REGULATION OF PROTEINS IN THE CHOLESTEROL SIDE-CHAIN CLEAVAGE SYSTEM IN JEG-3 AND Y-1 CELLS

被引:51
作者
BLACK, SM
SZKLARZ, GD
HARIKRISHNA, JA
LIN, D
WOLF, CR
MILLER, WL
机构
[1] UNIV CALIF SAN FRANCISCO, DEPT PEDIAT, BLDG MR-IV, ROOM 209, SAN FRANCISCO, CA 94143 USA
[2] UNIV CALIF SAN FRANCISCO, METAB RES UNIT, SAN FRANCISCO, CA 94143 USA
[3] UNIV EDINBURGH, IMPERIAL CANC RES FUND, EDINBURGH EH8 9YL, MIDLOTHIAN, SCOTLAND
关键词
D O I
10.1210/en.132.2.539
中图分类号
R5 [内科学];
学科分类号
1002 ; 100201 ;
摘要
The conversion of cholesterol to pregnenolone, the rate-limiting step in steroid hormone synthesis, occurs on mitochondrial cytochrome P450scc, which catalyzes this reaction by receiving electrons from NADPH via a flavoprotein [adrenodoxin reductase (AdRed)] and an iron sulfur protein [adrenodoxin (Adx)]. The behavior of the genes and mRNAs encoding these proteins has been studied in several systems, but little is known about the behavior of the human proteins. Using cloned cDNAs for human P450scc and AdRed, we constructed bacterial expression vectors to make milligram quantities of the corresponding proteins. These, plus purified human Adx similarly prepared by Dr. L. Vickery, were injected into rabbits to raise antiserum to each of the proteins. Each antiserum was highly specific and did not cross-react with other mitochondrial proteins detectable by Western blotting. Human JEG-3 choriocarcinoma cells and mouse Y-1 adrenocortical carcinoma cells were then incubated for 0-24 h with 1 mm 8-bromo-cAMP (8Br-cAMP) or 30 nm phorbol 12-myristate 13-acetate (PMA; phorbol ester) plus 1 Am A23187 (calcium ionophore) to activate the protein kinase-A and -C pathways, respectively. In JEG-3 cells, 8Br-cAMP increased and PMA/A23187 slightly decreased the abundance of P450scc and Adx, but neither treatment had a detectable effect on AdRed. The production of pregnenolone by these cells increased 3-fold in response to 8Br-cAMP and fell to one third in response to PMA/A23187. In Y-1 cells, 8Br-cAMP increased the abundance of all three proteins, while PMA/A23187 decreased the abundance of P450scc and Adx. The production of pregnenolone by these cells increased 9-fold in response to 8Br-cAMP and was unaffected by TPA/A23187. These studies show that the three proteins of the cholesterol side-chain cleavage system behave in response to 8Br-cAMP and PMA/A23187 as predicted from the study of their genes and mRNAs, indicating that the chronic regulation of steoridogenesis in these cell systems is regulated principally at the level of mRNA abundance.
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页码:539 / 545
页数:7
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