Essential role for p38alpha MAPK but not p38gamma MAPK in Igf2 expression and myoblast differentiation.

The muscle satellite cell is established as the major stem cell contributing to fiber growth and repair. p38 MAPK signaling is essential for myoblast differentiation and in particular for up-regulation of promyogenic Igf2 expression. p38 exists as four isoforms (alpha, beta, gamma, and delta), of which p38gamma is uniquely abundant in muscle. The aim of this study was to characterize p38 isoform expression and importance (using shRNA knockdown; demonstrated via both reduced protein and kinase activities) during myoblast differentiation. p38alpha and -gamma mRNA levels were most abundant in differentiating C2 cells with low/negligible contributions from p38beta and -delta, respectively. Increased phosphorylation of p38alpha and -gamma occurred during differentiation but via different mechanisms: p38alpha protein levels remained constant, whereas total p38gamma levels increased. Following shRNA knockdown of p38alpha, myoblast differentiation was dramatically inhibited [reduced myosin heavy chain (MHC), myogenin, pAkt protein levels]; significantly, Igf2 mRNA levels and promoter-reporter activities decreased. In contrast, knockdown of p38gamma induced a transient increase in both myogenin and MHC protein levels with no effect on Igf2 mRNA levels or promoter-reporter activity. Knockdown of p38alpha/beta markedly increased but that of p38gamma decreased caspase 3 activity, suggesting opposite actions on apoptosis. p38gamma was initially proposed to have a promyogenic function; however, p38gamma overexpression could not rescue reduced myoblast differentiation following p38alpha/beta inhibition. Therefore, p38alpha is essential for myoblast differentiation, and part of its action is to convert signals that indicate cell density into promyogenic gene expression in the form of the key peptide, IGF-II; p38gamma has a minor, yet opposing antimyogenic, function.

MEK5 and ERK5 are mediators of the pro-myogenic actions of IGF-2.

During the differentiation of muscle satellite cells, committed myoblasts respond to specific signalling cues by exiting the cell cycle, migrating, aligning, expressing muscle-specific genes and finally fusing to form multinucleated myotubes. The predominant foetal growth factor, IGF-2, initiates important signals in myogenesis. The aim of this study was to investigate whether ERK5 and its upstream MKK activator, MEK5, were important in the pro-myogenic actions of IGF-2. ERK5 protein levels, specific phosphorylation and kinase activity increased in differentiating C2 myoblasts. ERK5-GFP translocated from the cytoplasm to the nucleus after activation by upstream MEK5, whereas phospho-acceptor site mutated (dominant-negative) ERK5AEF-GFP remained cytoplasmic. Exogenous IGF-2 increased MHC levels, myogenic E box promoter-reporter activity, ERK5 phosphorylation and kinase activity, and rapidly induced nuclear localisation of ERK5. Transfection with antisense Igf2 decreased markers of myogenesis, and reduced ERK5 phosphorylation, kinase and transactivation activity. These negative effects of antisense Igf2 were rescued by constitutively active MEK5, whereas transfection of myoblasts with dominant-negative MEK5 blocked the pro-myogenic action of IGF-2. Our findings suggest that the MEK5-ERK5 pathway is a novel key mediator of IGF-2 action in myoblast differentiation.

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

Cell-cell contact is essential for appropriate co-ordination of development and it initiates significant signalling events. During myogenesis, committed myoblasts migrate to sites of muscle formation, align and form adhesive contacts that instigate cell-cycle exit and terminal differentiation into multinucleated myotubes; thus myogenesis is an excellent paradigm for the investigation of signals derived from cell-cell contact. PI3-K and p38 MAPK are both essential for successful myogenesis. Pro-myogenic growth factors such as IGF-II activate PI3-K via receptor tyrosine kinases but the extracellular cues and upstream intermediates required for activation of the p38 MAPK pathway in myoblast differentiation are not known. Initial observations suggested a correlation between p38 MAPK phosphorylation and cell density, which was also related to N-cadherin levels and Igf2 expression. Subsequent studies using N-cadherin ligand, dominant-negative N-cadherin, constitutively active and dominant-negative forms of RhoA, and MKK6 and p38 constructs, reveal a novel pathway in differentiating myoblasts that links cell-cell adhesion via N-cadherin to Igf2 expression (assessed using northern and promoter-reporter analyses) via RhoA and p38alpha and/or beta but not gamma. We thus define a regulatory mechanism for p38 activation that relates cell-cell-derived adhesion signalling to the synthesis of the major fetal growth factor, IGF-II.

