MEF2 Cooperates With Forskolin/cAMP and GATA4 to Regulate Star Gene Expression in Mouse MA-10 Leydig Cells

Abstract

In Leydig cells, steroidogenic acute regulatory protein (STAR) participates in cholesterol shuttling from the outer to the inner mitochondrial membrane, the rate-limiting step in steroidogenesis. Steroid hormone biosynthesis and steroidogenic gene expression are regulated by LH, which activates various signaling pathways and transcription factors, including cAMP/Ca(2+)/CAMK (Ca(2+)/calmodulin-dependent kinase)-myocyte enhancer factor 2 (MEF2). The 4 MEF2 transcription factors are essential regulators of cell differentiation and organogenesis in numerous tissues. Recently, MEF2 was identified in Sertoli and Leydig cells of the testis. Here, we report that MEF2 regulates steroidogenesis in mouse MA-10 Leydig cells by acting on the Star gene. In MA-10 cells depleted of MEF2 using siRNAs (small interfering RNAs), STAR protein levels, Star mRNA levels, and promoter activity were significantly decreased. On its own, MEF2 did not activate the mouse Star promoter but was found to cooperate with forskolin/cAMP. By chromatin immunoprecipitation and DNA precipitation assays, we confirmed MEF2 binding to a consensus element located at -232 bp of the Star promoter. Mutation or deletion of the MEF2 element reduced but did not abrogate the MEF2/cAMP cooperation, indicating that MEF2 cooperates with other DNA-bound transcription factor(s). We identified GATA4 (GATA binding protein 4) as a partner for MEF2 in Leydig cells, because mutation of the GATA element abrogated the MEF2/cAMP cooperation on a reporter lacking a MEF2 element. MEF2 and GATA4 interact as revealed by coimmunoprecipitation, and MEF2 and GATA4 transcriptionally cooperate on the Star promoter. Altogether, our results define MEF2 as a novel regulator of steroidogenesis and Star transcription in Leydig cells and identify GATA4 as a key partner for MEF2-mediated action.

Novel Targets for the Transcription Factors MEF2 in MA-10 Leydig Cells1

“…In agreement with this, we recently reported that the Star gene encoding the STAR protein, an essential component of the cholesterol transport machinery, is a direct target of MEF2 [7]. Consistent with this, in MEF2-deficient Leydig cells, steroidogenesis was significantly decreased [7], indicating that MEF2 is an important contributor to steroidogenesis in these cells.…”

“…The position of MEF2 upstream of a panregulator of steroidogenic genes, NR4A1, in a regulatory cascade suggests a broad role for MEF2 on several steroidogenic genes in Leydig cells [8]. In agreement with this, we recently reported that the Star gene encoding the STAR protein, an essential component of the cholesterol transport machinery, is a direct target of MEF2 [7]. Consistent with this, in MEF2-deficient Leydig cells, steroidogenesis was significantly decreased [7], indicating that MEF2 is an important contributor to steroidogenesis in these cells.…”

“…Two genes, Star and Nr4a1, previously described as targets for MEF2 in Leydig cells [7,8] were present in the microarray data and served as internal controls. As expected, Star mRNA levels were reduced in MEF2-deficient cells while Nr4a1 was not affected (Fig.…”

“…MEF2 factors mediate their effects on gene expression by binding to the palindromic AT-rich sequence (C/ T)TA (A/T) 4 TA (G/A) present in the promoter region of target genes. MEF2 factors are also known to cooperate with transcription factors, including members of the GATA family [6,7].…”

Calcium-dependent Nr4a1 expression in mouse Leydig cells requires distinct AP1/CRE and MEF2 elements

“…As the knockdown of MEF2A/2D has already been shown to reduce Nr4a1 expression and steroid hormone production in MA-10 Leydig cells (Daems et al 2014, Daems et al 2015, the contribution of the cJUN/CREB complex to these processes was next evaluated. Activated cJUN and CREB have been correlated with maximal steroidogenesis and activation of Nr4a1 promoter activity , Manna et al 2011.…”