ORIGINAL RESEARCH ARTICLE

Journal of

BMP-2 Induction of Dlx3 Expression Is Mediated by p38/Smad5 Signaling Pathway in Osteoblastic MC3T3-E1 Cells

Cellular Physiology

GUOBIN YANG, GUOHUA YUAN, XIAOYAN LI, PINGXIAN LIU, ZHI CHEN, AND MINGWEN FAN* The State Key Laboratory Breeding Base of Basic Science of Stomatology and Key Laboratory of Oral Biomedicine Ministry of Education, School and Hospital of Stomatology, Wuhan University, Wuhan, China Dlx3 is essential for osteoblast differentiation and bone formation, and its expression is regulated by bone morphogenetic protein-2 (BMP2). However, the intimate mechanism of BMP-2 regulation of Dlx3 transcription in osteoblasts is still unknown. Considering the important roles of Smad5 and p38 in osteoblast differentiation, we hypothesized that Smad5 and p38 mediated BMP-2-induced Dlx3 transcription in osteoblasts. We found activation of Smad5 and p38 increased the expression of Dlx3, whereas knocking down Smad5 or inactivation of p38 inhibited BMP-2-induced Dlx3 expression. Both Smad5 and p38 were able to activate Dlx3 promoter activity and p38/Smad5 response elements were located from
698 to
368 in Dlx3 promoter. Two Smad5 binding sites (SBEI and SBEII, TGTCT box) were identified in this region by EMSA and ChIP assay. Deletions and mutagenesis study of the Dlx3 promoter region indicated that the TGTCT boxes are crucial for p38/Smad5-induced Dlx3 promoter activity. At last, we found a cross-talk between p38 and Smad5, and that activation of p38 is necessary for BMP-2-induced Smad5 phosphorylation and nuclear translocation. Overall, we provide a novel insight that BMP-2-induced Dlx3 expression is regulated by p38/Smad5 signaling pathway in osteoblasts. J. Cell. Physiol. 229: 943–954, 2014. ß 2013 Wiley Periodicals, Inc.

Vertebrate development is regulated by hundreds of homeodomain (HD) proteins. Distal-less (Dlx) genes are a subfamily of homeobox genes that play crucial roles during skeletal formation and the development of the central nervous system (Panganiban and Rubenstein, 2002). In vertebrates, there are six Dlx genes organized into three clusters (Dlx1/2, Dlx3/4, and Dlx5/6; Ghanem et al., 2003). Paired genes are localized on three distinct chromosomes with an inverted configuration and are separated by a short intergenic region (Ghanem et al., 2003). Dlx3 is expressed in periosteum, osteoblasts, and chondrocytes of the developing limb and in osteogenic lineage cells (Choi et al., 2012) with especially strong expression in differentiating and differentiated osteoblasts (Ghoul-Mazgar et al., 2005), and is the only Dlx family member that is strongly up-regulated during late differentiation stage of osteoblasts (Li et al., 2008). As a transcription factor, Dlx3 recognizes a specific sequence with a central TAAT core motif, which was found on the promoter regions of several osteogenic genes including osteocalcin (Hassan et al., 2004), Runx2 (Shirakabe et al., 2001), bone sialoprotein (Roca et al., 2005), and regulates their transcription. Over-expression of Dlx3 in osteoprogenitor cells promotes the expression of osteoblastic differentiation markers such as type I collagen, bone sialoprotein, osteocalcin, and alkaline phosphatase (Hassan et al., 2004). Although Dlx3 null mice are embryonically lethal prior to the skeletal development due to placental failure (Beanan and Sargent, 2000), mutations in Dlx3 in human cause Tricho–Dento–Osseous (TDO) syndrome, which is an ectodermal dysplasia characterized by defects in ectodermal derivatives such as hair, teeth and bone (Price et al., 1998). The patients with TDO exhibit increased bone mineral density in craniofacial and appendicular bones, and often have prognathic mandible and aberrant skull shape (Nguyen et al., 2013). All of these findings suggest Dlx3 is essential for osteoblast differentiation and bone formation. However, despite its vital ß 2 0 1 3 W I L E Y P E R I O D I C A L S , I N C .

roles in osteoblast differentiation, the regulation of Dlx3 gene expression in osteoblasts is poorly understood. The bone morphogenetic proteins (BMPs), belonging to the transforming growth factor b (TGF-b) super-family, play important roles in many aspects of vertebrate embryogenesis and are identified as inducers of ectopic bone and cartilage formation (Reddi, 1997). In osteoblasts, transcription of Dlx3 is regulated by BMP signaling, especially BMP-2. Dlx family members are rapidly induced in response to BMP-2 mediated osteoblast differentiation (Balint et al., 2003; Harris et al., 2003). Among the Dlx family members, induction of Dlx3 after BMP-2 treatment was coincident with the onset of commitment of pluripotent C2C12 mesenchymal cells to the osteogenic lineage (Balint et al., 2003). However, the intimate mechanism of BMP-2 regulation of Dlx3 transcription in osteoblasts is still unknown. Thus, the purpose of the present

Guobin Yang and Guohua Yuan contributed equally to the study. Contract grant sponsor: National Nature Science Foundation of China; Contract grant numbers: 81000418, 81371105, 81000436. *Correspondence to: Mingwen Fan, The State Key Laboratory Breeding Base of Basic Science of Stomatology and Key Laboratory of Oral Biomedicine Ministry of Education, School and Hospital of Stomatology, Wuhan University, 237 Luoyu Road, Wuhan 430079, China. E-mail: [email protected] Manuscript Received: 16 September 2013 Manuscript Accepted: 4 December 2013 Accepted manuscript online in Wiley Online Library (wileyonlinelibrary.com): 16 December 2013. DOI: 10.1002/jcp.24525

