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Vol. 19, Issue 4, 1519-1528, April 2008

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Involvement of the p38 Mitogen-activated Protein Kinase , β, and Isoforms in Myogenic Differentiation

Haixia Wang^*, Qing Xu, Fang Xiao^*, Yong Jiang, and Zhenguo Wu^*

*Department of Biochemistry, The Hong Kong University of Science and Technology, Clearwater Bay, Kowloon, Hong Kong, China; Dana-Farber Cancer Institute, Boston, MA 02115; and Department of Pathophysiology, Southern Medical University, Guangzhou 510515, China

Submitted August 22, 2007; Revised January 14, 2008; Accepted January 25, 2008
Monitoring Editor: Marianne Bronner-Fraser

	ABSTRACT

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
We and others previously showed that p38 mitogen-activated protein kinase is indispensable for myogenic differentiation. However, it is less clear which of the four p38 isoforms in the mouse genome participates in this process. Using C2C12 myogenic cells as a model, we showed here that p38, β, and are expressed with distinct expression patterns during differentiation. Knockdown of any of them by small interfering RNA inhibits myogenic differentiation, which suggests that the functions of the three p38 isoforms are not completely redundant. To further elucidate the unique role of each p38 isoform in myogenic differentiation, we individually knocked down one p38 isoform at a time in C2C12 cells, and we compared the whole-genome gene expression profiles by microarrays. We found that some genes are coregulated by all three p38 isoforms, whereas others are uniquely regulated by one particular p38 isoform. Furthermore, several novel p38 target genes (i.e., E2F2, cyclin D3, and WISP1) are found to be required for myogenin expression, which provides a molecular basis to explain why different p38 isoforms are required for myogenic differentiation.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
Myogenic differentiation is one of the best models to study the molecular mechanisms by which the cellular programs switch from proliferation to differentiation. This is largely due to our extensive knowledge of key myogenic factors and the availability of both cell culture and animal models. Two families of transcription factors, namely, the myogenic regulatory factors (MRF) and myocyte enhancer binding factor 2s (MEF2s), act cooperatively as master transcription factors to control the expression of an array of cellular genes that collectively contribute to the initiation of the differentiation program and maintenance of the differentiated state (Molkentin and Olson, 1996; Puri and Sartorelli, 2000). MRFs consist of four basic helix-loop-helix (bHLH) proteins (i.e., Myf5, MyoD, myogenin, and MRF4), each heterodimerizing with E proteins (E12 or E47) to efficiently bind to the E box (i.e., CANNTG) in promoters of many muscle-specific genes (Tapscott, 2005). MEF2s consist of four MADS-box-containing proteins (i.e., MEF2A, 2B, 2C, and 2D) that are capable of forming both homo- and heterodimers to bind to a consensus AT-rich sequence (i.e., the MEF2 site) (Black and Olson, 1998).

Both MRFs and MEF2 are in turn regulated by distinct intracellular signaling pathways. We and others previously showed that the p38 mitogen-activated protein kinase (MAPK)-mediated signaling pathway is indispensable for myogenic differentiation (Cuenda and Cohen, 1999; Zetser et al., 1999; Wu et al., 2000b). Accumulating evidence indicates that p38 MAPK can regulate myogenic differentiation through multiple mechanisms. p38 MAPK directly phosphorylates MEF2 and enhances its transcriptional activity (Han et al., 1997; Zhao et al., 1999; Wu et al., 2000a). p38 MAPK also potently enhances the transcriptional activity of MyoD, which is probably due to an increased association of MyoD with E47, because the latter can be directly phosphorylated by p38 MAPK (Wu et al., 2000b; Lluis et al., 2005). Through MyoD and MEF2, which directly bind to the myogenin promoter, p38 MAPK also critically controls the expression of myogenin gene, which is absolutely essential for execution of the differentiation program (Xu et al., 2002). Recently, p38 MAPK was also found to directly phosphorylate BAF60, a component in the SWI/SNF complex, and to target the complex to selected muscle-specific loci (Simone et al., 2004). p38 MAPK also facilitates the recruitment of MyoD and MEF2D to selected promoters of muscle-specific genes (Penn et al., 2004). In addition, p38 MAPK can promote activation of the quiescent muscle satellite cells, and it is also required for proliferation of myoblasts (Jones et al., 2005).

