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Hypothermia-induced RNA-binding motif protein 3 (RBM3) stimulates osteoblast differentiation via the ERK signaling pathway
Accepted Manuscript Hypothermia-induced RNA-binding motif protein 3 (RBM3) stimulates osteoblast differentiation via the ERK signaling pathway Do-Young Kim, Kyeong-Min Kim, Eun-Jung Kim, Won-Gu Jang PII:
S0006-291X(18)30465-0
DOI:
10.1016/j.bbrc.2018.02.209
Reference:
YBBRC 39574
To appear in:
Biochemical and Biophysical Research Communications
Received Date: 21 February 2018 Accepted Date: 28 February 2018
Please cite this article as: D.-Y. Kim, K.-M. Kim, E.-J. Kim, W.-G. Jang, Hypothermia-induced RNAbinding motif protein 3 (RBM3) stimulates osteoblast differentiation via the ERK signaling pathway, Biochemical and Biophysical Research Communications (2018), doi: 10.1016/j.bbrc.2018.02.209. 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.
A.A + β-GP
A.A: Ascorbic acid β -GP: β−Glycerophosphate
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Hypothermia
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ERK
PD98059
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RBM3
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ERK
Cytoplasm Nucleus
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Runx2 and OC
Osteoblast differentiation
ACCEPTED MANUSCRIPT Hypothermia-induced RNA-binding motif protein 3 (RBM3) stimulates osteoblast differentiation via the ERK signaling pathway
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Do-Young Kima,b, Kyeong-Min Kima,b, Eun-Jung Kim b,c*, Won-Gu Janga,b,*
a
Department of Biotechnology, School of Engineering, Daegu University, Gyeongbuk, 38453, Republic of Korea Research Institute of Anti-Aging, Daegu University, Gyeongbuk, 38453, Republic of Korea
c
Department of Immunology, Kyungpook National University School of Medicine, Daegu,
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b
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41944, Republic of Korea
Running title: RBM3 increases osteoblast differentiation via ERK signaling.
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Authors’ e-mail addresses: D.Y. Kim: [email protected] K.M. Kim: [email protected] E.J Kim: [email protected] W.G. Jang: [email protected]
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*Corresponding authors: Won-Gu Jang, Department of Biotechnology, college of Engineering, Daegu University, Gyeongbuk 38453, Republic of Korea. Tel.: 82-53-850-6552; Fax: 82-53-850-6559; E-mail: [email protected]. Eun-Jung Kim, Department of Immunology, Kyungpook National University School of Medicine, Daegu, 41944, Republic of Korea. Tel: 82-53-420-4877; Fax: 82-53-423-4628; E-mail: [email protected]
Abbreviations A.A, ascorbic acid; β-GP, β-glycerophosphate; RBM3, RNA-binding motif protein 3; OC, osteocalcin; Runx2, Runt-related transcription factor 2; ERK, Extracellular signal-regulated kinase
ACCEPTED MANUSCRIPT ABSTRACT The RNA-binding motif protein 3 (RBM3) belongs to a small group of proteins whose synthesis increases during hypothermia while global protein production is slowed down.
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Bone homeostasis is maintained by a balance between bone resorption and bone formation. Osteoblasts are key components of the bone and have an important role in bone remodeling cycle. However, hypothermia-induced RBM3 between osteoblasts remains unclear. At 32°C,
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expression of RBM3 and Runx2 was increased in a time-dependent manner and mineralization was also increased. RBM3 was also increased in a time-dependent manner
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under osteogenic conditions. Overexpression of RBM3 increased the expression of osteogenic genes such as Runx2 and OC. The osteogenic condition-induced expressions of RBM3, Runx2 and OC gene were decreased by RBM3 siRNA. Moreover, RBM3 promoted ERK and p38 phosphorylation. The inhibitor of ERK decreased the expression of Runx2 but
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did not affect the expression of RBM3. Taken together, these results demonstrate that RBM3 stimulates osteoblast differentiation via the ERK signaling pathway.
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Keywords: Hypothermia, RBM3, ERK, Runx2, Osteoblast differentiation
ACCEPTED MANUSCRIPT 1. Introduction Metabolic diseases such as osteoporosis, degenerative arthritis, obesity, and diabetes are greatly influenced by daily habits due to changes in the diet culture of modern society and
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decrease in exercise [1,2]. Osteoporosis is caused by the collapse of the balance of cells in maintaining bone health [3]. Osteoporosis is a frequent occurrence in women after menopause, requiring osteoporosis treatment and development of health functional foods [4].
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The importance of prevention rather than treatment is emphasized because there is no drug that can completely restore the reduced bone mass [5]. Estrogen is administered as the most
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typical treatment [6,7]. This treatment modality enables estrogen to regulate bone resorption and bone formation through the regulation of cytokines [8]. However, long-term use of estrogen causes many side effects, and therefore natural substances are needed to overcome this problem [9,10]. In recent years, research has focused on searching for natural materials
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that can replace synthetic drugs that cause side effects.
Osteoblast differentiation is regulated by a range of hormones, cytokines and various transcription factors [11,12]. Ascorbic acid (A.A) and β-glycerophosphate disodium salt
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hydrate (β-GP) promote osteoblast differentiation [13,14]. A.A+β-GP increase gene
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expression of Distal-less homeobox 5 (Dlx5) through an intracellular signaling system. Increased Dlx5 increases gene expression of Runt-related transcription factor 2 (Runx2), gene expression of Runx2 is essential for osteoblast differentiation. In addition, Runx2 expression increases calcium deposits by inducing mineralization through gene and protein expression of osteocalcin (OC) [15,16]. Previous studies have shown that bone density is related to various factors such as age, body type, sex, and race [17,18,19]. However, few studies have focused on cold-related bone mineral density. In the case of polar bears and penguins, thicker subcutaneous fat layers are
ACCEPTED MANUSCRIPT developed in the body to withstand the cold than bears and penguins that live in relatively warmer regions. In addition, obese people are reported to have a higher bone density than normal people [20]. On the basis of these findings, we judge that people living in cold
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regions will evolve subcutaneous fat to withstand the cold, and bone density will also increase to endure the developing fat. Therefore, we hypothesized that bone mineral density would increase if exposed to hypothermia.
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RNA-binding motif protein 3 (RBM3) is a member of the glycine-rich RNA-binding protein family and is the most representative protein that induces expression under cold shock
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and low oxygen conditions [21,22]. RBM3 was first screened in humans in 1995 and is known to be expressed in almost all tissues [23]. Expression of RBM3 has been shown to be essential for proper cell cycle progression and mitosis [22,24]. Interestingly, RBM3 has been reported to be able to regulate mitogen-activated protein kinases (MAPKs) such as
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extracellular signal regulated kinase (ERK), p38 kinase (p38) and c-Jun N-terminal kinases (JNK) [25,26]. In mammalian cells, there are MAPK signaling pathways, such as ERK, p38 and JNK that mediate extracellular signaling to the nucleus to activate stimulatory reactive
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genes such as growth factors and oxidative stress. The MAPK signaling pathways are essential for controlling many cellular processes including inflammation, cell differentiation,
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cell proliferation and death [27].
The role of 32°C condition-induced RBM3 in osteoblast differentiation has not yet
been elucidated. In this study, we demonstrate that hypothermia-induced RBM3 regulates Runx2 expression and activation through the ERK phosphorylation pathway, and subsequently increases the expression of OC.
ACCEPTED MANUSCRIPT 2. Materials and Methods 2.1. Cell culture and differentiation MC3T3-E1 cells were obtained from ATCC CCL-2593 (ATCC, Manassas, VA, USA).