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study is to elucidate the molecular mechanism of BMP-2 induction of Dlx3 expression. BMPs signal via two transmembrane serine/threonine kinase receptors, the BMP receptor type I (BMPRIa, BMPRIb) and type II (BMPRII) (Heining et al., 2011). BMPs bind to preformed heterocomplexes of BMPRI and BMPRII, initiating Smaddependent signaling pathway. The Smad-dependent pathway is initiated by phosphorylation of R-Smad (Smad1/5/8), which subsequently interacts with the Co-Smad (Smad4), translocates into the nucleus, and regulates transcription of target genes (Massague et al., 2005). In addition to BMP/Smad signaling pathway, BMP ligand binding to the high affinity receptor BMPRI induces the formation of heteromeric BMP-induced signaling complexes (BISCs), which activates the mitogenactivated protein kinase (MAPK) signaling cascade, including p38, ERK, JNK (Sieber et al., 2009). Among the downstream mediators of BMP signal, Smad5 and p38 are essential for osteoblast differentiation. Smad5 plays a critical role in the BMP-2-induced osteoblastic differentiation of C2C12 cells (Nishimura et al., 1998). Runx2, as a common target of BMP-2 and TGF-b, activates osteoblastessential genes, including the transcription factors required for in vivo bone formation (Lee et al., 2000; Javed et al., 2008). Cooperation between Runx2 and Smad5 induces osteoblastspecific gene expression in C2C12 cells (Lee et al., 2000). Colocalization of PSmad5 (phospho-Smad5) and Dlx3 was found during hair morphogenesis (Hwang et al., 2008), which indicates that Smad5 may mediate the regulation of Dlx3 by BMP-2. Besides, many studies have reported that p38 activation is necessary for differentiation of osteoblasts (Guicheux et al., 2003; Hu et al., 2003). Studies using MC3T3E1 cells have shown that activation of p38 is critical for ALP expression (Lai and Cheng, 2002; Suzuki et al., 2002). p38 is activated at an early stage during osteoblast differentiation, and inhibition of p38 suppresses BMP-2-induced expressions of ALP and osteocalcin and matrix mineralization, which suggests BMP-2 is required to activate p38 in order to stimulate osteogenic differentiation and maturation (Gallea et al., 2001; Hu et al., 2003; No¨th et al., 2003). Based on these biological functions of Smad5 and p38 during osteoblast differentiation, we hypothesized that BMP-2 regulating Dlx3 transcription was mediated by Smad5 and/or p38 signaling pathway.

Materials and Methods Cell culture Mouse osteoblastic cells MC3T3-E1 were obtained from American Type Culture Collection (ATCC, Manassas, VA). Cells were maintained in a-minimal essential medium (MEM, Gibco, Grand Island, NY), with 2 mM L-glutamine, 1% penicillin/streptomycin and 10% fetal bovine serum (FBS, Gibco). For cell-signaling experiments, cells were starved overnight in FBS-free medium, then cells were treated with recombinant mouse BMP-2 protein (R&D Systems, Minnesota, MN) for the indicated time. In experiments using the p38 pharmacological inhibitor SB203580 (Cell Signaling Technology, Danvers, MA), cells were pre-treated in medium containing the inhibitor for 2 h before BMP-2 stimulation. Overexpression MC3T3-E1 cells cultured in six-well plates (nearly 75% confluent) were transfected with Smad5 expression vector (pCMV-Smad5) or Mkk6 expression vector (pCDNA3-Mkk6) or their respective empty vectors using Lipofectamine 2000 transfection reagent (Invitrogen, Grand Island, NY) according to the manufacture’s instructions. Cell lysates were harvested 48 h post-transfection and prepared for Western blot. JOURNAL OF CELLULAR PHYSIOLOGY

RNA interference Small interfering RNA (siRNA) against mouse Smad5 (Santa Cruz Biotechnology, Dallas, TX) and negative control siRNA (Santa Cruz Biotechnology) were transfected into MC3T3-E1 cells at a final concentration of 20 or 40 nM using Lipofectamine 2000 (Invitrogen). Forty-eight hours after transfection, cells were treated with recombinant BMP-2 protein for 2 h. After BMP-2 treatment, cells were prepared for Western blot. Western blot Cell cultures under different conditions were washed with phosphate-buffered saline (PBS) and then lysed with RIPA lysis buffer (Cell Signaling Technology). Insoluble materials were removed by centrifugation at 13,000 rpm for 10 min at 4˚C. Equal amounts of proteins were separated with SDS–PAGE gel and transferred to nitrocellulose membrane. The membrane was blocked in 5% non-fat milk for 30 min at room temperature with constant rocking and then incubated with anti-Smad5 (1:1,000, Rabbit monoclonal, Cell Signaling Technology), anti-PSmad5 (1:500, Rabbit monoclonal, Abcam, Cambridge, MA), anti-p38 (1:1,000, Rabbit monoclonal, Cell Signaling Technology), anti-Pp38 (phospho-p38, 1:500, Rabbit monoclonal, Cell Signaling Technology), anti-Dlx3 (1:2,000, Rabbit polyclonal, Abcam), anti-P-MAPKAPK2 (phospho-MAPKAPK2, 1:1,000, Rabbit monoclonal, Pierce, Rockford, IL), anti-MAPKAPK2 (1:1,000, Rabbit polyclonal, Pierce), or anti-b-actin (1:2,000, Rabbit polyclonal, Santa Cruz Biotechnology) antibodies overnight at 4˚C with constant rocking. The membrane was then washed three times and incubated with HRP-labeled secondary antibody (1:5,000, Goat anti-Rabbit IgG, Pierce) for 1 h. Immunoreactive bands were visualized using an ECL kit (Pierce). Western blot for each sample were repeated at least three times. Construction of reporter plasmids and mutagenesis Mouse genomic DNA was extracted from mouse tails using DNeasyTM Tissue Kit (Qiagen, Hilden, Germany) following the manufacturer’s instructions. The mouse Dlx3 promoter region from
1481 to þ56 relative to the position of the transcription start site (þ1) was amplified by PCR using mouse genomic DNA as template with the following primer sets (Luc-1481): forward: 50 cgcctcgagCCTGTCTGCCTGCTTAC CTCCTCC-30 and reverse: 50 -cgcaagcttGCTGTCGGTCAGTCGCTGCGTGCCT-30 . Other fragments with different lengths in the 50 -flanking region were amplified by PCR using the cloned 50 -flanking DNA (Luc-1481) as template with the same reverse primer and the following forward primers: Luc-698: 50 -cgcctcgagGCTCCAGTAGGGACTTGCAG30 ; Luc-368: 50 -cgcctcgagTGGGTAAATTCCCCTCCTT C-30 ; Luc-147: 50 -cgcctcgagAAGAGGGTTGAGAGGGTTGG-30 . All forward primers contain a 50 -cgcctc gag-XhoI overhang and reverse primer contains a 50 -cgcaagctt-HindIII overhang. All the PCR products were gel-purified, digested with XhoI and HindIII (New England Biolabs, Ipswich, MA), and subcloned into pGL3Basic luciferase reporter vector (Promega, Madison, WI). Site directed deletion targeting the Smad5 binding sites in Dlx3 promoter was completed using the QuickChange1 II SiteDirected Mutagenesis Kit (Agilent Technologies, Santa Clara, CA). Mutant plasmids, SBEI-Del and SBEII-Del, were generated by site-directed deletions at the
565/
561 and
432/
428 sites of Luc-698 construct as a template. Oligonucleotide primers designated for mutant plasmids were as follows: SBEI-Del, 50 -CAGGGCCATACGGAGAAACCCCGAAAAAAAACAACAAAAAAC-30 , and 50 -CCCTGGGTCTTTGCCACTTTCGTCATTTGCATAGACCACACA-30 ; SBEII-Del, 50 -CCCTGGGTCTTTGCCACTTTCGTCATTTGCATAGACCACACA-30 , and 50 -TGTGTGGTCTATGCAAAT GACGAAAGTGGCAAAGACCCAGGG-30 . All constructs were verified by DNA sequencing

