¹Department of Biochemistry, School of Medicine, Chungbuk National University, Cheongju, 361-763, South Korea

²Institute for Tumor Engineering, Chungbuk National University, Cheongju, 361-763, South Korea

Correspondence to: S-C Bae, E-mail: scbae@med.chungbuk.ac.kr

The Runx family of transcription factors plays pivotal roles during normal development and in neoplasias. In mammals, Runx family genes are composed of Runx1 (Pebp2B/Cbfa2/Aml1), Runx2 (Pebp2A/Cbfa1/Aml3) and Runx3 (Pebp2C/Cbfa3/Aml2). Runx1 and Runx3 are known to be involved in leukemogenesis and gastric carcinogenesis, respectively. Runx2, on the other hand, is a common target of transforming growth factor-1 (TGF-1) and bone morphogenetic protein-2 (BMP-2) and plays an essential role in osteoblast differentiation. Runx2 is induced by the receptor-activated Smad; Runx2 mediates the blockage of myogenic differentiation and induces osteoblast differentiation in C2C12 pluripotent mesenchymal precursor cells. However, Smad does not directly induce Runx2 expression; an additional step of de novo protein synthesis is required. Here we report that Smad-induced junB functions as an upstream activator of Runx2 expression. Furthermore, not only the Smad pathway but also the mitogen-activated protein kinase (MAPK) cascades are involved in the induction of Runx2 by TGF-1 and BMP-2. Our results demonstrate that following TGF- and BMP induction, both the Smad and p38 MAPK pathways converge at the Runx2 gene to control mesenchymal precursor cell differentiation.

Oncogene (2002) 21, 7156-7163. doi:10.1038/sj.onc.1205937

Runx2; junB; p38; TGF-1; BMP-2; osteoblast

Introduction

Members of the transforming growth factor- (TGF-) superfamily elicit diverse cellular responses, including inhibition of adipogenesis and myogenesis, and stimulation of chondrogenesis and osteogenesis (Hogan, 1996; Roberts et al., 1988). The most important members of the TGF- superfamily which effect on bone cell differentiation in vivo are TGF- and bone morphogenetic proteins (BMPs). TGF-1 induces new bone formation when injected in close proximity to bone tissue. On the other hand, BMP-2 induces bone formation even when injected ectopically (Wozney et al., 1988). In the pluripotent mesenchymal precursor cell line, C2C12, TGF-1 inhibits default differentiation of the cell into multinucleated myotubes without inducing osteoblast phenotypes. BMP-2 not only inhibits myogenic differentiation of C2C12 cells but also induces osteoblast phenotypes (Katagiri et al., 1994).

TGF- family members exert their cellular effects by binding to transmembrane receptors that possess serine/threonine kinase activity (Massague, 1998). Following ligand activation, the receptor kinase phosphorylates Smad proteins, which move into the nucleus to stimulate the transcription of a set of target genes. Smad2 and 3 are activated by TGF- receptors and mediate TGF- responses, whereas Smad1, 5 and 8 are activated by BMP receptors and transduce BMP signals (Heldin et al., 1997; Massague, 1998). In addition to the Smad group of proteins, activation of mitogen-activated protein kinase (MAPK) cascades is involved in TGF- superfamily signal transduction. Both TGF- and BMP have been shown to activate p38, a member of the stress-activated protein kinases (SAPKs), through MAPK kinase (MKK) 6 or MKK3 (Gallea et al., 2001; Hanafusa et al., 1999). It has been reported that both Smad and MAPK pathways are essential components of the TGF- superfamily signaling and affect osteoblast differentiation (Derynck et al., 2001; Fujii et al., 1999; Gallea et al., 2001; Nishimura et al., 1998; Yamamoto et al., 1997). However, how these signaling pathways affect osteoblast differentiation is poorly understood.

Runx family of transcription factors encoded by three distinct genes, Runx1 (Pebp2B/Cbfa2/Aml1), Runx2 (Pebp2A/Cbfa1/Aml3) and Runx3 (Pebp2C/Cbfa3/Aml2), plays pivotal roles during normal development and in neoplasias. Runx1 plays a critical role in the formation of hematopoietic stem cells (North et al., 1999; Okuda et al., 1996; Yokomizo et al., 2001). It is the most frequent target of chromosome translocation in leukemia and is responsible for about 30% of human acute leukemia cases (Look, 1997).

