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Keywords:

  • SPATA4;
  • OSTEOBLAST;
  • ERK;
  • RUNX2;
  • MINERALIZATION

Abstract

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Disclosures
  8. Acknowledgements
  9. References

The spermatogenesis associated 4 gene (Spata4, previously named TSARG2) was demonstrated to participate in spermatogenesis. Here we report that Spata4 is expressed in osteoblasts and that overexpression of Spata4 accelerates osteoblast differentiation and mineralization in MC3T3-E1 cells. We found that Spata4 interacts with p-Erk1/2 in the cytoplasm and that overexpression of Spata4 enhances the phosphorylation of Erk1/2. Intriguingly, we observed that Spata4 increases the transcriptional activity of Runx2, a critical transcription factor regulating osteoblast differentiation. We showed that Spata4-activated Runx2 is through the activation of Erk1/2. Consistent with this observation, we found that overexpression of Spata4 increases the expression of osteoblastic marker genes, including osteocalcin (Ocn), Bmp2, osteopontin (Opn), type 1 collagen, osterix (Osx), and Runx2. We concluded that Spata4 promotes osteoblast differentiation and mineralization through the Erk-activated Runx2 pathway. Our findings provided new evidence that Spata4 plays a role in regulation of osteoblast differentiation. © 2011 American Society for Bone and Mineral Research


Introduction

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Disclosures
  8. Acknowledgements
  9. References

Spermatogenesis associated 4 gene (Spata4, previously named TSARG2) was demonstrated to participate in the spermatogenesis and was identified initially in human testes.1 Spata4 has been reported to have an important function in the development of the cryptorchid testis.1 Spata4 has a calponin homology (CH) domain at the N-terminus, which was reported to interact with extracellular signal-regulated kinase (Erk), a critical kinase important in osteoblast differentiation.2, 3

The Erk pathway is important for many cellular responses and is also vital for the bone in response to multiple signals, including growth/hormone factor stimulation and matrix-integrin binding.4–7 The activity of the Erk–mitogen-activated protein kinase (MAPK) pathway is required for osteoblast differentiation.8 In particular, Erk is found to phosphorylate the bone-specific transcription factor Runx2 at Ser301 and Ser319 residues and to stimulate its transcriptional activity.4, 9–13 In calvarial osteoblasts from a transgenic mouse of constitutively active MAPK/Erk1, Runx2 transcriptional levels are increased, whereas the activity of Runx2 is repressed in a transgenic mouse of dominant-negative MEK1.8 Consistent with these lines of evidence, constitutively active MAPK was reported to increase mRNA levels of the osteoblast differentiation–related gene Ocn.8, 14 Since Spata4 has a CH domain, we speculated that it might play a role in osteoblast differentiation in relation to Erk-mediated signal transduction.

Although several studies showed the Spata4 mRNA transcript level in different species, little is known about the function in osteoblast differentiation. In this study, we report that Spata4 plays an important role in osteoblast differentiation by using MC3T3-E1 cells as a model.

Materials and Methods

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Disclosures
  8. Acknowledgements
  9. References

Plasmids, cell lines, and cell culture

Constructs with Runx2-binding element–Luc and 1.3mOG2-Luc were kindly provided by Dr RT Franceschi (School of Medicine, University of Michigan, Ann Arbor, MI, USA). Mouse Spata4 cDNA was cloned from a total cDNA library from mouse testes and subcloned into pcDNA3.1 vector.

MC3T3-E1 cells were maintained in α modified essential medium (α-MEM; Gibco, Carlsbad, CA, USA) supplemented with 10% fetal bovine serum (FBS), 100 units/mL of penicillin, and 100 µg/mL of streptomycin. The mineralization medium was prepared by addition of 50 µg/mL of ascorbic acid, 10 mM sodium β-glycerophosphate, and 10 nM dexamethasone into α-MEM.15

A Spata4-overexpression-stable cell line (MC3T3-E1-Spata4) was established by selecting with G418 (750 µg/mL) and maintained with G418 (300 µg/mL).

