Inhibins differentially antagonize activin and bone morphogenetic protein action in a mouse adrenocortical cell line

Inhibin, a member of the TGF-beta superfamily, has been proposed to act as an inhibitor of activin and bone morphogenetic protein (BMP) by sequestering their type II receptors in nonsignaling complexes with betaglycan. This mechanism of inhibin action was tested in a mouse adrenocortical (AC) cell line by examining the effects of inhibins A and B on cytochrome P450 17alpha-hydroxylase 17,20-lyase (Cyp17) expression and 17alpha-hydroxylase activity, measured by progesterone 17alpha-hydroxylation, in the absence and presence of activin or BMP isoforms. Cyp17 mRNA endogenously expressed by AC cells was suppressed by activins A and B and BMP-2, -6, and -7, and each ligand accordingly inhibited 17alpha-hydroxyprogesterone production (IC(50) of 0.24, 0.27, 0.4, 0.51, and 2.2 nm, respectively). Neither inhibin A nor inhibin B alone affected Cyp17 expression or 17alpha-hydroxyprogesterone production. Both inhibin A and inhibin B blocked the inhibitory actions of activins A and B in AC cells, supporting the antiactivin model of inhibin action. Inhibin A provided more potent and effective antagonism of both activins than did inhibin B, and activin A was less subject to antagonism by either inhibin than was activin B. In contrast to the major antagonism of activin by both inhibins, only inhibin A antagonized the actions of BMP-2, BMP-6, and BMP-7, whereas inhibin B was ineffective against all tested BMP isoforms except BMP-7 at high concentrations. These results provide limited support for the anti-BMP model of inhibin action and reveal that, relative to inhibin A, inhibin B essentially behaves as a selective activin antagonist in AC cells. In conclusion, inhibins A and B differentially antagonize the actions of activins and BMPs to control adrenocortical C(19) steroid production.     

Transforming growth factor beta inhibits steroidogenic acute regulatory (StAR) protein expression in human ovarian thecal cells

In this study, we investigated the effects of TGFβ1 on steroidogensis and expression of the steroidogenic acute regulatory (StAR) protein which regulates an important early step in the steroidogenic pathway. We utilized a human ovarian thecal like tumor (HOTT) cell model and investigated the effects of activin-A, inhibin-A, or TGFβ1 in the presence of forskolin and the effect of dibutyryl cyclic AMP (dbcAMP) on steroid accumulation in the culture medium. Cells were also treated with different concentration of TGFβ1 in the presence of forskolin, combined steroid production was measured at the end of 48 h and after 3 h incubation with 22R-hydroxycholesterol. In the presence of TGFβ1 there was a dose-dependent inhibition of androstenedione production. Inhibition in combined steroid production was apparent at the highest concentration of TGFβ1 tested. In the presence of 22R-hydroxycholesterol, combined steroid production was significantly inhibited at lower concentrations. TGFβ1 inhibited StAR protein expression in a concentration dependent manner. There was also a similar inhibition in StAR mRNA. These results suggest that the effect of TGFβ1 on steroid production and possibly follicular development may be in part due to its effects on StAR expression.     

Inhibin removes the inhibitory effects of activin on steroid enzyme expression and androgen production by normal ovarian thecal cells

Activin and inhibin are important local modulators of theca cell steroidogenesis in the ovary. Using a serum-free primary theca cell culture system, this study investigated the effects of inhibin on theca cell androgen production and expression of steroidogenic enzymes. Androstenedione secretion from theca cells cultured in media containing activin, inhibin and follistatin was assessed by RIA over 144?h. Activin (1-100?ng/ml) suppressed androstenedione production. Inhibin (1-100?ng/ml) blocked the suppressive effects of added activin, but increased androstenedione production when added alone, suggesting it was blocking endogenous activin produced by theca cells. Addition of SB-431542 (activin receptor inhibitor) and follistatin (500?ng/ml) increased androstenedione production, supporting this concept. Infection of theca cells with adenoviruses expressing inhibitory Smad6 or 7 increased androstenedione secretion, confirming that the suppressive effects of activin required activation of the Smad2/3 pathway. Activin decreased the expression levels of steroidogenic acute regulatory protein (STAR), whereas STAR expression was increased by inhibin and SB-431542, alone and in combination. CYP11A was unaffected. The expression of CYP17 encoding 17α-hydroxylase was unaffected by activin but increased by inhibin and SB-431542, and when added in combination the effect was further enhanced. The expression of 3β-hydroxysteroid dehydrogenase (3β-HSD) was significantly decreased by activin, while inhibin alone and in combination with SB-431542 both potently increased the expression of 3β-HSD. In conclusion, activin suppressed theca cell androstenedione production by decreasing the expression of STAR and 3β-HSD. Inhibin and other blockers of activin action reversed this effect, supporting the concept that endogenous thecal activin modulates androgen production in theca cells.     

