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. 2006 Apr;21(4):637-46.
doi: 10.1359/jbmr.060109. Epub 2006 Apr 5.

BMP signaling is required for RUNX2-dependent induction of the osteoblast phenotype

Mattabhorn Phimphilai  1 , Zhouran Zhao, Heidi Boules, Hernan Roca, Renny T Franceschi
Affiliations

BMP signaling is required for RUNX2-dependent induction of the osteoblast phenotype

Mattabhorn Phimphilai et al. J Bone Miner Res. 2006 Apr.

Abstract

RUNX2 expression in mesenchymal cells induces osteoblast differentiation and bone formation. BMP blocking agents were used to show that RUNX2-dependent osteoblast differentiation and transactivation activity both require BMP signaling and, further, that RUNX2 enhances the responsiveness of cells to BMPs.

Introduction: BMPs and the RUNX2 transcription factor are both able to stimulate osteoblast differentiation and bone formation. BMPs function by activating SMAD proteins and other signal transduction pathways to stimulate expression of many _target genes including RUNX2. In contrast, RUNX2 induces osteoblast-specific gene expression by directly binding to enhancer regions in _target genes. In this study, we examine the interdependence of these two factors in controlling osteoblast differentiation in mesenchymal progenitor cells.

Materials and methods: C3H10T1/2 mesenchymal cells and primary cultures of marrow stromal cells were transduced with a RUNX2 adenovirus and treated with BMP blocking antibodies or the natural antagonist, NOGGIN. Osteoblast differentiation was determined by assaying alkaline phosphatase and measuring osteoblast-related mRNA using quantitative RT/PCR. Activation of BMP-responsive signal transduction pathways (SMAD, extracellular signal-regulated kinase [ERK], p38, and c-jun-N-terminal kinase [JNK]) was assessed on Western blots.

Results and conclusions: C3H10T1/2 cells constitutively synthesize BMP2 and 4 mRNA and protein, and this BMP activity is sufficient to activate basal levels of SMAD phosphorylation. Inhibition of BMP signaling was shown to disrupt the ability of RUNX2 to stimulate osteoblast differentiation and transactivate an osteocalcin gene promoter-luciferase reporter in C3H10T1/2 cells. BMP blocking antibodies also inhibited RUNX2-dependent osteoblast differentiation in primary cultures of murine marrow stromal cells. Conversely, RUNX2 expression synergistically stimulated BMP2 signaling in C3H10T1/2 cells. However, RUNX2 did not increase the ability of this BMP to activate SMAD, ERK, p38, and JNK pathways. This study shows that autocrine BMP production is necessary for the RUNX2 transcription factor to be active and that BMPs and RUNX2 cooperatively interact to stimulate osteoblast gene expression.

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Conflict of interest statement

The authors state that they have no conflicts of interest.