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For maintenance, α-Minimal Essential Medium (α-MEM, Gibco, Grand Island, NY, USA) was used as the basal culture, and 10% Fetal Bovine Serum (FBS, Atlas biologicals, USA), 100 units/mL penicillin (Gibco) and 100 µg/mL streptomycin (Gibco) were mixed and
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incubated at 37°C and 5% CO2. Differentiation of osteoblasts was induced by addition of osteogenic condition containing 50 µg/mL ascorbic acid (A.A, Sigma Aldrich, St. Louis, MO,
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USA) and 5 mM β-glycerophosphate (β-GP, Sigma Aldrich). The culture medium was replaced every 2 days. To induce hypothermic conditions, cells were cultured in a 32°C 5% CO2 incubator.
RT-PCR and real-time PCR analysis
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2.2.
Total RNA was isolated from cells using TRI-solution (Bio Science Technology,
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Daegu, Korea) as per the manufacturer’s instructions. Reverse transcription was performed using 2.5 µg of total RNA. RT-PCR was performed using an emerald Amp GT PCR master
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mix (Takara, Rockland, ME, USA). Real-time PCR was performed on a LightCycler Nano Instrument (Roche, Mannheim, Germany) using AmpiGene™ qPCR Green Mix Hi-ROX (Enzo Life Sciences, Farmingdale, NY, USA). The RT-PCR and real-time PCR primer sequences are shown in Table 1.
2.3.
Transient transfection Overexpression vector "pCMV-RBM3 and pcDNA-Runx2-HA" used in the
ACCEPTED MANUSCRIPT experiment was distributed from the Korea Human Gene Bank (KRIBB, Korea). Potential RBM3 and Runx2 siRNAs were designed by Turbo si-Designer software (Bioneer, Korea). Negative control siRNA and potential RBM3 and Runx2 siRNA sequences are shown in
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Table 2. MC3T3-E1 cells were transiently transfected with the indicated plasmids using Lipofectamine 2000 (Invitrogen, Carlsbad, CA, USA) by using Opti-MEM® I (Gibco). After incubation at 37°C 5% CO2 incubator for 6 h, the medium for the basal culture was changed
ARS staining
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2.4.
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to α-MEM containing 10% FBS.
For mineralization analysis, MC3T3-E1 cells were cultured with or without A.A+βGP for 21 days. To compare with hypothermic conditions, one plate was transferred to the 32°C 5% CO2 incubator on the 19th day and cultured for 2 days. Staining was performed
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using standard protocols. Briefly, cultured cells were treated with 2% alizarin red solution
2.5.
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(Sigma Aldrich) for 10 min at room temperature.
Western blot analysis
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Total protein was extracted using EzRIPA Lysis kit (ATTO Technology, Tokyo, Japan).
Total protein was quantified using Bradford protein assay reagent. The protein samples were separated by SDS-PAGE and transferred onto PVDF membranes. Signals were detected using ECL reagent (Advansta, Menlo Park, CA, USA) according to the manufacturer's protocol. Densitometry analysis of the blotted membranes was performed using a Fusion Solo (Vilber Lourmat, Eberhardzell, Germany).
ACCEPTED MANUSCRIPT 2.6. Statistical analysis All experiments were repeated at least three times. Statistical analysis was performed using Prism 5 (version 5.0, GraphPad Software, La Jolla, CA, USA) to Student’s t-test. P
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values of < 0.05 were considered significant. Results are expressed as the mean ± SEM of
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triplicate independent samples.
ACCEPTED MANUSCRIPT 3. Results 3.1 Hypothermic condition induces osteoblast differentiation via RBM3 expression in MC3T3E1 cells
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To confirm the effect of hypothermic conditions on osteoblast differentiation, we cultured MC3T3-E1 cells respectively at 37°C or 32°C 5% CO2 incubator for 0 to 24 h. RTPCR analysis showed that hypothermic conditions increased expression of RBM3 and
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osteogenic marker genes such as Runx2 and OC (Fig. 1A). Hypothermic conditions also increased protein levels of RBM3 and Runx2 better than at 37°C (Fig. 1B). In addition, when
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treated with ascorbic acid and β-glycerophosphate (A.A+β-GP) were induced for osteoblast differentiation, it was confirmed that the extracellular matrix was mineralized by calcium deposition. In addition, it was confirmed that the mineralization by hypothermic condition was significantly increased (Fig. 1C). To confirm whether RBM3 expression was regulated
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under differentiation conditions instead of hypothermic condition, cells were treated A.A+βGP. According to Real-time PCR analysis, the A.A+β-GP increased RBM3 expression with osteogenic gene, such as Runx2 and OC, expression in a time-dependent manner (Fig. 1D).
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Western blot analysis showed that protein levels of Runx2 and RBM3 also increased (Fig.
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1E). Taken together, the RBM3 expression was increased by hypothermic or osteogenic conditions with osteogenic gene expressions such as Runx2 and OC. These results suggest that RBM3 plays an important role in osteoblast differentiation in MC3T3E1 cells.
3.2 Overexpression of RBM3 positively regulates a major osteogenic marker expression To identify the molecular mechanisms involved in the control of osteoblast differentiation by RBM3, we focused on the early stage marker gene of osteoblast differentiation such as Runx2. The Real-time PCR analysis demonstrated that overexpression
ACCEPTED MANUSCRIPT of RBM3 significantly increased expression of Runx2 and OC gene expression (Fig. 2A-C). Western blot analysis showed that overexpression of RBM3 increased Runx2 expression (Fig. 2D). Overall, these results suggest that expression of RBM3 induced Runx2 and OC
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expression.
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3.3 Inhibition of RBM3 specifically suppresses Runx2 expression
To confirm the regulatory role of RBM3 in osteoblasts, the effect of down-regulation
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of RBM3 on Runx2 gene expression was examined using an RBM3-specific siRNA (siRBM3). The RT-PCR and Western blot analysis showed that A.A+β-GP-induced Runx2, OC and RBM3 gene expressions were reduced by siRBM3 (Fig. 3A). The increased protein expression level of Runx2 by A.A+β-GP also decreased by siRBM3 (Fig. 3B). To further
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determine if Runx2 regulates RBM3 expression, the effects of Runx2 on RBM3 expression using real-time PCR analysis. As a result, Runx2 overexpression did not affect RBM3 expression (Fig. 3C). In addition, Runx2-specific siRNA (siRunx2) was used to confirm the
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expression of Runx2 and RBM3 during osteoblast differentiation. The real time-PCR and Western blot analysis showed that A.A+β-GP-induced Runx2 and RBM3 expression was
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reduced by siRunx2 (Fig. 3D and F). However, siRunx2 did not affect RBM3 expression (Fig. 5E and F). Overall, these results suggest that Runx2 expression is increased by RBM3 during osteoblast differentiation.
3.4 RBM3 regulates osteoblast differentiation through the ERK signaling pathway To identify the molecular mechanisms involved in the control of Runx2 expression by
ACCEPTED MANUSCRIPT RBM3, we focused on the role of MAPKs. According to previous reports, RBM3 is known to regulate MAPKs in neuroblastoma cells [25,26]. We investigated whether RBM3 induces phosphorylation
of MAPKs
in
osteoblasts. Overexpression
of RBM3
increased
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phosphorylation of ERK and p38 but decreased phosphorylation of JNK (Fig. 4A). To confirm whether RBM3 regulates Runx2 through phosphorylation of ERK, genes and protein expression of Runx2 and RBM3 was confirmed using ERK inhibitor (PD98059, Calbiochem,
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Poland). The expression of Runx2 and RBM3 was increased by hypothermic conditions and A.A+β-GP. Although ERK inhibitor decreased Runx2 expression, it did not affect RBM3
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expression (Fig. 4B-E). Taken together, these results demonstrate that RBM3 regulates osteoblast differentiation by promoting Runx2 expression through ERK phosphorylation in osteoblast differentiation. In summary, hypothermic conditions and A.A+β-GP induced RBM3 expression, and RBM3 increased expression of Runx2 through phosphorylation of
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ERK in MC3T3-E1 cells.
