine,
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Corresponding authors. Address: L. Chen, Department of Rehabilitation
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Abstract
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Objective: We previously reported that genetic ablation of Fgfr1 in knee cartilage
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attenuates the degeneration of articular cartilage in adult mice, which suggests that
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FGFR1 is a potential targeting molecule for osteoarthritis (OA). Here, we identified
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R1-P1, an inhibitory peptide for FGFR1 and investigated its effect on the
ACCEPTED MANUSCRIPT pathogenesis of OA in mice induced by destabilization of medial meniscus (DMM).
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Design: Binding ability between R1-P1 and FGFR1 protein was evaluated by ELISA
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and molecular docking. Alterations in cartilage were evaluated histologically. The
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expression levels of molecules associated with articular cartilage homeostasis and
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FGFR1
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immunohistochemistry. The chondrocyte apoptosis was detected by TUNEL assay.
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Results: R1-P1 had highly binding affinities to human FGFR1 protein, and efficiently
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inhibited ERK1/2 pathway in mouse primary chondrocytes. In addition, R1-P1
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attenuated the IL-1β induced significant loss of proteoglycan in full-thickness
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cartilage tissue from human femur head. Moreover, this peptide can significantly
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restore the IL-1β mediated loss of proteoglycan and type II collagen (Col II) and
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attenuate the expression of matrix metalloproteinase-13 (MMP13) in mouse primary
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chondrocytes. Finally, intra-articular injection of R1-P1 remarkably attenuated the
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loss of proteoglycan and the destruction of articular cartilage and decreased the
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expressions of ECM degrading enzymes and apoptosis in articular chondrocytes of
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mice underwent DMM surgery. Conclusions: R1-P1, a novel inhibitory peptide for
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FGFR1, attenuates the degeneration of articular cartilage in adult mice, which is a
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potential leading molecule for the treatment of OA.
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by
qRT-PCR,
Western
blotting
and
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analyzed
Key Words: FGFR1; Peptide; Osteoarthritis; Mice
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were
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signaling
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Introduction
ACCEPTED MANUSCRIPT Osteoarthritis (OA) is one of the most prevalent chronic joint diseases with
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progressive cartilage destruction and insufficient extracellular matrix synthesis.
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Currently, there are no effective biological therapies to prevent or treat OA, the most
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common final therapeutic option is the total joint replacement.
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In recent years, a variety of molecules and signaling pathways, such as mTOR, IHH
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and Wnt/β-catenin signaling have been found to be involved in cartilage homeostasis
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and OA development (1,2). Fibroblast growth factors (FGF) and their receptors
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(FGFRs) regulate the maintenance of cartilage and therefore, play a crucial role in
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joint homeostasis and OA development (3,4,5). FGF2 promotes cartilage catabolism
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via FGFR1-Ras-Raf-MEK1/2-ERK1/2 axis in human articular chondrocytes (6).
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FGFR1 and FGFR3 are highly expressed in human articular chondrocytes, and the
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expression ratio of FGFR1 to FGFR3 was significantly increased in OA chondrocytes
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compared to normal chondrocytes (7), which suggests the potential role of FGF
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signaling in cartilage homeostasis. FGF18 can promote chondrogenesis through
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FGFR3 and is a well-established anabolic growth factor for articular cartilage (8).
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Surgical DMM in mice is a well-established OA model (9,10), which is commonly
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used to screen biological and pharmacological agents for OA treatment (11,12).
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FGFR3 deficiency mice exhibited abnormal cartilage metabolism and early signs of
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OA, including increased cellular hypertrophy and loss of proteoglycan with
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upregulated expressions of MMP13, type X collagen (Col X) (13,14). Regarding to
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FGFR1, our previous study showed that conditional knockout (cKO) of Fgfr1 in
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cartilage attenuates articular cartilage degeneration, which is associated with
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ACCEPTED MANUSCRIPT down-regulation of MMP13 and increased proteoglycan synthesis. Thus, we expected
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that pharmacological down-regulation of the activity of FGFR1 or its downstream
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pathways may attenuate the development of OA. Since we previously found a short
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FGFR1-binding peptide (GPPDWHWKAMTH, named as R1-P1) using phage display
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and demonstrated that it can blunt the activity of ERK1/2 MAPK pathway. We thus
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tested whether it may exert a protective effect on OA in mouse model. Our results
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demonstrated that R1-P1 can attenuate the severity of OA induced by DMM in mice.
