okadaic-acid and Adrenal-Cortex-Neoplasms

Cytochrome P450c17 catalyzes 17 alpha-hydroxylation needed for cortisol synthesis and 17,20 lyase activity needed to produce sex steroids. Serine phosphorylation of P450c17 specifically increases 17,20 lyase activity, but the physiological factors regulating this effect remain unknown. Treating human adrenal NCI-H295A cells with the phosphatase inhibitors okadaic acid, fostriecin, and cantharidin increased 17,20 lyase activity, suggesting involvement of protein phosphatase 2A (PP2A) or 4 (PP4). PP2A but not PP4 inhibited 17,20 lyase activity in microsomes from cultured cells, but neither affected 17 alpha-hydroxylation. Inhibition of 17,20 lyase activity by PP2A was concentration-dependent, could be inhibited by okadaic acid, and was restored by endogenous protein kinases. PP2A but not PP4 coimmunoprecipitated with P450c17, and suppression of PP2A by small interfering RNA increased 17,20 lyase activity. Phosphoprotein SET found in adrenals inhibited PP2A, but not PP4, and fostered 17,20 lyase activity. The identification of PP2A and SET as post-translational regulators of androgen biosynthesis suggests potential additional mechanisms contributing to adrenarche and hyperandrogenic disorders such as polycystic ovary syndrome.

Topics: Adrenal Cortex Neoplasms; Androgens; Base Sequence; Chromosomal Proteins, Non-Histone; DNA Primers; DNA-Binding Proteins; Enzyme Inhibitors; Histone Chaperones; Humans; Hydroxylation; Kinetics; Microsomes; Okadaic Acid; Phosphoprotein Phosphatases; Phosphorylation; Phosphoserine; Protein Phosphatase 2; Proteins; Recombinant Proteins; Reverse Transcriptase Polymerase Chain Reaction; Steroid 17-alpha-Hydroxylase; Transcription Factors; Tumor Cells, Cultured

ACTH treatment of Y1 adrenocortical cells induces the synthesis of Nur77, an orphan nuclear receptor that can act as a potent transactivator for such genes as 21-hydroxylase (CYP21). Nur77 has thus been proposed to be a mediator of ACTH action in activating the expression of genes that encode steroidogenic enzymes. Here we show that ACTH regulates the activity of Nur77 at the level of phosphorylation. ACTH induces the synthesis of transcriptionally active, DNA-binding Nur77 that is unphosphorylated at Ser354, which resides within the DNA-binding domain. By contrast, the Nur77 population that is constitutively present in Y1 cells is phosphorylated at Ser354 and does not bind DNA. Substitutions of Ser354 with negatively charged amino acids, such as Asp or Glu, dramatically decreased Nur77 DNA-binding and trans-activation activities, whereas mutation to the neutral Ala had no effect. Aside from phosphorylation within the DNA-binding domain, ACTH treatment does not induce modifications in the N- and C-terminal domains of Nur77 that significantly affect activity. Although the specific kinases that phosphorylate Nur77 in vivo are not known, the mitogen-activated protein kinase/pp90RSK pathway is not critical to Nur77 regulation. We propose that ACTH treatment of Y1 cells results in modulation of the activities of both kinases and phosphatases, which, in turn, regulate the activities of such transcription factors as Nur77.

Topics: Adrenal Cortex Neoplasms; Adrenocorticotropic Hormone; Animals; Base Sequence; Calcium-Calmodulin-Dependent Protein Kinases; Chromatography, Affinity; DNA; DNA-Binding Proteins; Gene Expression Regulation; Mice; Nuclear Receptor Subfamily 4, Group A, Member 1; Okadaic Acid; Phosphorylation; Protein Processing, Post-Translational; Receptors, Cytoplasmic and Nuclear; Receptors, Steroid; Serine; Transcription Factors; Transcriptional Activation; Transfection; Tumor Cells, Cultured