Oxytocin inhibits LH-stimulated production of androstenedione by bovine theca cells

Oxytocin secretion by bovine granulosa cells increases dramatically after the LH/FSH surge. We have shown that oxytocin stimulates progesterone secretion and inhibits FSH-stimulated estradiol secretion in vitro by granulosa cells from bovine preovulatory follicles obtained before the LH/FSH surge. To determine if oxytocin regulates LH-stimulated steroid production by bovine theca interna cells, theca cells were isolated from preovulatory follicles obtained before the LH surge and were cultured for 4 days in the presence or absence of LH (2 or 4 ng/ml), without or with graded doses of oxytocin (125-1000 ng/ml). LH increased accumulation of androstenedione and progesterone. Oxytocin inhibited LH-stimulated androstenedione production, but had no effect on LH-stimulated progesterone production by cultured theca interna. The next objective was to determine if oxytocin regulates LH-stimulated steroidogenesis by modulating the levels of mRNA for steroidogenic enzymes and/or Steroidogenic Acute Regulatory protein (StAR). Low doses of LH alone increased the levels of mRNA for P450 17 alpha-hydroxylase (17 alpha-OH), 3beta-hydroxysteroid dehydrogenase (3beta-HSD) and cytochrome P450 side-chain cleavage, but not for StAR. In contrast, the effects of oxytocin on LH-stimulated androstenedione production were not associated with changes in the levels of mRNA for steroidogenic enzymes or StAR. These results suggest that oxytocin may play a paracrine role in regulating the follicular/luteal phase shift in steroidogenesis by decreasing androstenedione secretion by theca cells of the ovulatory follicle and that this effect is not mediated by changes in the levels of mRNA for steroidogenic enzymes and StAR.     

Localization and expression of low-density lipoprotein receptor, steroidogenic acute regulatory protein, cytochrome P450 side-chain cleavage and P450 17-alpha-hydroxylase/C17-20 lyase in developing swine follicles: in situ molecular hybridization and immunocytochemical studies

The present study utilizes in situ molecular hybridization and immunocytochemistry to investigate the follicular localization and expression of four thematically related sterol-metabolizing genes; low-density lipoprotein (LDL) receptor, steroidogenic acute regulatory protein (StAR), cytochrome P450 side-chain cleavage (CYP11A) enzyme, and cytochrome P450 17alpha-hydroxylase/C(17-20) lyase (CYP17). To this end, semiquantitative analyses were applied to individual nonatretic follicles (N=54) harvested from cycling gilts slaughtered on days 1, 3, 5, and 7 (N=3 per day) following withdrawal of the progesterone agonist, altrenogest. In situ and immunocytochemical signal intensities were assigned numerical values of 0-3 corresponding to the degree of expression of each mRNA and its corresponding protein. LDL receptor mRNA and protein content was undectable in theca and granulosa cells on days 1, 3, and 5, and then increased to low levels in theca cells on day 7. StAR message rose progressively in theca cells with follicular maturation, reaching a maximum on day 5, and then declining slightly on day 7 after the LH surge. In granulosa cells, small amounts of StAR mRNA and protein were detected on days 5 and 7. The amounts of CYP11A mRNA and protein were high in theca cells, and increased at each time point studied. Granulosa cells exhibited minimal CYP11A message on days 3, 5, and 7, while protein became detectable at low levels on day 7 only. Expression of CYP17 was localized exclusively in theca cells with protein and message content increasing unidirectionally to maxima on days 5 (RNA) and 7 (protein), respectively. Follicular fluid concentrations of androstenedione, and progesterone in contralateral ovaries correlated strongly and positively with accumulation of CYP17, and CYP11A proteins. In summary, these analyses demonstrate that preovulatory follicular development proceeds with the coordinate induction of pivotal genes and proteins that mediate the selective uptake, delivery and utilization of sterol substrate in granulosa and theca-cell steroidogenesis.     

Development-related effects of recombinant activin on steroid synthesis in rat granulosa cells.