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Materials and Methods
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Sequence analysis and molecular docking
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Sequence analysis and molecular docking were carried out according to previous
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protocols (15,16), The peptide sequence was analyzed with Bioedit and ProtParam
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programs (http://au.expasy.org/tools/protparam.html). The crystal structure of FGFR1
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was accessed from RCSB database (ID: 5AM6) and prepared as receptor with
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biopolymer package of crystal. We determined the binding site based on the crystal
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structure of FGFR1. Then, the peptide was docked into receptor by surflex-dock
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method and the pose with highest score was used to generate the peptide/FGFR1
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complex. Molecular dynamics simulation was performed as following steps: (1)
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Minimization (1000 steps, restraint receptor); (2) Heating (NVE, 50 ps, 0K-300 K,
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restraint receptor); (3) Density adjustment (NPT, 50 ps, 300 K, constraint receptor); (4)
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Equilibrium (NPT, 500 ps, 300 K); (5) Production (NPT, 120 ns, 300 K). The
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molecular dynamics simulation was performed by amber.
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Peptide synthesis
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The peptide was synthesized at Shanghai China Peptides Co., Ltd. by solid-phase
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synthesis and purified by high-performance liquid chromatography (HPLC). Peptide
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with high purity grade (95.43%) was obtained after purification. The synthetic
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product was further confirmed by mass spectrometry analysis.
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ELISA assay for binding ability
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Binding affinities were checked by ELISA using previous protocol (17). Briefly,
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microtiter plates were coated with 20 mg/ml FGFR1 overnight at 4°C. After being
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blocked at 37°C for 1 h, the plates were washed six times. R1-P1 at different
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concentrations (0,1,5,10,15,25,50,100 mM) were added and incubated at room
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temperature for 1 h. After the plates were washed six times, anti-FGFR1 antibody
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were added and incubated at 37°C for 1 h. The plates were washed six times. Goat
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anti-rabbit immunoglobulin G (IgG)-HRP was then added and incubated at 37°C for
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30 min. The plates were washed, and color development was initiated with addition of
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3,3’,5,5’-tetramethylbenzidine (TMB; 100 ml/well of TMB). The reaction was
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terminated 20 min later by adding 20 µl stopping buffer, and the absorbance was
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measured at 450 nm.
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Cell culture
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Mouse embryonic carcinoma-derived cell line ATDC5 cells were cultured in 12-well
ACCEPTED MANUSCRIPT plates until 90% confluence, then cells were pre-incubated with FGF2 (10 ng/ml) for
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30 min, followed by treatment with peptide R1-P1 (0, 1, 10, 25 or 50mM) for 2 hours.
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PD173074 (Selleck), an inhibitor of FGFR1, was used as the positive control. Mouse
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primary chondrocytes were isolated as previously described (18). Chondrocytes were
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cultured in 12-well plates until 90% confluence and were pre-incubated with R1-P1
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(25 or 50 mM) alone for 2 hours, before treatment with IL-1β (10 ng/ml) for 24 hours.
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Culture of human articular cartilage explants
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This project received approval from the Ethical and Protocol Review Committee of
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Daping Hospital, Chongqing, and the culture were carried out according to the
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previous protocols (19). Briefly, human full-thickness femur head cartilage tissue was
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cut into 4 mm diameters, following 48 hours culture, explants were treated with IL-1β
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(20 ng/ml) and peptide R1-P1 (25 or 50 mM) every 3 days for 14 days under
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serum-free conditions (plus mini-ITS™ + Premix). Following 14 days of incubation,
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the medium was collected, and all explants were fixed in 4% paraformaldehyde
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overnight, followed by paraffin embedding.
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Dimethylmethylene Blue (DMMB) assay
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DMMB dye solution and sample digestion solution were prepared following previous
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protocol (20). Briefly, 250 µl of DMMB reagent was added to 40 µl of culture
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medium and the absorbance was measured at 525 nm immediately. Chondroitin
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sulfate (Sigma-Adrich) with several concentrations was used to plot a standard curve,
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which was used to measure the amount of aggrecan released into culture medium.
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Aggrecan released into the medium was normalized as mass of GAG per milliliter (ml)
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of culture medium.
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RNA extraction and quantitative real-time PCR (qPCR)
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Total RNA of treated mouse primary chondrocytes was isolated by TRIZOL reagent
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(Invitrogen). Real-time PCR was repeated at least three times using Mx3000P PCR
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machine (Stratagene) and SYBR Premix Ex TaqTM kit (Takara). The primers for
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genes measured were listed in Table 1. Expression values were normalized to
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Cyclophilin A.