Four p38 isoforms, namely, p38 , β, , and , exist in both the human and mouse genomes (Lluis et al., 2005; Keren et al., 2006). So far, many studies on the role of p38 MAPK in myogenic differentiation have relied on the use of SB203580 and SB202190, two pyridinyl imidazole-based small-molecule inhibitors that mainly inhibit p38 and β, but they have no effect on p38 and (Kumar et al., 1997). As both SB203580 and SB202190 potently inhibit myogenic differentiation (Cuenda and Cohen, 1999; Zetser et al., 1999; Wu et al., 2000b), it suggests that p38 and do not play any significant role in myogenic differentiation. In contrast, a previous report showed that p38 is required for myogenic differentiation, because overexpression of a wild-type p38 promotes, whereas that of a dominant-negative form of p38 inhibits myogenic differentiation in C2C12 cells (Lechner et al., 1996). It remains unclear whether p38 plays any role in myogenic differentiation. To clarify the confusion about the role of p38 and to clearly understand the role of each p38 isoform in myogenic differentiation, we decide to take the short interfering RNA (siRNA) approach by individually knocking down each p38 isoform followed by assessment of the knockdown effect on myogenic differentiation. Except for p38, which is barely detectable in C2C12 cells, we found that p38, β, and are all required for myogenic differentiation. Microarray analysis reveals that some genes are uniquely controlled by a particular p38 isoform, whereas others are coregulated by multiple p38 isoforms. Importantly, several novel p38 target genes were indeed found to be involved in myogenic differentiation, which provides a molecular basis to explain why different p38 MAPKs are required for myogenic differentiation.

	MATERIALS AND METHODS

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Cell Culture and Antibodies
C2C12 cells (American Type Culture Collection, Manassas, VA) were cultured in DMEM supplemented with 20% fetal bovine serum, 100 U/ml penicillin, and 100 µg/ml streptomycin (growth medium, or GM) in a humidified incubator at 37°C with 5% CO₂. To induce differentiation, near confluent C2C12 cells were grown in DMEM supplemented with 2% horse serum (differentiation medium, or DM). Antibodies used in this work were from the following sources: anti-sarcomeric myosin heavy chain (MHC) (MF20) (Developmental Studies Hybridoma Bank, University of Iowa, Iowa City, IA); anti-p38 (Cell Signaling Technology, Danvers, MA); anti-p38β (Zymed Laboratories, South San Francisco, CA); and cyclin D3 (C-16), E2F2 (C-20), anti-myogenin (F5D), anti-β-actin (C-2), and anti-hemagglutinin (HA) (Santa Cruz Biotechnology, Santa Cruz, CA). The polyclonal anti-p38 was a kind gift from Dr. M. A. Bogoyevitch (University of Western Australia).

DNA Constructs, Transfection, and Cell Lysis
Expression vectors encoding Flag-p38, Flag-p38β, and Flag-p38 were kindly provided by Dr. J. Han (Scripps Research Institute, San Diego, CA). G133-luc, 4RE-luc, 3MEF2-luc, and HA-MKK6EE were described previously (Wu et al., 2000b; Xu et al., 2002). Flag-Wnt-induced secreted protein 1 (WISP1) was amplified from C2C12-derived cDNAs by polymerase chain reaction (PCR), and it was cloned into pcDNA3.0 vector. Transient transfection was performed by using LipofectAMINE/PLUS reagents following the manufacturer's instructions (Invitrogen, Carlsbad, CA). Cells were lysed in the lysis buffer (50 mM HEPES, pH 7.6, 10% glycerol, 1% Triton X-100, 150 mM NaCl, 1 mM EGTA, 1.5 mM MgCl₂, 100 mM NaF, 20 mM biscyclohexylammonium salt, 20 mM β-glycerol phosphate, 2 mM dithiothreitol, 50 µM sodium vanadate, 0.5 mM phenylmethylsulfonyl fluoride, 2 µg/ml aprotinin, 0.5 µg/ml leupeptin, and 0.7 µg/ml pepstatin) for 10 min on ice. Soluble whole cell extracts (WCEs) were prepared by centrifugation to remove cell debris.

RNA Interference (RNAi)
The following siRNAs (only the top-strand sequence is shown) were synthesized at either Dharmacon RNA Technologies (Lafayette, CO) or RIBOBIO (Guangzhou, China): enhanced green fluorescent protein (5'-GCT GAC CCT GAA GTT CAT C-3'); p38 (1#: 5'-AGC CCA GCA ACC TAG CTG T-3'; 2#: 5'-GAG CCT GAC CTA TGA TGA A-3'); p38β (1#: 5'-TGC TGG TAC TAG ACA GCG A-3'; 2#: 5'-ATT GAG CAG TGA GGC ATT G-3'); p38 (1#: 5'-GCA TGA GAC CCT GAG TGA A-3'; 2#: 5'-CCT GTT CTT CGG CTT TCG A-3'); E2F2 (1#: 5'-GCA CCT GAC CGA AGA TAA T-3'; 2#: 5'-GTG ACC TCT TCG ACT CCT A-3'); cyclin D3 (1#: 5'-CCA TTC ACC TGT AGC TTG A-3'; 2#:5'-CTG CTT AGC TTC TGT GAT T-3'); and WISP1 (1#:5'-CGG CAG GTC CTA TGG ATT A-3'; 2#:5'-CCA CTA GAG GAA ACG ACT A-3'). For siRNA transfection, 40–60% confluent C2C12 cells were transfected with 100 nM siRNA by LipofectAMINE 2000 following the manufacturer's instructions. Four to 6 h later, the siRNA-liposome mixtures were removed, and fresh GM was added into cell culture plates. Generally, 24 h after transfection, GM was replaced with DM to induce myogenic differentiation.

