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The lactogen-dependent rat Nb2 lymphoma is a useful model to investigate PRL signaling pathways that lead to regulation of gene transcription. A primary mechanism coupled to PRL receptor (PRLR) activation in Nb2 cells involves phosphorylation by Jak-family tyrosine kinases of one or more signal transducers and activators of transcription (Stat) factors which subsequently bind to γ-interferon activation sequences (GAS) within promoter regions of target genes. However, it is presently unclear whether this mechanism is operative as a means for regulating PRL-induced gene expression to the exclusion of other signaling pathways. Previously, we reported that PRL directly stimulated rapid expression of the protooncogene, pim-1, at the mRNA and protein levels in lactogen-dependent Nb2–11 cells. In the present study, experiments were conducted to evaluate signaling mechanisms by which PRL regulates transcription of pim-1. Toward this end, a 1,268-bp segment upstream of the transcription initiation site of the 5′-p...

This study tested the hypothesis that nitric oxide (NO) inhibits the rate-limiting catalytic step in steroidogenesis, cytochrome P450 cholesterol side-chain cleaving enzyme (CYP11A1), independent of soluble guanylyl cyclase (GC-S) stimulation. To assess CYP11A1 activity, pregnenolone levels were quantified in murine adrenocortical Y1 cells in the presence of the 3beta-hydroxy-Delta(5)-steroid dehydrogenase inhibitor, 2alpha-cyano-17beta-hydroxy-4,4',17alpha-trimethylandrost-5-ene-3-one. The NO donor, (Z)-1-[2-(2-aminoethyl-N-(2-ammonioethyl)amino]diazen-1-ium-1,2-diolate(deta nonoate), inhibited vasoactive intestinal peptide-, forskolin- and 22alpha-hydroxycholesterol (22HC)-facilitated pregnenolonogenesis in the absence of GC-S activation and in the presence of a GC-S inhibitor, 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ). CYP11A1 was also heterologously expressed in monkey COS7 cells. Deta nonoate inhibited 22HC-facilitated activity of the over-expressed enzyme in the absence of GC-S activation and in the presence of ODQ. The NO-independent, GC-S agonist, 1-benzyl-3-(5'-hydroxymethyl-2'-furyl)indazole did not inhibit steroidogenesis. The IC(50) for effects of free NO on CYP11A1 was potent and in the 0.4-2 microM range. These results support the hypothesis that NO inhibits the rate-limiting enzyme in steroidogenesis independent of GC-S activation.