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Western blotting
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Protein was extracted using ice-cold RIPA lysis buffer containing protease inhibitors
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(Roche). Equal amount of protein samples (30 ug) were dissolved by 10%
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SDS-PAGE gels and transferred onto a polyvinylidene difluoride membrane
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(Millipore). After being blocked with 8% nonfat milk in tris buffered saline tween
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buffer, the membrane was probed with antibody specific to ERK1/2 (1:1000 dilution;
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Cell Signaling Technology (CST)), p-ERK1/2 (1:1000 dilution; CST), MMP13
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(1:1000 dilution; Millipore), Aggrecan (1:1000 dilution; Abcam) and β-Actin
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(1:10000 dilution; Sigma) followed by chemiluminescent (Pierce) detection. All
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antibodies were solvated with antibody diluent (Beyotime). Intensity values were
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analyzed with the Image J software and were normalized to those of β-Actin. Each
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sample was analyzed three times and the mean gray values of immunoblot band were
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calculated (21).
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Surgical model of OA in mice
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All mice were maintained in the Animal Facility (specific pathogen free) of the
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Daping Hospital (Chongqing, China). All procedures were approved by the
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Institutional Animal Care and Use Committee of Daping Hospital (Chongqing, China).
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8-week-old male C57BL/6J mice (25-30 g) were purchased from the Beijing HFK
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Bioscience Co. Ltd.. According to previous protocol (22), DMM surgery was
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performed on the right knee joints, sham surgery was performed with medial
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capsulotomy only on the left knee joints of mice. Mice divided into groups at
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randomisation. After 1 week, mice in treatment group received R1-P1 (once a week,
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15µg/week) postoperatively via articular cavity injection for 4, 8 and 12 weeks. In
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control mice, their articular cavities were injected with physiological saline only.
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Histologic assessment
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Microscopic scoring of mouse cartilage degeneration was performed in accordance
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with the recommendations of the Osteoarthritis Research Society International
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(OARSI) (23). Briefly, the joints were fixed in 4% paraformaldehyde, then decalcified
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in 20% formic acid, and embedded in paraffin. Then serial sagittal sections were
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obtained across the entire knee joint. Histologic grading of cartilage degeneration was
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performed using the OARSI recommended subjective scoring system (on a scale of
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0-6) (23). In addition, cartilage aggrecan depletion was scored (on a scale of 0-5) as a
200
complement measure of cartilage degeneration
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agreement between the two blinded observers (Quan Wang and Wei Xu).
(24). There was a high level of
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Synovitis score
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Mouse articular joint sections were stained with hematoxylin and eosin (H&E) to
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assess the grade of synovitis. Scoring was performed by two blinded observers (25).
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Three features of synovitis (enlargement of lining cell layer, cellular density of
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synovial stroma, leukocytic infiltrate) were semiquantitatively evaluated (from 0,
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absent to 3, strong) and each feature was graded separately. The sum provided the
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synovitis score, which is interpreted as follows: 0-1, no synovitis; 2-4, low-grade
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synovitis; 5-9, high grade synovitis.
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Immunohistochemistry (IHC)
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IHC were carried out using SP-9000 Histostain-Plus kits (Zsgb Bio) as previous
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protocols (14). Incubated with antibody specific to Aggrecan (1:200 dilution; Abcam),
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Col II (1:400 dilution; Chondrex), Adamts5 (1:200 dilution; Abcam), MMP13 (1:200
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dilution; Abcam), Col X (1:200 dilution; Millipore) and FGFR3 (1:200 dilution; Santa
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Cruz Biotechnology) followed by diaminobenzidine (DAB) kit detection, all
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antibodies were solvated with antibody diluent (Origene).
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TUNEL assay
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Apoptotic cells in articular cartilage were detected by TdT mediated dUTP-X nick
ACCEPTED MANUSCRIPT 222
end labeling (TUNEL) assay using in situ cell death detection kit (peroxidase (POD))
223
of Roche according to the manual.
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Statistical analysis
226
We used 5-10 animals in each group for in vivo studies and three independent
227
biological replicates for in vitro studies. Individual cell experiments were also
228
performed in triplicate as technical replicates. The sample sizes for the groups of
229
interest were big enough based on a power of 80% and p < 0.05 using power and
230
sample size calculation online software (http://powerandsamplesize.com/Calculators/)
231
(Supplementary Table 1). All tested variables were tested for normality and
232
homogeneity using Shapiro-Wilk test and Levine’s test, respectively. Quantitative
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data of histologic grading of cartilage degeneration, IHC analysis of MMP13, ColX,
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FGFR3 and TUNEL assay were compared by using the non-parametric
235
Mann-Whitney U test. For the other data, all the tested variables did not violate the
236
assumptions of normality and equal variance. Differences between two groups were
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evaluated using Student’s unpaired t-test, and comparisons of multiple groups were
238
evaluated using analysis of variance (ANOVA) followed by Tukey’s test. All data
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were analyzed using GraphPad Prism v.6.01 software and depicted in univariate
240
scatter plots as previously described (26). P < 0.05 was considered to be statistically
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significant.
