Tissue inhibitor of metalloproteinase 1 suppresses growth and differentiation of osteoblasts and differentiation of osteoclasts by targeting the AKT pathway

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Tissue inhibitor of metalloproteinase 1 suppresses growth and differentiation of osteoblasts and differentiation of osteoclasts by targeting the AKT pathway Yongming Xia,∗∗, Hui Huangb, Zheng Zhaoa, Jinfeng Maa, Yan Chenc,∗ a

Department of Orthopaedics, Affiliated Hospital of Qingdao University, Qingdao, China Department of Anesthesia, Affiliated Hospital of Qingdao University, Qingdao, China c Princess Margaret Cancer Center, University Health Network, Toronto, Canada b

A R T I C LE I N FO

A B S T R A C T

Keywords: TIMP1 MMP AKT Osteoblast Osteoclast

Tissue inhibitor of metalloproteinase 1 (TIMP1) has various biological activities including the regulation of cell growth and differentiation. However, its role in bone homeostasis and remodeling remains poorly understood. In this study, we investigate the effects of TIMP1 on osteoblast and osteoclast activity at both cellular and molecular level using siRNA-mediated knockdown technique. Our results show that knockdown of TIMP1 stimulates proliferation and survival, but decreases apoptosis in osteoblastic MC3T3-E1 cells, suggesting that TIMP1 inhibits cell growth. TIMP1 also dampens differentiation of committed osteoblasts, as well as osteoblastogenesis of bone marrow-derived mesenchymal stem cells (BMSCs). We further show that the modulation of TIMP1 on osteoblast activity is independent of its MMP inhibition. Importantly, we uncover that TIMP1 suppresses osteoblast growth and differentiation by targeting the AKT pathway, and this is associated with TIMP1-mediated induction of PTEN via its binding to the cell surface receptor CD44. Therefore, our results highlight a novel TIMP1/CD44/PTEN/AKT signaling nexus that functions as a suppressor of osteoblast activity. Moreover, we show that TIMP1 also inhibits osteoclast differentiation in osteoclast precursor RAW 264.7 cells by targeting the AKT. In conclusion, our results demonstrate that TIMP1 can act as a suppressor of growth and differentiation of osteoblasts and differentiation of osteoclasts through the negative regulation of the AKT pathway. We propose that TIMP1 may serve as a potential target for low bone mass-related skeletal diseases, such as osteoporosis.

1. Introduction Bone is composed predominantly by collagen-rich, mineralized extracellular matrix (ECM), which is synthesized and degraded by osteoblasts and osteoclasts, respectively. In light of the dynamic remodeling of bone throughout the lifespan, coordinated osteoblastosteoclast function and regulation of bone matrix turnover are crucial to skeletal health in humans. Metalloproteinases including matrix metalloproteinases (MMPs) and a disintegrin and metalloproteinases (ADAMs) control appropriate assembly, organization, composition, as well as regulation of bone ECM during bone homeostasis and remodeling [1].

Tissue inhibitors of metalloproteinases (TIMPs) are widely distributed in the animal kingdom, and the human genome contains four paralogous genes encoding TIMP1 to 4. TIMPs have a broad influence on tissue microenvironment due to their capacity to inhibit activated MMPs and ADAMs [2] and are well-positioned to simultaneously affect matrix scaffolds and growth factors and cytokine bioavailability [3]. TIMPs also participate in the regulation of various biological activities such as cell growth, apoptosis, and differentiation that are independent of its metalloproteinase inhibitory activity [2]. In-depth knowledge of the TIMPs function in the bone and their associated signaling pathways will help advance key concepts governing skeletal growth, homeostasis, and aging.

Abbreviations: TIMP, tissue inhibitor of metalloproteinase; MMP, matrix metalloproteinase; ADAM, a disintegrin and metalloproteinase; ALP, alkaline phosphatase; BMD, bone mineral density; BMSC, bone marrow-derived mesenchymal stem cell; PI3K, phosphatidylinositol 3-kinase; PIP2, phosphatidylinositol (4,5)-bisphosphate; PIP3, phosphatidylinositol (3,4,5)-triphosphate; PTEN, phosphatase and tensin homolog deleted from chromosome 10; qPCR, quantitative real-time PCR; siRNA, short interfering RNA; TRAP, tartrate-resistant acid phosphatase ∗ Corresponding author: TMDT Building, 101 College Street, Toronto, Canada. ∗∗ Corresponding author: 16 Jiangsu Road, Qingdao, China. E-mail addresses: [email protected] (Y. Xi), [email protected] (Y. Chen). https://doi.org/10.1016/j.yexcr.2020.111930 Received 26 September 2019; Received in revised form 17 February 2020; Accepted 26 February 2020 0014-4827/ © 2020 Elsevier Inc. All rights reserved.

Please cite this article as: Yongming Xi, et al., Experimental Cell Research, https://doi.org/10.1016/j.yexcr.2020.111930

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Scientific) and routinely tested for mycoplasma with Mycoplasma Detection Kit (ATCC). All experimental animal procedures were reviewed and approved by the Animal Care Committee of Qingdao University. For transfection, a set of 3 different target-specific siRNA oligo duplexes of mouse TIMP1 or CD44 gene was purchased from MyBioSource. The 3 duplexes were pooled together and transfected into MC3T3-E1 and RAW 264.7 cells, respectively, using Lipofectamine 2000 Transfection Reagent (Invitrogen). Other MC3T3-E1 or RAW 264.7 cells transfected with the siRNA Negative Control (MyBioSource) were used as control cells.

