bSteroid hormones regulate essential physiological processes, and inadequate levels are associated with various pathological conditions. In testosterone-producing Leydig cells, steroidogenesis is strongly stimulated by luteinizing hormone (LH) via its receptor leading to increased cyclic AMP (cAMP) production and expression of the steroidogenic acute regulatory (STAR) protein, which is essential for the initiation of steroidogenesis. Steroidogenesis then passively decreases with the degradation of cAMP into AMP by phosphodiesterases. In this study, we show that AMP-activated protein kinase (AMPK) is activated following cAMP-to-AMP breakdown in MA-10 and MLTC-1 Leydig cells. Activated AMPK then actively inhibits cAMP-induced steroidogenesis by repressing the expression of key regulators of steroidogenesis, including Star and Nr4a1. Similar results were obtained in Y-1 adrenal cells and in the constitutively steroidogenic R2C cells. We have also determined that maximum AMPK activation following stimulation of steroidogenesis in MA-10 Leydig cells occurs when steroid hormone production has reached a plateau. Our data identify AMPK as a molecular rheostat that actively represses steroid hormone biosynthesis to preserve cellular energy homeostasis and prevent excess steroid production.

In the gonads and adrenal glands, the transient increase in steroidogenesis after hormonal stimulation requires modulation of steroidogenic acute regulatory protein (Star) expression and activity in a tightly regulated process involving cAMP and Ca(2+). In Leydig cells, the cAMP and Ca(2+) pathways account for most if not all of LH-induced steroidogenesis. Although the cAMP-activated molecular network has been well characterized in Leydig cells, little is known about the molecular cascade triggered by the Ca(2+) signaling pathway and the transcription factors responsible for mediating the genomic response. It is established that LH induces an increase in cytoplasmic Ca(2+) from the endoplasmic reticulum primarily through the ryanodine receptors. Previous reports also suggested a role of the Ca(2+) signaling pathway in Star expression based on the fact that inhibition of the Ca(2+)/calmodulin (CaM) protein kinase pathway greatly impaired Star expression in Leydig and adrenal cells. In this study, we used ryanodine receptors and CaM antagonists to show that the increase in intracellular Ca(2+) level is an essential modulator of progesterone synthesis through the regulation of Star gene expression in MA-10 Leydig cells. Furthermore, we mapped a Ca(2+)/CaM-sensitive element in the Star promoter, which led to the identification of the nuclear receptor 4A1 (NR4A1) as a key mediator of the Ca(2+)/CaM signaling pathway in these cells. These data provide new insights into the Ca(2+) molecular pathway essential for steroidogenesis in Leydig cells.

The CCAAT/enhancer-binding protein ␤ (C/EBP␤) is expressed as three isoforms (LAP*, liver-enriched activating protein (LAP), and liver-enriched inhibitory protein (LIP)) that differentially regulate gene expression. The interplay between LAP*, LAP, and LIP in regulating cellular processes is largely unknown, and LIP has been largely regarded to repress transcription through a passive heterodimerization-dependent mechanism. Recently, we have shown that p300/GCN5 and mSin3A/HDAC1 differentially regulate the ability of C/EBP␤ to stimulate preadipocyte differentiation through activation of C/ebp␣ transcription. Here, we have mapped requirements for binding of mSin3A/HDAC1 to LAP/LAP* and LIP to a 4-amino acid motif in the central region of LAP/LAP* (residues 153-156) and the N terminus of LIP. Reducing mSin3A/HDAC1 binding to LAP/LAP* and LIP through deletion of this motif reduced the recruitment of HDAC1 to the C/ebp␣ promoter and increased preadipocyte differentiation stimulated by insulin and 1-methyl-3-isobutylxanthine. Additional studies showed that the interaction of HDAC1 with LIP provides for active repression of C/ebp␣ transcription and is largely responsible for the ability of LIP and HDAC1 to repress preadipocyte differentiation. Thus, although mSin3A/HDAC1 interacted readily with LAP/LAP* in addition to LIP and that expression of LAP/LAP* was sufficient to recruit HDAC1 to the C/ebp␣ promoter, mutations in C/ebp␤ that abrogated HDAC1 association to LAP/LAP* in the absence of LIP provided no additional stimulation of differentiation or transcription beyond the deletion of LIP alone. The implication of these results for the interaction between p300/GCN5 and mSin3A/HDAC1 in regulating C/EBP␣ transcription and preadipocyte differentiation are discussed.The CCAAT/enhancer-binding protein ␤ (C/EBP␤) 2 is a basic leucine zipper (bZip) transcription factor that plays key roles in cellular differentiation, proliferation, stress responses, and inflammation. The C/ebp␤ gene encodes three isoforms, LAP* (liver-enriched activating protein*), LAP, and LIP (liverenriched inhibitory protein) translated from a single mRNA from three consecutive in-frame methionines (Fig. 1A) (1, 2). Relative expression of LAP*/LAP/LIP varies according to cell type, and the three isoforms dimerize interchangeably and heterodimerize broadly with other bZip factors. The shorter LIP isoform is distinguished by the absence of the transcriptional activation functions that occur in the N terminus of LAP*/LAP. Thus, LIP has been primarily hypothesized to be a passive repressor of transcription that reduces transcriptional activation through competition for DNA-binding sites and the formation of heterodimers in which only one partner contributes to activation (1). Although the relative level of LAP/LAP* in a cell is regulatable, generally the level of LIP expression is approximately equivalent to the level of LAP, with LAP* levels usually being significantly lower (2). C/EBP␤ has been shown to play an important role at the onset of the differenti...

The nuclear receptor NR4A1 is expressed in steroidogenic Leydig cells where it plays pivotal roles by regulating the expression of several genes involved in steroidogenesis and male sex differentiation including Star, HSD3B2, and Insl3. Activation of the cAMP and Ca 2+ signaling pathways in response to LH stimulation leads to a rapid and robust activation of Nr4a1 gene expression that requires the Ca 2+ /CAMKI pathway. However, the downstream transcription factor(s) have yet to be characterized. To identify potential Ca 2+ /CaM effectors responsible for hormone-induced Nr4a1 expression, MA-10 Leydig cells were treated with forskolin to increase endogenous cAMP levels, dantrolene to inhibit endoplasmic reticulum Ca 2+ release, and W7 to inhibit CaM activity. We identified Ca 2+ -responsive elements located in the discrete regions of the Nr4a1 promoter, which contain binding sites for several transcription factors such as AP1, CREB, and MEF2. We found that one of the three AP1/CRE sites located at -255 bp is the most responsive to the Ca 2+ signaling pathway as are the two MEF2 binding sites at -315 and -285 bp. Furthermore, we found that the hormone-induced recruitment of phospho-CREB and of the co-activator p300 to the Nr4a1 promoter requires the Ca 2+ pathway. Lastly, siRNAmediated knockdown of CREB impaired NR4A1 expression and steroidogenesis. Together, our data indicate that the Ca 2+ signaling pathway increases Nr4a1 expression in MA-10 Leydig cells, at least in part, by enhancing the recruitment of coactivator most likely through the MEF2, AP1, and CREB transcription factors thus demonstrating an important interplay between the Ca 2+ and cAMP pathways in regulating Nr4a1 expression.

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