Reporter Assays
For reporter assays, 50–70% confluent C2C12 cells in 12-well dishes were cotransfected with 0.5 µg of reporter plasmids and 100 nM siRNA together by LipofectAMINE 2000. All the samples were prepared in duplicate or triplicate. Luciferase activity was determined with a LB9507 luminometer (Berthold Technologies, Bad Wilbad, Germany) by adding 10 µl of WCE to 150 µl of freshly made luciferase buffer [0.4 µM luciferin, 13.3 mM ATP, 0.1 M Tris-HCl, pH 7.8, 1 mM EDTA, pH 8.0, and 10 mM Mg(OAc)₂]. Luciferase units were normalized against total protein amount present in each sample determined by Protein Assay Reagent (Bio-Rad, Hercules, CA).

Immunostaining
Cells were first fixed in 4% paraformaldehyde for 15 min, and then they were permeabilized in 0.2% Triton X-100 for 15 min and blocked in 5% bovine serum albumin/phosphate-buffered saline (PBS) for 1 h. Cells were then incubated with the mouse anti-MHC antibody overnight, washed three times with PBS, and reincubated with the rhodamine-conjugated donkey anti-mouse IgG (Jackson ImmunoResearch Laboratories, West Grove, PA) for 1 h. Then, 100 ng/ml 4,6-diamidino-2-phenylindole (DAPI) was then added for another 10 min. Fluorescence microscopy was performed using an Olympus IX70 microscope linked to a charge-coupled device digital camera (Spot RT; Diagnostic Instruments, Sterling Heights, MI).

Gene Expression Analysis by Microarrays
Duplicate C2C12 cells were transfected separately with siRNAs targeting p38, p38β, p38, or green fluorescent protein (GFP) (control). Twenty-four hours after transfection, cells were switched to DM for another 18 h before harvest. Total RNA was extracted using TRIzol reagent following the manufacturer's suggestions. The GeneChip Mouse Genome 430 2.0 arrays (Affymetrix, Santa Clara, CA) were used for hybridization, which provide 45,000 probe sets to analyze the expression levels of >39,000 transcripts and variants of mouse genes. The raw data were normalized and analyzed using dChip (www.dchip.org) (Li and Hung Wong, 2001). To obtain p38 isoform-specific genes, the combined comparisons were carried out. For example, the combined comparison (control vs. p38 and not control vs. p38β and not control vs. p38) was used to identify genes uniquely regulated by p38 with the criteria of group mean fold change >1.5, absolute group mean differences >20, and p 0.05 for testing equal group means using Student's t test. To identify genes controlled by all three p38 isoforms, we carried out the combined comparison (i.e., control vs. p38 AND control vs. p38β AND control vs. p38) by using the same criteria as mentioned above.

