cAMP‐Induced Expression of the Orphan Nuclear Receptor Nur77 in MA‐10 Leydig Cells Involves a CaMKI Pathway

Abstract

The Nur77 (Nr4a1) gene, encoding the orphan nuclear receptor NUR77 (NR4A1), is an immediate early response gene whose expression is rapidly induced by a variety of physiologic stimuli. Nur77 is expressed in several organs, including the classic steroidogenic tissues: gonads and adrenal. In MA-10 Leydig cells, NUR77 has been shown to regulate expression of several genes involved in steroidogenesis and male sex differentiation. In Leydig cells, androgen biosynthesis is controlled primarily by the pituitary gonadotropin luteinizing hormone (LH) acting via its receptor (LH-R), which in turn activates the adenylate cyclase/cyclic adenosine monophosphate (cAMP) signaling pathway. Even though Nur77 expression is induced both at the mRNA and protein levels in response to LH/forskolin/cAMP in Leydig cells, the mechanisms involved remain largely unknown. Here, we report that cAMP-mediated induction of Nur77 expression at the protein, mRNA, and promoter levels in MA-10 cells involves different mechanisms. We found that increased NUR77 protein requires transcription and translation, whereas increased Nur77 mRNA does not require de novo protein synthesis, and would therefore rely on transcription factors already present in the cell. In addition, our detailed analysis of the Nur77 promoter in MA-10 cells revealed that distinct regulatory elements are involved in basal and cAMP-induced Nur77 transcription. Finally, we found that maximal cAMP-mediated increase in Nur77 promoter activity involves a Ca

Expressions of Sox9, Sox5, and Sox13 transcription factors in mice testis during postnatal development

“…The pSox9-luc and pSox5-luc reporter plasmid constructs harboring 4 consensus regulatory elements for Sox9 or Sox5, respectively, were purchased from Signosis Inc. (Santa Clara, CA, USA), and the-902 bp murine StAR luciferase promoter construct has been described previously [57]. MA-10 cells were transfected using the jetPRIME Ò reagent according to the manufacturer's instructions (Polyplus Transfection, Illkirch, France) followed by treatments using medium without serum and analysis of lysates as previously described [56,58]. Treatments were done using increasing concentrations of 8Br-cAMP (0.1, 0.5, and 2.5 mM) for different times of incubation (3, 6, and 12 h) or selective inhibitors for importin (importazole at 4 lM) and calmodulin (CGS-9343B at 5 lM) in combination with activators of the cAMP/PKA pathway such as 0.5 mM 8Br-cAMP, 10 lM forskolin (an activator of the adenylate cyclase), and 1 mM IBMX (a non-specific inhibitor of cAMP and cGMP phosphodiesterases) for 6 h.…”

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

“…The MEF2D and MEF2-Eng expression vectors were previously described [8]. The Nr4a1 and Insl3 reporters were previously described [16,17]. The Amhr2 reporter [18] was kindly provided by Dr. Jose Teixeira (Michigan State University).…”

A Cell-Autonomous Molecular Cascade Initiated by AMP-Activated Protein Kinase Represses Steroidogenesis

“…Furthermore, de novo synthesis of the NR4A1 (nuclear receptor subfamily 4, group A, member 1) (NUR77) transcription factor is also required (10). Induction of Nr4a1 and Star in Leydig cells also requires Ca 2ϩ release from internal stores (11,12), leading to activation of the Ca 2ϩ /calmodulin-dependent kinase I (CAMKI) (10,13).…”