We have recently reported that homozygous, whole-body deletion of all four TIMP genes in transgenic mice results in defective osteoblastogenesis and osteoclast over-activity, triggering loss of bone mass [4]. However, it remains to be elucidated how individual TIMP molecule affects bone homeostasis. As a major regulator during ECM degradation, TIMP1 is ubiquitously expressed in numerous human cells and tissues. In the skeletal system, the expression of TIMP1 can be detected in osteoblasts, osteocytes, chondrocytes, as well as osteoclasts [5,6], implying that TIMP1 is implicated in bone homeostasis and remodeling. Using transgenic mice that overexpress TIMP1 in osteoblasts, Geoffroy et al. reported a slight increase of bone mineral density (BMD) and trabecular bone volume only in the 1-month-old female mice (but not male mice), and this seems attributed to decreased bone formation and bone resorption, leading to a low bone turnover rate [7]. The same research group further reported that the TIMP1 transgene prevented estrogen deficiency-induced bone loss, presumably resulting from reduced bone resorption activity [8]. TIMP1 was also reported to inhibit osteogenic differentiation of human bone marrow-derived mesenchymal stem cells (hBMSCs) by targeting the Wnt/β-catenin pathway [9] and suppress apoptosis of osteoblasts independent of its MMP inhibition [10]. Even though these studies reveal an important role of TIMP1 in the bone microenvironment, it remains poorly understood how this tissue inhibitor affects osteoblast and osteoclast activity, particularly the signaling pathway whereby TIMP1 regulates the bone cells. The phosphatidylinositol 3-kinase (PI3K)/AKT signaling pathway plays a central role in the control of cell survival and growth throughout the body [11]. PI3K leads to phosphorylation of PI (4,5)bisphosphate (PIP2) to PI (3,4,5)-triphosphate (PIP3), the latter acts as a second messenger to activate AKT [12]. Activation of AKT regulates several target proteins involved in the control of cell proliferation, apoptosis, survival, invasion, migration, and other processes [13]. Concerning bone, there is an increasing amount of evidence that this pathway functions as a crucial regulator of osteoblasts and osteoclasts by promoting their differentiation and survival to maintain bone mass and turnover [14–16]. However, it is still unknown whether the AKT pathway is implicated during TIMP-mediated regulation of bone homeostasis and remodeling. In this study, we aim at investigating the role of TIMP1 in osteoblasts and osteoclasts at both the cellular and molecular level. We try to figure out the modulation of TIMP1 on cell growth and differentiation, focusing on the signaling mechanism underlying TIMP1-mediated regulating effect. We hypothesize that TIMP1 exerts its inhibitory effect in osteoblasts and osteoclasts by targeting the AKT signaling pathway.

2.2. Proliferation, apoptosis & survival assay Cell proliferation was determined using the MTT-based Cell Proliferation Kit (Roche). Briefly, MC3T3-E1 or RAW 264.7 cells were plated in a series of 96-well plates at 5x103/well in 100 μl of culture medium. For treatment, 10 μM batimastat (BB94, a potent pan-MMP inhibitor, ApexBio) or 10 μM AKTi-1/2, (an AKT inhibitor, Abcam) in a volume of 1 μl was added to the culture every two days without changing the medium. After every 24 h, 10 μl of MTT labeling reagent was added to each well and incubated for 2 h. The formazan product in cells was dissolved in 100 μl of the solubilization solution, and absorbencies were read at OD 570 nm on a microtiter plate reader. The assay was repeated every 24 h for 7 days. For apoptosis assay, MC3T3-E1 cells were cultured under serum starvation condition (0.5% FBS) for 24 h, treated with 10 μM BB94 or 10 μM AKTi-1/2. Apoptotic activity was determined with Caspase-3 Colorimetric Assay Kit (BD Biosciences) according to the manufacturer's protocol. Cell survival was examined by determining the cell viability using CellTiter-Glo Luminescent Assay Kit (Promega). Briefly, MC3T3E1 cells were treated with 10 μM BB94 or 10 μM AKTi-1/2 for 24 h. Then 2x104, 4x104 and 8x104 cells in 100 μl medium were added in 96well-plate. An equal volume of CellTiter-Glo reagent was added to the medium and mixed for 2 min on a shaker to induce cell lysis. Luminescence was recorded after incubation for 10 min. 2.3. MMP & ADAM10/17 activity assay MMP activity in MC3T3-E1 or BMSC cell lysates was determined with MMP Activity Assay Kit (Abcam) using the manufacturer's protocol. Briefly, 10 μM BB94 or 100 ng/ml recombinant human TIMP1 (rhTIMP1, Perprotech) in a volume of 1 μl was added to the MC3T3-E1 or BMSCs medium and incubated for 24 h. Cell lysates were harvested using a modified RIPA buffer. Then, 50 μl cell lysate was mixed with the same volume of MMP Green Substrate solution for 1 h. The fluorescence intensity was measured at 490/525 nm excitation/emission. For combined ADAM10/17 activity assay, cell lysates were pre-treated with GW280264X (a dual inhibitor for both ADAM10 and ADAM17) at 10 μM for 2 h and then mixed with SMO-3670-PI (substrate for both ADAM10 and ADAM17) at 5 μM. The generation of the fluorescent cleavage product was monitored in a spectral fluorometer at 355/ 510 nm excitation/emission.

2. Materials and methods 2.1. Cell culture & transfection Murine osteoblastic MC3T3-E1 cells (ATCC) were cultured in growth medium containing Dulbecco's Modified Eagle's Medium (DMEM, Invitrogen), supplemented with 2% L-glutamine, 10% fetal bovine serum (FBS, Invitrogen), 100 units/ml penicillin and 100 ng/ml streptomycin, in an incubator under standard conditions (37 °C, 5% CO2). For osteoblast differentiation, MC3T3-E1 cells were cultured in osteogenic medium containing DMEM-10% FBS, antibiotics, 50 μg/ml ascorbic acid, 5 mM sodium β-glycerophosphate, 1 nM dexamethasone, and 100 ng/ml recombinant human BMP2 (Prospec). Osteoclast precursor RAW 264.7 cells (ATCC) were cultured in αMEM (Invitrogen) with 10% FBS and antibiotics. Primary BMSCs were established as previously reported [17]. Briefly, long bones including tibiae, femurs, and humeri were harvested from 6 to 8 weeks old C57BL/6 mice. The bone marrow was flushed out and was cultured in DMEM-15% FBS at 37 °C in 5% CO2 for 5 days. When cells reached 70–90% confluence, non-adherent cells were removed and cells were passaged at a split ratio of 1:3. Cells at passage 2–3 were used for experiments. All cells were authenticated using small tandem repeat (STR) analysis (Thermo Fisher

2.4. Alkaline phosphatase (ALP) activity & mineralization assay When MC3T3-E1 cells reached 80% confluence, the growth medium was replaced with osteogenic medium and cultured for 48 h. ALP activity in cell lysates was examined using a QuantiChrom ALP Assay Kit (BioAssay Systems) using the manufacturer's protocol. For mineralization assay, MC3T3-E1 cells were cultured in osteogenic medium for 3 weeks and stained with Alizarin red S reagent (Sigma). The visional results were verified using ARed-Q assay, a method developed by ScienCell Res Lab. Briefly, the stains were harvested using 10% acetic acid and neutralized by 10% ammonium hydroxide. The sample solution was measured using a microplate reader at OD 402 nm. 2

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cultured in DMEM-20% FBS with or without 100 ng/ml rhTIMP1 at 37 °C in 5% CO2. After 2 h, irradiated 2x105 3T3 fibroblasts were added as feeder cells. Cells were cultured for 16 days, and 100 ng/ml rhTIMP1 in a volume of 1 μl was added every two days without changing the medium. On day 16, cultures were washed with PBS, fixed with methanol and stained with 0.5% crystal violet. Adherent spindle-shaped cells clusters containing > 30 cells were counted as a colony. For osteoblastogenesis, BMSCs were cultured in StemPro Osteogenesis medium (Gibco) for 4 days and stained with ALP. The intensity of staining was quantified using an online software “Basic intensity quantification with ImageJ”. For mineralization assay, BMSCs were cultured in StemPro Osteogenesis medium for 4 weeks and stained with Alizarin red S reagent.