Semiquantitative Reverse Transcription (RT)-PCR and SYBR Green-based Quantitative (q)RT-PCR
RT-PCR was performed by a two-step method. Briefly, cDNA was generated from 0.5 µg of total RNA by Improm-II reverse transcription system (Promega, Madison, WI) with oligo(dT)₁₅ as a primer according to the manufacturer's instruction. PCR was performed in a reaction of 25 µl containing 40 ng of cDNA. PCR products were analyzed by 2% agarose gel electrophoresis. For qRT-PCR, 2x SYBR Green Supermix from Bio-Rad (Hercules, CA) was added to 25 µl of PCR reactions according to the manufacturer's instructions. Triplicate samples were subjected to qPCR using a Stratagene Mx3000P real-time PCR system with the maximum cycle number of 40. GAPDH was used as an internal control. The relative abundance of genes of interest was calculated after normalized to GAPDH. Three independent batches of RNA samples were used for qRT-PCR analysis, and data were presented as mean ± SD and analyzed by student t test (p < 0.05 was considered statistically significant). Primer pairs used were as follows: myogenin (forward: 5'-GAC TCC CCA CTC CCC ATT CAC ATA-3'; reverse: 5'-GGC GGC AGC TTT ACA AAC AAC ACA-3'); GAPDH (forward: 5'-CCC ACT CTT CCA CCT TCG-3'; reverse: 5'-TCC TTG GAG GCC ATG TAG GCC AT-3'); p38 (forward: 5'-GCA GGG ACC TTC TCA TAG AT-3'; reverse: 5'-GAG GGA TAG CCT CAG ACC-3'); p38β (forward: 5'-CTG CAA GGA AAG GCC CTC-3'; reverse: 5'-CAG GCA ATG CCT CAC TGC-3'); p38 (forward: 5'-GAT TAC TGG GAA GAT CCT G-3'; reverse: 5'-CGT CAC AGA GCC GTC TCC-3'); p38 (forward: 5'-GAC ACT CTT CAA GGG CAA G-3'; reverse: 5'-GCC ATC AAT CAC TGC AGC-3'); E2F2 (forward: 5'-GGT TCC TGT GGT CAG GAG-3'; reverse: 5'-CAG TTC CTG AGG GTG AAC-3'); and WISP1 (forward: 5'-GTC CAG GAC TTC ACA ATT GAG C-3'; reverse: 5'-CCA GGC TTT GCT TCC ATT G-3').

	RESULTS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
p38, β, and Are Expressed in Both Mature Muscles and C2C12 Myogenic Cells and Display Distinct Expression Profiles during Myogenic Differentiation
To explore the roles of p38 isoforms in myogenic differentiation, we first examined the mRNA expression patterns of four p38 isoforms in mouse muscle tissues by RT-PCR. The mouse brain, liver, and kidney tissues were used as controls. In agreement with previous reports, p38 and β were abundantly expressed in both muscles and many other tissues, whereas p38 was preferentially expressed in muscles (Figure 1A) (Lechner et al., 1996). In contrast, p38 was barely detectable in muscles, even though it was abundantly expressed in kidney tissues (Figure 1A) (Hu et al., 1999). Similarly, we found that p38, β, and , but not , were also expressed in C2C12 myogenic cells as judged by RT-PCR (data not shown). To examine the protein levels and expression patterns of p38 isoforms during C2C12 differentiation, we first characterized isoform-specific p38 antibodies. As shown in Figure 1B, each p38 isoform-specific antibody was indeed highly specific for that particular isoform and did not cross-react with other p38 isoforms. Using these isoform-specific antibodies, we found that, during C2C12 differentiation, the levels of p38 remained unchanged and the levels of p38 gradually increased, whereas the levels of p38β gradually decreased (Figure 1C). Myogenin expression was used here as a marker to monitor the progression of differentiation, whereas β-actin levels were used as a loading control.

View larger version (12K): [in this window] [in a new window]   Figure 1. Expression profiles of p38 isoforms in mature muscles and C2C12 cells. (A) Detection of different p38 isoforms in selected mouse tissues by semiquantitative RT-PCR analysis. GAPDH was used as a loading control. (B) 293T cells were separately transfected with plasmids encoding Flag-tagged p38, β, , and . Different Flag-p38 isoforms were immunoprecipitated (IP) from cell extracts with the anti-Flag antibody, and the immunoprecipitates were subjected to SDS-polyacrylamide gel electrophoresis (PAGE) and examined by Western blotting (WB) by using isoform-specific p38 antibodies. (C) C2C12 cells were let to grow and differentiate in cell culture. Cells were harvested at different time points as indicated, and WCEs were subjected to SDS-PAGE followed by Western blotting analysis.

View larger version (29K): [in this window] [in a new window]   Figure 2. p38, β, and are all required for C2C12 myogenic differentiation. (A) C2C12 cells were separately transfected with siRNAs targeting either GFP (control) or different p38 isoforms. After growing in GM for 24 h, cells were induced to differentiate in DM for another 24 or 48 h (for detection of MHC only). Cell extracts were harvested and subjected to Western blot analysis. 1# and 2# denote two different siRNAs against each p38 isoform. (B) After differentiating in DM for 48 h, C2C12 cells were fixed and immunostained with an anti-MHC antibody (MF20). Cell nuclei were counterstained with DAPI. The fusion index was calculated as the ratio of the number of MHC-positive cells with two or more nuclei over that of DAPI-positive nuclei in three randomly chosen fields. The results are presented as mean ± SD. Phase, phase-contrast images. (C) C2C12 cells were cotransfected with various siRNAs as indicated together with 4xRE-luc or 3xMEF2-luc. After growing in GM for 24 h, cells were switched to DM for another 24 h before harvest followed by luciferase assays. All experiments were done in triplicate, and the results are shown as mean ± SD. Fold change was the ratio of the luciferase activity in cells transfected with p38-siRNAs over that with GFP-siRNA.

