PubMed · 14434881
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J T PRIESTLEY. 1960. Hyperfunctioning adrenal lesions.. https://pubmed.ncbi.nlm.nih.gov/14434881/
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Post-translational modifications such as phosphorylation and specific proteolysis affect the steroidogenic acute regulatory protein (StAR) activity. We have found that in pcDNA3.1-StAR-transfected COS-1 cells, StAR was phosphorylated on S55, S56 and S194 (Fleury et al., unpublished). In this study, we are comparing the two-dimensional gel electrophoresis (2D-PAGE) characteristics of the WT StAR with those of the S194A and S55A/S56A/S194A-StAR mutants under control and (Bu)(2)-cAMP stimulation, using an anti-StAR antibody and an anti-phospho-(Ser/Thr) PKA substrate antibody. The 2D-PAGE migration pattern of the WT StAR analyzed by immunoblotting with the anti-StAR antibody revealed many StAR species with different pI and different molecular weights. In the (Bu)(2)-cAMP-WT preparations, except for three, all these StAR species were also recognized by the anti-phospho-(Ser/Thr) PKA substrate antibody; in contrast, less phosphorylated species were found in the non-stimulated WT preparations. The two-dimensional (2D) patterns of StAR revealed by the anti-StAR and the anti-phospho-(Ser/Thr) PKA substrate antibodies were modified for the S194A mutant and further modified for the S55A/S56A/S194A mutant. Whereas many species could still be detected by the anti-StAR antibody in the triple mutant S55A/S55A/S194A, none of these could be revealed by the anti-phospho-(Ser/Thr) PKA substrate antibody. Finally we found that, in addition to phosphorylation, the formation of different StAR species was also due to the hydrolysis of the molecule at its N-terminal and to a lesser degree at its C-terminal.
To identify genes that are induced by corticotropin (ACTH) in adrenal cortex cells, we carried out a differential hybridization screening of adrenal cortex cDNA libraries. Some of the clones we identified represented tissue inhibitor of metalloproteinase 1 (TIMP-1) mRNA. We examined ACTH dependence of the expression of TIMP-1 in vitro in cultured bovine adrenocortical cells, and in ACTH-treated rats. Northern blot analysis of total RNA from cells showed that the level of TIMP-1 mRNA increases sharply within 3h after ACTH stimulation. Since TIMP-1 inhibits some cell matrix metalloproteinases (MMPs) of the collagenase type, we examined the effect of ACTH on collagenase activity in bovine adrenocortical cells. Exposure of confluent cultures to ACTH for 24h showed dose-dependent inhibition of collagenase activity. Northern blot analysis of total RNA from rat adrenal zona fasciculata-reticularis and zona glomerulosa showed that in both of these zones TIMP-1 expression was induced within 12h after ACTH injection. Long-term (9 days) treatment with ACTH increased TIMP-1 mRNA levels nearly sixfold in zona fasciculata-reticularis. Overall, our results show that ACTH causes induction of TIMP-1 and suppression of collagenase activity, and suggest that ACTH may modulate the activities of MMPs and hence cell matrix remodeling.
Inner zone antigen (IZA) is a protein specifically expressed in the zona fasciculata and reticularis of the adrenal cortex. The cDNA encoding IZA was found to be identical to that encoding the previously reported putative membrane-associated progesterone receptor (MPR) and the TCDD-induced 25kDa protein (25-Dx). From its structure, MPR was classed as a member of a protein family containing a haem-binding domain, and progesterone was proposed to be a ligand of this domain. Indeed, when GST-tagged IZA was expressed in Escherichia coli and purified, the purified GST-IZA had a brown colour with maximum absorbance at 400 nm. The addition of dithionate shifted the absorbance peak to 420 nm, suggesting a haem-binding function. The possible role of IZA in steroidogenesis has been addressed, and the inhibition of adrenal steroidogenesis by the addition of an anti-IZA monoclonal antibody has been reported. When COS-7 cells were transformed with plasmids for appropriate steroidogenic enzymes in the presence or absence of an IZA expression plasmid and tested for their steroidogenic activities, 21-hydroxylation of progesterone was found to be specifically activated by IZA overexpression, suggesting the involvement of IZA in progesterone metabolism. Taken together, the available evidence suggests that IZA may have an important role in the functions of the adrenal zona fasciculata and reticularis.