REGULATION OF Dlx3 BY BMP-2-p38/Smad5 PATHWAY

and purified with Plasmid Midi Kit (Qiagen) according to the manufacturer’s protocol. Luciferase reporter assay Cells grown in 48-well plates were transfected with pGL3-Basic empty vector (Promega) or containing different length of Dlx3 promoter inserts and pRL-TK Renilla luciferase expression vector (Promega) as an internal control, as well as pCMV-Smad5 or pCDNA3-Mkk6 using the Lipofectamine 2000. The cells were collected 48 h after transfection and lysed in Passive lysis buffer (Promega). The luciferase assay was performed using the Dual Luciferase Reporter Assay System (Promega) according to the manufacture’s specifications. Firefly and Renilla luciferase activities were determined using the Glomax Luminometer (Promega). Luciferase expression was normalized against Renilla luciferase expression to determine relative luciferase activity. All luciferase assays were performed at least three times. Chromatin immunoprecipitation assays MC3T3-E1 cells (10-cm dish) were transiently transfected with pCMV-Smad5, pCDNA3-Mkk6, and Luc-1481 using Lipofectamine 2000. Chromatin immunoprecipitation assay was carried out according to the manufacturer’s instructions using the Chromatin Immunoprecipitation (ChIP) Assay Kit (Millipore, Billerica, MA). Briefly, 48 h after transfection the cells were cross-linked with 1% formaldehyde for 10 min, washed with cold PBS, and lysed in lysis buffer. Lysates were sonicated to shear DNA using an Ultrasonic Processor (Fisher Scientific, Waltham, MA). Supernatant was diluted 10-fold in ChIP dilution buffer. A proportion of the diluted supernatant was kept as input DNA. After preclearing for 30 min with Protein A Agarose/Salmon Sperm DNA, samples were incubated overnight at 4˚C with 5 mg of anti-PSmad5, anti-Pp38, or anti-Smad1 (Cell Signaling Technology) antibody. Negative control IgG (Santa Cruz Biotechnology) incubation was included as control for the immunoprecipitation. Protein A/G Agarose/Salmon Sperm DNA was then added for 1 h at 4˚C to collect the immune complexes. Then immune complexes were sequentially washed with low-salt, high-salt and LiCl washing buffers, then TE buffer for 5 min each with rotation at 4˚C. Immune complexes were eluted by addition of elution buffer for 30 min with rotation at room temperature and cross-links were reversed by addition of 5 M NaCl and heating at 65˚C for 4 h. Samples were then incubated with 0.5 M EDTA, 1 M Tris-HCl, pH 6.5, and proteinase K for 1 h at 45˚C. DNA was recovered by phenol/chloroform extraction, ethanol precipitated and resuspended in 25 ml of water. The recovered DNA was analyzed by PCR using the following primers: primer1, 50 -GCTCCAGTAGGGACTTGCAGGCCCAA-30 , primer2, 50 -TTGGGTG TTTGTGGAAGGAGGTGGTC-30 , primer3: 50 -GCAAAGACCCAGGGTCCATGAAAATA-30 , and primer4, 50 -TATTTTCATGGACCCTGGGTCTTTGC-30 . Primer1, 2 were designed to amplify the promoter regions of the Dlx3 gene from
698 to
369. While primer1, 3, and primer2, 4 amplify SBEI and SBEII region of Dlx3 promoter, respectively. Electrophoretic mobility shift assay (EMSA) Nuclear extracts were prepared from MC3T3-E1 cells using NEPER Nuclear and Cytoplasmic Extraction Kit (Pierce) according to the manufacturer’s instructions. Protein concentration of the nuclear fraction was determined using the Bradford’s assay (BioRad, Hercules, CA). Complementary oligonucleotides consisting Smad5 binding region on the Dlx3 promoter were synthesized: sense: 50 -CGGAGAAACCCTG TCTCGAAAAAAAACAAC-30 , antisense: 50 -GTTGTTTTTTTTCGAGACAGGGTTTCTCCG30 ; sense: 50 GTCTTTGCCACTTTCTGTCTGTCATTTGCATAG-30 , antisense: 50 -CTATGCAAATGAC JOURNAL OF CELLULAR PHYSIOLOGY