Insulin-like growth factor-binding protein-5 induces a gender-related decrease in bone mineral density in transgenic mice.

IGF-binding protein-5 (IGFBP-5) is abundant in serum and bone during normal skeletal development, but levels decrease in osteoporosis. Studies have shown that IGFBP-5 stimulates markers of bone formation by potentiating IGF actions and by IGF-independent actions. To test the hypothesis that IGFBP-5 promotes the acquisition of bone mineral density (BMD), we generated transgenic (Tg) mice overexpressing Igfbp5 using a cytomegalovirus enhancer and beta-actin promoter (CMV/betaA). Tg animals showed an increase in serum IGFBP-5 concentrations by 7.7- to 3.5-fold at 3-8 wk of age, respectively. Concentrations were 6-49% higher for males compared with females in both wild-type and Tg mice. Surprisingly, BMD decreased in a gender-dependent manner, with Tg male adults affected more severely than Tg females (31.3% vs. 19.2% reduction, respectively, compared with wild-type mice, assessed by dual energy x-ray absorptiometry). Significant gender differences in BMD were confirmed by peripheral quantitative computed tomography. Histomorphometry revealed that although the bone formation rate and mineralizing surface at the periosteum decreased in Tg mice, they increased at the endosteum, suggesting opposing effects of IGFBP-5 on periosteal and endosteal osteoblasts (by altering proliferation or survival). These findings differ from previous observations in Igf1- and Igf2-null animals. In conclusion, IGFBP-5 has a significant influence on BMD acquisition and maintenance that is dependent on gender and age. The phenotype of Igfbp5 mice cannot be explained solely by IGF inhibition; thus, this study provides the first in vivo evidence, by genetic manipulation, for IGF-independent actions of IGFBP-5 in bone function. These findings have implications for the gender-biased progression of osteoporosis.

Akt2, a novel functional link between p38 mitogen-activated protein kinase and phosphatidylinositol 3-kinase pathways in myogenesis.

Activation of either the phosphatidylinositol 3-kinase (PI 3-kinase)/Akt or the p38 mitogen-activated protein kinase (MAPK) signaling pathways accelerates myogenesis but only when the reciprocal pathway is functional. We therefore examined the hypothesis that cross-activation between these signaling cascades occurs to orchestrate myogenesis. We reveal a novel and reciprocal cross-talk and activation between the PI 3-kinase/Akt and p38 MAPK pathways that is essential for efficient myoblast differentiation. During myoblast differentiation, Akt kinase activity correlated with S473 but not T308 phosphorylation and occurred 24 h after p38 activation. Inhibition or activation of p38 with SB203580, dominant-negative p38, or MKK6EE regulated Akt kinase activity. Analysis of Akt isoforms revealed a specific increase in Akt2 protein levels that coincided with AktS473 phosphorylation during myogenesis and an enrichment of S473-phosphorylated Akt2. Akt2 promoter activity and protein levels were regulated by p38 activation, thus providing a mechanism for communication. Subsequent Akt activation by S473 phosphorylation was PI 3-kinase dependent and specific for Akt2 rather than Akt1. Complementary to p38-mediated transactivation of Akt, activation or inhibition of PI 3-kinase regulated p38 activity upstream of MKK6, demonstrating reciprocal communication and positive feedback characteristic of myogenic regulation. Our findings have identified novel communication between p38 MAPK and PI 3-kinase/Akt via Akt2.