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Results
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R1-P1 binds to human FGFR1 protein and inhibits MAPK/ERK signaling
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pathway
ACCEPTED MANUSCRIPT All 11 clones with high binding affinity to FGFR1 protein shared the
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“GPPDWHWKAMTH” amino acid sequences, which was named as peptide R1-P1.
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ProtParam programs (http://web.expasy.org/protparam/) were then applied to analyze
249
the sequence and predict its properties. The molecular weight of the R1-P1 is 1462.65,
250
theoretical isoelectric point of the peptide is 6.92, grand average of hydropathicity of
251
the R1-P1 is -1.35 (Table 2).
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To evaluate the binding ability between peptide R1-P1 and the extracellular region of
253
FGFR1 (5AM6) protein, we first determined the binding site based on the crystal
254
structure of FGFR1. Results indicate that there was a large groove in the surface of
255
FGFR1 (Figure 1A), which could accommodate the peptide. Then the peptide was
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docked into binding site by surface-dock. The best pose, total score and C score were
257
7.87 and 5 respectively, was combined with FGFR1 to generate FGFR1/pep complex.
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To achieve a stable structure, a 20 nanoseconds MD simulation was performed.
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According to the RMSD plot (Figure 1B), the RMSD was fluctuated close to 2.5Å
260
after 70 nanoseconds, which means the structure of complex was reasonable. The
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peptide could bind to FGFR1 stably (Figure 1C) relied on hydrogen bonds,
262
electrostatic and hydrophobic interaction (Figure 1D). Figure 1D shows that there
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were 8 hydrogen bonds between peptide with residue Arg493, Arg512, Asp513, Leu484,
264
Gly490, Asp527, Glu571, and Asn568.
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We examined the binding affinity of peptide R1-P1 at different concentrations
266
(0,1,5,10,15,25,50,100 mM) to FGFR1, ELISA result showed that R1-P1 can bind to
267
FGFR1 protein in a dose-response manner, and the binding affinities were not further
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ACCEPTED MANUSCRIPT increased from 50 to 100 mM (Figure 1E). In addition, ELISA results demonstrated
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that R1-P1 strongly bound to FGFR1, although it also slightly bound to FGFR2 and
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FGFR3 (Figure 1F). FGFs/FGFRs exert their diverse biological effects through
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multiple intracellular signaling pathways, among which the MAPK pathway has been
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found to inhibit chondrocyte differentiation and matrix synthesis (6). We examined
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whether R1-P1 could affect the protein level of total ERK1/2 and phosphorylated
274
ERK1/2 induced by FGF2. Furthermore, western blotting result showed that
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FGF2-mediated ERK1/2 phosphorylation were partially blocked by R1-P1 at 25 and
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50mM concentration (Figure 1G), indicating that R1-P1 inhibited ERK1/2 MAPK
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signaling pathway, a classic downstream signaling pathway of FGFR1 in
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chondrocytes.
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R1-P1 attenuates IL-1β induced proteoglycan loss in adult human articular
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cartilage
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To determine the effect of R1-P1 on proteoglycan loss, we treated cultured healthy
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human femoral head cartilage explant with IL-1β (20 ng/ml) with or without the
284
R1-P1 (25 or 50 mM) for 14 days. Safranin O staining showed that IL-1β induced
285
significant proteoglycan loss, which was partially blunted by peptide R1-P1 treatment
286
(Figure 2A).
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The GAG released into the culture medium was significantly increased after IL-1β
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treatment (P < 0.001), which were markedly decreased after being incubated with 25
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or 50 mM R1-P1 (all P < 0.001, Figure 2B), respectively. These data were consistent
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with the results of Safranin O staining. Together, these results indicate that R1-P1 can
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attenuate the catabolic effect of IL-1β on chondrocytes.