2.5. Quantitative real-time RT-PCR (qPCR), Western blot & coimmunoprecipitation (Co-IP) Total RNA was isolated from MC3T3-E1 cells using RNA Isolation Kit (Qiagen), and was reversed-transcribed to cDNA using qScript cDNA SuperMix (Quanta Biosciences). qPCR was performed with the Power TaqMan PCR Master Mix (Applied Biosystems) containing unlabeled PCR primers and FAM-labeled TaqMan MGB probes: TIMP1 Mm01341361_m1, ALP Mm00475834_m1, Runx2 Mm00501578_m1, COL1a1 Mm00801666_g1, RANKL Mm01313944 _g1, OPG Mm00441906_m1, MMP3 Mm00440295_ m1, MMP7 Mm00487724_m1, MMP13 Mm00439491_m1, MMP14 Mm01318969_g1, PTEN Mm05901471_g1, ACTIN 4352933E. The PCR products were analyzed with ABI PRISM 7900HT Sequence Detections System (Applied Biosystems). Relative mRNA concentrations of the target genes were determined with the ABI software (RQ Manager Version 1.2), which normalizes the target gene threshold cycle to that of endogenous housekeeping β-actin transcripts (ΔΔCt), using formula 2−ΔΔCt to determine fold change. For Western blot, cell lysate were isolated with modified RIPA buffer (10 mM Tris, pH 7.4, 100 mM NaCl, 1 mM EDTA, 1 mM EGTA, 1 mM NaF, 20 mM Na4P2O7, 2 mM Na3VO4, 1% Triton X-100, 10% glycerol, 0.1% SDS, 0.5% deoxycholate). An equal amount of protein (25 μg) was resolved by 10% SDS-PAGE and transferred to nitrocellulose membrane (Bio-Rad). The membrane was incubated with specific primary antibodies (Table 1) at 4 °C overnight and was then incubated with horseradish peroxidase-conjugated secondary antibodies (Table 1) for 1 h at room temperature. Immunoblots were visualized using Western ECL Blotting Substrates (Bio-Rad). Densitometry for protein bands was analyzed with ImageJ (NIH). For Co-IP analysis, 500 μg of MC3T3-E1 cell lysate was incubated with anti-CD44 or control IgG at 4 °C overnight. The immunocomplexes were then incubated with protein A agarose beads (Thermo Fisher Scientific) at room temperature for 30 min. The bound protein/complexes were eluted followed by western blotting.

2.8. Immunofluorescence (IF) For IF analysis, MC3T3-E1 cells were fixed with 4% PFA and permeabilized in 0.1% Triton X 100. After blocking with 10% serum, cells were incubated with mouse monoclonal anti-RANKL antibody (Abcam) for 2 h at room temperature. Cells were then treated with a Cy5-conjugated goat anti-mouse secondary antibody (Abcam) at room temperature for 30 min and was mounted using DAPI-containing mounting medium (Molecular Probes). 2.9. Tartrate-resistant acid phosphatase (TRAP) staining TIMP1-siRNA transfected RAW264.7 or control cells were cultured in αMEM with 10% FBS for 5 days, with or without 5 ng/ml recombinant human RANKL (rhRANKL, R&D Systems). TRAP staining was performed on day 5 using Acid Phosphatase, Leukocyte Kit (Sigma). The number of multinucleated osteoclasts was counted in each well. 2.10. Statistical analyses

2.6. Flow cytometry

Data are reported as mean ± SD (or mean ± SEM) of at least 3 independent experiments and were analyzed by two-tailed Student's ttest for two groups, or one-way ANOVA for multiple groups using GraphPad Prism software. The variance is similar between the groups in the same experiment. P < 0.05 was considered significant.

MC3T3-E1 cells were washed with PBS twice before incubation with 0.05% trypsin-EDTA. Cells were collected and stained with rat antimouse PE-conjugated CD63 or APC-conjugated CD44 (e-Bioscience) in the dark for 30 min on ice. Dead cells were excluded by DAPI (Sigma) staining. CD63+ and CD44+ cells were gated using FACSDiva software on an LSR II flow cytometer (BD Biosciences). Flow cytometry analysis was performed using FlowJo V10 (BD Biosciences).

3. Results 3.1. Knockdown of TIMP1 elevates endogenous osteoblastic MMP activity

2.7. Colony-forming unit-fibroblast (CFU–F) & osteoblastogenesis assay

To investigate the role of TIMP1 in osteoblasts, we established a TIMP1-knockdown cell line by transfection of osteoblastic MC3T3-E1 cells with mouse TIMP1-siRNA. Using qPCR analysis, we confirmed the knockdown of TIMP1 mRNA in these cells 24 h after the transfection (Fig. 1A). Western blot displayed the reduction of TIMP1 at the protein level. (Fig. 1B). It has been demonstrated that osteoblasts express MMP3, MMP7, MMP13 and MMP14 [6], and TIMP1 can inhibit all of these osteoblastic MMPs [18]. To investigate if knockdown of TIMP1 unleashes MMP in osteoblasts, we first examined the expression of these osteoblastic MMP molecules at the mRNA level. As shown in Fig. 1C, although MMP14 expression showed no difference, the expression of MMP3, MMP7, and MMP13 mRNA were all increased in TIMP1knockdown osteoblasts. The knockdown of TIMP1 also increased endogenous MMP activity (Fig. 1D). However, treatment with 10 μM BB94, a potent and broad-spectrum MMP inhibitor, reduced both basal level and TIMP1 knockdown-induced MMP activity (Fig. 1E). Interestingly, we found that knockdown of TIMP1 had little effect on combined ADAM10/17 activity (Fig. 1F). These results suggest that TIMP1 knockdown unleashes osteoblastic MMP activity.

For CFU-F assay, 2x106 BMSCs were seeded on a 60-mm dish and Table 1 Antibodies used. Antibodies

Manufactures

Cat No.

