Hormonal regulation of steroidogenic enzyme gene expression in Leydig cells

Abstract

In normal mouse Leydig cells, steady state levels of mRNA of CYP11A, 3β-hydroxysteroid dehydrogenase Δ 5- > Δ 4-isomerase (3βHSD), and CYP17 are differentially regulated. There is high basal expression of 3βHSD and CYP11A mRNA, while expression of CYP17 mRNA is absolutely dependent on cAMP stimulation. cAMP is required for maximal expression of all three enzymes. The expression of CYP11A in normal mouse Leydig cells is repressed by glucocorticoids. Glucocorticoids also repress both basal and cAMP-induced expression of 3βHSD mRNA, but do not repress the synthesis or mRNA levels of CYP17. cAMP induction of 3βHSD mRNA can be observed only when aminoglutethimide (AG), an inhibitor of cholesterol metabolism, is added to the Leydig cell cultures. The addition of AG also markedly increases cAMP induction of CYP17 mRNA levels. Addition of testosterone or the androgen agonist, mibolerone, to cAMP plus AG treated cultures reduced 3βHSD and CYP17 mRNA levels to levels comparable to those observed when cells were treated with cAMP only. These data indicate that testosterone acting via the androgen receptor represses expression of both CYP17 and 3βHSD. The role of protein synthesis in mediating the cAMP induction of 3βHSD, CYP17 and CYP11A was examined. The addition of cycloheximide, an inhibitor of protein synthesis, to cAMP treated cultures for 24 h completely suppressed both constitutive and cAMP-induced 3βHSD mRNA levels. Cycloheximide also repressed cAMP-induced levels of CYP17 to 12% of levels observed in the absence of cycloheximide. In sharp contrast, treatment for 24 h with cycloheximide did not suppress cAMP induction of CYP11A mRNA, but reduced basal levels by approx. 50%. These data indicate that newly synthesized protein(s) are required for cAMP induction of CYP17 and 3βHSD mRNA levels, but not for CYP11A mRNA. A mouse Cyp17 genomic clone containing the entire coding region plus 10 kb of 5′ flanking region has been isolated. Fragments of 5′ flanking sequences were subcloned into vectors containing the CAT reporter gene and transfected into MA-10 Leydig cells. Transfected cells were treated with cAMP and expression was determined by measuring CAT activity. A cAMP responsive element was identified in a region between -245 and -346 bp relative to the transcription initiation site of Cyp17. Cotransfection into MA-10 Leydig cells of constructs containing 4.5 kb of Cyp17 5′ flanking sequences together with a mouse androgen receptor expression vector demonstrate a dose dependent repression of cAMP-induced Cyp17 transcription by the androgen receptor. Studies with the mouse Cyp11a gene demonstrate that the 5′ flanking region of the gene contains sequences between 2.5 and 5 kb that are necessary for expression of mouse Cyp11a in Leydig cells but not in adrenal cells.