RUNX3 has been shown to be a tumor suppressor of gastric cancer (Li et al., 2002). RUNX3 was frequently inactivated in human gastric cancer tissues and tumorigenicity of gastric cancer cell lines in nude mice was inversely related to their level of RUNX3 expression. Furthermore, a mutation identified from a gastric cancer patient abolished the tumor suppressive effect of RUNX3.

Runx2, on the other hand, is an essential transcription factor required for osteogenesis (Ducy et al., 1997; Komori et al., 1997; Otto et al., 1997). Haploinsufficiency of the Runx2 gene was shown to be the cause of the human disease cleidocranial dysplasia (CCD), an autosomal dominant bone disorder (Lee et al., 1997; Mundlos et al., 1997). The oncogenic properties of Runx2 were demonstrated in transgenic mice where Runx2 overexpression pertubates T cell development and synergizes strongly with c-myc in lymphomagenesis (Vaillant et al., 1999).

Runx2 expression is transiently up regulated by TGF-1 and BMP-2-activated Smads and mediates the blockage of myogenic differentiation of C2C12 cells. Runx2 is essential for the common activities of by TGF-1 and BMP-2 and cooperation between Runx2 and receptor activated Smads is required for the ligand specific gene expression (Lee et al., 2000; Zhang et al., 2000). However, Smad does not directly induce Runx2 expression; an additional step of de novo protein synthesis is required (Lee et al., 2000). Our results suggest that junB is the newly synthesized protein required for the induction of Runx2 by TGF-1 and BMP-2. Furthermore, we show that activation of p38 MAPK is also involved in Runx2 induction. These results provide important insights into how the TGF-1 and BMP-2 signals are transmitted to their major target gene, Runx2.

Results

Involvement of junB in Runx2 induction by TGF-1 and BMP-2

Previously we reported that activation of Smads by TGF-1 and BMP-2 resulted in the induction of Runx2 and that Runx2 played an important role in blocking myogenic differentiation in C2C12 cells. However, in addition to the activation of Smad, induction of Runx2 requires de novo protein synthesis (Lee et al., 2000). Since Runx2 induction is observed as early as 2 h after stimulation, we assumed that an immediate early gene product might be involved in the Runx2 induction. Among the immediate early genes, junB is known to be induced by TGF-1 and BMP-2 in C2C12 cells, while expression of c-jun, junD and c-fos is not changed (Chalaux et al., 1998; Jonk et al., 1998). Since junB also mediates blockage of myogenic differentiation of C2C12 cells (Chalaux et al., 1998), we asked whether de novo synthesis of junB is required prior to the induction of Runx2. Firstly, we examined the time course of junB and Runx2 induction in response to TGF-1 stimulation. As shown in Figure 1a, the maximum level of junB mRNA was detected 1 h after TGF-1 stimulation, which is 1 h earlier than Runx2, implying that junB could be an upstream activator of Runx2. In contrast, the expression level of ATF-2, which plays an important role in osteoblast differentiation in vivo and makes heterodimers with junB (Reimold et al., 1996), was not altered under the same experimental conditions.

We further examined whether there is any correlation between the level of junB and Runx2 expression in response to various concentrations of BMP-2. The control C2C12 and Smad5 over-expressing C2C12 cells (C2C12-Sm5) were treated with serially diluted concentrations of BMP-2 for 1 h and junB mRNA levels were measured using Northern blot analysis. As shown in Figure 1b, junB was induced by 300 ng/ml of BMP in control C2C12 cells; however, in C2C12-Sm5 cells, junB expression was detected in the absence of BMP-2 and increased at higher concentrations of BMP-2. The expression pattern of junB in C2C12 and C2C12-Sm5 cells in response to various concentrations of BMP-2 is very similar to that of Runx2 (Lee et al., 2000). These results encouraged us to examine the effect of junB activation on Runx2 expression. C2C12 cells were treated with a potent junB activator, 12-O-tetradecanoylphorbol-13acetate (TPA) (Chiu et al., 1989), and the level of Runx2 mRNA was analysed. As shown in Figure 1c, treatment of TPA strongly induced Runx2 gene expression (Figure 1c). Therefore, we examined whether the exogenous expression of junB could induce Runx2 expression. For this purpose, C2C12 cells stably expressing junB (C2C12-junB) were obtained (Figure 2a), and the level of Runx2 expression in response to TGF-1 and BMP-2 was measured by Northern blotting (Figure 2b,c). In control C2C12 cells, only very low levels of Runx2 mRNA were detected and mRNA levels increased following TGF-1 and BMP-2 stimulation, as reported previously (Lee et al., 2000). In contrast, C2C12-junB cells showed high levels of Runx2 mRNA in the absence of stimulation, and the levels further increased following TGF-1 and BMP-2 treatment.