Immunofluorescence staining

MC3T3-E1 cells were grown on a 6-well dish with a density of 1 to 2 × 106 cells/well and fixed with methylate-acetone (1:1) for 30 minutes. After blocking with 10% bovine serum albumin (BSA) for 1 hour, a primary antibody against Spata4 was added for 2 hours and labeled with FITC (1:100). Propidium iodide (0.1 mg/mL) was added to label the nucleus for 10 minutes. A negative control without the primary antibody was used to monitor the staining background. Images were obtained through a confocal laser scanning microscope (TCS SP5, Leica, Mannheim, Germany).

Immunohistochemistry

Tissues from the calvaria and femurs of C57BL6 mice within 24 hours of birth were subjected to frozen sections and immersed in 0.3% H2O2 solution to inhibit endogenous perxidase activity. Following a blocking treatment with horse sera, an anti-Spata4 antibody (1:100) as the primary antibody was applied to the sections overnight at 4°C. A biotin-conjugated anti-rabbit IgG was used as secondary antibody and incubated for 20 minutes at 37°C. Horseradish peroxidase (HRP)–conjugated streptavidin was added for 15 minutes at 37°C. Immunoreaction was visualized by addition of 2% diaminobenzidine supplemented with 0.002% H2O2. Buffer without the primary antibody was used as a negative control.16 Sections were stained with hematoxylin for 1 minute.

Immunoprecipitation

Immunoprecipitation was performed by using antibodies against p-Erk1/2 (Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA) and protein A/G PLUS-Agarose (Santa Cruz Biotechnology, Inc.). MC3T3-E1-Spata4 cells were lysed in RIPA buffer (Biomiga, San Diego, CA, USA). Cell lysates were incubated with antibodies for 24 hours at 4°C and then with protein A/G PLUS-Agarose for 2 hours at room temperature.17 An IgG antibody was used as a negative control.

Quantitative real-time polymerase chain reaction (RT-qPCR)

Total mRNA was extracted from MC3T3-E1-Spata4, control (MC3T3-E1 transfected with an empty vector), and MC3T3-E1 cells transfected with a Spata4 siRNA using Trizol (Invitrogen, Carlsbad, CA, USA). cDNA was made with SuperScript III (Invitrogen) using oligo dTs as primers and was used subsequently for a RT-qPCR analysis with SYBR Green (Toyobo, Osaka, Japan). Primer information is listed in Table 1. Amplifications were performed in a program for 41 cycles as follows: first cycle (10 minutes at 95°C, 1 minute at 56°C, 30 seconds at 72°C), next 40 cycles (30 seconds at 95°C, 1 minute at 56°C, 30 seconds at 72°C).

Table 1. Primers Used for RT-PCR
GenePrimerBp
  1. bp = base pairs; Fw = forward; Rv = reverse.

GapdhFw: 5'-CATGGCCTTCCGTGTTCCTA-3'104
 Rv: 5'-CCTGCTTCACCACCTTCTTGAT-3' 
OsteocalcinFw: 5'-CAATAAGGTAGTGAACAGAC-3'95
 Rv: 5'-CTTCAAGCCATACTGGTCT-3' 
OsteopontinFw: 5'-TCCAAAGCCAGCCTGGAAC-3'142
 Rv: 5'-TGACCTCAGAAGATGAACTC-3' 
Bmp2Fw: 5'-CTGACCACCTGAACTCCAC-3'126
 Rv: 5'-CATCTAGGTACAACATGGAG-3' 
Type 1 collagenFw: 5'-CCTGGTAAAGATGGTGCC-3'222
 Rv: 5'-CACCAGG TTCACCTTTCGCACC-3' 
Runx2Fw: 5'-GAATGCACTACCCAGCCAC-3'98
 Rv: 5'-TGGCAGGTACGTGTGGTAG-3' 
OsterixFw: 5'-GTCAAGAGTCTTAGCCAAACTC-3'123
 Rv: 5'-AAATGATGTGAGGCCAGATGG-3' 
Spata4Fw: 5'-CAGATACAAGTCAAGAGGTTC-3'120
 Rv: 5'-GTGTTCTCACAAGGATTTCCAC-3' 