Use

TIMP1 RANKL PTEN CD44 Phospho-AKT AKT APC-conjugated CD44 PE-conjugated CD63 Beta-actin Anti Goat IgG Anti Rabbit IgG

Abcam Abcam Abcam Abcam Cell Signaling Cell Signaling BioLegend BioLegend Santa Cruz Cell Signaling Cell Signaling

ab38978 ab45039 ab31392 ab157107 9271 9272 103011 143903 SC-48888 7076 7074

WB IF WB WB, IP WB WB FC FC WB WB WB

WB, western blot; IF, immunofluorescence; FC, flow cytometry; IP, immunoprecipitation 3

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Fig. 1. The knockdown of TIMP1 stimulates osteoblastic MMP in MC3T3-E1 cells. (A) qPCR analysis for TIMP1 mRNA in TIMP1-knockdown (TIMP1_KD) and control cells; n = 3. (B) Western blot for TIMP1 in TIMP1-knockdown and control cells (i); densitometry analysis using ImageJ for the protein bands of TIMP1 normalized to that of β-actin (ii). Data are representative of three independent experiments. (C) qPCR analysis for osteoblastic MMP mRNA; n = 3. (D) MMP assay in TIMP1-knockdown and control cells; n = 3. (E) MMP assay in TIMP1-knockdown cells treated with/without BB94 (10 μM) for 24 h; n = 3. (F) Combined ADAM10/ 17 activity assay in MC3T3-E1 cells treated with/without GM280264X (a dual inhibitor for ADAM10 and ADAM17, 10 μM) for 24 h; n = 3. For A, C–F, data are shown as mean ± SEM. For B, data are shown as mean ± SD. *P < 0.05, **P < 0.01, ***P < 0.001.

3.2. TIMP1 inhibits osteoblast proliferation and survival

3.3. TIMP1 promotes osteoblast apoptosis

TIMPs have various biological activities including the modulation of cell proliferation, migration, invasion, as well as apoptosis. We then determined the function of TIMP1 in osteoblasts. Using MTT assay, we found that TIMP1 knockdown accelerated the proliferation rate of MC3T3-E1 cells when in comparison with control cells (Fig. 2A), suggesting that TIMP1 inhibits osteoblast proliferation. Given that many TIMP-mediated activities are independent of MMP inhibition [18], we tested the effect of BB94 on MC3T3-E1 cells. As we have described above, TIMP1-knockdown osteoblasts exhibited elevated endogenous MMP activity (Fig. 1D). We reasoned that if the inhibitory effect of TIMP1 on cell proliferation results from the inhibition of MMP, BB94 would also negatively regulate proliferation in TIMP1-knockdown cells. Interestingly, treatment with 10 μM BB94 in TIMP1-knockdown cells displayed no difference in cell proliferation (Fig. 2B), suggesting that TIMP1 inhibits osteoblast proliferation independent of MMP inhibition. Next, we evaluated the effect of TIMP1 on cell survival using a luminescent assay approach. Our results showed that TIMP1-knockdown MC3T3-E1 cells exhibited higher viability than control cells (Fig. 2C). Likewise, the addition of MMP inhibitor BB94 showed no effect on cell viability (Fig. 2D). These results suggest that TIMP1 inhibits the survival of osteoblasts, a mechanism that is independent of its metalloproteinase inhibitory activity.

Previous studies have reported that TIMP may exert anti- or proapoptotic activity depending on cell type-specific environment. To explore the modulation of TIMP1 on apoptosis of osteoblasts, we cultured MC3T3-E1 cells under serum starvation condition (0.5% FBS) and examined caspase 3 activity utilizing two approaches. Using a colorimetric assay, we showed that knockdown of TIMP1 decreased caspase 3 activity as compared to control cells (Fig. 3A). However, no difference in caspase 3 activity was observed between TIMP1-knockdown cells treated with and without BB94 (Fig. 3B). Consistent with this, Western blot analysis revealed that the expression level of cleaved caspase 3 was lower in TIMP1-knockdown cells than in control cells, whereas addition of BB94 exhibited no apparent effect (Fig. 3C). Our results suggest that TIMP1 promotes osteoblast apoptosis, which does not require MMP inhibition. 3.4. TIMP1 inhibits osteoblast differentiation in committed osteoblasts and BMSCs We have shown that TIMP1 acts as a suppressor of cell growth. Our next goal was to investigate whether TIMP1 influences osteoblast differentiation in committed osteoblasts, MC3T3-E1. We started by examining the ALP (early osteoblast differentiation marker) after 48 h in

Fig. 2. The knockdown of TIMP1 increases the proliferation and survival of MC3T3-E1 cells. (A) MTT assay in TIMP1-knockdown and control cells; n = 5. (B) MTT assay in TIMP1-knockdown cells, treated with/without BB94 (10 μM); n = 5. (C) Cell viability assay in TIMP1-knockdown and control cells; n = 3. (D) Cell viability assay in TIMP1-knockdown cells, treated with/without BB94 (10 μM) for 24 h; n = 3. Data are shown as mean ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001. 4

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Fig. 3. The knockdown of TIMP1 reduces apoptosis of MC3T3-E1 cells. (A) Caspase-3 activity assay in TIMP1-knockdown and control cells; n = 3. (B) Caspase-3 activity assay in TIMP1-knockdown cells, treated with/without BB94 (10 μM) for 24 h; n = 3. (C) Western blot for cleaved caspase 3 and total caspase 3 in TIMP1knockdown and control cells, treated with/without BB94 (10 μM) for 24 h (i); densitometry analysis using ImageJ for the ratio of cleaved caspase 3/total caspase 3 normalized to β-actin (ii). Data are representative of three independent experiments. For A and B, data are shown as mean ± SEM. For C, data are shown as mean ± SD. *P < 0.05, **P < 0.01.

Fig. 4. TIMP1 inhibits osteoblast commitment and differentiation in MC3T3-E1 cells. (A) ALP activity assay in TIMP1-knockdown and control cells; n = 3. (B) qPCR analysis for ALP, Runx2, collagen 1, and osteocalcin mRNA in TIMP1-knockdown and control cells; n = 3. (C) Alizarin red S staining in TIMP1-knockdown and control cells cultured for 3 weeks (i); quantification (ii); n = 3. (D) ALP activity assay in TIMP1-knockdown cells, treated with/without BB94 (10 μM) for 48 h; n = 3. (E) Alizarin red S staining in TIMP1-knockdown cells cultured for 3 weeks, treated with/without BB94 (10 μM) (i); quantification (ii); n = 3. (F) MMP activity assay in BMSCs, treated with/without rhTIMP1 (100 ng/ml) for 24 h; n = 3. (G) CFU-F assay in BMSCs, treated with/without rhTIMP1 (100 ng/ml) for 16 days (i); counting of colony numbers (ii); n = 3. (H) ALP staining in BMSCs cultured in osteogenic medium for 4 days, treated with/without rhTIMP1 (100 ng/ml), and/or BB94 (10 μM) (i); quantification of staining intensity (ii); n = 3. (I) Alizarin red S staining in BMSCs cultured in osteogenic medium for 4 weeks, treated with/without rhTIMP1 (100 ng/ml), and/or BB94 (10 μM) (i); quantification (ii); n = 3. For A, B, D, and F, data are shown as mean ± SEM. For C, E, G, H, and I, data are shown as mean ± SD. *P < 0.05, **P < 0.01, ***P < 0.001.