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4. Discussion This study demonstrates that hypothermia-induced RBM3 regulates Runx2 through phosphorylation of ERK in MC3T3-E1 cells. In addition, RBM3 is positively regulated by
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A.A+β-GP. RBM3 increases mineralization of the extracellular matrix by increasing expression of OC and the knockdown of RBM3 expression suppresses transcription of the
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Runx2 gene. These observations indicate that RBM3 plays a key role in osteoblast differentiation.
Therapeutic hypothermia is used to treat patients whose heart or brain function is temporarily stopped. By reducing the body temperature artificially to decrease metabolism
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and oxygen consumption, it is possible to withstand relatively long blood flow interruption, and it is possible to prevent brain cell destruction by suppressing the response of the body to unnecessary stress [28,29]. In addition, there is a report that muscular dysfunction due to
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ischemia in muscle graft surgery can protect muscles due to short duration of ischemia by
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hypothermic preservation [30,31,32]. However, there are no studies related to osteoporosis and fracture.
Previously, 35°C or 27°C conditions have been reported to increase expression of
transcription factor Runx2 and Osterix [33]. However, the role of 32°C condition-induced RBM3 in osteoblast differentiation is unknown. Therefore, we focused on the role of RBM3 in osteoblasts. Runx2 plays a major role in osteoblast differentiation and regulates a range of factors, such as ALP and OC [11,34]. RBM3 positively regulated osteoblast differentiation in MC3T3-E1 cells. In addition, hypothermia-induced RBM3 significantly increased expression
ACCEPTED MANUSCRIPT of mature osteoblast marker genes (Runx2 and OC) and mineralization. Moreover, expression of RBM3 increased during osteogenesis. These results suggest that RBM3 is closely related to osteoblast differentiation.
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To gain insights into the mechanisms by which RBM3 regulates osteoblast differentiation, we first evaluated MAPK signaling pathways because previous studies have shown that RBM3 modulates MAPKs [26,35]. Overexpression of RBM3 had been shown to
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increase phosphorylation of MAPKs such as ERK and p38. When ERK is phosphorylated and activated, it is known to increase the expression of Runx2 and osteoblast differentiation
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marker genes [36,37,38,39]. Therefore, it was confirmed that ERK inhibitor did not affect the expression of RBM3 when ERK phosphorylation was inhibited, but decreased the expression of Runx2. Although it had been shown that RBM3 promotes osteoblast differentiation and osteogenesis through ERK signaling, there is a need to further study whether RBM3
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promotes osteoblast differentiation through the p38 signaling pathway. There are several studies indicating that the p38 signaling pathway and the ERK signaling pathway can promote osteoblast differentiation by the same stimulus [40,41,42]. Therefore, RBM3 can
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also be considered to promote osteoblast differentiation through p38 signaling.
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In summary, this study clarifies the role of RBM3 in osteoblast differentiation. RBM3 increases Runx2 gene expression via phosphorylation of ERK in hypothermia and osteogenic condition. Further studies are required to elucidate the detailed upstream signaling pathway and to determine if RBM3 is a valid therapeutic target for osteoporosis. Hypothermic treatment may be considered for bone related diseases such as osteoporosis and fracture.
Conflict of interest None of the authors have a conflict of interest.
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Acknowledgements
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This study was supported by the Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Education, Science and Technology (NRF-2016R1D1A1B03930733) (W.G. Jang) and E.J Kim was supported by NRF funded by Ministry of Education, Science and Technology (NRF2017R1D1A1B03030274).
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Table 1. Specific primer sequences for RT-PCR and real time-PCR Primer Sequence Forward
5′ -AGATGACATCCCCATCCATC- 3′
Reverse
5′ -GTGAGGGATGAAATGCTGG- 3′
Forward
5′ -CTGACCTCACAGATGCCAAG- 3′
Reverse
5′ -GTAGCGCCGGAGTCTGTTC- 3′
Forward
5′ -CCTTCACAAACCCAGAGCAT- 3′
Reverse
5′ -CCTTCACAAACCCAGAGCAT- 3′
Forward
5′ -TTCTACAATGAGCTGCGTGTG- 3′
Reverse
5′ -GGGGTGTTGAAGGTCTCAAA- 3′
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Runx2
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RBM3
β-actin
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Table 2. Sequences for siRNA synthesis
RBM3-2
Antisense
5′ -UAGCACUAAGGUGACCUGC- 3′
Sense
5′ -GAUAGAUAACUGGAGGUAU- 3′
Antisense
5′ -AUACCUCCAGUUAUCUAUC- 3′
Sense
5′ -GAGCUAUUAAAGUGACAGU- 3′
Antisense
5′ -ACUGUCACUUUAAUAGCUC- 3′
Sense
5′ -UGAUGACUCUAAACCUAGU- 3′
Antisense
5′ -ACUAGGUUUAGAGUCAUCA- 3′
Sense
5′ -GAGGAUGUACUGUGAUCAU- 3′
Antisense
5′ -AUGAUCACAGUACAUCCUC- 3′
Sense
5′ -UUCUCCGAACGUGUCACGU- 3′
Antisense
5′ -ACGUGACACGUUCGGAGAA- 3′
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Runx2-1
5′ -GCAGGUCACCUUAGUGCUA- 3′
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RBM3-1
Sequence Sense
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Name
Runx2-2
Runx2-3
Negative control
ACCEPTED MANUSCRIPT Figure legends
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Fig. 1. Effect of hypothermic condition and RBM3 on osteoblast differentiation in MC3T3E1 cells. MC3T3-E1 cells were cultured in the 37°C or 32°C Temperature (Temp.) 5% CO2 incubator. (A) The cells were harvested to determine the mRNA expression of osteogenic gene and RBM3 by RT-PCR analysis. (B) Runx2 and RBM3 protein level were determined by Western bolt analysis. (C) Cells were cultured with osteogenic medium (described in the Materials and Methods sessions) in the 37°C or 32°C Temperature (Temp.) 5% CO2 incubator. After 21 days, Alizarin Red S staining were conducted to check the extracellular matrix mineralization. **, p < 0.01 compared to the control (37°C), respectively. Data represent the mean ± SEM of three individual experiments. (D and E) MC3T3-E1 cells were treated with A.A+β-GP for 0, 12, 24, 36, 48 h. (D) Real-time PCR analysis were performed using total RNA isolated from the cells. *, p < 0.05 and **, p < 0.01 compared to the untreated control (0 h), respectively. Data represent the mean ± SEM of three individual experiments. (E) Runx2 and RBM3 protein levels were determined by Western blot analysis.
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Fig. 2. Overexpression of RBM3 regulated expression of osteogenic markers in MC3T3-E1 cells. MC3T3-E1 cells were transfected with 2 µg of pCMV-RBM3. At indicated times after transfection, the cells were harvested. (A-C) mRNA expression of RBM3 and osteogenic genes were determined by real-time PCR analysis. Relative mRNA expression was normalized by β-actin. *, p < 0.05, **, p < 0.01 compared to the untreated control (0 h), respectively. (D) The cells were transfected with 4 µg of pCMV-RBM3. At indicated times after transfection, the cells were harvested. Protein expression of Runx2 and RBM3 was determined by Western blot analysis with the indicated antibodies.
