MyoD1 promoter autoregulation is mediated by two proximal E-boxes.

We show that in mouse myoblasts the MyoD1 promoter is highly stimulated by MyoD1 expression, suggesting that it is controlled by a positive feedback loop. Using deletion and mutation analyses, we identified the targets for MyoD1 promoter autoregulation as the two proximal E-boxes located close to the MyoD1 core promoter. Gel mobility shift competition assays with MyoD1 antibodies as competitor suggest that the MyoD1 protein is binding directly to these E-boxes. Autoregulation did not occur in fibroblasts cotransfected with the expression vector of MyoD1. It is assumed that autoregulation is controlled by the stoichiometry between the MyoD1 protein and negatively regulatory proteins like Id, which is known to be highly expressed in fibroblasts. When the MyoD1 promoter was methylated, autoregulation only occurred when the density of methylated sites was low. The density of DNA methylation, therefore, can determine the accessibility of the MyoD1 promoter to transcription factors and interfere with the auto- and crossregulatory loop. The MyoD1 promoter in vivo was found to be only partially methylated in all tissues tested except in skeletal muscle where it was demethylated. We propose that high level expression of the MyoD1 gene is a result of release from constraints such as negative regulatory factors and/or DNA methylation interfering with MyoD1 autoregulation

Estrogen receptor regulates MyoD gene expression by preventing AP-1-mediated repression.

Cell growth and differentiation are opposite events in the myogenic lineage. Growth factors block the muscle differentiation program by inducing the expression of transcription factors that negatively regulate the expression of muscle regulatory genes like MyoD. In contrast, extracellular clues that induce cell cycle arrest promote MyoD expression and muscle differentiation. Thus, the regulation of MyoD expression is critical for muscle differentiation. Here we show that estrogen induces MyoD expression in mouse skeletal muscle in vivo and in dividing myoblasts in vitro by relieving the MyoD promoter from AP-1 negative regulation through a mechanism involving estrogen receptor/AP-1 protein-protein interactions but independent of the estrogen receptor DNA binding activity

Phosphorylation by p38 MAPK as an alternative pathway for GSK3beta inactivation.

Glycogen synthase kinase 3beta (GSK3beta) is involved in metabolism, neurodegeneration, and cancer. Inhibition of GSK3beta activity is the primary mechanism that regulates this widely expressed active kinase. Although the protein kinase Akt inhibits GSK3beta by phosphorylation at the N terminus, preventing Akt-mediated phosphorylation does not affect the cell-survival pathway activated through the GSK3beta substrate beta-catenin. Here, we show that p38 mitogen-activated protein kinase (MAPK) also inactivates GSK3beta by direct phosphorylation at its C terminus, and this inactivation can lead to an accumulation of beta-catenin. p38 MAPK-mediated phosphorylation of GSK3beta occurs primarily in the brain and thymocytes. Activation of beta-catenin-mediated signaling through GSK3beta inhibition provides a potential mechanism for p38 MAPK-mediated survival in specific tissues

Galectin-1 Co-clusters CD43/CD45 on Dendritic Cells and Induces Cell Activation and Migration through Syk and Protein Kinase C Signaling*

Galectin-1 is a galactoside-binding lectin expressed in multiple tissues that has pleiotropic immunomodulatory functions. We previously showed that galectin-1 activates human monocyte-derived dendritic cells (MDDCs) and triggers a specific genetic program that up-regulates DC migration through the extracellular matrix, an integral property of mucosal DCs. Here, we identify the galectin-1 receptors on MDDCs and immediate downstream effectors of galectin-1-induced MDDC activation and migration. Galectin-1 binding to surface CD43 and CD45 on MDDCs induced an unusual unipolar co-clustering of these receptors and activates a dose-dependent calcium flux that is abrogated by lactose. Using a kinome screen and a systems biology approach, we identified Syk and protein kinase C tyrosine kinases as mediators of the DC activation effects of galectin-1. Galectin-1, but not lipopolysaccharide, stimulated Syk phosphorylation and recruitment of phosphorylated Syk to the CD43 and CD45 co-cluster on MDDCs. Inhibitors of Syk and protein kinase C signaling abrogated galectin-1-induced DC activation as monitored by interleukin-6 production; and MMP-1, -10, and -12 gene up-regulation; and enhanced migration through the extracellular matrix. The latter two are specific features of galectin-1-activated DCs. Interestingly, we also found that galectin-1 can prime DCs to respond more quickly to low dose lipopolysaccharide stimulation. Finally, we underscore the biological relevance of galectin-1-enhanced DC migration by showing that intradermal injection of galectin-1 in MRL-fas mice, which have a defect in skin DC emigration, increased the in vivo migration of dermal DCs to draining lymph nodes