Transforming growth factor-beta inhibits ovarian 17 alpha-hydroxylase activity by a direct noncompetitive mechanism.
Transforming growth factor-beta1 (TGF-beta1) inhibits theca-interstitial cell (TIC) androgen biosynthesis while enhancing progesterone production without altering P45017 alpha protein content. The purpose of the present study was to define the mechanism of TGF-beta 1 inhibition of ovarian androgen production by determining the effects of TGF-beta 1 on steroidogenic enzyme messenger RNA (mRNA) expression and 17 alpha-hydroxylase activity in TIC in vitro. TIC isolated from hypophysectomized immature rat ovaries by Percoll gradient centrifugation were cultured with and without LH and TGF-beta 1 up to 6 days. At various times, cytoplasmic mRNA was extracted from the TIC, and P450scc, 3 beta-HSD and P450(17 alpha) mRNA were measured by specific assays, using RT-PCR. Treatment with TGF-beta 1 alone (0.1-100 ng/ml) had no effect on mRNA expression at 2 days but increased P450scc and 3 beta-HDS mRNA at 4 days. TGF-beta did not alter the LH stimulation of P450scc and 3 beta-HSD mRNA up to 6 days but caused a modest (2.5-fold) increase in P450 (17 alpha) mRNA at 2 days. Specificity studies with inhibin-A (30 ng/ml), activin-A (100 ng/ml), and MIS (300 ng/ml) demonstrated that the effects of TGF-beta 1 were unique within this family of peptides. We next examined the effect of TGF-beta 1 on 17 alpha-hydroxylase activity. Kinetic analysis revealed that the 17 alpha-hydroxylase enzyme has an apparent Michaelis-Menten constant of 3.42 mumol/liter and maximum velocity of 0.23 pmol/min x mg protein. TGF-beta 1 inhibited 17 alpha-hydroxylase activity by a noncompetitive mechanism with an apparent inhibin constant (Ki) of 46.4 pM. The results of our studies demonstrate that TGF-beta 1 directly inhibits TIC androgen production by a noncompetitive mechanism. This novel mechanism may be important in preventing excessive androgen production in developing ovarian follicles without preventing differentiation of the TIC.
Department of Obstetrics and Gynecology, Cedars-Sinai Research Institute/University of California, Los Angeles School of Medicine 90048, USA.
N
N
Fournet
S R
SR
Weitsman
R J
RJ
Zachow
D A
DA
Magoffin
D000446:Aldehyde-Lyases / Q000037:antagonists & inhibitors*; D000728:Androgens / Q000096:biosynthesis; D000818:Animals; D001483:Base Sequence; D065607:Cytochrome P-450 Enzyme Inhibitors; D005260:Female; D007700:Kinetics; D015335:Molecular Probes / Q000235:genetics; D008969:Molecular Sequence Data; D010053:Ovary / Q000201:enzymology*; D016133:Polymerase Chain Reaction; D012333:RNA, Messenger / Q000378:metabolism; D051381:Rats; D017207:Rats, Sprague-Dawley; D013254:Steroid 17-alpha-Hydroxylase; D013256:Steroids / Q000096:biosynthesis; D013799:Theca Cells / Q000378:metabolism; D014158:Transcription, Genetic; D016212:Transforming Growth Factor beta / Q000494:pharmacology*
D000728:Androgens; D065607:Cytochrome P-450 Enzyme Inhibitors; D015335:Molecular Probes; D012333:RNA, Messenger; D013256:Steroids; D016212:Transforming Growth Factor beta; D013254:Steroid 17-alpha-Hydroxylase; D000446:Aldehyde-Lyases