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Fig. 3. Expression of RBM3 induced Runx2 expression in MC3T3-E1 cells. (A and B) The cells were transfected with 100 nM of siRBM3-1 and siRBM3-2. 48 h after transfection, cells were harvested. (A) mRNA expression of osteogenic gene was determined by RT-PCR analysis. (B) Protein expression of Runx2 and RBM3 was determined by Western blot analysis using the indicated antibodies, and were quantified using image J. **, p < 0.01 compared to the untreated control. #, p < 0.05, ##, p < 0.01 compared to the positive control (A.A+β-GP), respectively. Data represent the mean ± SEM of three individual experiments. (C) MC3T3-E1 cells were transfected with 2 µg of HA-Runx2. 48 h after transfection, cells were harvested to determine the mRNA expression of Runx2 and RBM3. Relative mRNA expression was normalized by β-actin. *, p<0.05 compared with the untreated control. Data represent the mean ± SEM of three individual experiments. The cells were transfected with 100 nM of siRunx2-1, siRunx2-2 and siRunx2-3. 48 h after transfection, cells were harvested. (D and E) mRNA expression of osteogenic gene was determined by real-time PCR analysis. *, p < 0.05, **, p < 0.01 compared to the untreated control. #, p < 0.05 compared to the positive control (A.A+β-GP), respectively. Data represent the mean ± SEM of three individual
ACCEPTED MANUSCRIPT experiments. (F) Protein expression of Runx2 and RBM3 was determined by Western blot analysis using the indicated antibodies.
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Fig. 4. Block of ERK signaling decreases osteoblast differentiation. (A) MC3T3-E1 cells were transfected with 4 µg of pCMV-RBM3. At indicated times after transfection, the cells were harvested. Phosphorylation of ERK, p38 and JNK was determined by Western blot analysis with the indicated antibodies. (B and C) The cells were cultured with or without PD98059 (+: 10 µM, ++: 50 µM) in the 37‐ or 32‐ Temperature (Temp.) 5% CO2 incubator. (D and E) Cells were treated with or without A.A+β-GP and PD98059 (+: 50 µM). After 48 h, mRNA expression was measured by RT-PCR analysis. Western blot analysis also was performed with the indicated antibodies.
ACCEPTED MANUSCRIPT Figure 1.
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1.0 0.5 0.0
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siRunx2-3 Runx2
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Highlights Hypothermic condition induces the expression of RBM3 and osteogenic marker genes. Expression of RBM3 increases during the osteoblast differentiation. RBM3 plays a key role the phosphorylation of ERK in osteoblast differentiation.
S0006-291X(18)30465-0
DOI:
10.1016/j.bbrc.2018.02.209
Reference:
YBBRC 39574
To appear in:
Biochemical and Biophysical Research Communications
Received Date: 21 February 2018 Accepted Date: 28 February 2018
Please cite this article as: D.-Y. Kim, K.-M. Kim, E.-J. Kim, W.-G. Jang, Hypothermia-induced RNAbinding motif protein 3 (RBM3) stimulates osteoblast differentiation via the ERK signaling pathway, Biochemical and Biophysical Research Communications (2018), doi: 10.1016/j.bbrc.2018.02.209. 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.
A.A + β-GP
A.A: Ascorbic acid β -GP: β−Glycerophosphate
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Hypothermia
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ERK
PD98059
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Cytoplasm Nucleus
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Runx2 and OC
Osteoblast differentiation
ACCEPTED MANUSCRIPT Hypothermia-induced RNA-binding motif protein 3 (RBM3) stimulates osteoblast differentiation via the ERK signaling pathway
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Do-Young Kima,b, Kyeong-Min Kima,b, Eun-Jung Kim b,c*, Won-Gu Janga,b,*
a
Department of Biotechnology, School of Engineering, Daegu University, Gyeongbuk, 38453, Republic of Korea Research Institute of Anti-Aging, Daegu University, Gyeongbuk, 38453, Republic of Korea
c
Department of Immunology, Kyungpook National University School of Medicine, Daegu,
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b
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41944, Republic of Korea
Running title: RBM3 increases osteoblast differentiation via ERK signaling.
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Authors’ e-mail addresses: D.Y. Kim: [email protected] K.M. Kim: [email protected] E.J Kim: [email protected] W.G. Jang: [email protected]
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*Corresponding authors: Won-Gu Jang, Department of Biotechnology, college of Engineering, Daegu University, Gyeongbuk 38453, Republic of Korea. Tel.: 82-53-850-6552; Fax: 82-53-850-6559; E-mail: [email protected]. Eun-Jung Kim, Department of Immunology, Kyungpook National University School of Medicine, Daegu, 41944, Republic of Korea. Tel: 82-53-420-4877; Fax: 82-53-423-4628; E-mail: [email protected]
Abbreviations A.A, ascorbic acid; β-GP, β-glycerophosphate; RBM3, RNA-binding motif protein 3; OC, osteocalcin; Runx2, Runt-related transcription factor 2; ERK, Extracellular signal-regulated kinase
ACCEPTED MANUSCRIPT ABSTRACT The RNA-binding motif protein 3 (RBM3) belongs to a small group of proteins whose synthesis increases during hypothermia while global protein production is slowed down.
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Bone homeostasis is maintained by a balance between bone resorption and bone formation. Osteoblasts are key components of the bone and have an important role in bone remodeling cycle. However, hypothermia-induced RBM3 between osteoblasts remains unclear. At 32°C,
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expression of RBM3 and Runx2 was increased in a time-dependent manner and mineralization was also increased. RBM3 was also increased in a time-dependent manner
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under osteogenic conditions. Overexpression of RBM3 increased the expression of osteogenic genes such as Runx2 and OC. The osteogenic condition-induced expressions of RBM3, Runx2 and OC gene were decreased by RBM3 siRNA. Moreover, RBM3 promoted ERK and p38 phosphorylation. The inhibitor of ERK decreased the expression of Runx2 but
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did not affect the expression of RBM3. Taken together, these results demonstrate that RBM3 stimulates osteoblast differentiation via the ERK signaling pathway.
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Keywords: Hypothermia, RBM3, ERK, Runx2, Osteoblast differentiation
ACCEPTED MANUSCRIPT 1. Introduction Metabolic diseases such as osteoporosis, degenerative arthritis, obesity, and diabetes are greatly influenced by daily habits due to changes in the diet culture of modern society and
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decrease in exercise [1,2]. Osteoporosis is caused by the collapse of the balance of cells in maintaining bone health [3]. Osteoporosis is a frequent occurrence in women after menopause, requiring osteoporosis treatment and development of health functional foods [4].
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The importance of prevention rather than treatment is emphasized because there is no drug that can completely restore the reduced bone mass [5]. Estrogen is administered as the most
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typical treatment [6,7]. This treatment modality enables estrogen to regulate bone resorption and bone formation through the regulation of cytokines [8]. However, long-term use of estrogen causes many side effects, and therefore natural substances are needed to overcome this problem [9,10]. In recent years, research has focused on searching for natural materials
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that can replace synthetic drugs that cause side effects.