The essential role of junB in the induction of Runx2 was further confirmed by suppression of AP-1 activity following stable expression of a dominant negative c-fos (A-fos), which inhibits jun family dependent transactivation (Olive et al., 1997). Two independent C2C12 cell clones that express either high level of A-fos (C2C12-A-fos-1) or low level (C2C12-A-fos-2) were obtained (Figure 3a), and the level of Runx2 mRNA in the presence or absence of TGF-1 and BMP-2 was analysed using Northern blotting. As shown in Figure 3b, exogenous expression of A-fos significantly inhibited induction of Runx2 by TGF-1 and BMP-2 stimulation. Notably, the inhibitory effect correlated with the level of A-fos protein. Collectively, these results indicate that induction of junB is essential for the induction of Runx2 expression by TGF-1 and BMP-2.

Involvement of the p38 MAPK signaling pathway in the induction of Runx2 expression

To determine whether any other kinase is also involved in the induction of Runx2 expression following TGF-1 and BMP-2 stimulation, C2C12 cells were treated with various kinase inhibitors in the presence or absence of BMP-2, and the level of Runx2 mRNA was analysed using Northern blotting. As shown in Figure 4a, treatment with SB203580, a specific inhibitor of p38 MAPK, effectively inhibited Runx2 induction by TGF-1 and BMP-2, while other kinase inhibitors had no significant effect. To examine whether the activation of p38 could induce Runx2 expression, C2C12 cells were treated with anisomycin, a potent activator of p38, in the presence or absence of SB203580, and the level of Runx2 mRNA was measured using Northern blotting. As shown in Figure 4b, Runx2 expression was significantly induced by anisomycin, and induction was effectively blocked by SB203580. To further investigate the involvement of the p38 signaling pathway in the induction of Runx2, C2C12 cells stably expressing dominant negative p38 [p38(AGF)] (Noguchi et al., 2000), were obtained [C2C12-p38(AGF)] (Figure 5a). We confirmed that p38 was activated by TGF-1 and over-expression of p38(AGF) effectively blocked the p38 activation (Figure 5b). Control C2C12 and C2C12-p38(AGF) cells were treated with TGF-1 and BMP-2, and the level of Runx2 mRNA was measured using Northern blotting. As shown in Figure 5c, the induction of Runx2 was significantly inhibited in the C2C12-p38(AGF) cells. These results indicate that activation of p38 is involved in the induction of Runx2 by TGF-1 and BMP-2.

The effect of junB and p38 on osteoblast differentiation

TGF- and BMP are known to induce preosteoblast stage specific gene expression (collagen (1) and fibronectin) in C2C12 cells. However, osteoblast-specific genes (for example, alkaline phosphatase and osteocalcin) are induced only by BMP. Previously, we showed that Runx2 alone can induce preosteoblast stage specific genes, but fails to fully induce osteoblast-specific genes (Lee et al., 2000). Since we found that induction of junB and activation of p38 are involved in the induction of Runx2, we examined the effect of junB and p38 pathways on differentiation-specific gene expression. C2C12 cells were treated with serially diluted concentrations of BMP-2, and alkaline phosphatase activities were determined. Control C2C12 cells required 300 ng/ml of BMP-2 for the induction of alkaline phosphatase. On the other hand, in both C2C12-Rx2 and C2C12-JunB cells, alkaline phosphatase was weakly expressed even in the absence of BMP-2 and strongly expressed at very low concentrations of BMP-2 (Figure 6). The induction of alkaline phosphatase by BMP-2 was inhibited by A-fos and p38(AGF) (Figure 6). These results, together with the observation that junB induces Runx2 expression, suggest that junB is involved in the process of osteoblast differentiation by inducing Runx2 expression, and that the activation of p38 MAPK is an additional essential component for the TGF-1 and BMP-2-dependent induction of Runx2.

