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First published online 14 November 2006
doi: 10.1242/jcs.03278

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Convergence of Igf2 expression and adhesion signalling via RhoA and p38 MAPK enhances myogenic differentiation

¹ The Babraham Institute, Babraham Research Campus, Cambridge, CB2 4AT, UK
² Department of Oncology, Cambridge University, MRC-Hutchison Centre, Cambridge, CB2 2XZ, UK

View larger version (45K): [in this window] [in a new window]   Fig. 1. Increased cell density enhances myogenic differentiation of C2 cells. (A) H and E staining of C2 myoblasts seeded at the different cell densities of subconfluency (1x105 cells/60-mm-diameter plate) and confluency (4x105 cells/60-mm-diameter plate), and also at confluency in the presence of 1.75 mM EGTA; cells were stained 48 hours after the initiation of differentiation induced by transfer of cells from growth medium (GM) to differentiation medium (DM), as described in the Materials and Methods. Magnification, x50. (B) Western blot detection of MHC, phospho-p38 MAPK (pp38), total p38 MAPK and ß-actin in differentiating C2 cells at subconfluency, confluency, and confluency in the presence of 1.75 mM EGTA, sampled at the times indicated after transfer from GM to DM. (C) Northern blot of Igf2 mRNA levels in differentiating myoblasts sampled as in B; 18S rRNA ethidium bromide staining is shown as a loading control. (D) Western blot detection of N-cadherin during myogenesis sampled as in B; ß-actin is shown as a loading control.

View larger version (37K): [in this window] [in a new window]   Fig. 2. Dominant-negative N-cadherin delays myogenesis and decreases p38 MAP kinase activation and Igf2 expression. (A) Schematic of the dominant-negative cN390 N-cadherin construct. (B) H and E staining of differentiating C2 cells retrovirally transduced with cN390 or pBabe only. Magnification, x50. (C) Western blot of MHC in cN390 or pBabe stably transfected cells; total p38 is shown as a loading control. (D) Western blot to detect the HA tag (cN390 cells only), phospho-p38 (pp38), total p38, phospho-ERK1/2 and total ERK1/2 in vector alone (pcDNA) and cN390 transiently transfected cells. (E) p38 MAPK kinase activity and total p38 levels by western blot analysis in vector alone (pcDNA) and cN390 transiently transfected cells; MBP, myelin basic protein. (F) Northern blot analysis of Igf2 expression in C2 cells stably transfected with either cN390 or pBabe alone; 18S rRNA is shown as a loading control. (G) Igf2 P3 promoter luciferase reporter activity in C2 cells transiently transfected with either cN390 or pcDNA alone. Fold activation levels are expressed as the ratio of luciferase activity of cN390 transfected cells to that of cells transfected with vector alone: values were normalised to the relative GFP expression levels per unit of protein; *P<0.05 compared with an activation value of 1.0.

View larger version (44K): [in this window] [in a new window]   Fig. 3. Stimulation of cadherin with the NcadFc ligand activates p38 and induces Igf2 expression. (A) Schematic of the N-cadherin/Fc fusion protein (termed NcadFc ligand). (B) Western blot detection of phospho-p38 (pp38), phospho ERK1/2 (pERK1/2), total p38 and total ERK1/2 in myoblasts treated with extract from control Cos7 cells or NcadFc derived from transfected Cos7 cells. (C) p38 MAP kinase activity of myoblasts treated as in B; total p38 levels were determined by western blotting. (D) Northern blot analysis of Igf2 expression in myoblasts treated as in B; ethidium bromide staining of the 18S rRNA bands is shown as a loading control. (E) Igf2 P3 promoter luciferase reporter activity in C2 cells treated as in B. Igf2 promoter activity was expressed as a ratio of luciferase activity of the control secretion treated cells compared with NcadFc-treated cells; ***P<0.001 compared with levels in the control. Western blot analysis of the lysates shows the GFP expression levels.

View larger version (58K): [in this window] [in a new window]   Fig. 4. RhoA activation during myoblast differentiation and in response to cadherin signalling. (A) Western blot determination of activated (GTP-bound) RhoA and total RhoA during myoblast differentiation. (B) Western blot analysis of MHC and ß-actin in differentiating C2 myoblasts treated with the ROCK inhibitor Y-27632. (C) Western blot analysis of total and activated RhoA in lysates from C2 cells transfected with cN390 or vector alone (left panels) or with NcadFc and control secretion (right panels).

View larger version (36K): [in this window] [in a new window]   Fig. 5. RhoA activity regulates p38 MAPK phosphorylation and kinase activity. (A) Western blot determination of active and total RhoA in lysates from C2 cells transiently transfected with empty vector (pcDNA), constitutively active RhoA (L63 RhoA) and dominant-negative RhoA (T19N RhoA). The band shifts observed for L63 and T19N RhoA are due to their Myc and HA tags (shown in B); thus the lower molecular weight bands show endogenous levels of RhoA. (B) Western blot determination of phospho-p38 (pp38), phospho-ERK1/2 (pERK1/2), total ERK1/2, Myc and HA tags and total p38 in whole cell lysates of C2 cells transfected and sampled as described in A. Phospho- and total p38 were semi-quantified by scanning densitometry and p38 phosphorylation normalised for total p38 calculated (lower panel); data are expressed as fold increases of p38 phosphorylation above pcDNA control levels; *P<0.05, ***P<0.001 compared with levels in the controls. (C) p38 MAP kinase activity in myoblasts transfected with pcDNA, L63 RhoA or T19N RhoA; total p38 levels were determined by Western blotting. (D) SRF promoter reporter activity in lysates extracted from C2 cells transiently transfected with pcDNA3, cN390, L63 RhoA, WT RhoA or N19 RhoA (left panel) or treated with control extract or NcadFc (right panel); Western blot analysis of lysates shows transfected GFP and ß-actin levels.