Osteoblast differentiation is regulated by a range of hormones, cytokines and various transcription factors [11,12]. Ascorbic acid (A.A) and β-glycerophosphate disodium salt
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hydrate (β-GP) promote osteoblast differentiation [13,14]. A.A+β-GP increase gene
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expression of Distal-less homeobox 5 (Dlx5) through an intracellular signaling system. Increased Dlx5 increases gene expression of Runt-related transcription factor 2 (Runx2), gene expression of Runx2 is essential for osteoblast differentiation. In addition, Runx2 expression increases calcium deposits by inducing mineralization through gene and protein expression of osteocalcin (OC) [15,16]. Previous studies have shown that bone density is related to various factors such as age, body type, sex, and race [17,18,19]. However, few studies have focused on cold-related bone mineral density. In the case of polar bears and penguins, thicker subcutaneous fat layers are
ACCEPTED MANUSCRIPT developed in the body to withstand the cold than bears and penguins that live in relatively warmer regions. In addition, obese people are reported to have a higher bone density than normal people [20]. On the basis of these findings, we judge that people living in cold
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regions will evolve subcutaneous fat to withstand the cold, and bone density will also increase to endure the developing fat. Therefore, we hypothesized that bone mineral density would increase if exposed to hypothermia.
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RNA-binding motif protein 3 (RBM3) is a member of the glycine-rich RNA-binding protein family and is the most representative protein that induces expression under cold shock
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and low oxygen conditions [21,22]. RBM3 was first screened in humans in 1995 and is known to be expressed in almost all tissues [23]. Expression of RBM3 has been shown to be essential for proper cell cycle progression and mitosis [22,24]. Interestingly, RBM3 has been reported to be able to regulate mitogen-activated protein kinases (MAPKs) such as
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extracellular signal regulated kinase (ERK), p38 kinase (p38) and c-Jun N-terminal kinases (JNK) [25,26]. In mammalian cells, there are MAPK signaling pathways, such as ERK, p38 and JNK that mediate extracellular signaling to the nucleus to activate stimulatory reactive
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genes such as growth factors and oxidative stress. The MAPK signaling pathways are essential for controlling many cellular processes including inflammation, cell differentiation,
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cell proliferation and death [27].
The role of 32°C condition-induced RBM3 in osteoblast differentiation has not yet
been elucidated. In this study, we demonstrate that hypothermia-induced RBM3 regulates Runx2 expression and activation through the ERK phosphorylation pathway, and subsequently increases the expression of OC.
ACCEPTED MANUSCRIPT 2. Materials and Methods 2.1. Cell culture and differentiation MC3T3-E1 cells were obtained from ATCC CCL-2593 (ATCC, Manassas, VA, USA).
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For maintenance, α-Minimal Essential Medium (α-MEM, Gibco, Grand Island, NY, USA) was used as the basal culture, and 10% Fetal Bovine Serum (FBS, Atlas biologicals, USA), 100 units/mL penicillin (Gibco) and 100 µg/mL streptomycin (Gibco) were mixed and
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incubated at 37°C and 5% CO2. Differentiation of osteoblasts was induced by addition of osteogenic condition containing 50 µg/mL ascorbic acid (A.A, Sigma Aldrich, St. Louis, MO,
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USA) and 5 mM β-glycerophosphate (β-GP, Sigma Aldrich). The culture medium was replaced every 2 days. To induce hypothermic conditions, cells were cultured in a 32°C 5% CO2 incubator.
RT-PCR and real-time PCR analysis
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2.2.
Total RNA was isolated from cells using TRI-solution (Bio Science Technology,
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Daegu, Korea) as per the manufacturer’s instructions. Reverse transcription was performed using 2.5 µg of total RNA. RT-PCR was performed using an emerald Amp GT PCR master
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mix (Takara, Rockland, ME, USA). Real-time PCR was performed on a LightCycler Nano Instrument (Roche, Mannheim, Germany) using AmpiGene™ qPCR Green Mix Hi-ROX (Enzo Life Sciences, Farmingdale, NY, USA). The RT-PCR and real-time PCR primer sequences are shown in Table 1.
2.3.
Transient transfection Overexpression vector "pCMV-RBM3 and pcDNA-Runx2-HA" used in the
ACCEPTED MANUSCRIPT experiment was distributed from the Korea Human Gene Bank (KRIBB, Korea). Potential RBM3 and Runx2 siRNAs were designed by Turbo si-Designer software (Bioneer, Korea). Negative control siRNA and potential RBM3 and Runx2 siRNA sequences are shown in
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Table 2. MC3T3-E1 cells were transiently transfected with the indicated plasmids using Lipofectamine 2000 (Invitrogen, Carlsbad, CA, USA) by using Opti-MEM® I (Gibco). After incubation at 37°C 5% CO2 incubator for 6 h, the medium for the basal culture was changed
ARS staining
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2.4.
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to α-MEM containing 10% FBS.
For mineralization analysis, MC3T3-E1 cells were cultured with or without A.A+βGP for 21 days. To compare with hypothermic conditions, one plate was transferred to the 32°C 5% CO2 incubator on the 19th day and cultured for 2 days. Staining was performed
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using standard protocols. Briefly, cultured cells were treated with 2% alizarin red solution
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(Sigma Aldrich) for 10 min at room temperature.
Western blot analysis
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Total protein was extracted using EzRIPA Lysis kit (ATTO Technology, Tokyo, Japan).
Total protein was quantified using Bradford protein assay reagent. The protein samples were separated by SDS-PAGE and transferred onto PVDF membranes. Signals were detected using ECL reagent (Advansta, Menlo Park, CA, USA) according to the manufacturer's protocol. Densitometry analysis of the blotted membranes was performed using a Fusion Solo (Vilber Lourmat, Eberhardzell, Germany).
ACCEPTED MANUSCRIPT 2.6. Statistical analysis All experiments were repeated at least three times. Statistical analysis was performed using Prism 5 (version 5.0, GraphPad Software, La Jolla, CA, USA) to Student’s t-test. P
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values of < 0.05 were considered significant. Results are expressed as the mean ± SEM of
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triplicate independent samples.
ACCEPTED MANUSCRIPT 3. Results 3.1 Hypothermic condition induces osteoblast differentiation via RBM3 expression in MC3T3E1 cells
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To confirm the effect of hypothermic conditions on osteoblast differentiation, we cultured MC3T3-E1 cells respectively at 37°C or 32°C 5% CO2 incubator for 0 to 24 h. RTPCR analysis showed that hypothermic conditions increased expression of RBM3 and
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osteogenic marker genes such as Runx2 and OC (Fig. 1A). Hypothermic conditions also increased protein levels of RBM3 and Runx2 better than at 37°C (Fig. 1B). In addition, when
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treated with ascorbic acid and β-glycerophosphate (A.A+β-GP) were induced for osteoblast differentiation, it was confirmed that the extracellular matrix was mineralized by calcium deposition. In addition, it was confirmed that the mineralization by hypothermic condition was significantly increased (Fig. 1C). To confirm whether RBM3 expression was regulated
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under differentiation conditions instead of hypothermic condition, cells were treated A.A+βGP. According to Real-time PCR analysis, the A.A+β-GP increased RBM3 expression with osteogenic gene, such as Runx2 and OC, expression in a time-dependent manner (Fig. 1D).
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Western blot analysis showed that protein levels of Runx2 and RBM3 also increased (Fig.
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1E). Taken together, the RBM3 expression was increased by hypothermic or osteogenic conditions with osteogenic gene expressions such as Runx2 and OC. These results suggest that RBM3 plays an important role in osteoblast differentiation in MC3T3E1 cells.
