Regulation and function of myocyte enhancer factor 2 (MEF2) in myogenic and neurogenic cells

"In eukaryotic organisms, regulation of gene expression at the transcriptional level is a fundamental mechanism which is evolutionary conserved in all cellular systems. It tightly regulates the diversification in expression patterns of genes and proteins required for biological complexity and function. Transcriptional regulation is mediated by the physical interaction between transcription factors and specific cis-acting regulatory elements in gene promoter regions. Myocyte enhancer factor 2 (MEF2) is a transcription factor highly conserved in eukaryotes involved in differentiation, proliferation, and survival/apoptosis. The MEF2 gene family (MEF2 A-D) regulates development of various tissue types including muscle (skeletal, cardiac, and smooth muscle), bone, lymphocytes and neurons. The regulation of MEF2 activity is complex, and is coordinated at multiple levels including posttranslational modifications and protein-protein interaction, that together modulate MEF2's function. Conversely dysregulation of MEF2 activity underlies pathogenesis in muscle and neuronal cells. MEF2 is responsive to various signaling cascades which provide a way for distinct stimuli to differentially regulate MEF2-dependent gene expression. It is known that phosphorylation by kinases is an important process through which the activity of MEF2 is up- or down-regulated. Several kinases (p38MAPK, CDK5, PKC, and ERK5) have been linked to muscle and neuronal development, as well as survival in part due to their modulation of MEF2 function. In addition MEF2 is known to be targeted by co-repressors, such as class Ila histone deacetylases (HDAC 4, 5, 7 and 9). This interaction contributes to repression of MEF2-dependent gene expression. Although MEF2 family members are critical regulators of skeletal muscle differentiation and cardiovascular function their individual roles within nervous system are less well characterized.

In current studies, we attempted to investigate the posttranslational regulation of MEF2 both in myogenic and neurogenic cells. The cAMP/protein kinase A (PKA) signaling pathway regulates a variety of cellular functions and numerous important biological processes. Many of the effects of cAMP/PKA are mediated via changes in gene expression. We have previously documented that cAMP/PKA signaling negatively regulates MEF2 activity and inhibits myogenes1s by direct phosphorylation of MEF2 proteins. MEF2 A-D are highly expressed in multiple regions of the brain, including cortex, cerebellum, and hippocampus. Distinct patterns of expression during pre- and postnatal development suggest specific functions for MEF2 proteins at different stages of neuronal maturation and survival. However, whether the cAMP/PKA pathway inhibits MEF2 mediated gene expression in neurons was unclear. Recently, we evaluated whether cAMP/PKA signaling can inhibit MEF2-dependent gene expression directly or indirectly and survival role of MEF2D in hippocampal neurons. We performed survival assays to determine PKA effects in neuronal cells. We observed that experimental induction of cAMP/PKA signaling promotes apoptosis in primary hippocampal neurons as indicated by TUNEL and FACS analysis. Luciferase reporter gene assays revealed that PKA potently represses MEF2D trans-activation properties in neurons. Kruppel-like factor 6 (KLF6) was identified as a key transcriptional target of MEF2 in hippocampal neurons and siRNA mediated knockdown of KLF6 expression promotes neuronal cell death and also antagonizes the pro-survival role of MEF2D. In this study, we found that cAMP/PKA signaling represses KLF6 transcriptional activity and induce neuronal apoptosis by phosphorylating MEF2 and preventing HDAC4 export from the nucleus. These observations characterize a potent inhibitory effect of PKA on the transactivation properties of MEF2D leading to repression of KLF6 expression and compromising neuronal survival (Chapter III).

Next, we were interested to determine how MEF2 controls diverse cellular processes in muscle development in the presence/absence of cofactors. Tandem Affinity Purification (TAP) combined with mass spectrometry analysis was employed in the current studies to identify MEF2 interacting cofactors. We identified Strawberry notch 1(Sbno1) as a novel interacting factor of MEF2D which is known to be downstream effecter of Notch signaling. Notch signaling is known to block the expression and activity of myogenic factors such as MEF2s. We therefore characterized the mechanism of myogenic inhibition by Notch-Sbnol signaling. C2C 12 myoblasts provide a useful in vitro model to study skeletal muscle differentiation. We determined the expression patterns, by western blot analysis, of muscle specific gene expression during myogenesis. Sbno1 represses MEF2 transactivation properties and plays a critical role in inhibition of skeletal muscle differentiation. Immunocytochemistry analysis suggests that Notch-Sbno1 might be involved in maintaining the ""reserve"" cell population. Our data suggested that protein-protein interactions between Sbno1 and MEF2D result in interference with the function of myogenic factors (Chapter IV).

MEF2D is a known transcriptional regulator of muscle differentiation. Current studies identified KLF6 as a novel MEF2D target gene which is involved in hippocampal neuronal survival. TGF? has been reported as a potent inhibitor of myogenic differentiation by maintaining myoblasts in a proliferative state (undifferentiated myoblasts). Further, TGF? and KLF6 regulate each other's expression in other cell types. We therefore sought to investigate the possible role of KLF6 in a myogenic context and assessed whether TGF? activation regulates KLF6 protein expression and function in a MEF2 dependent manner in C2C12 myoblasts (Chapter V).

Taken together, these studies indicate that differential activation of signaling cascades and co-factors regulate the MEF2 transcriptional complex which has profound effects on gene expression in myogenic and neurogenic cells."