View this table: [in this window] [in a new window]   Table 2. Selected genes uniquely regulated by p38

View this table: [in this window] [in a new window]   Table 4. Selected genes uniquely regulated by p38

E2F2 Is Coregulated by p38, β, and and Essential for Myogenin Expression

View larger version (40K): [in this window] [in a new window]   Figure 3. E2F2 is coregulated by p38, β, and and required for myogenic differentiation. (A) C2C12 cells were separately transfected with various p38-siRNAs and induced to differentiate in DM for 24 h. Total RNA was extracted and the E2F2 mRNA level was analyzed by both RT-PCR (bottom) and qRT-PCR (top). (B) Near-confluent C2C12 cells were induced to differentiate for 12 and 24 h with or without SB202190 (10 µM) (added at the time of medium change). WCEs were subjected to Western blotting analysis. + and – signs denote the presence and absence of SB202190, respectively. (C and D) Total RNA (C) and WCEs (D) were harvested from C2C12 cells at different times as indicated. The mRNA and protein levels of E2F2 were analyzed by RT-PCR, qRT-PCR, and Western blotting, respectively. (E–G) C2C12 cells were separately transfected with E2F2-siRNA and GFP-siRNA, and they were induced to differentiate for various times as indicated. In E and G, WCEs were prepared and analyzed by SDS-PAGE and Western blotting. In F, cells were fixed after growing in DM for 48 h followed by immunostaining with an anti-MHC antibody (MF20). Cell nuclei were counterstained with DAPI. The calculation of fusion index and data presentation were essentially the same as described in the legend of Figure 2B. (H) C2C12 cells were cotransfected with various siRNAs as indicated together with 4xRE-luc or 3xMEF2-luc. After growing in GM for 24 h, cells were switched to DM for another 24 h before harvest followed by luciferase assays. All experiments were done in duplicate, and the results are shown as mean ± SD. Fold change was the ratio of the luciferase activity in cells transfected with E2F2-siRNAs over that with GFP-siRNA.

View larger version (28K): [in this window] [in a new window]   Figure 4. Cyclin D3 is uniquely regulated by p38β and required for myogenic differentiation. (A) C2C12 cells were transfected with various siRNAs and induced to differentiate for various times. (B) C2C12 cells were induced to differentiate for various times. (C and D) C2C12 cells were separately transfected with either GFP-siRNA or two different cyclin D3-siRNAs, then induced to differentiate for various times. WCEs from A–D were subjected to SDS-PAGE and Western blotting. (E) C2C12 cells were cotransfected with siRNAs and reporter plasmids as indicated. The measurement of the luciferase activity, calculation of the fold change and data presentation were the same as described in the legend of Figure 3H.

WISP1 Is Specifically Regulated by p38 and Required for Myogenic Differentiation

View larger version (20K): [in this window] [in a new window]   Figure 5. WISP1 is uniquely regulated by p38 and required for myogenic differentiation. (A and B) Total RNA was isolated from C2C12 cells either transfected with various siRNAs (A) or left untreated and induced to differentiate for various times (B). The expression of WISP1 mRNA was analyzed by qRT-PCR (top) and RT-PCR (bottom). (C) C2C12 cells were separately transfected with various siRNAs as indicated. 24 h after transfection, cells were induced to differentiate in DM for another 18 or 36 h (for detection of MHC) before harvest. (D) C2C12 cells were cotransfected with various siRNAs together with reporter plasmids as indicated. After growing in GM for 24 h, cells were switched to DM for another 24 h before harvest followed by luciferase assays. All experiments were done in duplicate, and the results are shown as mean ± SD. (E) C2C12 cells were transfected with GFP-siRNA or WISP1-siRNA. Twenty-four hours after transfection, the culture media were replaced with conditioned media collected from C2C12 cells transfected with either an empty vector or Flag-WISP1. Cells were induced to differentiate in conditioned media for another 24 h before harvest. WCEs from C and E were subjected to SDS-PAGE and Western blotting.