Osteoblast lineage is initiated from MSCs [19]. To further explore whether TIMP1 has an impact on osteoblastogenesis of undifferentiated stem cells, we established primary cultures of murine BMSCs. First, we treated cells with 100 ng/ml recombinant human TIMP1 (rhTIMP1) and found this treatment decreased endogenous MMP activity of BMSCs (Fig. 4F). Then we evaluated the capacity of BMSCs to generate CFU-F by plating 2x106 cells and cultured for 16 days. We showed that rhTIMP1 diminished CFU-F formation in BMSCs, in that these cells generated only 52 ± 6.8 colonies, whereas the control cells generated 87 ± 7.9 colonies (Fig. 4G). Next, we induced osteoblastogenesis by incubating BMSCs with a potent osteogenic medium. ALP staining analysis on day 4 showed that rhTIMP1-treated cells displayed lower

the osteogenic medium. We found that knockdown of TIMP1 led to an induction of ALP activity (Fig. 4A). qPCR analysis showed upregulation of several osteoblast differentiation markers including ALP, collagen I, and osteocalcin in TIMP1-knockdown osteoblasts, yet Runx2 mRNA was slightly decreased (Fig. 4B). Alizarin red S staining revealed that TIMP1 knockdown resulted in extensive mineralized nodule formation after 3 weeks. In contrast, the control cells exhibited less mineralized nodules (Fig. 4C). Notably, when treated with BB94, both ALP activity and mineralization assay failed to show any differences in TIMP1-knockdown cells (Fig. 4D, E). These results suggest that TIMP1 inhibits osteoblast differentiation and mineralization in committed osteoblasts independent of MMP inhibition. 5

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difference between the two groups (Fig. 5N). These data imply that CD44 might function as a binding protein for TIMP1 on the surface of MC3T3-E1 cells. Taken together, all these results suggest that TIMP1 induces PTEN, and this leads to the inactivation of AKT and subsequent suppression of osteoblast function.

staining intensity than control cells (Fig. 4H), indicating that rhTIMP1 inhibits the commitment of BMSCs towards osteoblast lineage. Four weeks after culture, Alizarin red S staining showed a reduced mineralization capacity in rhTIMP1-treated BMSCs as compared to control cells (Fig. 4I). Notably, in rhTIMP1-treated BMSCs, co-treatment with BB94 didn't further reduce ALP staining intensity and mineralization level when in comparison with cells treated with rhTIMP1 alone (Fig. 4H, I). These observations suggest that TIMP1 inhibits osteoblast commitment and differentiation from BMSCs independent of its MMP inhibitory activity.

3.6. TIMP1 inhibits osteoclast differentiation Finally, we explored the modulation of TIMP1 on osteoclast growth and differentiation. MTT assay showed that knockdown of TIMP1 in osteoclast precursor RAW 264.7 cells exhibited no effect (P > 0.05) in the proliferation rate when in comparison with control cells (Fig. 6A). To investigate the regulation of TIMP1 on osteoclast differentiation, we started by determining the expression of the receptor activator of nuclear factor kappa-B ligand (RANKL), which is expressed by osteoblasts and stromal cells and is the key molecule that triggers osteoclast differentiation and bone resorption [22]. With the qPCR technique, we found that the expression of RANKL mRNA was higher in TIMP1knockdown MC3T3-E1 cells than in control cells (Fig. 6Bi). However, knockdown of TIMP1 resulted in a slight decrease in the expression of osteoprotegerin (OPG) (Fig. 6Bii), a molecule that protects the skeleton from excessive bone resorption by binding to RANKL and preventing it from binding to its receptor, RANK [23]. These opposing changes led to an increase of the RANKL/OPG mRNA ratio by 3.3-fold (Fig. 6Bii). IF analysis further showed that TIMP1-knockdown MC3T3-E1 cells exhibited a higher level of RANKL than the control osteoblasts (Fig. 6C). We then cultured RAW 264.7 cells that had been transfected with TIMP1-siRNA and treated with 5 ng/ml rhRANKL to stimulate osteoclast differentiation for 5 days. TRAP staining analysis showed no formation of multinucleated osteoclasts in either control or TIMP1knockdown RAW 264.7 cells. In stark contrast, rhRANKL dramatically enhanced osteoclast differentiation, in that the control cells generated 48 ± 4.5 osteoclasts, whereas 85 ± 12 osteoclasts were detected in cells with TIMP1 knockdown. Notably, treatment with 10 μM BB94 exhibited little effect in the formation of multinucleated osteoclasts (Fig. 6D). These findings suggest that TIMP1 suppresses osteoclast differentiation via a mechanism independent of its MMP inhibition activity. In light of AKT signaling that has been previously reported to promote the differentiation and survival of osteoclasts [14,24], we assumed that TIMP1 could inhibit osteoclast differentiation by targeting the AKT. For this purpose, we treated RAW 264.7 cells with 5 ng/ml rhRANKL and analyzed AKT phosphorylation status using Western blot. We found that TIMP1-knockdown RAW 264.7 cells expressed a higher level of pho-AKT than the control cells. Importantly, the addition of rhRANKL further enhanced AKT phosphorylation in both groups (Fig. 6E). Functionally, treatment with 10 μM AKTi-1/2 attenuated TIMP1 knockdown-stimulated osteoclast formation (Fig. 6F). Hence, our results suggest that TIMP1 promotes osteoclast differentiation by targeting the AKT pathway.

