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First published online 7 August 2007
doi: 10.1242/jcs.006619

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The RhoA effector mDiaphanous regulates MyoD expression and cell cycle progression via SRF-dependent and SRF-independent pathways

¹ Center for Cellular and Molecular Biology, Uppal Road, Hyderabad, 500 007, India
² Department of Pharmacology, Kyoto University, Kyoto, Japan

View larger version (22K): [in this window] [in a new window]   Fig. 1. Knockdown of mDia1 suppresses SRF activity, MyoD expression and cell cycle progression. (A) Quantitative real-time RT-PCR analysis of mDia1 mRNA in cells transfected with control GFP shRNA (sh-GFP) and mDia1 shRNA (sh-mDia). Values represent normalized fold differences between mDia1 and GAPDH mRNA in each sample (n=3) ± s.d. (B,C) MyoD expression in myoblasts co-transfected with empty vector or mDia1 shRNA and a GFP reporter, 24 hours after transfection (mean ± s.d., n=4, P<0.0013). (D) Knockdown of mDia1 reduces SRF activity. Normalized SRF activity in C2C12 myoblasts co-transfected with mDia1 shRNA or empty vector, the SRF reporter 3D.Aluc and -gal (mean ± s.d., n=4, P<0.0001). (E) FACS analysis of mDia1-knockdown cells shows an increased G1 population compared with control cells.

View larger version (21K): [in this window] [in a new window]   Fig. 2. mDia1 regulates MyoD by an SRF-independent pathway. (A) Schematic of full-length mDia1 (FL) and five truncation derivatives. (B,C) mDia1N3 activates SRF. C2C12 myoblasts were co-transfected with full-length mDia1 (FL), mDia1 mutants or a GFP control, the SRF reporter and a -gal plasmid. To minimize effects of serum on SRF activity, transfected cells were incubated in 0.5% serum for 24 hours before assay. mDiaN3 increased SRF activity >25 fold (mean ± s.e.m., n=4, P<0.0041). Western blotting with anti-GFP (panel B, inset) showed that all mutants were expressed at relatively equal levels. (C) Quantification of MyoD expression detected by immunofluorescence assay in cells overexpressing GFP (control), full-length mDia1 (FL) or mDia1 mutants (N3, Hind3, F2, H+P, CC). Despite strongly activating SRF, mDia1N3 suppresses MyoD expression maximally (mean ± s.e.m., n=7, P<0.0001). (D) Immunodetection of MyoD expression in C2C12 myoblasts transiently transfected with GFP-tagged mDia1 truncation mutants (N3, H+P, CC).

View larger version (48K): [in this window] [in a new window]   Fig. 3. mDia1 mutants that affect MyoD expression cause G1 arrest without differentiation. (A) Cell cycle analysis of C2C12 myoblasts transfected with GFP-tagged N3 or N3HindIII 24 hours after transfection. Transfected (T) and untransfected (U) cells were distinguished by gating on GFP. (B) A greater proportion of N3 and N3HindIII transfected cells showed a G1 DNA content compared with untransfected cells (mean ± s.e.m., n=4, P<0.0002). (C,D) N3 transfected cells show reduced BrdU incorporation. Immunodetection of BrdU (green) in cells transfected with Flag-tagged mDiaN3 (red) (mean ± s.d., n=3). (E,F) N3 transfected cells do not differentiate. N3 transfected cells were stained for Myogenin and p21, after 24 hours in differentiation medium (mean ± s.e.m., n=2). Arrows indicate transfected cells that are negative for two markers of differentiation: p21 (top) or Myogenin (middle), but positive for p27, a marker of reversible arrest (bottom). (G) Quantification of p27 induction: mDia mutants that affect MyoD expression (N3, N3HindIII, H+P) induce expression of p27 (mean ± s.e.m., n=4).

View larger version (28K): [in this window] [in a new window]   Fig. 4. APC inhibits MyoD expression: Microtubule-association of APC is not essential. (A) APC inhibits MyoD expression: Confocal analysis of cells transfected with full-length APC (APC-FL-GFP) or APC lacking the microtubule-binding domain (APCMT-GFP). Full-length APC is microtubule associated whereas APCMT-GFP localizes to the cytoplasm; both forms effectively inhibit MyoD expression (red). Bar, 20 µm. (B) Quantification of MyoD expression in cells transfected with GFP alone (control) APC-FL-GFP or APCMT-GFP (mean ± s.e.m., n=2).

View larger version (65K): [in this window] [in a new window]   Fig. 5. mDia negatively regulates -catenin localization. (A) The specific GSK3 inhibitor BIO induces nuclear localization of endogenous -catenin (red) and increased cell-cell contact (phase) in myoblasts after a 24-hour treatment in growth medium. Bar, 10 µm (25 µm in phase). (B) mDiaN3 inhibits -catenin nuclear localization in BIO-treated cells. Myoblasts were transiently transfected with control (EGFP), mDia1-FL, N3, HIND3, H+P or F2 constructs (all GFP-tagged), treated with 2.5 µM BIO for 24 hours and stained for -catenin (red). In N3 and HIND3-transfected cells, accumulation of -catenin at cell-cell contacts correlates with loss of nuclear staining. (C) Confocal analysis shows absence of -catenin staining in the nuclei of N3-transfected cells. Bar, 10 µm. (D) Quantification of the effects of GFP-tagged mDia constructs on -catenin localization (mean ± s.e.m., n=2).