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R1-P1 inhibits the phosphorylation of ERK1/2 and rescues IL-1β induced
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dysregulated ECM metabolism in mouse primary chondrocytes
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To explore the mechanisms for the anti-catabolic effects of R1-P1 on chondrocytes,
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real-time qPCR and western blotting were performed to examine the effects of R1-P1
297
on the synthesis and breakdown of extracellular matrix in mouse primary
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chondrocytes. The mRNA expressions of Adamts5 (P < 0.001) and MMP13 (P <
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0.001) were significantly increased in mouse primary chondrocytes after IL-1β
300
treatment. R1-P1 treatment resulted in a marked reduction in the expressions of these
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two catabolic markers (P < 0.001 and P = 0.006, respectively). Meanwhile, the
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mRNA expressions of Aggrecan (P < 0.001) and Col II (P < 0.001) were decreased
303
after IL-1β treatment, which were partially recovered (P < 0.001 and P < 0.001,
304
respectively) by peptide R1-P1 (Figure 3A).
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Western blotting results showed that following the stimulation of IL-1β, ERK1/2
306
MAPK signaling pathway was activated, the protein levels of Aggrecan was
307
decreased and MMP13 was increased (all P < 0.001). Treatment with 25 or 50 mM
308
R1-P1 significantly decreased the protein levels of MMP13 and phosphorylated
309
ERK1/2, while up-regulated the protein levels of Aggrecan in mouse primary
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chondrocytes (all P < 0.001, Figure 3B and 3C). Taken together, these results
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demonstrate that R1-P1 may help to maintain the homeostasis of mouse primary
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chondrocytes.
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R1-P1 delays articular cartilage degradation in a mouse OA model
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We determined the effect of R1-P1 on the development of OA in mice induced by
316
DMM. There is no remarkable toxicity observed in the R1-P1 treatment group, as
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evidenced by no signs of abnormal gross phenotypes including weight loss during
318
observation period. Histologic examination at 4 weeks after DMM surgery revealed
319
the loss of proteoglycan content (data not shown). Furthermore, 8 weeks after DMM
320
mice showed an early OA-like manifestations including loss of proteoglycan content
321
and cartilage tissue (Figure 4A-B). The OA-like phenotype became more profound in
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mice at 12 weeks after DMM surgery, which exhibited as more severe destruction of
323
the articular cartilage associated with greater loss of proteoglycan content (Figure
324
5A-B). However, intra-articular injection of R1-P1 significantly decreased the
325
cartilage destruction and proteoglycan loss in mouse knee joint after DMM surgery
326
(Figure 4A-B and 5A-B).
327
Accordingly, the OARSI scores were significantly increased in mice at 8 and 12
328
weeks after DMM surgery compared to those of sham operation mice (all P < 0.001,
329
Figure 4C-F and 5C-F). R1-P1 treated mice had a significantly lower sum score and
330
maximal score in medial femoral condyle and medial tibial plateau than those in the
331
vehicle treated mice at 8 weeks (P = 0.001, P = 0.014, P = 0.002 and P = 0.008,
332
respectively, Figure 4C-F) and 12 weeks (P < 0.001, P = 0.002, P = 0.021 and P =
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0.006, respectively, Figure 5C-F) following DMM surgery. In addition, H&E
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ACCEPTED MANUSCRIPT staining and scoring of synovitis showed that treatment with R1-P1 attenuated the
335
severity of synovitis at 8 weeks (P = 0.002, Figure 4G-H) and 12 weeks (P < 0.001,
336
Figure 5G-H) following DMM surgery compared with vehicle. Taken together, these
337
results demonstrate that intra-articular injection of R1-P1 partially delayed OA
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development.
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R1-P1 attenuates ECM loss and chondrocyte hypertrophy and apoptosis in knee
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joints of DMM mice
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To explore the cellular mechanisms underlying the delayed progression of cartilage
343
degradation in peptide R1-P1 treated mice with DMM, we performed IHC to examine
344
the expressions of Aggrecan, Col II, Col X, Adamts5 and MMP13. The results
345
showed that the expressions of Adamts5, MMP13 were significantly increased and the
346
expressions of Aggrecan, Col II were decreased in the articular cartilage after DMM
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surgery (Figure 6A-D). In addition, after DMM surgery, the ratio of cells with
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positive Col X in the articular cartilage was significantly increased (Figure 6E).
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However, peptide R1-P1 largely lowered the ratio of Adamts5, MMP13, Col X
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positive cells and significantly increased the ratio of Aggrecan, Col II positive cells in
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the articular cartilage after DMM surgery compared to vehicle treatment (all P <
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0.001, Figure 6H). Furthermore, the ratio of FGFR3 positive cells was
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down-regulated in the articular cartilage when OA was induced by DMM surgery.