Discussion

JunB is an upstream regulator of Runx2 expression following induction by TGF-1 and BMP-2

JunB is a direct target gene of Smads activated by TGF-1 and BMP-2 and mediates the blockage of myogenic differentiation in C2C12 cells (Chalaux et al., 1998; Jonk et al., 1998). We found that junB not only blocks myogenic differentiation of C2C12 cells but also makes the cells highly sensitive to BMP-2-induced osteoblast differentiation (Figure 6). This activity may be mediated by osteoblast-specific transcription factors induced by junB, since junB is a general transcription factor that is not specific for osteoblast differentiation. Runx2 encodes an osteoblast-specific transcription factor and is transiently induced by TGF-1 and BMP-2 (Ducy, 2000; Lee et al., 2000). The induction of Runx2 is responsible for the inhibition of myogenic differentiation and over-expression of Runx2 makes the cells highly sensitive to BMP-2-induced osteoblast differentiation (Lee et al., 2000). Even though the induction of Runx2 by TGF-1 or BMP-2 was observed very early after stimulation, it still required de novo synthesis of at least one protein. In this study, we showed that junB expression is induced earlier than Runx2 following TGF-1 stimulation. Furthermore, activation and over-expression of junB resulted in the induction of Runx2 and inhibition of AP-1 blocked TGF-1 and BMP-2-dependent Runx2 induction. These results suggest that junB is one of the upstream regulators of Runx2 and provide an important signal transduction pathway from receptor-activated Smads to the osteogenic master switch gene Runx2.

Involvement of the p38 MAPK signal pathway in the induction of Runx2

We showed that exogenous expression of junB by itself resulted in the induction of Runx2. Interestingly, the level of Runx2 mRNA was further increased by TGF-1 and BMP-2 stimulation (Figure 2b,c). This result suggests that the induction of junB might not be the only mechanism involved in Runx2 induction; there could be other independent pathways that function synergistically to induce Runx2. Our results demonstrate that the activation of p38 MAPK is also involved in the induction of Runx2 by TGF-1 and BMP-2 stimulation. The activation of p38 by TGF-1 and BMP-2 stimulation in C2C12 cells and the crucial role of p38 in osteoblast differentiation have been reported (Gallea et al., 2001; Hanafusa et al., 1999). However, target transcription factors of p38 pathway responsible for the osteoblast differentiation have been poorly understood. Our results showing that the activation of p38 is involved in the induction of Runx2 suggest that p38 pathway could contribute to the osteoblast differentiation by inducing Runx2.

So far, the mechanism for the induction of Runx2 by TGF- and BMP-activated p38 is unclear. Activation of ATF-2 could be involved in the induction of Runx2, since ATF-2 is known as a major target of p38 and plays an important role in bone formation in vivo (Reimold et al., 1996). Induction of junB by p38 might also be responsible for the induction of Runx2. Further study will be required to fully understand the signaling pathway regulating Runx2 expression through p38 MAPK.

The effect of junB and p38 on osteoblast differentiation

The sequential expression of specific genes for osteoblast differentiation has been determined (Lian and Stein, 1995). During the preosteoblast stage, several genes associated with the formation of the extra-cellular matrix (for example, collagen I and fibronectin) are actively expressed. Following the down-regulation of proliferation, proteins associated with the bone cell phenotype are detected (for example, alkaline phosphatase). Runx2 is known to be an osteogenic master gene, and regulates the osteoblast specific marker genes (Lee et al., 2000; Xiao et al., 1999). The effect of exogenous expression of junB on the induction of alkaline phosphatase was almost equivalent to that of Runx2 and inhibition of AP-1 or p38 effectively blocked BMP-induced marker gene expression (Figure 6). These results strongly support our claim that junB is an upstream regulator of Runx2 and that p38 is an additional essential component for the induction of Runx2.