Western blotting

MC3T3-E1-Spata4, control, and primary mouse calvarial and femur cells were harvested and lysated with TEN-T buffer (150 mM NaCl, 10 mM Tris-HCl, pH 7.4, 5 mM EDTA, pH 8.0, 1% Triton X-100, 1 mM PMSF, and 2 µg/mL of aprotinin). Cell lysates were centrifuged at 12,000g for 15 minutes at 4°C to discard cell debris. An amount of 30 µg total protein was fractioned by 10% SDS-polyacrylamide gels. Proteins were transferred to polyvinylidene fluoride (PVDF) membranes for blotting. An antibody against Spata4 was used at 1:1000 dilution and p-Erk1/2 at 1:1000 (Biosource, Camarillo, CA, USA), Erk1/2 at 1:1000 (Chemicon, Temecula, CA, USA), and Runx2 at 1:500 (Abcam, Cambridge, UK) for blotting overnight at 4°C, respectively. Second antibodies were used at 1:10,000 dilution. Immunoreactivity was visualized using an enhanced chemiluminescent substrate for detection of HRP (Pierce, Waltham, MA, USA).18

Cell transfection and luciferase assays

Cells were transfected using lipofectamin 2000 (Invitrogen) according to the manufacturer's instruction. The amounts of transfected DNA were balanced in each transfection. Cells were cultured in α-MEM or mineralization medium for different times as indicated. Luciferase activities were measured using a Dual Luciferase Assay Kit (Promega, San Luis Obispo, CA, USA) on a Luminometer (Berthold, Bad Wildbad, Germany). A relative value was calculated based on an internal control and presented as an average with standard error from three repeats.

Alkaline phosphatase (ALP) activity

Cells were plated in 96-well plates at a density of 1 × 104 cells/well for different days as indicated. Cell lysates from the same treatment were divided into two parts for measurements of protein concentration and ALP activity. Protein concentration was determined using a BCA protein assay kit (Pierce) with the absorbance at 550 nm. ALP assays were measured by incubation in 0.1 M NaHCO3-Na2CO3 buffer (pH 10.0) containing 0.1% Triton X-100, 2 mM MgSO4, and 6 mM p-nitrophenol inorganic phosphate (PNPP) for 30 minutes at 37°C under absorbance at 405 nm. The relative ALP activity was calculated as the value of absorbance at 405 nm divided by the value of absorbance at 550 nm (A405/A550).19

Mineralization analysis

Cells were plated into a 6-well dish at a density of 1 × 104 cells/cm2 for osteoblast differentiation. The mineralization medium was added after cells were incubated overnight with normal medium. The mineralization medium was changed every 3 days. Mineralization was examined after incubating for 14 days for Alizarin red staining and 21 days for von Kossa staining. For Alizarin red staining, cells were fixed in 70% ethanol for 10 minutes, and calcium deposits were stained for 15 minutes at room temperature with Alizarin red solution (40 mM, pH 4.2). Nonspecific staining was removed by several washes in water.19 For von Kossa staining, cells were fixed in ice-cold 10% formalin for 15 minutes and stained with 5% silver nitrate for 30 minutes. Then cells were washed with 5% sodium carbonate in 10% formalin for at least 5 minutes and 5% sodium thiosulfate for 5 minutes. Reactions were stopped by washing three times with water.20

Statistical analysis

Data are expressed as mean ± SE from at least three independent experiments. Statistical significance was subject to an unpaired Student's t test. A p value of less than .05 was used for a threshold of a statistical significance.

