Regulation by Adrenocorticotropin (ACTH), Angiotensin II, Transforming Growth Factor-b, and Insulin-Like Growth Factor I of Bovine Adrenal Cell Steroidogenic Capacity and Expression of ACTH Receptor, Steroidogenic Acute Regulatory Protein, Cytochrome P450c17, and 3b-Hydroxysteroid Dehydrogenase*

The purpose of this study was to evaluate the time-course effect of a 36-h treatment with ACTH (10 M), transforming growth factor-b1 (TGFb1; 10 M), angiotensin II (AngII; 10 M), and insulin-like growth factor I (IGF-I; 10 M) on the steroidogenic capacity of bovine adrenocortical cells (BAC) and on messenger RNA (mRNA) levels of ACTH receptor, cytochrome P450c17, 3b-hydroxysteroid dehydrogenase (3bHSD), steroidogenic acute regulatory protein (StAR), and StAR protein. ACTH and IGF-I enhanced, in a time-dependent manner, the acute 2-h ACTH-induced cortisol production, whereas TGFb1 and AngII markedly reduced it. ACTH, IGF-I, and AngII increased ACTH receptor mRNA, but the opposite was observed after TGFb1 treatment. ACTH and IGF-I increased P450c17 and 3bHSD mRNAs, whereas AngII and TGFb1 had the opposite effects. However, the effects of the four peptides on ACTH-induced cortisol production appeared before any significant alterations of the mRNA levels occurred. The most marked and rapid effect of the four peptides was on StAR mRNA. The stimulatory effect of ACTH was seen within 1.5 h, peaked at 4–6 h, and declined thereafter, but at the end of the 36-h pretreatment, the levels of StAR mRNA and protein were higher than those in control cells. IGF-I also enhanced StAR mRNA levels within 1.5 h, and these levels remained fairly constant. The effects of AngII on StAR mRNA expression were biphasic, with a peak within 1.5–3 h, followed by a rapid decline to almost undetectable levels of both mRNA and protein. TGFb1 had no significant effect during the first 3 h, but thereafter StAR mRNA declined, and at the end of the experiment the StAR mRNA and protein were almost undetectable. Similar results were observed when cells were treated with ACTH plus TGFb1. A 2-h acute ACTH stimulation at the end of the 36-h pretreatment caused a higher increase in StAR mRNA and protein in ACTHor IGF-I-pretreated cells than in control cells, which, in turn, had higher levels than cells pretreated with TGFb1, ACTH plus TGFb1, or AngII. These results and the fact that the stimulatory (IGF-I) or inhibitory (AngII and TGFb1) effects on ACTH-induced cortisol production were more pronounced than those on the ability of cells to transform pregnenolone into cortisol strongly suggest that regulation of StAR expression is one of the main factors, but not the only one, involved in the positive (IGF-I) or negative (TGFb1 and AngII) regulation of BAC for ACTH steroidogenic responsiveness. A high correlation between steady state mRNA level and acute ACTH-induced cortisol production favors this conclusion. (Endocrinology 141: 1599–1607, 2000) T BIOSYNTHESIS OF corticosteroids involves the participation of various steroidogenic enzymes (1). The first step in steroidogenesis is the conversion of cholesterol to pregnenolone catalyzed by the cytochrome P450 side-chain cleavage (P450scc) enzymes, which reside in the inner mitochondrial membrane (2). However, many studies have shown that the limiting step was not due to P450scc activity, but, rather, to the translation of cholesterol from the outer to the inner mitochondrial membrane. Several proteins, including the sterol carrier protein-2 (3), the steroidogenesis activator polypeptide (4), and the peripheral benzodiazepine receptor (5), are able to promote steroidogenesis under some experimental conditions and therefore become candidates for mediators of cholesterol transport between the mitochondrial membranes. Although data supporting an important role for these proteins in cholesterol transport are convincing, the time course and regulation of their expression as well as the magnitude and specificity of their responses indicate that they are unlikely to be the primary determinant of the acute steroidogenic response in adrenals. More recently, a protein, namely StAR (steroidogenic acute regulatory protein), expressed exclusively in adrenals and gonads, the synthesis of which is rapidly induced by the steroidogenic hormones, has been purified and cloned (6). The biochemical and genetic evidence for the key role of this protein in the hormonally induced acute steroidogenic response of steroidogenic cells has been reviewed (7–9). Received September 17, 1999. Address all correspondence and requests for reprints to: Dr. José M. Saez, INSERM, U-369, and IFREL, Faculté de Medecine Laennec, rue Guillaume Paradin, 69372 Lyon Cedex 07, France. E-mail: [email protected]. * This work was supported by grants from INSERM and University Claude Bernard Lyon 1. † Supported by a predoctoral fellowship from Ministère de l’Education et de la Recherche et de l’Education National. 0013-7227/00/$03.00/0 Vol. 141, No. 5 Endocrinology Printed in U.S.A. Copyright © 2000 by The Endocrine Society