AGACAGAAAGTGGCAAAGAC-30 . These oligonucleotides were biotin labeled according to the Biotin 30 End DNA Labeling Kit (Pierce). To generate double-stranded probes, complementary oligonucleotides were mixed together at a 1:1 molar ratio, heated to 95˚C for 5 min and then gradually cooled down to room temperature. EMSA were performed using the LightShift Chemiluminescent EMSA Kit (Pierce). Briefly, 0.1 pmol of each biotin end-labeled double-stranded probe was incubated for 25 min at room temperature in 20 ml of EMSA binding buffer containing 2.5% Glycerol, 5 mM MgCl2, 50 ng/ml poly(dI–dC), 0.05% Nonidet P-40, and 6 mg of nuclear proteins. For competition binding reactions, 200-fold (20 pmol) excess unlabeled, doublestranded probe was added to the binding reaction. For antibody super-shift experiment, Smad5 antibody was incubated with nuclear extract at 4˚C for 30 min prior to the probe addition. The DNA-nuclear protein complexes were resolved by electrophoresis in 5% non-denaturing polyacrylamide gel. The protein-DNA complex was then transferred to nylon membrane and cross-linked using a UV crosslinker, and visualized using the Chemiluminescent Nucleic Acid Detection Module (Pierce). Co-immunoprecipitation Cells (10-cm dish) were transfected with pCMV-Smad5 and pCDNA3-Mkk6 plasmids. After 72 h of transfection, cells were lysed in 800 ml of RIPA lysis buffer (Cell Signaling Technology) with protease inhibitor (Roche Diagnostics, Mannheim, Germany) for 15 min at 4˚C. The lysates were centrifuged at 12,000 rpm at 4˚C for 20 min to remove cellular debris. The supernatant was precleared with 40 ml of protein A/G plus agarose beads (Santa Cruz Biotechnology) at 4˚C for 30 min. The beads were collected by centrifugation at 1,000g for 5 min at 4˚C. Anti-PSmad5, antiPp38 antibody or negative control IgG (3 mg each) was added to the precleared lysates following incubation at 4˚C overnight, and then with 50 ml of protein A/G plus agarose beads for 2 h to precipitate immunocomplexes. Beads were washed three times with wash buffer (10 mM Tris-HCl, pH 8.0, 150 mM NaCl, 10% glycerol, 1% NP-40, and 2 mM EDTA). Fifty microliters of sample buffer was added to the beads and samples were heated to 95˚C for 10 min. PSmad5 and Pp38 were detected in the protein complex using Western blot. Immunofluorescence Cells grown on glass cover-slips were fixed with 4% paraformaldehyde for 10 min and permeabilized with 0.5% TritonX for 15 min. Non-specific binding of antibodies was blocked by 10% goat serum for 30 min. Samples were incubated overnight at 4˚C with primary antibodies. After washing with PBS, secondary IgG antibodies conjugated to Alexa-Fluor 568 (1:500, Goat antiRabbit IgG, Invitrogen) were added and incubated for 1 h. DAPI (Invitrogen) was used as nuclear counterstain. Statistical analysis Student’s t-test or one-way analysis of variance (ANOVA) followed by post-hoc Bonferroni test was applied to investigate any significant difference between the groups using SPSS11.0 (SPSS, Inc., Chicago, IL). Results BMP-2 stimulates phosphorylation of Smad5 and p38 as well as expression of Dlx3 in MC3T3-E1 cells

BMP-2 transduces signals through activation of Smad1/5/8, and also activates p38 kinase pathway in a variety of cell types. We first examined whether phosphorylation of Smad5 and p38 occurred in MC3T3-E1 cells after BMP-2 treatment. As illustrated in Figure 1, PSmad5 and Pp38 increased and reached

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Fig. 1. Effect of BMP-2 on phosphorylation of Smad5 and p38 as well as expression of Dlx3 in MC3T3-E1 cells. A: Cells were serum-starved overnight and treated with BMP-2 (100 ng/ml) for the indicated time. The protein expressions of Smad5, PSmad5, p38, Pp38, Dlx3 and b-actin were showed by a representative Western blot. B–D: Quantitative analysis of Western blot normalized to control (0 min) from three independent repeats.  P < 0.05;  P < 0.01.

the maximum level at 15–30 min, then declined. In contrast, total levels (both phosphorylated and un-phosphorylated protein) of Smad5 or p38 remained unchanged after BMP-2 treatment. Meanwhile, the expression of Dlx3 was significantly up-regulated 2 h after BMP-2 treatment. Therefore, phosphorylation of Smad5 and p38 occurred earlier than up-regulation of Dlx3 after BMP-2 treatment, indicating that Smad5 and p38 probably participate in BMP-2-regulated Dlx3 expression. Activation of Smad5 and p38 increases the expression of Dlx3, and p38 is able to induce Smad5 phosphorylation

To evaluate the influence of Smad5 on Dlx3 expression, Smad5 was over-expressed by transfection of Smad5 expression plasmid pCMV-Smad5. The expression of Dlx3 was significantly enhanced (Fig. 2A,B). Then the effects of p38 on Dlx3 expression was examined by over-expression of p38 kinase activator—MKK6, which is well known to phosphorylate p38 on Thr-180 and Tyr-182 and to activate p38 (Raingeaud et al., 1996). With activation of p38, the expression of Dlx3 increased as well as the expression of PSmad5 (Fig. 2C,D). These results showed that activation of either Smad5 or p38 was able to increase the expression of Dlx3, and p38 was able to induce Smad5 phosphorylation in MC3T3-E1 cells. BMP-2 induces Dlx3 expression via Smad5 and p38 activation in MC3T3-E1 cells

To examine whether BMP-2-induced Dlx3 expression was dependent on Smad5, we down-regulated Smad5 expression using Smad5-targeted small interfering RNA (siRNA) before BMP-2 induction. Transfection of Smad5 siRNA significantly reduced Smad5 expression level compared with transfection of control siRNA (Fig. 3A). At the same time, BMP-2-induced Dlx3 expression was significantly decreased in the presence of Smad5 siRNA (Fig. 3B). We next examined if BMP-2-induced Dlx3 expression is also mediated by activation of p38. SB203580 is well recognized as a potent and highly specific inhibitor of p38 kinase. Cells were treated with SB203580 before BMP-2 induction. Because SB203580 inhibits the catalytic activity of Pp38, but not the phosphorylation of p38 JOURNAL OF CELLULAR PHYSIOLOGY

(Kumar et al., 1999), we first detected the expression of PMAPKAPK2, which is directly phosphorylated by Pp38, to confirm the inhibition efficiency. As shown in Figure 3C, expression of P-MAPKAPK2 was highly repressed in the presence of 10 and 20 mM SB203580. At the same time, BMP-2induced Dlx3 expression was attenuated when cells were pretreated with SB203580, and reduced to a lower level with the presence of both SB203580 and Smad5 siRNA (Fig. 3D). These results suggested that Smad5 and p38 are mediators for BMP-2-induced Dlx3 expression in osteoblastic MC3T3-E1 cells. Smad5 and p38 stimulate Dlx3 promoter activity