Results

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Disclosures
  8. Acknowledgements
  9. References

Spata4 is localized in the cytoplasm and expressed in the calvaria and femurs

To study the role of Spata4 in osteoblasts, we performed immunofluorescence and cellular fractionation experiments in MC3T3-E1 cells to illustrate its cellular localization. The results showed that Spata4 is localized in the cytoplasm (Fig. 1A). The Spata4 protein is found only in the fraction of the cytoplasma, whereas there is no distribution in the nucleus (Fig. 1B). Furthermore, the expression of Spata4 is abundant in primary cells harvested from mouse calvaria and trabecular bone (femurs) on postnatal days 1, 5, 10, and 20 (Fig. 1C). The endogenous protein of Spata4 in the cytoplasm was observed in calvaria and femurs from newborn mice in vivo by immunofluorescence staining (Fig. 1D) and immunohistochemistry (Fig. 1E). The Spata4 protein is also observed in the cytoplasm of hypertrophic cartilage of femur growth plates. All the data indicated that Spata4 is expressed and localized in the cytoplasm in osteoblasts.

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Figure 1. Spata4 protein localization in MC3T3-E1 cells and its expression in bone. (A) Spata4 protein localization in MC3T3-E1 cells via immunofluorescence. Fluorescein isothiocyanate (FITC) staining of Spata4 (green; anti-Spata4) is shown. Propidium iodide (PI) staining of cell nuclei (red), a composite image (Merge), and a negative control (Neg) are presented. Images shown were captured at ×630 magnification via confocal laser scanning microscopy. (B) Spata4 protein localization in MC3T3-E1 cells confirmed via subcellular fractionation and Western blotting. Cell fragments were examined by a Western blot with an anti-Spata4 antibody. Tublin was used as a marker for the fragment of cytoplasm and c-Jun for the nucleus. (C) Spata4 expression in primary calvaria and femurs. Calvaria and femurs were harvested from mice on postnatal days 1, 5, 10, and 20 assessed via a Western blotting. β-Actin was used as a loading control. (D, E) Expression of Spata4 in osteoblasts in vivo. Immunofluorescence staining (D, ×400) and immunohistochemistry (IHC; E, ×200) were performed using sections of calvaria (top) and trabecular (femur) bone (bottom) from newborn mice. Enlarged = enlarged sections from a merged image; GP = growth plate; HE = hematoxylin and eosin staining; Neg = negative control.

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Spata4 enhances osteoblast differentiation and mineralization in vitro

To investigate whether Spata4 has an effect on osteoblast differentiation, we stably overexpressed Spata4 in MC3T3-E1 cells and induced the cells with a conditional medium for osteoblast differentiation. We determined the ability of osteoblast differentiation by measuring the activity of ALP, an early marker for osteoblast differentiation. The results showed that the ALP activity was significantly higher in MC3T3-E1-Spata4 cells than that in control cells at different time points after the cells were induced into osteoblasts. The ALP activity was the highest on day 3 and decreased thereafter in both MC3T3-E1-Spata4 and control cells (Fig. 2A). These data indicated that Spata4 facilitates MC3T3-E1 cell differentiation into osteoblasts.

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Figure 2. Influence of Spata4 on osteoblastic differentiation and mineralization in MC3T3-E1 cells. (A) ALP activity in MC3T3-E1-Spata4 and control cells after culturing for 3, 6, and 9 days. MC3T3-E1 cells transfected with pcDNA 3.1 were used as controls. (B) Calcium mineralization of MC3T3-E1-Spata4 cells. Spata4-overexpressing and control cells were induced for mineralization on day 14, and microscopic observations of mineralization were indicated by Alizarin red staining (×100). Mineral nodules were stained with Alizarin red solution. MC3T3-E1 cells transfected with pcDNA 3.1 were used as a control. (C) Calcium mineralization of MC3T3-E1-Spata4 cells on day 21. Cells were induced for mineralization for 21 days, and von Kossa staining (×100) was performed with silver nitrate. Mineral nodules are shown as dark dots. Data are expressed as mean ± SE from all experiments, as indicated. **p < .01.