Our results indicate that the independent Smad-induced junB pathway and the receptor-activated MAP kinase pathway both play a crucial role in the induction of Runx2 by TGF-1 and BMP-2. It is worth noting that p38 is not the only MAP kinase activated by TGF-1 and BMP-2. Erk is also a common target of TGF-1 and BMP-2 and activates Runx2-dependent transcription without affecting the expression of Runx2 (Xiao et al., 2000). Therefore, concerted actions of the TGF- superfamily signaling pathways converge at the Runx2 gene for the C2C12 pluripotent mesenchymal precursor cell differentiation.

Materials and methods

Materials

Bioactive recombinant human BMP-2 was kindly provided by J Wozney (Genetics Institute, Cambridge, Massachusetts). Recombinant human TGF-1 was purchased from Sigma. Reagents were purchased from the following vendors: Restriction enzymes from Takara (Tokyo, Japan) or New England Biolabs; cell culture reagents, G418 and Lipofectamin from Gibco/BRL; anti-junB rabbit polyclonal, anti-ATF-2 rabbit polyclonal antibody from Santa Cruz; anti-p38 and anti-phospho-p38 MAPK rabbit polyclonal antibodies from New England Biolabs; monoclonal anti-FLAG antibody, alkaline phosphatase (ALP)-conjugated anti-goat IgG monoclonal antibody from Sigma; horseradish peroxidase (HRP)-conjugated anti-rabbit goat antibody from Pierse; anti--tubulin monoclonal antibody from Oncogene; ECL Western blotting kit including HRP-conjugated anti-mouse IgG goat antibody, Hybond-N+ nylon membrane and rediprime DNA labeling kit from Amersham; Immobilon from Milipore; SB203580, Wortmanin, PD98059, Rapamycin and Anisomycin from Sigma. All other chemicals of the purest grade available were obtained from commercial sources.

Plasmids

The plasmid expressing dominant negative Fos (A-Fos) (Olive et al., 1997) was kindly provided by Dr C Vinson. (National Institute of Health, Bethesda, MD, USA). JunB expressing plasmid (pcDNA-JunB) was kindly provided by Dr F Ventura (Universitat de Barcelona, Hospitalet de Llobregat, Spain). Dominant negative p38 MAP kinase (Noguchi et al., 2000) expressing plasmid (pcDNA3-p38-AGF) was kindly provided by Dr Y Kuchino (National Cancer Center Research Institute, Tokyo, Japan).

Cell lines and cultures

The mouse pluripotent mesenchymal precursor cell line, C2C12, was purchased from the American Type Culture Collection. C2C12 cells were maintained in Dulbecco Modified Eagle Medium (DMEM) containing 5-15% fetal bovine serum (FBS), penicillin G (100 U/ml) and streptomycin (100 g/ml) at 37°C in a humidified atmosphere of 5% CO₂ in air. To avoid cell density dependent fluctuations, all the treatments were performed after cells became completely confluent.

Stable transfection

Runx2 and Smad5 over-expressing C2C12 cells (C2C12-Rx2 and C2C12-Sm5, respectively) have been described previously (Lee et al., 2000). A-fos, p38(AGF) and junB over-expressing cells were obtained by stable transfection of pcDNA-A-fos, pcDNA3-p38-AGF and pcDNA-JunB, respectively, into C2C12 via the Lipofectamine method according to the manufacturer's instructions (Gibco/BRL). The stably transfected cells were screened for 2 weeks in selection medium containing 600 g/ml G418, and viable colonies were further screened by Western blotting.

Northern blot analysis

Northern blot analysis was performed as described previously (Sambrook et al., 1988). The DNA probes were either the PCR product or cloned cDNA of mouse Runx2, junB and ATF-2. All probes were labeled with [-³²P]dCTP (3000 Ci/mmol; NEN) using the rediprime DNA labeling kit.

Western blot analysis

Proteins from cell lysates were resolved by SDS-PAGE (8-10%) and transferred to Immobilon membrane (Milipore). The blots were blocked in BP solution (50 mM Tris pH 7.5, 150 mM NaCl, 0.1% Tween 20) containing 5% nonfat dry milk. Primary antibody (against FLAG, junB, or p38) was added to the BP solution at a 1 : 1000 dilution and incubated for 1 h at 25°C. The blots were washed three times with the BP solution and incubated with the secondary antibody (1 : 10 000) conjugated with HRP for 1 h at 25°C. After three washes with the BP solution, the blots were developed with ECL and exposed on Kodak XAR-5 film.