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However, inhibition of FGFR1 activity led to increased ratio of FGFR3 positive cells
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in the articular cartilage (P < 0.001, Figure 6F and Figure 6H).
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ACCEPTED MANUSCRIPT Since it has been reported that chondrocyte apoptosis is strongly related to the OA
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development (27,28). We performed TUNEL assay to analyze the apoptosis of
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chondrocytes in knee joints. Our results showed that peptide R1-P1 treatment largely
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decreased the apoptosis of chondrocytes in the articular cartilage 8 weeks after DMM
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surgery compared to vehicle treatment (as shown in red arrows, P < 0.001, Figure 6G
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and Figure 6H). Taken together, these results demonstrate that local intra-articular
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injection of R1-P1 reduced articular cartilage damage, at least in part, through its
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inhibition on the hypertrophy and apoptosis process of articular chondrocytes.
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Discussion
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OA is a degenerative disease characterized with cartilage degradation, synovial
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inflammation and dysregulated subchondral bone remodeling. The mechanisms
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underlying OA is not well understood. Currently, there is no effective biological
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treatment for OA. Finding targeting cells and/or molecules for effective therapies of
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OA is urgently needed.
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Decades of studies have demonstrated that FGFs/FGFRs play vital roles in cartilage
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homeostasis and OA development (11). We previously demonstrated that conditional
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deletion of Fgfr1 in mouse articular chondrocytes delays the progression of cartilage
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degeneration (29), suggesting that pharmacological down-regulation of FGFR1
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activity is a potential therapy for OA. Over the past decades, several FGFR inhibitors,
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such as PD173074, SU5402 and PD166866 have been developed as candidates for the
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treatment of FGF signaling related diseases (30). Because of the low-specificity and
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ACCEPTED MANUSCRIPT toxicity, majority of them failed to enter clinical application. Peptides can be easily
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synthesized and have low immunogenicity. Furthermore, peptide drugs have been
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successfully used in clinic such as a1-thymosin (31). Here, we, for the first time,
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provide evidence showing that a novel FGFR1 antagonist, peptide R1-P1, has high
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binding affinity to FGFR1 protein and can alleviate cartilage degradation in a mouse
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model of OA.
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The major characteristic of OA is an imbalance between anabolic effects mediated
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extracellular matrix synthesis and catabolic effects regulated matrix degradation (32).
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We demonstrated that R1-P1 inhibited the proteoglycan degradation induced by IL-1β
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in cultured human cartilage explants. Furthermore, we demonstrated that
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R1-P1attenuated the IL-1β induced up-regulation of the mRNA levels of Adamts5 and
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MMP13 and down-regulation of Aggrecan and Col II mRNA levels, in mouse articular
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chondrocytes. We speculate that the mechanism for the protective effect of R1-P1 on
391
OA is mainly via its inhibition on the activities of ERK1/2 MAPK signaling pathway.
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Chondrocyte hypertrophy, which can result in increased metabolic activity of articular
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chondrocytes and trigger unbalanced cartilage homeostasis favoring degenerative
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changes (33). We found that R1-P1 treatment reduced the expressions of MMP13 and
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Col X, markers for hypertrophic chondrocytes, in mouse OA model induced by DMM
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(12,34). These findings revealed that intra-articular injection of R1-P1 prevented
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articular chondrocytes from hypertrophy, which may contribute to the inhibitory
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effect of R1-P1 on OA development. The inhibitory effect of R1-P1 on chondrocyte
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hypertrophy is consistent with our previous findings showing that Fgfr1 deficiency in
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ACCEPTED MANUSCRIPT chondrocytes decreased chondrocyte hypertrophy in articular cartilage (29). FGF
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signaling pathway is highly associated with chondrocyte apoptosis (35).
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Gain-of-function mutation of Fgfr3 promotes chondrocyte apoptosis in thanatophoric
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dysplasia (TD) mice (36). Fgf2 transgenic mice exhibit chondrodysplasic phenotype
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resulting from both reduced proliferation and increased apoptosis of growth plate
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chondrocytes (37). FGF18 markedly reduces chondrocyte apoptosis and enhances the
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repair response of cartilage following cartilage insult (38). In current study, we
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showed that pharmacologically inhibiting FGFR1 activity by R1-P1 decreased
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chondrocyte apoptosis in mouse OA model, indicating that R1-P1 positively
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maintains cartilage homeostasis partially through downregulating chondrocyte
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apoptosis.