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We next observed mineralized nodules by staining the cells with Alizarin red solution. Mineralized nodules were seen on day 14 after the induction of differentiation. More mineralized nodules were observed in MC3T3-E1-Spata4 cells than in control cells (Fig. 2B). Consistent with Alizarin red staining results, von Kossa staining also showed more mineralized nodules in MC3T3-E1-Spata4 cells than in control cells after 21 days of induction (Fig. 2C). These results indicated that Spata4 enhances mineralized nodule formation in MC3T3-E1 cells in vitro. Taken together, our data indicated that Spata4 enhances osteoblast differentiation.

Spata4 interacts with p-Erk1/2 in vitro

Based on our prediction that Spata4 may interact with Erk because Spata4 has a calponin homology (CH) domain at the N-terminus that has been reported to interact with Erk,2, 3 we determined to examine whether these two proteins interact in osteoblasts. For this purpose, a Spata4-overexpressing stable cell line, MC3T3-E1-Spata4, was established. The results showed that both the mRNA (Fig. 3A) and protein (Fig. 3B) of ectopically expressed Spata4 remained at high levels in the cells. An immunoprecipitation (IP) result showed that Spata4 was precipitated by p-Erk1/2 in MC3T3-E1-Spata4 cells (Fig. 3C) and that p-Erk1/2 was precipitated down by Spata4 antibodies in MC3T3-E1 cells with constitutively active MEK (Fig. 3D), suggesting that Spata4 interacts with p-Erk1/2. To determine whether a signal from upstream of Erk regulates the interaction of Spata4 and p-Erk1/2, we either activated the Erk1/2 pathway by using fibroblast growth factor (FGF) and constitutively active MEK or inhibited Erk1/2 phosphorylation by using U0126, an inhibitor of MEK1/2, in MC3T3-E1-Spata4 cells and MC3T3-E1 cells with constitutively active MEK. The results showed that the interaction between Spata4 and p-Erk 1/2 was increased either when the Erk pathway was activated by addition of FGF or in the situation of constitutively active MEK, whereas addition of U0126 decreased the interaction (Fig. 3E). To determine whether the CH domain of Spata4 is critical for the interaction with p-Erk1/2, we deleted the CH domain to generate a mutant Spata4 protein (Spata4-CHΔ). The results showed that there was no interaction between the Spata4 mutant and p-Erk1/2 when the CH domain was deleted (Fig. 3F). These results indicated that activation of the Erk signaling pathway increases the interaction of Spata4 and p-Erk1/2 via the CH domain.

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Figure 3. Spata4 interacts with p-Erk1/2 in MC3T3-E1 cells. mRNA levels (A) and protein levels (B) of ectopically expressed Spata4 in MC3T3-E1-Spata4 cells are shown by RT-PCR and Western blot analysis. MC3T3-E1 cells transfected with pcDNA 3.1 and wild-type cells were used as controls. (C) Spata4 interacts with p-Erk1/2 in MC3T3-E1-Spata4 cells. The lysates from cells overexpressing Spata4 were used for an immunoprecipitation (IP) assay with a p-Erk1/2 antibody and blotted with a Spata4 antibody (IB). An anti-IgG antibody was used as a negative control, and the totals of Erk1/2 and Spata4 protein were used as loading controls. (D) Spata4 interacts with p-Erk1/2 in constitutively active MEK MC3T3-E1 cells. Cells were transfected with a construct to overexpress constitutively active MEK. (E) Spata4 interacted with p-Erk1/2 on stimulating the Erk pathway. MC3T3-E1-Spata4 cells were either treated with FGF (50 ng/mL) or MEK inhibitor U0126 (20 µM) or transfected with a constitutively active (CA) MEK. (F) Spata4 interacts with p-Erk1/2 via the CH domain. MC3T3-E1 cells were transfected with pcDNA3.1-Spata4-CHΔ and pcDNA3.1-Spata4, respectively.