Figures

FIG. 1
FIG. 1
Autocrine BMP synthesis in C3H10T1/2 cells. Cells were transduced with AdLacZ (open bars) or AdRUNX2 (gray bars) at 100 pfu/cell and harvested after 6 days for analysis of BMP (A) mRNA and (B) protein. Relative mRNA levels were measured by Q-RT/PCR and normalized to GAPDH mRNA. BMP protein in whole cell extracts was measured on Western blots. BMP-2, -4, and -7 standards (STD) were prepared from COS7 cell-conditioned medium.
FIG. 2
FIG. 2
Effect of BMP inhibition on SMAD activation and AdRUNX2-induced ALP activity. C3H10T1/2 cells or primary cultures of murine MSCs were transduced with AdLacZ or AdRUNX2 as in Fig. 1. (A) Inhibition of SMAD phosphorylation. Forty-eight hours after transduction, C3H10T1/2 cells were treated with preimmune serum or combined BMP2/4 and BMP7 blocking antibodies at 5 ηg/ml. After 4 h, whole cell extracts were prepared for Western blot detection of phospho-SMADs 1/5/8, total SMADs 1/5, and RUNX2 as indicated. (B) Inhibition of ALP induction in C3H10T1/2 cells. Cells were treated with NOGGIN (50 ηg/ml), BMP2/4 blocking antibody (5 ηg/ml), BMP7 blocking antibody (5 ηg/ml), or combined BMP2/4 and BMP7 blocking antibodies (5 ηg/ml each) as indicated for 5 days and harvested for measurement of ALP activity. (C) Inhibition of ALP induction in MSCs. Cells were treated with BMP blocking antibodies as indicated. AdLacZ, open bars; AdRUNX2, gray bars.
FIG. 3
FIG. 3
Effect of BMP inhibition on AdRUNX2-induced late osteoblast differentiation markers. C3H10T1/2 cells were transduced with AdLacZ (open bars) or AdRUNX2 (gray bars) at 100 pfu/cells and treated with combined BMP2/4 and BMP7 blocking antibodies as indicated in Fig. 2. After 6 days, cells were harvested for mRNA analysis by Q-RT/PCR. (A) OCN mRNA. (B) BSP mRNA. Values are expressed as fold increase relative to Ad-LacZ-treated controls.
FIG. 4
FIG. 4
Effect of BMP inhibition on AA-induced osteoblast differentiation markers in MC3T3-E1 pre-osteoblast cells. (A) Clone 4 MC3T3-E1 cells were growth in the presence (gray bars) or absence of AA (open bars) and treated with combined BMP2/4 and BMP7 blocking antibodies (5 ηg/ml each) for 5 days and assayed for ALP activity. Total RNA was isolated from replicate samples and assayed for (B) OCN, (C) BSP, and (D) RUNX2 mRNA using Q-RT/PCR.
FIG. 5
FIG. 5
Effect of BMP inhibition on RUNX2 transcriptional activity. (A) Inhibition of RUNX2-activated OCN promoter activity. C3H10T1/2 cells were transfected with a 1.3-kb mOG2-luc reporter plasmid, renilla luciferase plasmid (to assess transfection efficiency), and either LacZ (open bars) or RUNX2 (gray bars) expression plasmids as indicated. Cells were treated with combined BMP2/4 and BMP7 blocking antibodies (5 ηg/ml each) and harvested 4 days later for measurement of luciferase activity. All data were normalized for transfection efficiency and expressed as fold increase relative to LacZ control samples. The result was reported as fold increase of luciferase activity normalized with Renilla activity. (B) Inhibition of AA-induced OCN promoter activity. MC42 cells were grown in control (open bars) or AA-containing medium (gray bars) and treated with combined BMP2/4 and BMP7 blocking antibodies (5 ηg/ml each) for 5 days before luciferase activity was measured. Results were normalized to DNA and reported as fold increase relative to untreated controls.
FIG. 6
FIG. 6
Effect of RUNX2 on cellular responsiveness to BMP2. (A) Synergistic induction of ALP activity. C3H10T1/2 cells were transduced with either AdLacZ or AdRUNX2 at 100 pfu/cells, treated with rh-BMP2 at 50 ηg/ml, and harvested for ALP activity at the indicated time. AdLacZ control (○), AdLacZ control plus rhBMP2 (●), AdRUNX2 (Δ), AdRUNX2 plus rhBMP2 (▲). (B) BMP dose-dependence. C3H10T1/2 cells were tranduced as in A, treated with the indicated concentration of rhBMP2 for 5 days, and harvested for ALP activity. Ad-LacZ control (○), AdLacZ control plus rh-BMP2 (●). (C and D) Induction of OCN and BSP mRNA. C3H10T1/2 cells were transduced as in A and B, treated with rhBMP2 at 100 ηg/ml for 5 days, and harvested for measurement of (C) OCN and (D) BSP mRNA levels by Q-RT/PCR. AdLacZ, open bars; AdRUNX2, gray bars.
FIG. 7
FIG. 7
Western blot analysis of BMP2-responsive signal transduction pathways. C3H10T1/2 cells were transduced with Ad-LacZ or AdRUNX2 at 100 pfu/cell. Forty-eight hours after transduction, cells were treated with rhBMP2 for 30 minutes, harvested, and assayed on Western blots for the following signaling intermediates: phospho-SMAD 1/5/8, total SMADs 1/5, phospho-JNK, total JNK, phospho-p38, total p38, phospho-ERK, total ERK, and total RUNX2.
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