Jennifer M. Pell

Insulin-like growth factor-binding protein 5 (Igfbp5) compromises survival, growth, muscle development, and fertility in mice.

The insulin-like growth factors (IGFs) are essential for development; bioavailable IGF is tightly regulated by six related IGF-binding proteins (IGFBPs). Igfbp5 is the most conserved and is developmentally up-regulated in key lineages and pathologies; in vitro studies suggest that IGFBP-5 functions independently of IGF interaction. Genetic ablation of individual Igfbps has yielded limited phenotypes because of substantial compensation by remaining family members. Therefore, to reveal Igfbp5 actions in vivo, we generated lines of transgenic mice that ubiquitously overexpressed Igfbp5 from early development. Significantly increased neonatal mortality, reduced female fertility, whole-body growth inhibition, and retarded muscle development were observed in Igfbp5-overexpressing mice. The magnitude of the response in individual transgenic lines was positively correlated with Igfbp5 expression. Circulating IGFBP-5 concentrations increased a maximum of only 4-fold, total and free IGF-I concentrations increased up to 2-fold, and IGFBP-5 was detected in high Mr complexes; however, no detectable decrease in the proportion of free IGF-I was observed. Thus, despite only modest changes in IGF and IGFBP concentrations, the Igfbp5-overexpressing mice displayed a phenotype more extreme than that observed for other Igfbp genetic models. Although growth retardation was obvious prenatally, maximal inhibition occurred postnatally before the onset of growth hormone-dependent growth, regardless of Igfbp5 expression level, revealing a period of sensitivity to IGFBP-5 during this important stage of tissue programming.

Akt2, a Novel Functional Link between p38 Mitogen-Activated Protein Kinase and Phosphatidylinositol 3-Kinase Pathways in Myogenesis

ABSTRACT 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.

Partitioning of IGFBP-5 actions in myogenesis: IGF- independent anti-apoptotic function

Igfbp5 is upregulated during the differentiation of several key cell lineages and in some tumours; the function of IGFBP-5 in these physiological and pathological situations is unknown. Since IGFBP-5 contains sequence motifs consistent with IGF-independent actions, the aim of these studies was to distinguish between IGF-dependent and -independent actions of IGFBP-5. Myc-tagged wild-type (termed wtIGFBP-5) and non-IGF binding mouse Igfbp5 (termed mutIGFBP-5) cDNAs were generated and used to transfect C2 myoblasts, a cell line that undergoes differentiation to myotubes in an IGF- and IGFBP-5-regulated manner. WtIGFBP-5, but not mutIGFBP-5, inhibited myogenesis, as assessed by cell morphology, MHC immunocytochemistry and caveolin 3 expression. However, both wt- and mutIGFBP-5 increased cell survival and decreased apoptosis, as indicated by decreased caspase-3 activity and cell surface annexin V binding. Further examination of apoptotic pathways revealed that wt- and mutIGFBP-5 ameliorated the increase in caspase-9 but not the modest increase in caspase-8 during myogenesis, suggesting that IGFBP-5 increased cell survival via inhibition of intrinsic cell death pathways in an IGF-independent manner. The relationship between IGF-II and IGFBP-5 was examined further by cotransfecting C2 myoblasts with antisense Igf2 (previously established to induce increased cell death) and Igfbp5; both wt- and mutIGFBP-5 conferred equivalent protection against the decreased cell survival and increased apoptosis. In conclusion, we have partitioned IGFBP-5 action in myogenesis into IGF-dependent inhibition of differentiation and IGF-independent cell survival. Our findings suggest that, by regulation of cell survival, IGFBP-5 has an autonomous role in the regulation of cell fate in development and in tumourigenesis.