To assess whether Smad5 and p38-induced Dlx3 expression was mediated by stimulating Dlx3 promoter activity, Luc-1481, a luciferase reporter containing the basal Dlx3 promoter region (from
1481 to þ56), was transiently transfected into MC3T3-E1 cells, and transcriptional activity was measured with or without transfection of pCMV-Smad5 or pCDNA3Mkk6 plasmid. Cells transfected with Luc-1481 reporter construct gave about 40- to 60-fold transcriptional activity after 48 h of transfection compared with transfection with pGL3-Basic vector (Fig. 4A,B) with the presence of pCMVSmad5 or pCDNA3-Mkk6 plasmid. This result indicated that both Smad5 and p38 were able to stimulate Dlx3 promoter activity. Identification of the Smad5/p38 responsive region in the Dlx3 promoter

To identify the Smad5/p38 responsive region in the Dlx3 promoter, we constructed a series of luciferase reporters containing various promoter fragments of Dlx3 gene: Luc-698, Luc-368, Luc-147 (Fig. 4A). Deletion of the sequence between
1481 and
698 (Luc-698) did not change the transcription activity compared with Luc-1481 after 48-h induction by transfection of pCMV-Smad5 or pCDNA3-Mkk6 plasmid. However, deletion of the sequence between
698 and
368 (Luc-368) resulted in significant loss of basal transcription activity (Fig. 4B). These results indicated that PSmad5 and Pp38

REGULATION OF Dlx3 BY BMP-2-p38/Smad5 PATHWAY

Fig. 2. Dlx3 is a down-stream target of Smad5 and p38. A: Cells were transiently transfected with pCMV-Smad5 plasmid or corresponding empty vector (control). After 48 h of transfection, protein expression levels of Smad5 and Dlx3 were detected by Western blot. C: Cells were transiently transfected with pCDNA3-Mkk6 plasmid or corresponding empty vector (control). After 48 h of transfection, protein expression levels of Pp38, p38, PSmad5, Smad5, and Dlx3 were detected by Western blot. B,D: Quantitative analysis of Western blot versus empty vector control from three independent repeats.  P < 0.05;  P < 0.01.

response elements resided within the region between
698 and
368 in the mouse Dlx3 promoter. To further test whether PSmad5 or Pp38 binds directly or indirectly with the nucleotides
698 to
368 of Dlx3 promoter, we performed ChIP assay. Antibodyimmunoprecipitated protein-DNA complexes were decrosslinked, and the purified DNA was used as a template for PCR, using primers corresponding to the 50 -flanking region (from
698 to
368) of mouse Dlx3 gene. As expected, 330bp PCR products were amplified from the DNA fragment immunoprecipitated by anti-PSmad5 and Pp38 antibodies (Fig. 4C, Lanes 1 and 2, upper). Negative control was done using negative control IgG antibody (Fig. 4C, Lane 4, upper) and positive control was set up using total DNA before immunoprecipitation as the input DNA for PCR (Fig. 4C, Lanes 1–4, lower). Since BMP-2 treatment stimulates the association JOURNAL OF CELLULAR PHYSIOLOGY

of Smad5 and Smad1, Smad1 antibody was also used in ChIP assay. However, no PCR products were amplified from the DNA fragment immunoprecipitated by anti-Smad1 antibody (Fig. 4C, Lane 3, upper). These results indicated that PSmad5 and Pp38 can physically bind to Dlx3 promoter region from
698 to
368 in vivo, and this procedure is independent of Smad1. Smad5 and p38 stimulate Dlx3 expression through a TGTCT box

Smad5 is able to bind to a unique consensus sequence of TGTCT, which is different from the common binding sites of Smad1 and Smad8 (Li et al., 2001). Analysis of the 50 -flanking region from
698 to
368 of the mouse Dlx3 gene sequence revealed two putative Smad5 binding elements (SBE): SBEI and

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Fig. 3. BMP-2 induces Dlx3 expression via Smad5 and p38 activation. A: Interference efficiency of Smad5 siRNA was detected using Western blot after transfection of control siRNA or Smad5 siRNA for 48 h with indicated final concentrations. Quantitative analysis is shown in the lower part. B: Cells were transfected with Smad5 siRNA or control siRNA for 48 h, then the cells were treated with or without BMP-2 (100 ng/ ml) for 2 h. Protein expression level of Dlx3 was detected by Western blot. Quantitative analysis is shown in the lower part. C: Inhibition efficiency of SB203580 was detected using Western blot after treatment with DMSO or SB203580 for 2 h with indicated final concentrations. Protein expression levels of P-MAPKAPK2 and total MAPKAPK2 were detected. Quantitative analysis is shown in the lower part. D: Cells were treated with SB203580 for 2 h and with or without Smad5 siRNA (S5 si) transfection, then the cells were treated with or without BMP-2 (100 ng/ml) for 2 h. Protein expression level of Dlx3 was detected by Western blot. Quantitative analysis is shown in the lower part.  P < 0.05;  P < 0.01;  P < 0.001.

SBEII (Fig. 5A). First, we examined whether Smad5 is able to bind to TGTCT box in the Dlx3 promoter using EMSA. For EMSA, we prepared two oligonucleotide probes, SBE1 (
576/
547) and SBE2 (
447/
415) covering the SBEI and SBEII binding sites (Fig. 5B). Following incubation, proteinprobe binding complexes were detected (Fig. 5C, Lanes 1 and 4, black arrow). The specificity of the complex was confirmed by the competition assay. A 200-fold unlabeled cold probe substantially competed with the binding complex (Fig. 5C, Lanes 3 and 6). The identity of the specifically shifted protein was further confirmed using anti-Smad5 antibody. When the antibody was included in the binding reaction, a super-shift for antibody-protein-probe complex was detected (Fig. 5C, Lanes 2 and 5, white arrow). These results indicated Smad5 was JOURNAL OF CELLULAR PHYSIOLOGY

able to bind to the SBE binding sites on the Dlx3 promoter in vitro. To assess the essential role of TGTCT box for Smad5/p38 mediated Dlx3 promoter activity, we generated two Luc-698 mutant constructs with deletion of either SBE sites (Fig. 6A): SBEI-Del, SBEII-Del. Then Luc-698 or Luc-698 mutant constructs were transiently transfected into MC3T3-E1 cells, and their transcriptional activity was determined in the presence of pCMV-Smad5 and/or pCDNA3-MKK6 cotransfection. The results showed that deletion of SBEI binding site suppressed the Luc-698 response to both PSmad5 and Pp38, while deletion of SBEII binding site only suppressed the Luc-698 response to PSmad5 but not to Pp38 (Fig. 6B). These results indicate that PSmad5 induced an increase of the Luc-698