	DISCUSSION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

Identification of Novel p38 Target Genes Involved in Myogenic Differentiation
Many novel p38 target genes have been identified through our genome-wide gene expression profiling, and some of them are found to be required for early myogenic differentiation, which provides a mechanism to explain why different p38 isoforms are required. E2F2, a member of the E2F family, is coregulated by p38, β, and . Although E2F2 is normally involved in cell proliferation in many cellular systems (Lukas et al., 1996; Wu et al., 2001), unexpectedly, we find that it also plays an important role during early myogenic differentiation (Figure 3, E and F). Because E2F2 is already present in proliferating myoblasts (Figure 3, C and D), and it is required for myogenin induction upon differentiation, it suggests that, in addition to MEF2, MyoD, and Six proteins (Xu et al., 2002), E2F2 is another key mediator involved in p38 MAPK-mediated myogenin expression. However, it is noteworthy that these mediators are differentially regulated by p38 MAPK: whereas MEF2 serves as a direct substrate of p38 MAPK (Han et al., 1997; Wu et al., 2000b), E2F2 is regulated by p38 MAPK at either the transcriptional or posttranscriptional level. For MyoD and Six, it remains unclear how exactly p38 MAPK activates them. At present, we do not know whether E2F2 regulates myogenin expression through direct binding to the myogenin promoter or through other intermediate molecules that in turn bind to the myogenin promoter. Our initial analysis suggests that E2F2 is required for MRF- and MEF2-dependent gene transcription. In addition, E2F2 may facilitate cell cycle exit by promoting cyclin D1 down-regulation, because knockdown of E2F2 enhances cyclin D1 levels.

Unlike E2F2, cyclin D3 is uniquely regulated by p38β during myogenic differentiation. Consistently, the involvement of cyclin D3 in myogenic differentiation has been recognized in several previous reports (Kiess et al., 1995; Rao and Kohtz, 1995; Cenciarelli et al., 1999). Unlike cyclin D1, which is rapidly down-regulated upon differentiation, cyclin D3 levels gradually increase and peak in postmitotic myotubes (Figure 4B). Unphosphorylated Rb is found to interact with and stabilize cyclin D3 protein, which in turn forms a multiprotein complex containing inactive CDK2 and CDK4, p21, and proliferating cell nuclear antigen (Kiess et al., 1995; Cenciarelli et al., 1999). Such a complex is thought to contribute to irreversible cell cycle exit and maintenance of the differentiated state. In addition to these previous findings, our current study shows for the first time that cyclin D3 is required for myogenin induction. We suggest that cyclin D3 may facilitate cell cycle exit by promoting cyclin D1 down-regulation, because knockdown of cyclin D3 prominently up-regulates cyclin D1 levels (Figure 4D). Furthermore, we show that cyclin D3 is required for MRF-dependent gene transcription. Because cyclin D3 is a transcription target of MyoD (Cenciarelli et al., 1999), this suggests the existence of a positive regulatory loop between MyoD and cylin D3. It remains unclear how exactly cyclin D3 exerts its effect on cyclin D1- and MRF-dependent gene transcription.

WISP1, a known target gene induced by Wnt-1 (Pennica et al., 1998; Xu et al., 2000), is a member of the CCN family proteins that consists of Cysteine-rich 61 (CCN1), connective tissue growth factors (CTGF or CCN2), nephroblastoma overexpressed (Nov or CCN3), WISP1 (CCN4), WISP2 (CCN5), and WISP3 (CCN6) (Brigstock, 2003). The receptors for members of the CCN family remain poorly characterized. In several cases, certain integrins are thought to be the receptors for CCN1, CCN2, and CCN3 (Brigstock, 2003). We show here that WISP1 is specifically induced by p38 during myogenic differentiation (Figure 5). As a secreted molecule, we show that WISP1 can regulate myogenin expression in an autocrine manner (Figure 6). Furthermore, WISP1 enhances both MRF- and MEF2-dependent gene transcription. Interestingly, CCN3, another member of the CCN family, is found to be specifically induced by p38β (Table 3). However, the functional significance of CCN3 induction by p38 β remains unclear, because knockdown of CCN3 does not affect myogenin expression (data not shown). It also remains unclear how WISP1 affects myogenin expression. Because WISP1 is also induced by Wnt-1, it may serve as a converging point to integrate signals from both the p38 pathway and the Wnt pathway.

View larger version (9K): [in this window] [in a new window]   Figure 6. Schematic on how different p38 isoforms regulate myogenin induction through distinct target genes. Three p38 isoforms can regulate myogenin expression through both common target genes (e.g., E2F2) and unique isoform-specific target genes (e.g., cyclin D3 and WISP1). Unlike E2F2 and cyclin D3, WISP1 participates in myogenin regulation in an autocrine manner. G0, G1, G2, S, and M denote different phases of the cell cycle.

	ACKNOWLEDGMENTS

 
We thank Dr. J. Han for p38 expression vectors, Dr. J. R. Nevins (Duke University) for E2F2 expression vector, Dr. M. A. Bogoyevitch for p38 antibody, and Carol Wong for technical assistance. This project is supported by Hong Kong Research Grant Council grants HKUST6412/05M, HKUST6496/06M, and CA06/07.SC02 (to Z.W.); Area of Excellence Scheme AoE/B-15/01; and a 973 project from the Ministry of Science and Technology of China (2002 CB513005) (to Z.W. and Y.J.).