3.5. TIMP1 impacts osteoblasts by targeting the AKT signaling pathway We have shown that TIMP1 acts as a suppressor of growth and differentiation in osteoblasts at the cellular level, we next investigated the molecular mechanism underlying TIMP1-mediated inhibitory effect. We hypothesized that TIMP1 inhibits osteoblasts by inactivating the AKT pathway, in that there is increasing evidence showing that this pathway acts as a crucial regulator of osteoblasts by promoting their differentiation and survival to maintain bone mass and turnover [14–16]. Using Western blot analysis, we found that the expression of phosphorylated AKT (pho-AKT) was higher in TIMP1-knockdown MC3T3-E1 cells than in control cells, suggesting that TIMP1 negatively regulates AKT. Notably, treatment with BB94 did not change the expression of pho-AKT in TIMP1-knockdown cells (Fig. 5A), indicating that TIMP1 inactivates AKT independent of MMP inhibition. To verify that TIMP1 inhibits osteoblast growth and differentiation by targeting the AKT pathway, we treated TIMP1-knockdown MC3T3E1 cells with 10 μM AKTi-1/2, a specific AKT inhibitor. MTT assay showed that treatment with AKTi-1/2 strongly suppressed osteoblast proliferation (Fig. 5B). This AKT inhibitor also reversed TIMP1 knockdown-inhibited apoptosis (Fig. 5C). Besides, the induction of osteoblast differentiation and mineralization by TIMP1 knockdown was attenuated by AKTi-1/2, and this was evidenced by a decrease in ALP activity and mineralized nodule formation, respectively (Fig. 5D, E). These functional assays support the notion that TIMP1 exerts its suppressive role in osteoblasts through its inactivation of the AKT pathway. To find out the mechanism whereby TIMP1 targets AKT, we focused on the tumor suppressor PTEN. This is because PTEN is the only known lipid phosphatase that can convert PIP3 to PIP2, thus negatively regulating the PI3K/AKT pathway [12]. PTEN is also crucial to control cell growth under both physiologic and pathologic situations. Here, we showed that knockdown of TIMP1 in osteoblasts reduced PTEN mRNA (Fig. 5F). In stark contrast, treatment with 100 ng/ml rhTIMP1 in normal MC3T3-E1 cells substantially increased PTEN expression at both mRNA and protein levels (Fig. 5G, H), suggesting that TIMP1 antagonizes AKT pathway through its induction of PTEN. As we described above, the suppressive effect of TIMP1 on osteoblasts is independent of MMP inhibition, this suggests that TIMP1 may induce PTEN through its direct binding to the cell surface receptor(s). CD63 and CD44 have been identified as important binding proteins of TIMP1 on the cell surface [9,20]. Using flow cytometry analysis, we found that only 1.6% of MC3T3-E1 cells were positive for CD63 (Fig. 5Ii). This is consistent with Lizuka et al., who reported that CD63 is not expressed by MC3T3-E1 cells [21]. We then analyzed CD44 surface receptor and found that 42.7% of MC3T3-E1 cells were positive for CD44. Interestingly, treatment with rhTIMP1 increased CD44+ population up to 60% (Fig. 5Iii). Western blot analysis also revealed the upregulation of CD44 by rhTIMP1 treatment (Fig. 5J). Furthermore, we transfected MC3T3-E1 cells with CD44-siRNA and validated CD44 as a binding protein using Co-IP analysis. The knockdown of CD44 was confirmed by qPCR (Fig. 5K). As shown in Fig 5L, the amount of TIMP1 that co-precipitated with the anti-CD44 antibody greatly reduced following knockdown of CD44. Western blot analysis showed that CD44knockdown cells exhibited slightly decreased cellular TIMP1 as compared to control cells (Fig. 5M), whereas qPCR analysis found no

4. Discussion In this study, we have demonstrated that TIMP1 functions as a suppressor of osteoblast growth and differentiation that is independent of its MMP inhibition capacity. Importantly, we uncover that this inhibitory modulation by TIMP1 is mediated via targeting the AKT pathway, a mechanism associated with the induction of PTEN. TIMP1 also suppresses osteoclast differentiation by targeting the AKT. As such, our data highlight TIMP1 as an important negative regulator in bone homeostasis and remodeling. In the present study, knockdown of TIMP1 in osteoblasts unleashes endogenous MMP activity, reflecting TIMP1 as a potent inhibitor of osteoblastic MMP. However, ADAM10/17 activity is not affected following TIMP1 knockdown. This is not surprising, in that TIMP1 only inhibits ADAM10 whereas TIMP3 is the most effective and broad6

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Fig. 5. TIMP1 modulates MC3T3-E1 osteoblasts by targeting the AKT pathway. (A) Western blot for phosphorylated AKT (pho-AKT) and total AKT in TIMP1knockdown and control cells, treated with/without BB94 (10 μM)for 24 h (i); densitometry analysis for the ratio of pho-AKT/total AKT normalized to β-actin (ii). Data are representative of three independent experiments. (B) MTT assay for proliferation in TIMP1-knockdown cells, treated with/without AKTi-1/2 (10 μM); n = 5. (C) Caspase 3 activity assay in TIMP1-knockdown cells, treated with/without AKTi-1/2 (10 μM) for 24 h; n = 3. (D) ALP activity assay in TIMP1-knockdown cells, treated with/without AKTi-1/2 (10 μM) for 48 h; n = 3. (E) Alizarin red S staining in TIMP1-knockdown cells cultured for 3 weeks, treated with/without AKTi-1/2 (5 μM) (i); quantification (ii). (F) qPCR for PTEN mRNA in TIMP1-knockdown or control cells; n = 3. (G) qPCR for PTEN mRNA in MC3T3-E1 cells treated with/ without rhTIMP1 (100 ng/ml) for 24 h; n = 3. (H) Western blot for PTEN in cells treated with/without rhTIMP1 (100 ng/ml) for 24 h (i); densitometry analysis (ii). Data are representative of three independent experiments. (I) Flow cytometry for CD63 (i) or CD44 (ii) in cells treated with/without rhTIMP1 (100 ng/ml) for 24 h; n = 3. (J) Western blot for CD44 in MC3T3-E1 cells treated with/without rhTIMP1 (100 ng/ml) for 24 h (i); densitometry analysis (ii). Data are representative of three independent experiments. (K) qPCR for CD44 mRNA in CD44-knockdown or control cells; n = 3. (L) Co-IP assay in CD44-knockdown or control cells. Data are representative of three independent experiments. (M) Western blot for TIMP1 in CD44-knockdown or control cells (i); densitometry analysis (ii). Data are representative of three independent experiments. (N) qPCR for TIMP1 mRNA in CD44-knockdown or control cells; n = 3. For B-D, F, G, K, and N, data are shown as mean ± SEM. For A, E, H, I, J, L, and M, data are shown as mean ± SD. *P < 0.05, **P < 0.01, ***P < 0.001.