REGULATION OF Dlx3 BY BMP-2-p38/Smad5 PATHWAY

Fig. 4. Smad5 and p38 activate Dlx3 promoter activity. A: Schematic representation of the constructs used in the luciferase assay. B: Smad5 and p38 stimulated Dlx3 promoter activity. The value of luciferase activity was obtained by firefly/Renilla luciferase activity. Fold increases were showed for individual value versus the control group value and plotted as a graph showing the mean  SEM from the three independent experiments ( P < 0.001). C: ChIP assay. The upper bands of Lanes 1–4 were amplified products using the purified DNA fragments as templates which were immunoprecipitated by anti-PSmad5, Pp38, Smad1 antibodies, and negative control IgG (()CTL), respectively. The lower bands were amplified products with input DNA as templates.

promoter activity mediated by both SBEI and SBEII, while Pp38induced Luc-698 promoter activity was mediated only by SBEI but not SBEII, which suggests SBEII is not needed for Pp38mediated Dlx3 promoter activity. To further confirm that SBEII is not necessary for Pp38mediated Dlx3 promoter activity, ChIP assay was performed using primer pairs covering SBEI or SBEII only (Fig. 6C). A 259bp PCR product was amplified from the DNA fragment immunoprecipitated by anti-Pp38 antibody using primer1, 3 (Fig. 6D, Lane 2), but no PCR products were amplified using primer2, 4 (Fig. 6D, Lane 3). This result further confirmed Pp38 did not bind to SBEII site. The result that Pp38 promoted Dlx3 promoter activity though SBEI site implied that Pp38 may act as a component of transcription factor complexes, forming a complex with PSmad5 to increase transcription activity of PSmad5. We used co-immunoprecipitation to detect the protein–protein interaction between PSmad5 and Pp38. As expected, using anti-PSmad5 antibody as IP antibody, Pp38 was detected in the immunoprecipitation complex by Western blot, and using antiPp38 antibody as IP antibody, PSmad5 was also detected in the immunoprecipitation complex (Fig. 6E), which indicated Pp38 did interact with PSmad5 forming a complex. JOURNAL OF CELLULAR PHYSIOLOGY

To further confirm Pp38 may act as a component of transcription factor complexes, we detected the subcellular localization of Pp38 using immunofluorescence. In untreated cells, Pp38 showed diffuse staining throughout the cytoplasm and nuclei. While, with BMP-2 treatment, Pp38 staining in nuclei was dramatically increased (Fig. 6F). This result further supported that Pp38 interacted with PSmad5 in nucleus to act on Dlx3 promoter after BMP-2 induction. Activation of p38 is necessary for BMP-2-induced Smad5 phosphorylation and nuclear translocation

It was reported that inhibition of p38 with SB203580 suppresses Smad1 phosphorylation and nuclear translocation in osteoblasts (No¨th et al., 2003). It is yet not known if this also applies to Smad5. We therefore next tested the effect of SB203580 on Smad5 phosphorylation and nuclear translocation. SB203580 treatment caused a dose-dependent decrease of BMP-2-induced Smad5 phosphorylation (Fig. 7A). Then the subcellular localization of PSmad5 was detected using immunofluorescence in MC3T3-E1 cells following treatment with and without SB203580 or BMP-2 (Fig. 7B). In untreated cells, PSmad5 staining appeared mainly in the cytoplasm and

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Fig. 5. Determination of Smad5 binding sites in mouse Dlx3 promoter by EMSA. A: Mouse Dlx3 proximal promoter nucleotide sequences from 574 to 421 are shown. Two putative Smad5 binding sites (SBEI and SBEII, marked with underline and bold) are present in this region. B: The sequences of probes used in EMSA. SBE1 and SBE2 probes covered the SBEI and SBEII binding sites respectively. C: EMSA showed Smad5 was able to bind to the SBEI and SBEII binding sites in vitro. Black arrow indicates the protein-probe binding complex and white arrow indicates super-shift for antibody-protein-probe complex.

faintly in nucleus. Following treatment with BMP-2, the expression level of PSmad5 obviously increased, especially in the nuclei, compared to that in the untreated cells, suggesting that BMP-2 induced the phosphorylation and nuclear translocation of PSmad5. In those cells pretreated with SB203580 followed by BMP-2 induction, both the intensity and nuclear translocation were substantially suppressed, suggesting that treatment with SB203580 inhibited BMP-2-induced phosphorylation and nuclear translocation of PSmad5. Discussion

BMP-2 has been reported to induce Dlx3 expression, but until now, the molecular mechanisms whereby BMP-2 promotes Dlx3 transcription remain largely unknown. The present investigation provides a novel insight in molecular mechanisms about the regulation of Dlx3 expression by BMP-2 in MC3T3E1 cells. p38 is a kinase, together with JNK and ERK, belonging to the MAPK family. BMP-2 has been shown not only to activate Smad signaling but also to stimulate p38 kinase pathway. Previous studies have reported that the direct association between Smads and p38 kinase signaling is involved in BMP-2-induced bone matrix gene expression, ALP activity, and cell JOURNAL OF CELLULAR PHYSIOLOGY

differentiation in osteoblastic cells (Guicheux et al., 2003; Hsu et al., 2007). The regulation of Smads by MAPKs appears to be highly complex and remains debated. The Smad protein consists of two conserved domains (MH1 and MH2) connected by a linker region. In Smad1, this linker region contains four MAPK phosphorylation sites, which are conserved in Smad5 and Smad8 (Sapkota et al., 2007). The linker region of Smads phosphorylated by MAPKs, plays an important role in the regulation of BMP signaling (Sapkota et al., 2007). Linker phosphorylation induces the interaction of Smad with Smurf1, and subsequent Smad degradation (Fuentealba et al., 2007; Sapkota et al., 2007). It has also been reported that the phosphorylation of Smad1, Smad2, and Smad3 by ERK blocks their translocation to the nucleus resulting in inhibition of Smad-mediated transcription (Kretzschmar et al., 1997). However, in the present study, we used an antibody detecting the C-terminal phosphorylation of Smad5 and found that inhibition of p38 by SB203580 suppressed BMP-2-induced phosphorylation and nuclear translocation of Smad5, which means p38 promotes Smad5 C-terminal phosphorylation and nuclear translocation. This result is consistent with previous investigation (No¨th et al., 2003), which showed inhibition of p38 kinase activity suppressed BMP-2-induced Smad1 phosphorylation, as well as its translocation to nucleus.