	Footnotes

 
This was published online ahead of print in MBC in Press (http://www.molbiolcell.org/cgi/doi/10.1091/10.1091/mbc.mbc.E07-08-0817) on February 6, 2008.

Address correspondence to: Yong Jiang (jiang48231{at}163.com) or Zhenguo Wu (bczgwu{at}ust.hk).

	REFERENCES

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
Black, B. L., and Olson, E. N. (1998). Transcriptional control of muscle development by myocyte enhancer factor-2 (MEF2) proteins. Annu. Rev. Cell Dev. Biol 14, 167–196.[CrossRef][Medline]

Brigstock, D. R. (2003). The CCN family: a new stimulus package. J. Endocrinol 178, 169–175.[Abstract]

Cenciarelli, C., De Santa, F., Puri, P. L., Mattei, E., Ricci, L., Bucci, F., Felsani, A., and Caruso, M. (1999). Critical role played by cyclin D3 in the MyoD-mediated arrest of cell cycle during myoblast differentiation. Mol. Cell. Biol 19, 5203–5217.[Abstract/Free Full Text]

Cuenda, A., and Cohen, P. (1999). Stress-activated protein kinase-2/p38 and a rapamycin-sensitive pathway are required for C2C12 myogenesis. J. Biol. Chem 274, 4341–4346.[Abstract/Free Full Text]

Davies, S. P., Reddy, H., Caivano, M., and Cohen, P. (2000). Specificity and mechanism of action of some commonly used protein kinase inhibitors. Biochem. J 351, 95–105.[CrossRef][Medline]

Dimova, D. K., and Dyson, N. J. (2005). The E2F transcriptional network: old acquaintances with new faces. Oncogene 24, 2810–2826.[CrossRef][Medline]

Han, J., Jiang, Y., Li, Z., Kravchenko, V. V., and Ulevitch, R. J. (1997). Activation of the transcription factor MEF2C by the MAP kinase p38 in inflammation. Nature 386, 296–299.[CrossRef][Medline]

Hu, M. C., Wang, Y. P., Mikhail, A., Qiu, W. R., and Tan, T. H. (1999). Murine p38-delta mitogen-activated protein kinase, a developmentally regulated protein kinase that is activated by stress and proinflammatory cytokines. J. Biol. Chem 274, 7095–7102.[Abstract/Free Full Text]

Jones, N. C., Tyner, K. J., Nibarger, L., Stanley, H. M., Cornelison, D. D., Fedorov, Y. V., and Olwin, B. B. (2005). The p38alpha/beta MAPK functions as a molecular switch to activate the quiescent satellite cell. J. Cell Biol 169, 105–116.[Abstract/Free Full Text]

Keren, A., Tamir, Y., and Bengal, E. (2006). The p38 MAPK signaling pathway: a major regulator of skeletal muscle development. Mol. Cell. Endocrinol 252, 224–230.[CrossRef][Medline]

Kiess, M., Gill, R. M., and Hamel, P. A. (1995). Expression of the positive regulator of cell cycle progression, cyclin D3, is induced during differentiation of myoblasts into quiescent myotubes. Oncogene 10, 159–166.[Medline]

Kumar, S., McDonnell, P. C., Gum, R. J., Hand, A. T., Lee, J. C., and Young, P. R. (1997). Novel homologues of CSBP/p38 MAP kinase: activation, substrate specificity and sensitivity to inhibition by pyridinyl imidazoles. Biochem. Biophys. Res. Commun 235, 533–538.[CrossRef][Medline]

Lali, F. V., Hunt, A. E., Turner, S. J., and Foxwell, B. M. (2000). The pyridinyl imidazole inhibitor SB203580 blocks phosphoinositide-dependent protein kinase activity, protein kinase B phosphorylation, and retinoblastoma hyperphosphorylation in interleukin-2-stimulated T cells independently of p38 mitogen-activated protein kinase. J. Biol. Chem 275, 7395–7402.[Abstract/Free Full Text]

Lechner, C., Zahalka, M. A., Giot, J. F., Moller, N. P., and Ullrich, A. (1996). ERK6, a mitogen-activated protein kinase involved in C2C12 myoblast differentiation. Proc. Natl. Acad. Sci. USA 93, 4355–4359.[Abstract/Free Full Text]

Li, C., and Hung Wong, W. (2001). Model-based analysis of oligonucleotide arrays: model validation, design issues and standard error application. Genome Biol 2, RESEARCH0032.[Medline]