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Fig. 6. TIMP1 inhibits osteoclast differentiation by targeting the AKT pathway. (A) MTT assay in TIMP1-knockdown and control RAW 264.7 cells; n = 5. (B) qPCR for RANKL (i) and OPG (ii) mRNA, as well as the ratio of RANKL/OPG (iii) in TIMP1-knockdown and control MC3T3-E1 cells; n = 3. (C) IF for RANKL in TIMP1-knockdown and control MC3T3-E1 cells; n = 3; scale bar = 100 μm. (D) TRAP staining in TIMP1-knockdown and control RAW 264.7 cells, treated with/ without rhRANKL (5 ng/ml), and/or BB94 (10 μM) for 5 days. Arrowheads indicate differentiated multinucleated osteoclasts (OCs) (i); multinucleated OCs quantification (ii); n = 3. (E) Western blot for pho-AKT and total AKT in TIMP1-knockdown or control RAW 264.7 cells, treated with/without rhRANKL (5 ng/ml) (i); densitometry analysis (ii). Data are representative of three independent experiments. (F) Quantification of multinucleated OCs in TIMP1-knockdown RAW 264.7 cells, treated with/without rhRANKL (5 ng/ml), and/or AKTi-1/2 (10 μM); n = 3. For A and B, data are shown as mean ± SEM. For D-F, data are shown as mean ± SD. *P < 0.05, **P < 0.01, ***P < 0.001.

four TIMP genes [4]. One possibility for the discrepancy is that individual TIMP may have divergent, or even opposing effects on cell proliferation or death [30,31]. Therefore, the downregulation of osteoblast growth in the entire TIMPs-knockout mice may only reflect the balanced effect of all four TIMP members on osteoblast proliferation. In support of our findings, Schiltz et al. also reported a reduced proliferation rate in primary osteoblasts that overexpress TIMP1 [8]. The modulation of TIMP1 on osteoblast differentiation remains to be clarified. Geoffroy et al. reported that the differentiation and function of osteoblasts from TIMP1-overexpressing transgenic mice appear normal [7]. However, Molloy et al. showed that TIMP1 steadily increases throughout the whole differentiation process of MSCs towards osteoblast maturation, indicating that TIMP1 positively regulates osteoblastogenesis [32]. In contrast, using a series of in vitro studies, we report here that TIMP1 inhibits osteoblast differentiation in committed osteoblasts (i.e., MC3T3-E1), and this is evidenced by 1) TIMP1

spectrum inhibitor of ADAMs (e.g., ADAM10, ADAM17, etc.) [18]. The N-terminal domain of TIMP4, although not in full-length, also inhibits ADAM17 [25]. Hence, the knockdown of TIMP1 alone is not powerful enough to inhibit ADAM10/17 in the context of functionally intact TIMP3 and TIMP4. The regulation of TIMP1 on cell growth varies among different cell types. TIMP1 possesses potent growth-stimulating activity by promoting proliferation yet inhibiting apoptosis in granulocytes and fibroblasts [26,27]. On the other hand, TIMP1 has also been reported to attenuate proliferation and promote apoptosis in mammary epithelial cells and pancreatic cancer cells [28,29]. In our study, we report that TIMP1 knockdown increases proliferation and survival but decreases apoptosis of MC3T3-E1 cells, indicating a growth-inhibiting effect of TIMP1 in osteoblasts. However, these results seem different from our recent study, in which a dramatic decrease of osteoblast proliferation has been observed in transgenic mice with homozygous knockout of all 8

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reported that CD63 is not expressed by MC3T3-E1 cells, although it is expressed by another osteoblastic cell line Saos-2 [21]. In our study, we show that only 1.6% of MC3T3-E1 cells are positive for this surface protein, suggesting that it is unlikely that TIMP1 binds to CD63 to induce PTEN. However, CD44 is also a surface receptor for TIMP1 [20,39], which can be expressed by MC3T3-E1 cells [40]. Here, we find that treatment with recombinant TIMP1 increased CD44+ population of MC3T3-E1 cells. More importantly, Co-IP analysis reveals that the amount of TIMP1 that co-precipitated with anti-CD44 antibody substantially reduces in CD44-knockdown MC3T3-E1 cells. Therefore, it is plausible that TIMP1 might induce PTEN expression through its binding to CD44 on the surface of MC3T3-E1 cells, and this leads to the inactivation of AKT and subsequently, suppression of osteoblast activities. Consequently, our results highlight a TIMP1/CD44/PTEN/AKT signaling nexus that acts as a suppressor of osteoblast functions. Besides, we demonstrate that TIMP1 has little effect in osteoclast proliferation, whereas this tissue inhibitor negatively modulates osteoclast differentiation independent of MMP inhibition. The effect of TIMP1 on osteoclasts is still controversial. Consistent with our findings, Hill et al. showed that TIMP1 inhibits PTH or 1,25-dihydroxyvitamin D3 induced bone resorption in cultured neonatal mouse calvariae [41]. Merciris et al. also showed that osteoclastic surfaces that were increased in PTH-treated wildtype mice remain unchanged in TIMP1-overexpressing mice, suggesting that TIMP1 decreases osteoclastic differentiation [42]. However, Sobue et al. reported that TIMP1 directly stimulates the bone-resorbing activity of isolated mature osteoclasts [43]. We assume this discrepancy may be due to different dosages of recombinant TIMP1 used in these studies. TIMP1 stimulates osteoclastic bone resorption at concentrations < 50 ng/ml, which is the same level as that of their cell growth-stimulating activity [43,44]. In contrast, significantly higher concentrations (μg/ml) are required to inhibit osteoclastic differentiation and bone resorption [41,45]. Using the TIMP1 knockdown technique, we support that TIMP1 negatively regulates osteoclast differentiation. Importantly, this inhibition is also mediated through the inactivation of AKT, suggesting that the TIMP1/AKT pathway is implicated in both osteoblast and osteoclast differentiation. Our results are supported by a previous study, in which global AKT1knockout mice can cause impairment of bone resorption via dysfunctions of osteoclasts [14]. Although our results support that TIMP1 functions as a negative regulator of osteoblasts and osteoclasts by targeting the AKT pathway that is independent of its MMP inhibition, we do not exclude that TIMP1 may also impact bone homeostasis by inhibiting MMP. Normal bone homeostasis is maintained by a balanced bone formation and bone degradation, yet an imbalance in the regulation of bone remodeling results in many metabolic bone diseases (e.g., osteoporosis). Several MMPs, such as MMP2, MMP7, MMP9, MMP13, MMP14 are expressed in bone and cartilage and play an important role in bone modeling and remodeling by the degradation of skeletal ECM (e.g., collagen and aggrecan) [1]. Among those MMPs, MMP13 (Collagenase 3) has particular significance in skeletal biology, in that this collagenase is expressed exclusively in the bone and cartilage during skeletal development [46]. A point mutation in the human MMP13 gene causes spondyloepimetaphyseal dysplasia (SEMD), characterized by defective growth and modeling of the long bones [47]. Conditional inactivation of MMP13 in osteoblasts display normal bone formation but impaired bone degradation, leading to increased trabecular bone volume [48]. We report here that knockdown of TIMP1 in osteoblasts increases the expression of several MMPs including MMP13. TIMP1 knockdown also unleashes MMP activity in osteoblasts, whereas this enhancement can be greatly suppressed by BB94. Therefore, it seems highly plausible that TIMP1 may play disparate roles in regulating bone homeostasis via its MMP inhibition-independent or dependent mechanism, such that 1) TIMP1 functions as a suppressor of growth and differentiation in osteoblasts via CD44/PTEN/AKT signaling axis; 2) TIMP1 suppresses osteoclast differentiation by targeting AKT pathway; both of which are