REGULATION OF Dlx3 BY BMP-2-p38/Smad5 PATHWAY

Fig. 6. The SBE binding sites are essential for Smad5/p38 mediated Dlx3 promoter activity. A: Illustration of wild-type Luc-698 and mutant Luc-698 luciferase reporter gene constructs. The crossings represent the deleted nucleotides in the mutant constructs. B: Cells were transfected with wild-type Luc-698 or mutant Luc-698 constructs, with the presence of pCMV-Smad5 or/and pCDNA3-MKK6 plasmid. The value of luciferase was obtained by firefly/Renilla luciferase activity. Fold increases were showed for individual value versus the corresponding control group value and plotted as a graph showing the mean  SEM from the three independent experiments. The values were compared with wild-type Luc-698 construct in the interior pCDNA3-MKK6 or/and pCMV-Smad5 transfection group, respectively.  P < 0.05;  P < 0.01. C: Schematic diagram shows the primers used in ChIP. D: ChIP assay showed Pp38 did not bind to SBEII site. Lanes 2 and 3 showed the PCR results using the purified DNA fragments as templates which were immunoprecipitated with Pp38 antibody. Lanes 3 and 4 were PCR results with input DNA as templates. E: IP showed interaction between PSmad5 and Pp38. F: Cells were serum-starved overnight and treated with 50 ng/ml BMP-2 for 15 min. Pp38 was detected by immunofluorescence staining.

BMP-induced Smad linker phosphorylation lags behind C-terminal phosphorylation (Kaneko et al., 2011), and is a negative feedback mechanism for specifically removing activated C-terminal phosphorylated Smads (Fuentealba JOURNAL OF CELLULAR PHYSIOLOGY

et al., 2007; Sapkota et al., 2007). Therefore, p38 may be able to induce Smads phosphorylation at both C-terminal and linker region, and regulate the balance of Smads activation and degradation.

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Fig. 7. Effects of the p38 inhibitor SB203580 on BMP-2-induced Smad5 phosphorylation and nuclear translocation. A: Cells were serum-starved overnight, then pretreated with the indicated concentrations of SB203580 (SB) or DMSO for 2 h followed by treatment with or without 100 ng/ml BMP-2 for additional 30 min. Levels of PSmad5 and total Smad5 were determined by Western blot. Quantitative analysis is shown in the right part.  P < 0.05;  P < 0.01. B: Cells were serum-starved overnight, then pretreated with 10 mM of SB203580 or DMSO for 2 h followed by treatment with or without 100 ng/ml BMP-2 for additional 15 min. PSmad5 was detected by immunofluorescence staining.

Smads mediate their effects through protein–protein and protein–DNA interactions and several DNA-binding motifs for Smads have been identified. A binding sequence for Smad was originally identified in Drosophila. GCCGnCGC is a consensus binding sequence for Mad (Drosophila Smad1) (Kim et al., 1997). In mammals, similar GC-rich sequences, for example, GCCG or GGCGCC, have been found in the promoter regions of BMP target genes, such as Id1 and Herp2 (Korchynskyi and ten Dijke, 2002; Itoh et al., 2004). Recombinant protein containing the DNA binding domain of mouse Smad1 (Smad1 MH1) has been shown to bind to the GGCGCC sequence in vitro (BabuRajendran et al., 2010). This GGCGCC sequence is widely accepted as a binding sequence for BMP-specific receptor-regulated Smad subfamily (BR-Smads, Smad1/5/8). Also it was reported that a GCAT motif was responsible for Smad1 binding in the Xvent-2B promoter (Henningfeld et al., 2000). EMSA-based assay was used to select random oligonucleotide sequences that bind with Smad5 (Li et al., 2001). A 62-bp oligonucleotide with 16 random sequences in the center was used in EMSA with the GST-fusion protein that contained the MH1 domain of Smad5. All the putative Smad5-bound oligonucleotides contained a consensus sequence TGTCT. This consensus DNA sequence was previously identified for Smad3 and Smad4 (Dennler et al., 1998). In the present study we demonstrated that Smad5 was able to bind to the TGTCT sequence on Dlx3 promoter and regulate Dlx3 gene transcription. However, this consensus JOURNAL OF CELLULAR PHYSIOLOGY

TGTCT sequence is unable to bind Smad1 and Smad8 (Li et al., 2001). Although Smad1, Smad5, and Smad8 have been identified to be involved in signaling of BMP family receptors to down-stream genes, the results from the present investigation and Li et al.’s (2001) indicate Smad5 is distinct from the other two BMP-specific Smads in DNA binding and has different function from Smad1 in transducing BMP-2 signaling. MAPKs play important roles in cellular response to growth factors, cytokines, or environmental stress. Endogenous p38 is distributed both in the cytoplasm and nucleus in resting cells, and cytoplasmic p38 translocates into the nucleus upon activation to access its nuclear substrates. Without stimulation, inactivated p38 is exported to the cytoplasm to receive the next stimulation (Lu et al., 2006; Zeidan et al., 2008). Classical stresses, such as ultraviolet (UV) radiation, arsenite, anisomycin, LPS, hydrogen peroxide (H2O2), and sorbitol, are able to induce the nuclear translocation of p38, suggesting its nuclear accumulation is a common phenomenon upon stressinduced activation (Gong et al., 2010). Besides those classical stress, it was reported that BMP-7 caused a clear nuclear translocation of Pp38 in human proximal tubule epithelia cells (Motazed et al., 2008). Consistent with this report, we found marked nuclear accumulation of Pp38 with 15 min of BMP-2 treatment. MAPK signaling pathway plays important roles in regulating gene expression. In addition to their role in phosphorylating transcription factors, MAPKs may also act as components of transcriptional activation complexes and alter the activity of transcription factors (Yang et al., 2003). The affinity of Smads with DNA is weak and Smads need to cooperate with other factors to bind efficiently to the promoters of target genes (Korchynskyi and ten Dijke, 2002). It was reported that the 30zinc finger nuclear protein OAZ associates with BR-Smads in response to BMP (Hata et al., 2000). Runx2 was also shown to interact directly and cooperated with Smad1 and Smad5 in transcriptional regulation during BMP-induced osteoblast differentiation (Hanai et al., 1999; Lee et al., 2000; Zhang et al., 2000). In the present investigation, we found deletion of SBEI binding site on Dlx3 promoter suppressed the Luc-698 response both to Pp38 and to PSmad5, while deletion of SBEII binding site only suppressed the Luc-698 response to PSmad5 but not to Pp38, which indicates Pp38 may act a co-activator to interact with PSmad5 forming a complex to increase the transcription activity of Smad5 binding to SBEI site on Dlx3 promoter. The results that nuclear translocation of Pp38 with BMP-2 induction and protein–protein interaction between Pp38 and Pp38 shown by co-IP further confirmed this inference. Park and Morasso (2002) delineated the BMP-2-responsive sequence in the Dlx3 promoter at the region between
1917 and
1747 in keratinocytes and found a GCAT sequence, which is able to bind Smad1 and Smad4. A CCAAT box motif was identified between
77 and
64 in the Dlx3 promoter and is required for basal expression of the Dlx3 promoter (Holland et al., 2004). This CCAAT box binds NF-Y or CCAAT/ enhancer-binding protein b (C/EBPb) depending on cell type (Park and Morasso, 1999; Holland et al., 2004). In addition to CCAAT box, analysis of the proximal promoter region revealed a putative Sp1-binding site (located between
13 to 13), which plays a positive regulatory role, and its function is independent of the CCAAT box (Park and Morasso, 1999). Analysis of the Dlx3 proximal promoter region also revealed a sequence with two p63-like overlapping binding sites immediately upstream of the CCAAT box, located from
89 to
80 bp and from
84 to
75 bp of the transcriptional start site (Radoja et al., 2007). Dlx3 might be transcriptionally activated or repressed depending on the specific p63 isoform bound to the promoter (Radoja et al., 2007). The hairless (HR), a transcription factor, is expressed in the hair follicle epithelium.