3.2 Overexpression of RBM3 positively regulates a major osteogenic marker expression To identify the molecular mechanisms involved in the control of osteoblast differentiation by RBM3, we focused on the early stage marker gene of osteoblast differentiation such as Runx2. The Real-time PCR analysis demonstrated that overexpression
ACCEPTED MANUSCRIPT of RBM3 significantly increased expression of Runx2 and OC gene expression (Fig. 2A-C). Western blot analysis showed that overexpression of RBM3 increased Runx2 expression (Fig. 2D). Overall, these results suggest that expression of RBM3 induced Runx2 and OC
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expression.
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3.3 Inhibition of RBM3 specifically suppresses Runx2 expression
To confirm the regulatory role of RBM3 in osteoblasts, the effect of down-regulation
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of RBM3 on Runx2 gene expression was examined using an RBM3-specific siRNA (siRBM3). The RT-PCR and Western blot analysis showed that A.A+β-GP-induced Runx2, OC and RBM3 gene expressions were reduced by siRBM3 (Fig. 3A). The increased protein expression level of Runx2 by A.A+β-GP also decreased by siRBM3 (Fig. 3B). To further
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determine if Runx2 regulates RBM3 expression, the effects of Runx2 on RBM3 expression using real-time PCR analysis. As a result, Runx2 overexpression did not affect RBM3 expression (Fig. 3C). In addition, Runx2-specific siRNA (siRunx2) was used to confirm the
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expression of Runx2 and RBM3 during osteoblast differentiation. The real time-PCR and Western blot analysis showed that A.A+β-GP-induced Runx2 and RBM3 expression was
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reduced by siRunx2 (Fig. 3D and F). However, siRunx2 did not affect RBM3 expression (Fig. 5E and F). Overall, these results suggest that Runx2 expression is increased by RBM3 during osteoblast differentiation.
3.4 RBM3 regulates osteoblast differentiation through the ERK signaling pathway To identify the molecular mechanisms involved in the control of Runx2 expression by
ACCEPTED MANUSCRIPT RBM3, we focused on the role of MAPKs. According to previous reports, RBM3 is known to regulate MAPKs in neuroblastoma cells [25,26]. We investigated whether RBM3 induces phosphorylation
of MAPKs
in
osteoblasts. Overexpression
of RBM3
increased
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phosphorylation of ERK and p38 but decreased phosphorylation of JNK (Fig. 4A). To confirm whether RBM3 regulates Runx2 through phosphorylation of ERK, genes and protein expression of Runx2 and RBM3 was confirmed using ERK inhibitor (PD98059, Calbiochem,
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Poland). The expression of Runx2 and RBM3 was increased by hypothermic conditions and A.A+β-GP. Although ERK inhibitor decreased Runx2 expression, it did not affect RBM3
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expression (Fig. 4B-E). Taken together, these results demonstrate that RBM3 regulates osteoblast differentiation by promoting Runx2 expression through ERK phosphorylation in osteoblast differentiation. In summary, hypothermic conditions and A.A+β-GP induced RBM3 expression, and RBM3 increased expression of Runx2 through phosphorylation of
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ERK in MC3T3-E1 cells.
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4. Discussion This study demonstrates that hypothermia-induced RBM3 regulates Runx2 through phosphorylation of ERK in MC3T3-E1 cells. In addition, RBM3 is positively regulated by
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A.A+β-GP. RBM3 increases mineralization of the extracellular matrix by increasing expression of OC and the knockdown of RBM3 expression suppresses transcription of the
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Runx2 gene. These observations indicate that RBM3 plays a key role in osteoblast differentiation.
Therapeutic hypothermia is used to treat patients whose heart or brain function is temporarily stopped. By reducing the body temperature artificially to decrease metabolism
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and oxygen consumption, it is possible to withstand relatively long blood flow interruption, and it is possible to prevent brain cell destruction by suppressing the response of the body to unnecessary stress [28,29]. In addition, there is a report that muscular dysfunction due to
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ischemia in muscle graft surgery can protect muscles due to short duration of ischemia by
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hypothermic preservation [30,31,32]. However, there are no studies related to osteoporosis and fracture.
Previously, 35°C or 27°C conditions have been reported to increase expression of
transcription factor Runx2 and Osterix [33]. However, the role of 32°C condition-induced RBM3 in osteoblast differentiation is unknown. Therefore, we focused on the role of RBM3 in osteoblasts. Runx2 plays a major role in osteoblast differentiation and regulates a range of factors, such as ALP and OC [11,34]. RBM3 positively regulated osteoblast differentiation in MC3T3-E1 cells. In addition, hypothermia-induced RBM3 significantly increased expression
ACCEPTED MANUSCRIPT of mature osteoblast marker genes (Runx2 and OC) and mineralization. Moreover, expression of RBM3 increased during osteogenesis. These results suggest that RBM3 is closely related to osteoblast differentiation.
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To gain insights into the mechanisms by which RBM3 regulates osteoblast differentiation, we first evaluated MAPK signaling pathways because previous studies have shown that RBM3 modulates MAPKs [26,35]. Overexpression of RBM3 had been shown to
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increase phosphorylation of MAPKs such as ERK and p38. When ERK is phosphorylated and activated, it is known to increase the expression of Runx2 and osteoblast differentiation
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marker genes [36,37,38,39]. Therefore, it was confirmed that ERK inhibitor did not affect the expression of RBM3 when ERK phosphorylation was inhibited, but decreased the expression of Runx2. Although it had been shown that RBM3 promotes osteoblast differentiation and osteogenesis through ERK signaling, there is a need to further study whether RBM3
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promotes osteoblast differentiation through the p38 signaling pathway. There are several studies indicating that the p38 signaling pathway and the ERK signaling pathway can promote osteoblast differentiation by the same stimulus [40,41,42]. Therefore, RBM3 can
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also be considered to promote osteoblast differentiation through p38 signaling.
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In summary, this study clarifies the role of RBM3 in osteoblast differentiation. RBM3 increases Runx2 gene expression via phosphorylation of ERK in hypothermia and osteogenic condition. Further studies are required to elucidate the detailed upstream signaling pathway and to determine if RBM3 is a valid therapeutic target for osteoporosis. Hypothermic treatment may be considered for bone related diseases such as osteoporosis and fracture.
Conflict of interest None of the authors have a conflict of interest.
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Acknowledgements
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This study was supported by the Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Education, Science and Technology (NRF-2016R1D1A1B03930733) (W.G. Jang) and E.J Kim was supported by NRF funded by Ministry of Education, Science and Technology (NRF2017R1D1A1B03030274).