knockdown induces activity of ALP, an early osteoblast differentiation marker; 2) TIMP1 knockdown upregulates the expression of osteoblast differentiation markers, including ALP, collagen type I (the major product of osteoblasts), as well as osteocalcin (the late osteoblast differentiation marker); and 3) TIMP1 knockdown enhances osteoblast mineralization. Further, we show that TIMP1 also inhibits osteoblastogenesis of undifferentiated BMSCs towards osteoblast lineage. Therefore, TIMP1 functions as a suppressor during the entire differentiation process, including osteoblast commitment, early and late differentiation, as well as maturation. Our results are consistent with two previous studies that TIMP1 suppresses osteogenic differentiation of human MSCs [9,33]. Although TIMP1 has been implicated in proliferation or osteogenic differentiation of osteoblasts and MSCs, little is known regarding the molecular pathway underlying its regulating effect. So far, only inactivation of the Wnt/β-catenin signaling has been reported responsible for TIMP1-modulated MSC activity [9,33]. In our study, we focus on the AKT pathway, in that increasing amounts of evidence suggest that the PI3K/AKT is a central nexus in the extensive network of extracellular signaling pathways that control osteoblasts [16]. AKT1 deficiency in osteoblasts has been reported to increase susceptibility to apoptosis and suppress differentiation and function [14]. The PI3K/AKT signaling cascade is also essential for BMP2-activated osteoblast differentiation and maturation, bone development and growth [15]. Importantly, we uncover here for the first time that TIMP1 acts as a suppressor of osteoblast growth, survival and differentiation by targeting the AKT pathway, supported by a series of observations: 1) TIMP1 knockdown stimulates AKT phosphorylation in osteoblasts; 2) TIMP1 knockdowninduced proliferation and reduced apoptosis can be reversed by the AKT inhibitor, AKTi-1/2; 3) TIMP1 knockdown-promoted ALP activity and mineralization capacity can also be attenuated by AKTi-1/2. These results highlight an important suppressive role of TIMP1/AKT in osteoblast activity. Another interesting finding is the identification of tumor suppressor PTEN as a mediator that links between TIMP1 and AKT. Being the only known lipid phosphatase that antagonizes PI3K/AKT pathway by converting PIP3 to PIP2, PTEN not only inhibits tumor cell proliferation and survival but also impacts cell behaviors in other physiological and pathological situations [34]. For example, osteoblastic PTEN knockout mice exhibit increased bone formation due to elevated AKT-mediated cell survival [35]. In our study, we report that TIMP1 knockdown in osteoblasts decreases PTEN mRNA, whereas recombinant TIMP1 induces PTEN expression, suggesting that TIMP1 negatively regulates the AKT pathway through its induction of PTEN. Interestingly, Akahane et al. reported that TIMP1 stimulates the expression of PTEN to reduce phosphorylation of focal adhesion kinase (FAK) and inhibit endothelial cell migration [36]. Here, we provide another mechanism that TIMP1 induces PTEN to suppress osteoblast activity by targeting the AKT. Being multifunctional factors, TIMPs may exert biological functions partially arising from their MMP inhibition capacity, because MMP can modulate cell proliferation, migration, apoptosis, as well as differentiation by degradation of ECM proteins as well as non-matrix proteins [1,37]. However, many of TIMPs’ activities are also independent of their metalloproteinase inhibition. In our study, although knockdown of TIMP1 in osteoblasts enhances endogenous MMP activity, administration of BB94, a potent and broad-spectrum MMP inhibitor, does not affect TIMP1 knockdown-modulated osteoblast growth, differentiation, and mineralization. Notably, BB94 displays no effect on TIMP1 knockdown-activated AKT. These results not only suggest that the negative regulation of TIMP1 on osteoblasts via targeting AKT is independent of MMP inhibition, but also indicate that the effect of TIMP1 may be mediated by its direct binding to the cell surface receptor(s). CD63 has been identified as a major binding protein of TIMP1 on the cell surface [38]. Egea et al. demonstrated that TIMP1 can bind to CD63 and lead to inactivation of the Wnt/β-catenin pathway, thus inhibiting osteogenic differentiation of MSCs [9]. Unfortunately, it has been 9

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independent of MMP inhibition capacity and consequently lead to reduced bone formation and bone resorption (low bone turnover); 3) TIMP1 in osteoblasts may also directly inhibit MMP (particularly MMP13), and this results in impaired bone degradation and subsequently, compromised bone remodeling. As such, the balanced or imbalanced bone homeostasis is to be achieved by the overall expression of TIMPs (e.g., TIMP1), MMPs (e.g., MMP13), and/or MMP/TIMP ratio. In general, our study has demonstrated for the first time that TIMP1 induces PTEN expression and this leads to the negative regulation of the AKT pathway. Consequently, TIMP1 suppresses osteoblast growth and differentiation independent of its MMP inhibition. Our results highlight a novel TIMP1/CD44/PTEN/AKT signaling nexus in the modulation of osteoblast activity. TIMP1 also inhibits osteoclast differentiation by targeting AKT. These findings pinpoint TIMP1 as an important negative regulator for both osteoblasts and osteoclasts and suggest that TIMP1 might serve as a potential target for low bone mass-related skeletal disorders, such as osteoporosis.

[10]

[11] [12] [13] [14]

[15] [16] [17]

[18]

Author contributions

[19]

YX and YC designed the study and wrote the manuscript. YX, HH, ZZ, JM, and YC collected data and performed experiments. YX, HH, and YC analyzed the data.

[20]

CRediT authorship contribution statement [21]

Yongming Xi: Conceptualization, Methodology, Funding acquisition, Writing - review & editing, Supervision. Hui Huang: Methodology, Investigation, Formal analysis. Zheng Zhao: Methodology, Investigation, Formal analysis. Jinfeng Ma: Methodology, Investigation. Yan Chen: Conceptualization, Methodology, Writing - review & editing.

[22] [23] [24]

Declaration of competing interest

[25]

The authors declare that there are no competing interests associated with the manuscript.

[26]

Acknowledgments [27]

This work was supported by research funding from the National Natural Science Foundation of China (NSFC, 81470104) to YX; YX was also supported by the Taishan Scholars Program, China (No. TS20190985).

[28]

[29]

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