Lluis, F., Ballestar, E., Suelves, M., Esteller, M., and Munoz-Canoves, P. (2005). E47 phosphorylation by p38 MAPK promotes MyoD/E47 association and muscle-specific gene transcription. EMBO J 24, 974–984.[CrossRef][Medline]

Lukas, J., Petersen, B. O., Holm, K., Bartek, J., and Helin, K. (1996). Deregulated expression of E2F family members induces S-phase entry and overcomes p16INK4A-mediated growth suppression. Mol. Cell. Biol 16, 1047–1057.[Abstract]

Molkentin, J. D., and Olson, E. N. (1996). Defining the regulatory networks for muscle development. Curr. Opin. Genet. Dev 6, 445–453.[CrossRef][Medline]

Penn, B. H., Bergstrom, D. A., Dilworth, F. J., Bengal, E., and Tapscott, S. J. (2004). A MyoD-generated feed-forward circuit temporally patterns gene expression during skeletal muscle differentiation. Genes Dev 18, 2348–2353.[Abstract/Free Full Text]

Pennica, D. et al. (1998). WISP genes are members of the connective tissue growth factor family that are up-regulated in wnt-1-transformed cells and aberrantly expressed in human colon tumors. Proc. Natl. Acad. Sci. USA 95, 14717–14722.[Abstract/Free Full Text]

Perdiguero, E. et al. (2007). Genetic analysis of p38 MAP kinases in myogenesis: fundamental role of p38alpha in abrogating myoblast proliferation. EMBO J 26, 1245–1256.[CrossRef][Medline]

Puri, P. L., and Sartorelli, V. (2000). Regulation of muscle regulatory factors by DNA-binding, interacting proteins, and post-transcriptional modifications. J. Cell. Physiol 185, 155–173.[CrossRef][Medline]

Rao, S. S., and Kohtz, D. S. (1995). Positive and negative regulation of D-type cyclin expression in skeletal myoblasts by basic fibroblast growth factor and transforming growth factor beta. A role for cyclin D1 in control of myoblast differentiation. J. Biol. Chem 270, 4093–4100.[Abstract/Free Full Text]

Simone, C., Forcales, S. V., Hill, D. A., Imbalzano, A. N., Latella, L., and Puri, P. L. (2004). p38 pathway targets SWI-SNF chromatin-remodeling complex to muscle-specific loci. Nat. Genet 36, 738–743.[CrossRef][Medline]

Tapscott, S. J. (2005). The circuitry of a master switch: Myod and the regulation of skeletal muscle gene transcription. Development 132, 2685–2695.[Abstract/Free Full Text]

Trimarchi, J. M., and Lees, J. A. (2002). Sibling rivalry in the E2F family. Nat. Rev. Mol. Cell Biol 3, 11–20.[CrossRef][Medline]

Wu, H. et al. (2000a). MEF2 responds to multiple calcium-regulated signals in the control of skeletal muscle fiber type. EMBO J 19, 1963–1973.[CrossRef][Medline]

Wu, L. et al. (2001). The E2F1–3 transcription factors are essential for cellular proliferation. Nature 414, 457–462.[CrossRef][Medline]

Wu, Z., Woodring, P. J., Bhakta, K. S., Tamura, K., Wen, F., Feramisco, J. R., Karin, M., Wang, J. Y., and Puri, P. L. (2000b). p38 and extracellular signal-regulated kinases regulate the myogenic program at multiple steps. Mol. Cell. Biol 20, 3951–3964.[Abstract/Free Full Text]

Xu, L., Corcoran, R. B., Welsh, J. W., Pennica, D., and Levine, A. J. (2000). WISP-1 is a Wnt-1- and beta-catenin-responsive oncogene. Genes Dev 14, 585–595.[Abstract/Free Full Text]

Xu, Q., Yu, L., Liu, L., Cheung, C. F., Li, X., Yee, S. P., Yang, X. J., and Wu, Z. (2002). p38 Mitogen-activated protein kinase-, calcium-calmodulin-dependent protein kinase-, and calcineurin-mediated signaling pathways transcriptionally regulate myogenin expression. Mol. Biol. Cell 13, 1940–1952.[Abstract/Free Full Text]

Zetser, A., Gredinger, E., and Bengal, E. (1999). p38 mitogen-activated protein kinase pathway promotes skeletal muscle differentiation. Participation of the Mef2c transcription factor. J. Biol. Chem 274, 5193–5200.[Abstract/Free Full Text]

Zhao, M., New, L., Kravchenko, V. V., Kato, Y., Gram, H., di Padova, F., Olson, E. N., Ulevitch, R. J., and Han, J. (1999). Regulation of the MEF2 family of transcription factors by p38. Mol. Cell. Biol 19, 21–30.[Abstract/Free Full Text]

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