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Spata4 enhances Erk phosphorylation and Runx2 activity

We sought to determine whether the interaction of Spata4 and p-Erk affects the phosphorylation of Runx2 based on the fact that Erk signaling is required for osteoblast differentiation.8 We first examined the phosphorylation level of Erk under overexpression of Spata4. The result indicated that Erk phosphorylation was increased when Spata4 was stably overexpressed in MC3T3-E1 cells for 6 days (Fig. 4A). The phosphorylated Erk1/2 level by overexpression of Spata4 was inhibited by U0126 (Fig. 4B, comparing lane 4 with lane 3 or lane 8 with lane 7). These results indicated that Spata4 stimulates the phosphorylation of Erk in osteoblast-like cells.

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Figure 4. Effect of Spata4 on Erk phosphorylation and Runx2 transcriptional activity. (A) Erk1/2 phosphorylation is increased by Spata4. MC3T3-E1-Spata4 cells were transfected with a Spata4 expression plasmid and cultured for 3 or 6 days. A Western blot was used to show phosphorylated Erk1/2, and the total Erk1/2 protein was used as a loading control. (B) Spata4-activated Erk1/2 phosphorylation is inhibited by U0126. MC3T3-E1-Spata4 cells were treated with U0126 (20 µM) for 30 minutes. (C–F) Runx2 activity is increased with overexpression of Spata4. MC3T3-E1-Spata4 cells were transfected with 1.3-kb mOG2-Luc (C, E) or Runx2-binding element–Luc (D, F) plasmids in culture with α-MEM (C, D) and mineralization medium (E, F). Runx2 activity was examined by the relative luciferase activity in parallel in MC3T3-E1-Spata4 cells treated with U0126 (20 µM). Renilla luciferase plasmid pRL-SV40 was used as an internal control. (G) mRNA levels of Spata4 in MC3T3-E1 cells. Cells were transfected with a Spata4 siRNA, and the mRNA level was examined by an RT-qPCR analysis. A scramble siRNA was used as a control. (H, I) Depletion of Spata4 decreases the Runx2 activity. MC3T3-E1 cells were transiently transfected with a Spata4 siRNA and the 1.3-kb mOG2-Luc (H) and a Runx2- binding element–Luc (I) plasmid. Luciferase activities were measured on days 2 and 4 after transfection. A scramble siRNA was used as a control. Data are presented as mean ± SE from three repeated experiments. A statistical analysis was done as indicated. *p < .05; **p < .01.

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Next, we examined the activity of Runx2, a transcription factor that is directly activated by Erk signaling pathway and binds to DNA at the 1.3-kb mOG2 fragment.21 The Runx2 transcriptional activity was measured either by a 1.3-kb mOG2-Luc reporter, where a minimal 1.3-kb mOG2 promoter was linked to the luciferase reporter gene, or by a Runx2-binding element–Luc reporter, where 6 copies of the Runx2-binding site was linked to the luciferase reporter gene.8, 22 The results showed that the Runx2 transcriptional activity was increased significantly on day 14, assayed in either the 1.3-kb mOG2-Luc (Fig. 4C, E) or the Runx2-binding element–Luc (Fig. 4D, F) reporter under cultures in both a general medium (Fig. 4C, D) and a mineralization medium (Fig. 4E, F). Intriguingly, pretreatment with an Erk phosphorylation inhibitor, U0126, inhibited the Runx2 transcriptional activity in MC3T3-E1-Spata4 cells (Fig. 4C–F, black columns). In contract, the Runx2 activity was markedly suppressed when Spata4 was depleted by transient transfection of a Spata4 siRNA compared with a scramble siRNA in MC3T3-E1 cells (Fig. 4G–I). These results indicated that Spata4 has an ability to facilitate the Runx2 transcriptional activity through the Erk pathway.