ACCEPTED MANUSCRIPT 5. References
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loss of metacarpal cortex in relation to postmenopausal cigarette smoking and lack of obesity, Arch Intern Med 136 (1976) 298-304. [18] D.M. Smith, M.R. Khairi, J. Norton, C.C. Johnston, Jr., Age and activity effects on rate of bone mineral loss, J Clin Invest 58 (1976) 716-721. [19] S.-N. Choi, N.-Y. Chung, C.-H. Song, S.-R. Kim, Bone density and nutrient intake of university students, Journal of the Korean Society of Food Culture 22 (2007) 841-847. [20] S.-J.K. Jang, Jeoung-Yeun; Yook, Tae-Han, A Clinical Study on the Correlation between Bone Mineral Density (BMD) and Ovesity in 480 normal adults., The Journal Of Korean Acupuncture & Moxibustion Medicine Society 15 (1998) 383-392. [21] S. Danno, H. Nishiyama, H. Higashitsuji, H. Yokoi, J.H. Xue, K. Itoh, T. Matsuda, J. Fujita, Increased transcript level of RBM3, a member of the glycine-rich RNAbinding protein family, in human cells in response to cold stress, Biochem Biophys Res Commun 236 (1997) 804-807. [22] S. Wellmann, M. Truss, E. Bruder, L. Tornillo, A. Zelmer, K. Seeger, C. Bührer, The RNA-binding protein RBM3 is required for cell proliferation and protects against serum deprivation-induced cell death, Pediatric research 67 (2010) 35-41. [23] J.M. Derry, J.A. Kerns, U. Francke, RBM3, a novel human gene in Xp11.23 with a putative RNA-binding domain, Hum Mol Genet 4 (1995) 2307-2311. [24] S. Sureban, S. Ramalingam, G. Natarajan, R. May, D. Subramaniam, K. Bishnupuri, A. Morrison, B. Dieckgraefe, D. Brackett, R. Postier, Translation regulatory factor RBM3 is a proto-oncogene that prevents mitotic catastrophe, Oncogene 27 (2008) 4544-4556. [25] H.-J. Yang, F. Ju, X.-X. Guo, S.-P. Ma, L. Wang, B.-F. Cheng, R.-J. Zhuang, B.-B. Zhang, X. Shi, Z.-W. Feng, RNA-binding protein RBM3 prevents NO-induced apoptosis in human neuroblastoma cells by modulating p38 signaling and miR-143, Scientific Reports 7 (2017). [26] R.J. Zhuang, J. Ma, X. Shi, F. Ju, S.P. Ma, L. Wang, B.F. Cheng, Y.W. Ma, M. Wang, T. Li, Z.W. Feng, H.J. Yang, Cold-Inducible Protein RBM3 Protects UV IrradiationInduced Apoptosis in Neuroblastoma Cells by Affecting p38 and JNK Pathways and Bcl2 Family Proteins, J Mol Neurosci 63 (2017) 142-151. [27] M. Cargnello, P.P. Roux, Activation and function of the MAPKs and their substrates, the MAPK-activated protein kinases, Microbiology and molecular biology reviews 75 (2011) 50-83. [28] A.E. Bennett, R.E. Hoesch, L.D. DeWitt, P. Afra, S.A. Ansari, Therapeutic hypothermia for status epilepticus: A report, historical perspective, and review, Clin Neurol Neurosurg 126 (2014) 103-109. [29] Y.-M. Kim, H.-W. Yim, S.-H. Jeong, M.L. Klem, C.W. Callaway, Does therapeutic hypothermia benefit adult cardiac arrest patients presenting with non-shockable initial rhythms?: A systematic review and meta-analysis of randomized and non-randomized studies, Resuscitation 83 (2012) 188-196. [30] E.E. Dupont-Versteegden, R. Nagarajan, M.L. Beggs, E.D. Bearden, P.M. Simpson, C.A. Peterson, Identification of cold-shock protein RBM3 as a possible regulator of skeletal muscle size through expression profiling, American Journal of PhysiologyRegulatory, Integrative and Comparative Physiology 295 (2008) R1263-R1273. [31] A.L. Ferry, P.W. Vanderklish, E.E. Dupont-Versteegden, Enhanced survival of skeletal muscle myoblasts in response to overexpression of cold shock protein RBM3, Am J Physiol Cell Physiol 301 (2011) C392-402. [32] M.P. Bolognesi, L.E. Chen, A.V. Seaber, J.R. Urbaniak, Protective effect of hypothermia on contractile force in skeletal muscle, J Orthop Res 14 (1996) 390-395.
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[33] M.D. Aisha, M.N. Nor-Ashikin, A.B. Sharaniza, H.M. Nawawi, M.Y. Kapitonova, G.R. Froemming, Short-term moderate hypothermia stimulates alkaline phosphatase activity and osteocalcin expression in osteoblasts by upregulating Runx2 and osterix in vitro, Exp Cell Res 326 (2014) 46-56. [34] R.T. Franceschi, C. Ge, G. Xiao, H. Roca, D. Jiang, Transcriptional regulation of osteoblasts, Ann N Y Acad Sci 1116 (2007) 196-207. [35] Y. Wang, S. Luo, D. Zhang, X. Qu, Y. Tan, Sika pilose antler type I collagen promotes BMSC differentiation via the ERK1/2 and p38-MAPK signal pathways, Pharm Biol 55 (2017) 2196-2204. [36] Z. Hamidouche, O. Fromigue, J. Ringe, T. Haupl, P. Vaudin, J.C. Pages, S. Srouji, E. Livne, P.J. Marie, Priming integrin alpha5 promotes human mesenchymal stromal cell osteoblast differentiation and osteogenesis, Proc Natl Acad Sci U S A 106 (2009) 18587-18591. [37] G. Xiao, D. Jiang, P. Thomas, M.D. Benson, K. Guan, G. Karsenty, R.T. Franceschi, MAPK pathways activate and phosphorylate the osteoblast-specific transcription factor, Cbfa1, J Biol Chem 275 (2000) 4453-4459. [38] C. Ge, G. Xiao, D. Jiang, R.T. Franceschi, Critical role of the extracellular signalregulated kinase-MAPK pathway in osteoblast differentiation and skeletal development, J Cell Biol 176 (2007) 709-718. [39] C.B. Khatiwala, S.R. Peyton, M. Metzke, A.J. Putnam, The regulation of osteogenesis by ECM rigidity in MC3T3-E1 cells requires MAPK activation, J Cell Physiol 211 (2007) 661-672. [40] C. Ge, Q. Yang, G. Zhao, H. Yu, K.L. Kirkwood, R.T. Franceschi, Interactions between extracellular signal‐regulated kinase 1/2 and P38 Map kinase pathways in the control of RUNX2 phosphorylation and transcriptional activity, Journal of Bone and Mineral Research 27 (2012) 538-551. [41] M.B. Greenblatt, J.-H. Shim, W. Zou, D. Sitara, M. Schweitzer, D. Hu, S. Lotinun, Y. Sano, R. Baron, J.M. Park, The p38 MAPK pathway is essential for skeletogenesis and bone homeostasis in mice, The Journal of clinical investigation 120 (2010) 2457. [42] N. Artigas, C. Ureña, E. Rodríguez-Carballo, J.L. Rosa, F. Ventura, Mitogen-activated protein kinase (MAPK)-regulated interactions between Osterix and Runx2 are critical for the transcriptional osteogenic program, Journal of Biological Chemistry 289 (2014) 27105-27117.
