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

Abstract

Testosterone production by Leydig cells is a tightly regulated process requiring synchronized expression of several steroidogenic genes by numerous transcription factors. Myocyte enhancer factor 2 (MEF2) are transcription factors recently identified in somatic cells of the male gonad. In other tissues, MEF2 factors are essential regulators of organogenesis and cell differentiation. So far in the testis, MEF2 factors were found to regulate Leydig cell steroidogenesis by controlling Nr4a1 and Star gene expression. To expand our understanding of the role of MEF2 in Leydig cells, we performed microarray analyses of MEF2-depleted MA-10 Leydig cells, and the results were analyzed using Partek and Ingenuity Pathway Analysis software. Several genes were differentially expressed in MEF2-depleted Leydig cells, and 16 were validated by quantitative RT-PCR. A large number of these genes are known to be involved in fertility, gonad morphology, and steroidogenesis. These include Ahr, Bmal1, Cyp1b1, Hsd3b1, Hsd17b7, Map2k1, Nr0b2, Pde8a, Por, Smad4, Star, and Tsc22d3, which were all downregulated in the absence of MEF2. In silico analyses revealed the presence of MEF2-binding sites within the first 2 kb upstream of the transcription start site of the Por, Bmal1, and Nr0b2 promoters, suggesting direct regulation by MEF2. Using transient transfections in MA-10 Leydig cells, small interfering RNA knockdown, and a MEF2-Engrailed dominant negative, we found that MEF2 activates the Por, Bmal1, and Nr0b2 promoters and that this requires an intact MEF2 element. Our results identify novel target genes for MEF2 and define MEF2 as an important regulator of Leydig cell function and male reproduction. fertility, gonad morphology, Leydig cells, microarray, myocyte enhancer factor 2, steroidogenesis

The Transcription Factor MEF2 Is a Novel Regulator of Gsta Gene Class in Mouse MA-10 Leydig Cells

“…In our recent transcriptomic analyses of MEF2-depleted mouse MA-10 Leydig cells, we observed that the mRNA levels of several Gsta (1 to 4), Gstm (1 to 7) and Gstp (1 and 2) were significantly decreased suggesting a previously undescribed role for MEF2 in the protection against oxidative stress (46). Our current work expands on this by showing an important decrease in global GST activity in Leydig cells deficient in MEF2 factors.…”

“…Ϫ1.79 4.98E-3 with an expression vector encoding MEF2A ( Figure 4A) or MEF2-Engrailed (MEF2-Eng), a MEF2 dominant negative protein known to compete endogenous MEF2 activity (31,37,46,47). As shown in Figure 4B, endogenous Gsta1 mRNA levels were increased by 50% when MEF2 was overexpressed while expression of MEF2-Eng decreased Gsta1 mRNA levels by 20%.…”

“…Analyses of transcriptomic data from MEF2-depleted MA-10 Leydig cells (46) revealed that expression of all four Gsta genes was significantly reduced in the absence of MEF2 factors (Table 2). To further assess the role of MEF2 factors in Gsta gene expression, quantitative PCRs were performed in control and MEF2-depleted MA-10 Leydig cells.…”

Analysis of specific RNA in cultured cells through quantitative integration of q-PCR and N-SIM single cell FISH images: Application to hormonal stimulation of StAR transcription

“…The stimulation of StAR transcription has been extensively studied in MA10 Leydig cells that constitutively express Cyp11a1 and Hsd3b, but not Star at basal levels (Clark et al, 1994; Hales et al, 1990; Hiroi et al, 2004; Manna et al, 2011). cAMP regulates steroidogenic enzyme genes and steroid synthesis through collective modulation of chromatin and a shared cohort of transcription factors/cofactors that include SF1, GATA4, CREB/CBP, C/EBPβ, and NR4A1 (Clem et al, 2005; Di-Luoffo et al, 2015; Feng et al, 2000; Reinhart et al, 1999). Br-cAMP initiates a robust increase in StAR, beginning with primary RNA (p-RNA) transcripts (Lee et al, 2015).…”

Molecular regulation of steroidogenesis in endocrine Leydig cells

“…1, the transcription factors already present in the cell include SF1 (NR5A1, Ad4BP) [123,[125][126][127], LRH1 (NR5A2) [128], COUP-TFII (NR2F2) [9], GATA4 [129,130], C/EBPb [130][131][132], SP1 [133], CREB/CREM [134,135], members of the AP1 family (cFOS and cJUN) [136][137][138][139], and MEF2 [140][141][142]. All these factors were shown to stimulate Star gene expression and thus steroidogenesis in Leydig cells.…”