Spata4 increases osteoblast-specific marker gene expression

We finally examined mRNA levels of osteoblast-specific marker genes in MC3T3-E1 cells when Spata4 was stably overexpressed or transiently depleted by an siRNA. The results showed that mRNA levels of Ocn (Fig. 5A), Bmp2 (Fig. 5B), Opn (Fig. 5C), collagen type 1 (Fig. 5D), Osx (Fig. 5E), and Runx2 (Fig. 5F) were increased after culturing for 3 to 14 days. At the same time, treatment of MC3T3-E1-Spata4 cells with U0126 resulted in decreased mRNA levels of the genes (Fig. 5A–F, black columns). In contact, mRNA levels of all six osteoblast-specific marker genes were decreased when Spata4 was transiently depleted in MC3T3-E1 cells by a Spata4 siRNA compared with a scramble siRNA for 3 days (Fig. 5G). Consistent with increased Runx2 mRNA levels, Runx2 protein also was increased when Spata4 was overexpressed in MC3T3-E1-Spata4 cells (Fig. 5H). Taken together, all the data suggested that Spata4 increases the expression of osteoblast-specific marker genes at the transcriptional level through the Erk1/2 pathway.

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Figure 5. Effect of Spata4 on MC3T3-E1 cell differentiation. (A–F) Spata4 increases mRNA levels of osteoblast-specific marker genes in MC3T3-E1-Spata4 cells. MC3T3-E1 cells transfected with pcDNA3.1-Sapta4 or pcDNA 3.1 under treatment with U0126 (20 µM). Ocn (A), Bmp2 (B), Opn (C), collagen type I (D), Osx (E), and Runx2 (F) mRNA levels were detected on the indicated days. (G) Depletion of Spata4 decreased osteoblastic gene expression. mRNA levels of osteoblast-specific marker genes were examined by RT-qPCR in MC3T3-E1 cells transiently transfected with a Spata4 siRNA. A scramble siRNA was used as a control. (H) Spata4 increases the Runx2 protein level. Protein levels of Runx2 were examined in MC3T3-E1-Spata4 cells induced for osteoblast differentiation for the indicated days. Data from panels A through G are expressed as mean ± SE from all experiments, as indicated. *p < .05; **p < .01.

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Discussion

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Disclosures
  8. Acknowledgements
  9. References

Previous studies showed that Erk interacts with proteins containing the CH domain.2, 3 Since Spata4 has a CH domain, we speculated that Spata4 interacts with Erk. Indeed, we observed that Spata4 interacted with p-Erk in the stimulation of FGF or in the presence of constitutively active MEK. Intriguingly, we found that overexpression of Spata4 resulted in an elevated level of Erk in MC3T3-E1 cells. To our knowledge, this is the first line of evidence that Spata4 promotes Erk phosphorylation during osteoblast differentiation. Importantly, since the phosphorylated Erk, by translocating into the nucleus,23–25 regulates the transcriptional activity of Runx2,8, 26 we proposed that Spata4 could regulate osteoblast differentiation. By using cell differentiation and luciferase assays, we demonstrated that overexpression of Spata4 promotes osteoblast differentiation. Furthermore, the expression of osteoblastic marker genes, including Ocn, Bmp2, Opn, collagen type 1, Osx, and Runx2, was increased significantly in a cell line under stable expression of Spata4. We also provided data that depletion of Spata4 by siRNA impaired the osteoblast differentiation. All the data suggest that Spata4 is a novel regulator of osteoblast differentiation.

Spata4 was identified originally in human testis.1 However, in this study, we found that Spata4 plays an important role in osteoblast differentiation. To reveal the physiologic function, we examined the endogenous expression of Spata4 in both cell lines and mouse bone tissues. The results showed that Spata4 expression in osteoblast cells remains at high levels. In particular, we observed that Spata4 is located in the cytoplasm in the osteoblasts in calvaria and femurs of mice. Together with the osteoblast differentiation and mineralization data, we concluded that Spata4 positively regulates osteoblast differentiation.