Activin is structurally related to polypeptide growth factors such as transforming-growth factor-beta, which may have paracrine and/or autocrine functions in the ovaries. We have investigated the action of activin on granulosa cell steroidogenesis in vitro in relation to preovulatory follicular development in vivo. Estrogen-primed immature female rats received no other treatment (nondifferentiated granulosa cells), treatment with ovine (o) FSH (differentiated granulosa cells), or treatment with oFSH followed by human (h) CG (preovulatory granulosa cells) to stimulate preovulatory follicular development. Granulosa cells were isolated and cultured in the presence and absence of recombinant human activin-A using serum-free medium supplemented with 1.0 microM testosterone as an aromatase substrate and hFSH, hLH, forskolin, or 8-bromo-cAMP to stimulate steroid synthesis in vitro. After 48 h, medium was collected for measurement of estradiol (aromatase activity), progesterone, and cAMP. Basal steroid synthesis in nondifferentiated granulosa cells was unaffected by activin, but both aromatase activity and progesterone production induced by treatment with FSH in vitro were dosedependently enhanced up to 10-fold by the presence of activin. FSH-stimulated cAMP production was not measurably altered by activin; however, steroidogenesis induced by forskolin or 8-bromo-cAMP was significantly enhanced by the factor. Thus the effect of activin on steroidogenesis includes action at a subcellular level(s) distal to the production of cAMP. After gonadotropin treatment in vivo, granulosa cell aromatase activity and progesterone production showed divergent responses to activin in vitro. Basal-, FSH-, and LH-stimulated aromatase activity were all enhanced by activin in cultures of differentiated and preovulatory granulosa cells. However, whereas basal progesterone production was stimulated by activin in cultures of differentiated granulosa cells, in preovulatory granulosa cells it was inhibited. Moreover, in vitro stimulation of progesterone production by treatment of both differentiated and preovulatory granulosa cells with FSH or LH was suppressed by the presence of activin. Thus rat granulosa cells display development-related steroidogenic responses to activin, aromatase production becoming enhanced and progesterone production suppressed as follicular maturation progresses. These results further implicate activin as a local modulator of granulosa cell steroid synthesis in the ovaries, although its functional significance has yet to be established.     

Interactive stimulation by luteinizing hormone and insulin of the steroidogenic acute regulatory (StAR) protein and 17alpha-hydroxylase/17,20-lyase (CYP17) genes in porcine theca cells

LH and insulin are postulated to jointly stimulate theca-cell androgen biosynthesis in patients with hyperthecosis or polycystic ovarian syndrome. To explore the mechanisms of putative LH and insulin steroidogenic synergy in primary culture of normal theca cells, we have implemented an in vitro serum-free monolayer culture system of Percoll-purified, porcine theca cells harvested from immature ovaries. Initial dose and time course analyses revealed that a maximally effective concentration of LH (100 ng/ml) or insulin (100 ng/ml) individually will drive androstenedione production (at 6 to 48 h) by 1.5- to 2.6- and 1.1- to 1.7-fold, respectively, while combined agonists act synergistically over the interval 12 to 48 h yielding a 3- to 4-fold joint effect. Coadministration of LH and insulin can augment theca-cell concentrations of CYP17 and StAR messenger RNA (mRNA) resulting in 3.4- to 3.9- and 3.8- to 4.1-fold increases at 24 to 48 h, respectively (P < 0.01). Combined LH and insulin stimulation also amplified the nuclear content of intron-specific heterogeneous nuclear (hn)RNAs encoding CYP17 and StAR. Insulin significantly enhanced LH-driven but not basal cAMP accumulation (14-18 vs. 3-5.5 pmol/microg DNA/12-48 h) (P < 0.01). A stable exogenous analog of cAMP, 8 Br-cAMP, mimicked LH's effect on steroidogenesis and StAR and CYP17 gene expression and with insulin stimulated StAR mRNA and hnRNA accumulation synergistically. However, unlike LH, 8 Br-cAMP did not synergize with insulin on theca-cell androstenedione biosynthesis or CYP17 mRNA and hnRNA expression. In summary, the present in vitro data identify molecular interactions of LH and insulin on StAR and CYP17 gene expression, thus establishing potent signaling interfaces between these distinct hormonal agonists in regulating theca-cell steroidogenesis.     