View larger version (24K): [in this window] [in a new window]   Fig. 6. Constitutively active RhoA rescues p38 activity in dominant-negative N-cadherin transfected myoblasts. (A) Western blot detection of phospho-p38 (pp38), phospho-ERK1/2 (pERK1/2), total ERK1/2, HA and Myc tags, total p38 and ß-actin in lysates from C2 cells that were transfected with pcDNA, cN390, or co-transfected with cN390 and L63 RhoA. Phospho- and total p38 were semi-quantified by scanning densitometry, and p38 phosphorylation normalised for total p38 (lower panel); data are expressed as fold increases of p38 phosphorylation above pcDNA control levels; *P<0.05; ***P<0.001 compared with levels in the control. (B) p38 MAP kinase activity (MBP phosphorylation) in lysates from C2 cells treated as in A; total p38 levels were determined by western blotting.

View larger version (55K): [in this window] [in a new window]   Fig. 7. RhoA lies downstream of N-cadherin in the regulation Igf2 expression. (A) Northern blot analysis (upper panels) of Igf2 mRNA levels in C2 cells transfected with either empty vector (pcDNA), L63 RhoA or T19N RhoA; total RNA is shown by ethidium bromide staining of 18S rRNA. Igf2 P3 promoter luciferase reporter activity (lower panels), in cells co-transfected as above with L63 RhoA, T19N RhoA or control vector (pcDNA), and the Igf2 P3 promoter-reporter construct. Fold activation levels are expressed as the ratio of luciferase activity of L63 and T19N RhoA transfected cells to that in cells transfected with pcDNA: values were normalised to the relative GFP expression levels per unit of protein; *P<0.05, ***P<0.001 (compared with an activation value of 1.0). Western immunoblotting to detect ß-actin is shown as a loading control. (B) Northern blot analysis of Igf2 mRNA levels (upper panels) and Igf2 P3 promoter-reporter activity (lower panels) in C2 cells transfected with cN390, pcDNA or co-transfected with cN390 and L63 RhoA, and examined exactly as for A.

View larger version (44K): [in this window] [in a new window]   Fig. 8. p38 MAP kinase lies downstream of RhoA in the regulation of Igf2 expression. (A) Western blot detection of caveolin-3, phospho-ATF2, HA tag (MKK6EE expression), Flag tag (p38dn expression) and ß-actin in lysates from C2 cells that were transfected with control vector (pcDNA), constitutively active upstream activator of p38 MAPK, MKK6 (MKK6EE) and p38 dominant-negative (p38dn) constructs. (B) Northern blot analysis of Igf2 mRNA levels in C2 cells transiently transfected as in A; ethidium bromide staining of 18S rRNA is shown as a loading control. (C) Igf2 P3 promoter transactivation in C2 cells treated as in A but co-transfected with Igf2 P3 promoter and GFP. The lower panels additionally show western blot detection of MHC, HA tag (MKK6EE expression), flag tag (p38dn expression) and ß-actin. *P<0.05, ***P<0.001 (compared with an activation value of 1.0). (D) Northern blot analysis of Igf2 mRNA levels in C2 cells transiently transfected with control vector (pcDNA), cN390, T19N RhoA alone or co-transfected with MKK6EE; GAPDH mRNA levels are shown as a loading control. (E) Igf2 P3 promoter-reporter activity in C2 cells treated as in C but co-transfected with Igf2 P3 promoter and GFP. Western immunoblot determination (lower panel) of HA tag (detects cN390, T19N RhoA and MKK6EE expression), and total p38 (loading control). There is non-specific binding at 50 kDa in the HA immunoblot which is present in all lanes. *P<0.05; **P<0.01; ***P<0.001 compared with an activation value of 1.0.

View larger version (54K): [in this window] [in a new window]   Fig. 9. Differential phosphorylation of p38 isoforms by N-cadherin, LPA and activated RhoA. (A) Western blot analysis of pp38 using antibodies that recognise pp38/ß and pp38, total p38/ß and total p38 in C2 cells transiently transfected with pcDNA, cN390, L63 RhoA, T19N RhoA, MKK6EE or combinations of these. (B) Western blot analysis of pp38 using antibodies that recognise pp38/ß and pp38, total p38/ß and total p38 in C2 cells treated with either control extract or NcadFc ligand. (C) As B, except that C2 cells were transfected with pcDNA or p38HA. (D) Western blot of total and activated RhoA in response to LPA treatment. (E) Western blot analysis of pp38 using antibodies that recognise pp38/ß, total p38/ß and HA in C2 cells transfected with pcDNA or p38HA and treated with LPA.