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Table 1. Specific primer sequences for RT-PCR and real time-PCR Primer Sequence Forward
5′ -AGATGACATCCCCATCCATC- 3′
Reverse
5′ -GTGAGGGATGAAATGCTGG- 3′
Forward
5′ -CTGACCTCACAGATGCCAAG- 3′
Reverse
5′ -GTAGCGCCGGAGTCTGTTC- 3′
Forward
5′ -CCTTCACAAACCCAGAGCAT- 3′
Reverse
5′ -CCTTCACAAACCCAGAGCAT- 3′
Forward
5′ -TTCTACAATGAGCTGCGTGTG- 3′
Reverse
5′ -GGGGTGTTGAAGGTCTCAAA- 3′
RI PT
Runx2
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RBM3
β-actin
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Table 2. Sequences for siRNA synthesis
RBM3-2
Antisense
5′ -UAGCACUAAGGUGACCUGC- 3′
Sense
5′ -GAUAGAUAACUGGAGGUAU- 3′
Antisense
5′ -AUACCUCCAGUUAUCUAUC- 3′
Sense
5′ -GAGCUAUUAAAGUGACAGU- 3′
Antisense
5′ -ACUGUCACUUUAAUAGCUC- 3′
Sense
5′ -UGAUGACUCUAAACCUAGU- 3′
Antisense
5′ -ACUAGGUUUAGAGUCAUCA- 3′
Sense
5′ -GAGGAUGUACUGUGAUCAU- 3′
Antisense
5′ -AUGAUCACAGUACAUCCUC- 3′
Sense
5′ -UUCUCCGAACGUGUCACGU- 3′
Antisense
5′ -ACGUGACACGUUCGGAGAA- 3′
AC C
Runx2-1
5′ -GCAGGUCACCUUAGUGCUA- 3′
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RBM3-1
Sequence Sense
EP
Name
Runx2-2
Runx2-3
Negative control
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Fig. 1. Effect of hypothermic condition and RBM3 on osteoblast differentiation in MC3T3E1 cells. MC3T3-E1 cells were cultured in the 37°C or 32°C Temperature (Temp.) 5% CO2 incubator. (A) The cells were harvested to determine the mRNA expression of osteogenic gene and RBM3 by RT-PCR analysis. (B) Runx2 and RBM3 protein level were determined by Western bolt analysis. (C) Cells were cultured with osteogenic medium (described in the Materials and Methods sessions) in the 37°C or 32°C Temperature (Temp.) 5% CO2 incubator. After 21 days, Alizarin Red S staining were conducted to check the extracellular matrix mineralization. **, p < 0.01 compared to the control (37°C), respectively. Data represent the mean ± SEM of three individual experiments. (D and E) MC3T3-E1 cells were treated with A.A+β-GP for 0, 12, 24, 36, 48 h. (D) Real-time PCR analysis were performed using total RNA isolated from the cells. *, p < 0.05 and **, p < 0.01 compared to the untreated control (0 h), respectively. Data represent the mean ± SEM of three individual experiments. (E) Runx2 and RBM3 protein levels were determined by Western blot analysis.
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Fig. 2. Overexpression of RBM3 regulated expression of osteogenic markers in MC3T3-E1 cells. MC3T3-E1 cells were transfected with 2 µg of pCMV-RBM3. At indicated times after transfection, the cells were harvested. (A-C) mRNA expression of RBM3 and osteogenic genes were determined by real-time PCR analysis. Relative mRNA expression was normalized by β-actin. *, p < 0.05, **, p < 0.01 compared to the untreated control (0 h), respectively. (D) The cells were transfected with 4 µg of pCMV-RBM3. At indicated times after transfection, the cells were harvested. Protein expression of Runx2 and RBM3 was determined by Western blot analysis with the indicated antibodies.
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Fig. 3. Expression of RBM3 induced Runx2 expression in MC3T3-E1 cells. (A and B) The cells were transfected with 100 nM of siRBM3-1 and siRBM3-2. 48 h after transfection, cells were harvested. (A) mRNA expression of osteogenic gene was determined by RT-PCR analysis. (B) Protein expression of Runx2 and RBM3 was determined by Western blot analysis using the indicated antibodies, and were quantified using image J. **, p < 0.01 compared to the untreated control. #, p < 0.05, ##, p < 0.01 compared to the positive control (A.A+β-GP), respectively. Data represent the mean ± SEM of three individual experiments. (C) MC3T3-E1 cells were transfected with 2 µg of HA-Runx2. 48 h after transfection, cells were harvested to determine the mRNA expression of Runx2 and RBM3. Relative mRNA expression was normalized by β-actin. *, p<0.05 compared with the untreated control. Data represent the mean ± SEM of three individual experiments. The cells were transfected with 100 nM of siRunx2-1, siRunx2-2 and siRunx2-3. 48 h after transfection, cells were harvested. (D and E) mRNA expression of osteogenic gene was determined by real-time PCR analysis. *, p < 0.05, **, p < 0.01 compared to the untreated control. #, p < 0.05 compared to the positive control (A.A+β-GP), respectively. Data represent the mean ± SEM of three individual
ACCEPTED MANUSCRIPT experiments. (F) Protein expression of Runx2 and RBM3 was determined by Western blot analysis using the indicated antibodies.
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Fig. 4. Block of ERK signaling decreases osteoblast differentiation. (A) MC3T3-E1 cells were transfected with 4 µg of pCMV-RBM3. At indicated times after transfection, the cells were harvested. Phosphorylation of ERK, p38 and JNK was determined by Western blot analysis with the indicated antibodies. (B and C) The cells were cultured with or without PD98059 (+: 10 µM, ++: 50 µM) in the 37‐ or 32‐ Temperature (Temp.) 5% CO2 incubator. (D and E) Cells were treated with or without A.A+β-GP and PD98059 (+: 50 µM). After 48 h, mRNA expression was measured by RT-PCR analysis. Western blot analysis also was performed with the indicated antibodies.
ACCEPTED MANUSCRIPT Figure 1.
A.
B. 37 ℃ 0
Time(h)
12
32 ℃
24
48
0
12
24h
24
48
37
Temp. (℃ ℃)
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C. -
Temp. (℃ ℃)
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*
**
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β -GP 12 24 36 48 (h) A.A+β
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β-actin
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β-actin
β-actin
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RBM3
RBM3
Runx2
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Runx2
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A.A+β β -GP
48h
1.5
**
** **
1.0 0.5 0.0
A.A+β β -GP
0
12 24 36 48 (h)
**
12 24 36 48 (h)
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A.
0
1
3
6 12 24 (h)
3
6 12 24 (h)
SC
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RBM3
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A.
siRBM3-2 Runx2
+ -
+ + -
+ +
**
B.
siRBM3-2 Runx2
-
+ -
+ + -
+ +
1.5
#
1.0 ##
SC
RBM3/β -actin
RBM3 β-actin
A.A+β β -GP siRBM3-1
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-
0.5 0.0
β-actin
si-RBM3-2
-
+ -
+ + -
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A.A+β β -GP si-RBM3-1
Runx2/β -actin
A.A+β β -GP siRBM3-1
**
2.0
#
1.5
##
1.0 0.5 0.0
-
+ +
+ -
+ + -
+ +
D.
HA-Ruxn2
+
HA-Ruxn2
-
+
E.
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**
** **
** **
1.5 1.0 0.5 0.0
A.A+β β -GP siRunx2-1 siRunx2-2 siRunx2-3
##
-
+ -
+ + -
+ +
+ -
-
-
-
-
+
##
1.0 0.5
-
+ -
+ + -
+ +
+ -
-
-
-
-
+
A.A+β β -GP siRunx2-1 siRunx2-2
-
+ -
+ + -
+ +
+ -
siRunx2-3 Runx2
-
-
-
-
+
siRunx2-2 siRunx2-3
**
**
1.5
0.0
RBM3
2.0
Runx2 2.0
A.A+β β -GP siRunx2-1
EP
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*
Rel. RNA expression
C.
F.
RBM3 β-actin
ACCEPTED MANUSCRIPT Figure 4. A. RBM3
0
3
6
9
(h)
p-ERK ERK
RI PT
p-p38 p38 p-JNK JNK β-actin
37
32
-
PD98059
-
+
++
Runx2 RBM3 β-actin
C. Temp. (℃)
37
32
-
PD98059 p-ERK
-
+
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t-ERK Runx2 RBM3 β-actin
D. -
+
PD98059
-
-
+
+
+
AC C
RBM3 β-actin
-
EP
A.A+β β -GP Runx2
E.
++
A.A+β β -GP
-
+
-
+
PD98059
-
-
+
+
p-ERK t-ERK Runx2 RBM3 β-actin
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B.
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Highlights Hypothermic condition induces the expression of RBM3 and osteogenic marker genes. Expression of RBM3 increases during the osteoblast differentiation. RBM3 plays a key role the phosphorylation of ERK in osteoblast differentiation.