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During OA development, the expression ratio of FGFR1 to FGFR3 was up-regulated
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in articular chondrocytes from patients with OA (7), suggesting that down-regulation
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of FGFR3 is at least partially responsible for the development of OA in FGFR1
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deficiency related OA. We found that intra-articular injection of R1-P1 can increase
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ratio of FGFR3 positive cells in mouse OA model.
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Whether R1-P1 have the same effects in other animal model need to be studied. In
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addition, inflammation also plays essential role in the pathogenesis of OA by inducing
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catabolic effects on ECM, etc. Inhibition of inflammation leads to attenuated OA
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severity and progression (39,40). As a classic signaling pathway in development and
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homeostasis of a variety of tissues/organs, FGF signaling appears to have crosstalk
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with inflammation related signaling pathways. For example, inhibition of FGF23
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ACCEPTED MANUSCRIPT might serve as a novel anti-inflammatory strategy in chronic obstructive pulmonary
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disease (COPD) (41). So, we speculate that R1-P1 and anti-inflammatory drug may
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have overlapping but different effects on attenuating OA progression.
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In summary, we found a novel inhibitory peptide, R1-P1, for FGFR1 that can
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maintain the ECM of chondrocytes through its down-regulation of matrix-degrading
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enzymes and prevent articular chondrocytes from hypertrophy and apoptosis, which
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will facilitate the development of effective therapeutic agents for OA.
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Author contributions
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All authors listed have read and approved all versions of the manuscript.
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Conception and design: Qiaoyan Tan, Xiaolan Du, Yangli Xie, Lin Chen.
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Analysis and interpretation of the data: Qiaoyan Tan, Quan Wang, Wei Xu, Yuanqiang
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Wang, Huabing Qi, Yangli Xie.
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Drafting of the article: Qiaoyan Tan, Yangli Xie, Lin Chen.
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Critical revision of the article for important intellectual content: Chuxia Deng, Yangli
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Xie, Lin Chen.
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Final approval of the article: Qiaoyan Tan, Xiaolan Du, Yangli Xie, Lin Chen.
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Provision of study materials or patients: Qiaoyan Tan, Shuo Huang, Zhenhong Ni,
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Liang Kuang, Nan Su, Xiaolan Du.
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Statistical expertise: Qiaoyan Tan, Liang Kuang, Wei Xu.
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ACCEPTED MANUSCRIPT Administrative, technical, or logistic support: Yin Zhu, Wanling Jiang, Hangang Chen,
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Shuai Chen, Fangfang Li, Bin Zhang, Junlan Huang, Ruobing Zhang, Kexin Jin, Can
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Li, Min Jin, Xiaolan Du.
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Collection and assembly of data: All authors.
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Fundings
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This work was supported by Special Funds for Major State Basic Research Program
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of China (973 program) (No.2014CB942904), National Natural Science Foundation
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of China (No. 81530071, No.81472074), State Key Laboratory of Trauma, Burn and
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Combined Injury, China (No.201601).
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Conflict of interest
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The authors declare that they have no competing interests.
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Figure Legends
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Figure 1. Specific binding of the R1-P1 to FGFR1. (A) The binding site base
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crystal structure of FGFR1. (B) R1-P1 was docked into binding site by surface-dock.
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(C) R1-P1 could bind to FGFR1 stably. (D) Showed there were 8 hydrogen bonds
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between peptide with residue. (E) Binding affinities of R1-P1 at different
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concentrations (0,1,5,10,15,25,50,100 mM) to FGFR1 revealed by ELISA (n = 3). (F)
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Binding affinities of R1-P1 to FGFRs measured by ELISA (n = 6). (G) Results
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showed peptide R1-P1 inhibited phosphorylation of ERK1/2 induced by FGF2 (n = 3).
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Data in were expressed as the mean ± 95% confidence intervals.
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Figure 2. Effects of R1-P1 on proteoglycan loss in adult human articular
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cartilage. Full thickness cartilage samples from human femur were cultured in the
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absence or presence of IL-1β (20 ng/ml) and in the presence or absence of R1-P1 (25
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or 50 mM) for 14 days. (A) Sections were stained with Safranin O to identify the
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cartilage proteoglycan loss (n = 3). (B) Culture medium were collected, and the
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amount of GAG released into the medium were quantified by DMMB assay. GAG
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released into the medium was normalized as mass of GAG per ml of culture medium
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(n = 9). Scale bar: 100 µm. Data were expressed as the mean ± 95% confidence
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intervals.