REGULATION OF Dlx3 BY BMP-2-p38/Smad5 PATHWAY

Fig. 8. Schematic model for BMP-2 inducing Dlx3 expression via Smad5/p38 signaling pathway. BMP-2 binds to the BMP receptors and activates both p38 and Smad5 phosphorylation and nuclear translocation. Also, p38 is able to induce Smad5 phosphorylation and nuclear translocation. In nucleus, Psmad5 interacts with Pp38, forming a protein complex, and this complex binds to SBEI binding site (565/561) on Dlx3 gene promoter, and stimulates Dlx3 transcription. Besides, PSmad5 interacting with other proteins except Pp38, is able to bind to SBEII binding site (432/428) on Dlx3 promoter, to stimulate Dlx3 transcription.

HR specifically binds to the region spanning from
613 to
268 bp of the Dlx3 promoter and down-regulates Dlx3 expression at a transcriptional level in mouse keratinocytes (Kim et al., 2012). We found two Smad5 binding sites (SBEI, from
565 to
561; SBEII, from
432 to
428) on Dlx3 promoter region and confirmed Smad5 was able to bind to these two SBE sites both in vitro and in vivo, using EMSA and ChIP. Furthermore, with deletion of these binding sites, we found Smad5 activated Dlx3 transcription is mediated through these binding sites. In summary, our data demonstrated that BMP-2 induces Dlx3 expression through activation of p38 and Smad5 signaling pathways and there is a cross-talk between these two signaling pathways in osteoblasts (Fig. 8). Furthermore, we characterized two Smad5 binding elements in the Dlx3 promoter, which are essential for p38 and Smad5 mediated Dlx3 expression. Literature Cited BabuRajendran N, Palasingam P, Narasimhan K, Sun W, Prabhakar S, Jauch R, Kolatkar PR. 2010. Structure of Smad1 MH1/DNA complex reveals distinctive rearrangements of BMP and TGF-beta effectors. Nucleic Acids Res 38:3477–3488. Balint E, Lapointe D, Drissi H, van der Meijden C, Young DW, van Wijnen AJ, Stein JL, Stein GS, Lian JB. 2003. Phenotype discovery by gene expression profiling: Mapping of biological processes linked to BMP-2-mediated osteoblast differentiation. J Cell Biochem 89:401– 426. Beanan MJ, Sargent TD. 2000. Regulation and function of Dlx3 in vertebrate development. Dev Dyn 218:545–553. Choi YH, Choi HJ, Lee KY, Oh JW. 2012. Akt1 regulates phosphorylation and osteogenic activity of Dlx3. Biochem Biophys Res Commun 425:800–805. Dennler S, Itoh S, Vivien D, ten Dijke P, Huet S, Gauthier JM. 1998. Direct binding of Smad3 and Smad4 to critical TGF beta-inducible elements in the promoter of human plasminogen activator inhibitor-type 1 gene. EMBO J 17:3091–3100. Fuentealba LC, Eivers E, Ikeda A, Hurtado C, Kuroda H, Pera EM, De Robertis EM. 2007. Integrating patterning signals: Wnt/GSK3 regulates the duration of the BMP/Smad1 signal. Cell 131:980–993. Gallea S, Lallemand F, Atfi A, Rawadi G, Ramez V, Spinella-Jaegle S, Kawai S, Faucheu C, Huet L, Baron R, Roman-Roman S. 2001. Activation of mitogen-activated protein kinase cascades is involved in regulation of bone morphogenetic protein-2-induced osteoblast differentiation in pluripotent C2C12 cells. Bone 28:491–498. Ghanem N, Jarinova O, Amores A, Long Q, Hatch G, Park BK, Rubenstein JL, Ekker M. 2003. Regulatory roles of conserved intergenic domains in vertebrate Dlx bigene clusters. Genome Res 13:533–543. Ghoul-Mazgar S, Hotton D, Lezot F, Blin-Wakkach C, Asselin A, Sautier JM, Berdal A. 2005. Expression pattern of Dlx3 during cell differentiation in mineralized tissues. Bone 37:799– 809. Gong X, Ming X, Deng P, Jiang Y. 2010. Mechanisms regulating the nuclear translocation of p38 MAP kinase. J Cell Biochem 110:1420–1429. Guicheux J, Lemonnier J, Ghayor C, Suzuki A, Palmer G, Caverzasio J. 2003. Activation of p38 mitogen-activated protein kinase and c-Jun-NH2-terminal kinase by BMP-2 and their

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