It has been well documented that Erk phosphorylation enhances osteoblast differentiation.8 The molecular mechanism for Erk-induced osteoblast differentiation has been attributed to the activation of Runx2, a critical transcriptional factor regulating expression of many osteoblastic genes.12, 27 In this study, we observed that Spata4 activates Erk phosphorylation and thereafter promotes the transcriptional activity of Runx2 in regulating downstream gene expression. While we showed that overexpressed Spata4 results in activation of Runx2, we observed that inhibition of Erk by addition of U0126, an inhibitor of MEK1/2, significantly impaired the role of Spata4. We conclude that Spata4 regulates osteoblast differentiation through regulation of Erk phosphorylation.

Osteoblasts go through cell proliferation, differentiation, and matrix mineralization stages. Runx2 is a critical transcriptional factor that is regulated by several factors in osteoblast differentiation.12, 28, 29 It appears that Runx2 plays a positive role in the early stage of osteoblast differentiation together with many factors. One of the major factors is Erk as it phosphorylates Runx2. In this study, we observed that Erk1/2 is activated by Spata4 on days 3 and 6 after the osteoblast differentiation. Since the osteoblast differentiation takes a long time,30 we speculate that activation of Erk1/2 by Spata4 will remain at a high level during the process. Indeed, we observed that the phosphorylated Erk1/2 protein on day 6 was much higher than that on day 3 under osteoblast differentiation (Fig. 4A, B). It turned out that the accumulated phosphorylation of Erk1/2 boosts Runx2 transcriptional activity. Consistent with our speculation, a study indicated that mOG2 promoter was activated on day 7 in osteoblasts from calvaria.8, 28 On the other hand, it seems that Runx2 activity is also upregulated by expression of the mRNA because we observed an obvious increased mRNA of Runx2 (Fig. 5F). Since Runx2 could be activated by p-Erk and bind to its own promoter,12, 31 we consider that both the protein levels and the transcriptional activity of Runx2 are regulated by Spata4 during osteoblast differentiation.

A question remains unclear on how Spata4 regulates the phosphorylation of Erk. In our study, we observed that Spata4 interacts with p-Erk because Spata4 contains a CH domain, which has been reported to be critical for the association with Erk. However, it is unclear why association of Spata4 with p-Erk increases the phosphorylation level of Erk. Since Spata4 contains no kinase domain, we speculated that Spata4 may mediate an unknown kinase to associate with Erk. This speculation fits with our observation that Spata4 highly activated Erk1/2 after 6 days of osteoblast differentiation because this unknown kinase may be induced at 6 days after osteoblast differentiation. Further study is required to identify such a kinase. Since MEK1/2 is an upstream event of Erk activation in the canonical MAPK pathway, whether Spata4 mediates the association of MEK with Erk is of interest to study.

In conclusion, we have described a role for Spata4 in regulation of osteoblast differentiation and subsequent matrix mineralization. Interestingly, we observed that the function of Spata4 in osteoblast differentiation relies on Erk phosphorylation. We concluded that Spata4 regulates osteoblast differentiation through the Erk-activated Runx2 signaling pathway. We prospect that Spata4 may be a promising target for development of diagnostic reagents or therapeutic outcomes in bone aging–related diseases.

Disclosures

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Disclosures
  8. Acknowledgements
  9. References

All the authors state that they have no conflicts of interest.

Acknowledgements

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Disclosures
  8. Acknowledgements
  9. References

We thank Dr RT Franceschi for kindly providing Runx2-binding element–Luc and 1.3mOG2-Luc plasmids. This work was supported by the National Basic Research Program (973 Project) of China (2007CB507406) and the Tsinghua Yue-Yuen Medical Sciences Fund (THYY20070008) and NSFC grants (30871286, 30470888). We thank Dr Ying Qiu (Tsinghua University) for comments on the manuscript.

References

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Disclosures
  8. Acknowledgements
  9. References
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