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Figure 3. Effects of R1-P1 on mRNA levels of MMP13, Adamts5, Col II and
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Aggrecan and on the protein levels of MMP13, Aggrecan and p-ERK1/2 in IL-1β
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treated mouse primary chondrocytes. (A) Total RNA was isolated, and levels of
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mRNA of the catabolic markers (MMP13 and Adamts5) and chondrocyte markers
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(Col II and Aggrecan) were detected by Real-time qPCR (n = 9). (B) Cell lysates
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were analyzed by western blotting using antibodies specific for MMP13, Aggrecan
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and p-ERK1/2 (n = 3). (C) The signal intensities of MMP13, Aggrecan and
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phospho-ERK1/2 were quantitatively analyzed (n = 3). Data were expressed as the
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mean ± 95% confidence intervals.
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Figure 4. Histologic features of mouse articular at 8 weeks following DMM
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surgery. (A) The articular cartilage was stained with Safranin O-fast green at 8 weeks
622
after surgery to assess the extent of articular cartilage degeneration. (B) Expansion of
623
the region occupied by articular cartilage. (C-F) OARSI scoring system showed more
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severe articular cartilage destruction in the medial femur and tibia of mice at 8 weeks
625
following DMM surgery compared with sham operation (n = 8 or 10 mice per group).
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(G-H) H&E staining and scoring of synovitis showed that treatment with R1-P1 can
627
attenuate the severity of synovitis after DMM surgery compared with vehicle (n = 8
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or 10 mice per group). MFC: medial femoral condyle; MTP: medial tibial plateau.
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Scale bar: 100 µm. Data were expressed as the mean ± 95% confidence intervals.
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Figure 5. Histologic features of mouse articular cartilage at 12 weeks following
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DMM surgery. (A) The articular cartilage was stained with Safranin O-fast green at
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12 weeks after surgery to assess the extent of articular cartilage degeneration. (B)
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Expansion of the region occupied by articular cartilage. (C-F) OARSI scoring system
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showed more severe articular cartilage destruction in the medial femur and tibia of
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mice at 12 weeks following DMM surgery compared with sham operation (n = 5 mice
637
per group). (G-H) H&E staining and scoring of synovitis showed that treatment with
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peptide R1-P1 can attenuate the severity of synovitis after DMM surgery compared
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with vehicle (n = 5 mice per group). MFC: medial femoral condyle; MTP: medial
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tibial plateau. Scale bar: 100 µm. Data were expressed as the mean ± 95% confidence
641
intervals.
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Figure 6. Effects of peptide R1-P1 on cartilage ECM levels and degradation
644
enzymes and chondrocyte apoptosis in mice at 8 weeks after DMM. IHC analysis
645
of Aggrecan (A), Col II (B), Adamts5 (C), MMP13 (D), Col X (E), FGFR3 (F)
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protein expression in articular cartilage of mice from either sham operation group or
647
surgery group at 8 weeks after DMM surgery with intra-articular injection of peptide
648
R1-P1 or vehicle (n = 3). (G) TUNEL assay was performed on knee joints to measure
649
chondrocyte apoptosis either sham operation or at 8 weeks after DMM surgery with
650
intra-articular injection of peptide R1-P1 or vehicle (as shown in red arrows) (n = 3).
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(H) The percentage of cells that are positive for MMP13, Col X, FGFR3 and TUNEL
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in the articular cartilage were calculated (n = 3). Scale bar: 100 µm. Data were
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expressed as the mean ± 95% confidence intervals.
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ACCEPTED MANUSCRIPT Table 1. Primers and sequences for real-time PCR analysis. Forward primer
5'-CTGTGGAGGTCACTGTAGACT-3'
Adamts5
5'-GGAGCGAGGCCATTTACAAC-3'
5'-CGTAGACAAGGTAGCCCACTTT-3'
Col II
5'-CTGGTGGAGCAGCAAGAGCAA-3'
5'-CAGTGGACAGTAGACGGAGGAAAG-3'
Aggrecan
5'-CCTGCTACTTCATCGACCCC-3'
5'-AGATGCTGTTGACTCGAACCT-3'
Cyclophilin A
5'-CGAGCTCTGAGCACTGGAGA-3'
5'-TGGCGTGTAAAGTCACCACC-3'
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5'-CTTCTTCTTGTTGAGCTGGACTC-3'
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Reverse primer
Table 2. Properties of peptides displayed by positive phages
Clones
Sequence
Molecular weight
Theoretical pI a
GRAVY b
R1-P1
11
GPPDWHWKAMTH
1462.65
6.92
-1.35
PI, Isoelectric Point.
b
GRAVY, Grand Average of Hydropathicity.
AC C
EP
TE D
a
M AN U
Heptapeptide