IGF-independent effects of insulin-like growth factor binding protein-5 (Igfbp5) in vivo

IGF activity is regulated tightly by a family of IGF binding proteins (IGFBPs). IGFBP‐5 is the most conserved of these and is up‐regulated significantly during differentiation of several key lineages and in some cancers. The function of IGFBP‐5 in these physiological and pathological situations is unclear, however, several IGFBP‐5 sequence motifs and studies in vitro suggest IGF‐independent actions. Therefore, we aimed to compare the phenotypes of mice overexpressing wild‐type Igfbp5 or an N‐terminal mutant Igfbp5 with negligible IGF binding affinity. Both significantly inhibited growth, even at low expression levels. Even though wild‐type IGFBP‐5 severely disrupted the IGF axis, we found no evidence for interaction of mutant IGFBP‐5 with the IGF system. Further, overexpression of wildtype IGFBP‐5 rescued the lethal phenotype induced by “excess” IGF‐II in type 2 receptor‐null mice;mutant IGFBP‐5 overexpression could not. Therefore, wildtype IGFBP‐5 provides a very effective mechanism for the inhibition of IGF activity and a powerful in vivo mechanism to inhibit IGF activity in pathologies such as cancer. This study is also the first to suggest significant IGF‐independent actions for IGFBP‐5 during development.— Tripathi, G., Salih, D. A. M., Drozd, A. C., Cosgrove, R. A., Cobb, L. J., Pell, J. M. IGF‐independent effects of insulin‐like growth factor binding protein‐5 (Igfbp5) in vivo. FASEBJ. 23, 2616–2626 (2009)

Ezh2 maintains a key phase of muscle satellite cell expansion but does not regulate terminal differentiation

Summary Tissue generation and repair requires a stepwise process of cell fate restriction to ensure that adult stem cells differentiate in a timely and appropriate manner. A crucial role has been implicated for Polycomb-group (PcG) proteins and the H3K27me3 repressive histone mark in coordinating the transcriptional programmes necessary for this process, but the targets and developmental timing for this repression remain unclear. To address these questions, we generated novel genome-wide maps of H3K27me3 and H3K4me3 in freshly isolated muscle stem cells. These data, together with the analysis of two conditional Ezh2-null mouse strains, identified a critical proliferation phase in which Ezh2 activity is essential. Mice lacking Ezh2 in satellite cells exhibited decreased muscle growth, severely impaired regeneration and reduced stem cell number, due to a profound failure of the proliferative progenitor population to expand. Surprisingly, deletion of Ezh2 after the onset of terminal differentiation did not impede muscle repair or homeostasis. Using these knockout models and the RNA-Seq and ChIP-Seq datasets, we show that Ezh2 does not regulate the muscle differentiation process in vivo. These results emphasise the lineage and cell-type-specific functions of Ezh2 and Polycomb repressive complex 2.

POINT: IGF IS THE MAJOR PHYSIOLOGICAL REGULATOR OF MUSCLE MASS

The insulin-like growth factors (IGFs)-I and -II are homologous peptides that are structurally related to insulin. However, IGFs retain the C-peptide, enabling binding to their main signaling receptor, the IGF type 1 receptor (IGF1R). IGF-II, however, can also bind to and signal via the A isoform of

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 p38α and/or β but not γ. 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.

p38α MAPK Regulates Adult Muscle Stem Cell Fate by Restricting Progenitor Proliferation During Postnatal Growth and Repair

Stem cell function is essential for the maintenance of adult tissue homeostasis. Controlling the balance between self‐renewal and differentiation is crucial to maintain a receptive satellite cell pool capable of responding to growth and regeneration cues. The mitogen‐activated protein kinase p38α has been implicated in the regulation of these processes but its influence in adult muscle remains unknown. Using conditional satellite cell p38α knockout mice we have demonstrated that p38α restricts excess proliferation in the postnatal growth phase while promoting timely myoblast differentiation. Differentiation was still able to occur in the p38α‐null satellite cells, however, but was delayed. An absence of p38α resulted in a postnatal growth defect along with the persistence of an increased reservoir of satellite cells into adulthood. This population was still capable of responding to cardiotoxin‐induced injury, resulting in complete, albeit delayed, regeneration, with further enhancement of the satellite cell population. Increased p38γ phosphorylation accompanied the absence of p38α, and inhibition of p38γ ex vivo substantially decreased the myogenic defect. We have used genome‐wide transcriptome analysis to characterize the changes in expression that occur between resting and regenerating muscle, and the influence p38α has on these expression profiles. This study provides novel evidence for the fundamental role of p38α in adult muscle homeostasis in vivo. STEM Cells 2013;31:1597–1610

Essential Role for p38α MAPK But Not p38γ 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.