View larger version (24K): [in this window] [in a new window]   Fig. 6. mDia negatively regulates TCF. (A) TCF activity is suppressed by mDia1N3 and activated by mDia knockdown. (i) Myoblasts were co-transfected with the TCF reporter plasmid TOP-flash + GFP (control) or mDia1N3 (N3) and TCF-dependent luciferase activity measured. Values represent normalized ratios of TOP-flash activity to the respective FOP-flash control (mean ± s.e.m., n=11, P<0.0001). (ii) Myoblasts were co-transfected with TOP-flash + mU6 vector (control) or mDia1 shRNA (shRNA) and luciferase activity measured as in (i) (mean ± s.e.m., n=5, P<0.0021). (iii) Comparison of effects of full-length (FL) and different mDia mutants on TCF activity (values represent normalized TCF activity, mean ± s.e.m., n=2). N3 is the most effective at suppressing TCF activity. (B) Inhibition of TCF suppresses MyoD expression. Cells were transfected with GFP alone (control) or along with dominant negative TCF-1E (DN TCF-lacking the -catenin-binding domain) and MyoD expression quantified (mean ± s.e.m., n=3, P<0.0021). (C) SRF activity is not affected by -catenin S37A and dnTCF. Myoblasts were co-transfected with the SRF reporter with control (pBS), N3, S37A or DN TCF constructs and luciferase activity measured. (mean ± s.e.m., n=4).

View larger version (45K): [in this window] [in a new window]   Fig. 7. MyoD DRR activity is inhibited by mDia. (A) Gel-shift assays of the putative TCF/LEF sites in the MyoD DRR. 32P-end-labeled oligo probes representing a TCF consensus site or two of the three sites from the MyoD DRR (denoted DRR site A or DRR site B) were incubated with extracts prepared from control C2C12 myoblasts (indicated as `c') or myoblasts treated with 2.5 µM BIO for 24 hours. Assays were performed in the presence or absence of 100-fold molar excess of the respective cold competitor oligo. The consensus TCF site participated in the formation of complexes that were competed by excess cold probe, and induced by BIO (black arrow) consistent with the binding of -catenin–TCF. The gray arrow indicates a nonspecific complex. However, DRR sites A and B bound a nonspecific complex (indicated by *) that was neither competed nor BIO-inducible. Thus, the TCF consensus sites in the MyoD DRR do not appear to function as targets of specific nuclear factor binding. (B) MyoD DRR activity is inhibited by mDia. Myoblasts were transiently transfected in growth medium with a mouse MyoD DRR-pGL3 promoter construct along with pBS (control) or mDia constructs, and a gal plasmid. Luciferase activity was quantified after 24 hours and normalized for transfection efficiency (mean ± s.e.m., n=3). N3 is the most effective at suppressing DRR activity, and overall the DRR suppressive activity of the different forms of mDia1 correlated with suppression of TCF activity.

View larger version (30K): [in this window] [in a new window]   Fig. 8. Overexpression of an APC-independent form of -catenin leads to functional bypass of mDiaN3 inhibition. (A) Myoblasts were co-transfected with a control plasmid (GFP), N3+GFP or N3+ -catenin S37A and TCF activity determined (mean ± s.e.m., n=5, P<0.0046). (B) MyoD expression in cells transfected as in A. Note that N3+ -catenin S37A transfected cells retain the elongated morphology typical of N3 transfectants but are MyoD+. (C) Quantification of MyoD expression in myoblasts transfected with either N3+ -catenin S37A or N3HindIII+ -catenin S37A. The degradation-resistant -catenin S37A mutant partially reverses the inhibition of MyoD expression mediated by both mDia derivatives. (mean ± s.e.m., **P<0.0002, n=6; *P<0.046, n=3).

View larger version (29K): [in this window] [in a new window]   Fig. 9. mDiaN3 controls MyoD by reciprocal regulation of two transcription factors. A model for dual signaling to the MyoD gene by mDia via positive regulation of a directly acting Rho-actin-SRF pathway and negative regulation of an indirect APC–-catenin–TCF pathway. Actin assembly factors bind to the FH1 and FH2 domains, drive polymerization of microfilaments to activate SRF via MAL release and thereby induce MyoD expression. The interaction of mDia with APC may increase cytoplasmic degradation of -catenin or reduce nuclear shuttling of APC and thereby promote cytoplasmic retention of -catenin. As dnTCF inhibits MyoD, TCF may play an activating role by inducing positive upstream factors (X). Taken together, the data suggest that MyoD expression is suppressed by any perturbation of mDia, because SRF and TCF are reciprocally regulated by this key signaling adaptor protein.