Expression of enzyme associated with steroid hormone synthesis and local production of steroid hormone in endometrial carcinoma cells

Endometrial carcinoma is a common malignancy of the genital tract. In the present study, we examined the expression of human steroidogenic acute regulatory (StAR) protein, P450 side-chain cleavage enzyme (P450 scc), 17alpha-hydroxylase/17,20-desmolase (P45017alpha) and aromatase (P450 arom) in endometrial carcinoma cells to clarify the ability of these cells to produce steroid hormones. The results of RT-PCR analysis showed that StAR, P450 scc and P45017alpha genes were expressed in endometrial carcinoma cells. To examine the protein expression of StAR and P450 scc, we performed Western blotting using extracts from carcinoma cells. StAR protein and P450 scc were detected in both HHUA and HOUA-1 cells. The production of pregnenolone in HHUA cells increased 2.4-fold with transfection of a StAR expression vector and 4.3-fold with transfection of an F2 side-chain cleavage system. The RT-PCR product of 3beta-hydroxysteroid dehydrogenase was present in endometrial carcinoma cells. In endometrial carcinoma cells, the production of progesterone also increased with over-expression of StAR and the F2 system. Results of steroid metabolic assays showed that endometrial carcinoma cells produced not only 17alpha-hydroxyprogesterone but also androstenedione. Endometrial carcinoma cells express enzymes that are associated with the production of steroid hormones. Locally produced steroid hormones may have effects on tumor proliferation and tumorigenesis.     

A novel role for bone morphogenetic proteins in the synthesis of follicle-stimulating hormone

FSH is produced in pituitary gonadotropes as an alpha/beta heterodimer, and synthesis of the beta-subunit is the rate-limiting step in overall FSH production. Synthesis of FSHbeta can be regulated by activin and inhibin, both of which are members of the transforming growth factor-beta superfamily. Bone morphogenetic proteins (BMPs) also belong to the transforming growth factor-beta family and are multifunctional growth factors involved in many aspects of tissue development and morphogenesis, including regulation of FSH action in the ovary. Here we report a novel function for BMP-7 and BMP-6 in regulating FSH synthesis in the pituitary. Using primary pituitary cell cultures derived from transgenic mice that carry the ovine FSHbeta promoter linked to a luciferase reporter gene (oFSHbetaLuc), BMP-7 or BMP-6 was found to stimulate oFSHbetaLuc expression by 6-fold. Transient expression of the oFSHbetaLuc in a transformed gonadotrope cell line, LbetaT2, was induced 4-fold by BMP-7 or BMP-6 treatment. More importantly, BMP-7 and BMP-6 increased endogenous FSH secretion by 10- and 14-fold, respectively, from LbetaT2 cells, demonstrating for the first time that a functional signaling BMP system is present in gonadotropes. Two bioneutralizing antibodies to BMP-7, which cross-react with BMP-6, but not with activin A, decreased basal oFSHbetaLuc expression and FSH secretion from transgenic mouse pituitary cultures by 83-88% and 47-48%, respectively, suggesting an autocrine or paracrine role for BMP-7 or BMP-6 in FSH synthesis. Neither bioneutralizing antibody to activin A or activin B decreased basal oFSHbetaLuc expression or mouse FSH secretion significantly. Dose-dependent inhibition of FSH synthesis by anti-BMP7 was also observed in rat and sheep pituitary cultures. These results combined with the fact that the messenger RNAs for BMP-7 and BMP-6 were detected in mouse pituitaries and LbetaT2 cells indicate that BMP-7 and/or BMP-6 can function as FSH stimulators and may be significant physiological factors maintaining basal FSH expression in vivo.     

Expression of transforming growth factor-beta1, activin A, and their receptors in thyroid follicle cells: negative regulation of thyrocyte growth and function.

Thyroid growth and function are intricately regulated by both positive and negative factors. In the present study, we have investigated the expression of transforming growth factor-beta (TGF-beta) super-family members and their receptors in normal porcine thyroid follicle cells. In tissue sections of porcine thyroids, we observed an expression of TGF-beta1, activin A, and bone morphogenetic protein (BMP)-7 proteins. The staining was localized to the follicular epithelium. In affinity cross-linking experiments, TGF-beta1 was found to bind to heteromeric complexes of TGF-beta type I and type II receptors, and activin A bound most efficiently to heteromeric complexes of activin type IB and type II receptors. We were unable to detect any BMP receptors (BMPRs) in attempts to perform affinity cross-linking with BMP-7. However, expression of BMPR-IA and BMPR-II messenger RNA (mRNA) was detected by Northern blot analysis. Both TGF-beta1 and activin A, but not BMP-7, increased the phosphorylation of Smad2, induced nuclear translocation of Smad2, Smad3, and Smad4, and inhibited thyrocyte cell growth as well as TSH-stimulated cAMP response. TGF-beta1 was more potent, compared with activin A, to induce these cellular responses. Taken together, our findings indicate a role for several members of the TGF-beta family in regulation of thyroid growth and function.