Acknowledgements

This work was supported by a grant to S-C Bae from the Molecular Medicine Research Group Program (M1-0106-00-0064) of the Ministry of Science and Technology of Korea. This work was also supported by Korean Research Foundation grant KRF-2001-042-D0067.

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Figure 1 Induction of Runx2 mRNA by TGF-1 and BMP-2. (a) C2C12 cells were treated with TGF-1 (5 ng/ml) for the indicated times, and total RNA was prepared. Northern blotting was performed using Runx2, junB, and ATF-2 cDNA as probes. (b) Total RNAs were prepared from C2C12 cells and C2C12-Sm5 cells treated with serially diluted BMP-2 for 1 h. junB expression was analysed by Northern blot hybridization. (c) C2C12 cells were treated with TPA (100 ng/ml) for the indicated times, and total RNA was prepared. Runx2 mRNA expression was measured using Northern blotting. Probes prepared from GAPDH and -actin coding sequences were used as loading controls

Figure 2 Induction of Runx2 by junB. (a) Western blotting showing over-expressing of junB in C2C12-junB cells. Total cellular protein extracts were prepared from control C2C12 and C2C12-junB cells, and the junB protein was detected by a specific monoclonal antibody. For loading control, levels of -tubulin were measured by anti--tubulin antibody. (b) Control C2C12 and C2C12-junB cells were treated with BMP-2 (300 ng/ml) for 2 h and Runx2 mRNA levels were analysed using Northern blotting. (c) Control C2C12 and C2C12-junB cells were treated with the indicated concentrations of TGF-1 for 2 h. Total RNA was prepared from the cells, and Runx2 mRNA levels were analysed using Northern blotting

Figure 3 Inhibition of Runx2 induction by A-fos. (a) Western blotting showing exogenous expression of A-fos in C2C12-A-fos cells. Total cellular protein extracts were prepared from control C2C12, C2C12-A-fos-1 and C2C12-A-fos-2 cells, and A-fos protein was detected by an anti-FLAG antibody. (b) C2C12, C2C12-A-fos-1 and C2C12-A-fos-2 cells were treated with TGF-1 (5 ng/ml) and BMP-2 (300 ng/ml) for 2 h. Total RNA was prepared from the cells, and Runx2 mRNA levels were analysed using Northern blotting

Figure 4 Involvement of p38 MAPK in the induction of Runx2. (a) C2C12 cells were pretreated with SB203580 (50 M, 1 h), Wortmanin (0.5 M, 1 h), PD98059 (50 M, 20 min) or Rapamycin (20 ng/ml, 1 h) and then treated with BMP-2 (300 ng/ml) or TGF-1 (5 ng/ml) for 2 h. Total RNA was prepared from the cells, and Runx2 mRNA levels were analysed using Northern blotting. (b) C2C12 cells were treated with anisomycin (5 ng/ml) in the presence or absence of SB203580 for the indicated times, and total RNA was prepared. Runx2 mRNA levels were analysed using Northern blotting. An: Anisomycin; SB: SB203580

Figure 5 Inhibition of Runx2 induction by dominant negative p38 [p38(AGF)]. (a) Western blotting showing exogenous expression of p38(AGF) in C2C12-p38(AGF) cells. The arrows indicate p38 and p38(AGF) proteins. (b) C2C12 and C2C12-p38(AGF) cells were cultured in the presence or absence of TGF-1 (5 ng/ml) for 2 h, and phosphorylated p38 was detected with a phospho-p38-specific antibody. Note that p38(AGF) effectively blocked TGF-1-dependent p38 phosphorylation. (c) C2C12, and C2C12-p38(AGF) cells were treated with TGF-1 (5 ng/ml) or BMP-2 (300 ng/ml) for 2 h. Total RNA was prepared from the cells, and Runx2 mRNA levels were analysed using Northern blotting

Figure 6 The effect of Runx2 and JunB on osteoblast-specific gene expression. Cells were treated with the indicated concentration of BMP-2 for 2 days, and alkaline phosphatase activity was assayed as described previously (Katagiri et al., 1994)