Vascular endothelial growth factor is essential for corpus luteum angiogenesis.

The development and endocrine function of the ovarian corpus luteum (CL) are dependent on the growth of new capillary vessels. Although several molecules have been implicated as mediators of CL angiogenesis, at present there is no direct evidence for the involvement of any. Here we report the unexpected finding that treatment with truncated soluble Flt-1 receptors, which inhibit vascular endothelial growth factor (VEGF) bioactivity, resulted in virtually complete suppression of CL angiogenesis in a rat model of hormonally induced ovulation. This effect was associated with inhibition of CL development and progesterone release. Failure of maturation of the endometrium was also observed. Areas of ischemic necrosis were demonstrated in the corpora lutea (CLs) of treated animals. However, no effect on the preexisting ovarian vasculature was observed. These findings demonstrate that, in spite of the redundancy of potential mediators, VEGF is essential for CL angiogenesis. Furthermore, they have implications for the control of fertility and the treatment of ovarian disorders characterized by hypervascularity and hyperplasia.

Heparin induces dimerization and confers proliferative activity onto the hepatocyte growth factor antagonists NK1 and NK2.

Hepatocyte growth factor (HGF) is a potent epithelial mitogen whose actions are mediated through its receptor, the proto-oncogene c-Met. Two truncated variants of HGF known as NK1 and NK2 have been reported to be competitive inhibitors of HGF binding to c-Met, and to function as HGF antagonists (Lokker, N.A., and P.J. Godowski. 1993. J. Biol. Chem. 268: 17145-17150; Chan, A.M., J.S. Rubin, D.P. Bottaro, D.W. Hirschfield, M. Chedid, and S.A. Aaronson. 1991. Science (Wash. DC). 254:1382-1387). We show here, however, that NK1 acts as a partial agonist in mink lung cells. Interestingly, NK1, which is an HGF antagonist in hepatocytes in normal conditions, was converted to a partial agonist by adding heparin to the culture medium. The interaction of NK1 and heparin was further studied in BaF3 cells, which express little or no cell surface heparan sulfate proteoglycans. In BaF3 cells transfected with a plasmid encoding human c-Met, heparin and NK1 synergized to stimulate DNA synthesis and cell proliferation. There was no effect of heparin on the IL-3 sensitivity of BaF3-hMet cells, and no effect of NK1 plus heparin in control BaF3 cells, indicating that the response was specific and mediated through c-Met. The naturally occurring HGF splice variant NK2 also stimulated DNA synthesis in mink lung cells and exerted a heparin-dependent effect on BaF3-hMet cells, but not on BaF3-neo cells. The activating effect of heparin was mimicked by a variety of sulfated glycosaminoglycans. Mechanistic studies revealed that heparin increased the binding of NK1 to BaF3-hMet cells, stabilized NK1, and induced dimerization of NK1. Based on these studies, we propose that the normal agonist activity of NK1 and NK2 in mink lung cells is due to an activating interaction with an endogenous glycosaminoglycan. Consistent with that model, a large portion of the NK1 binding to mink lung cells could be blocked by heparin. Moreover, a preparation of glycosaminoglycans from the surface of mink lung cells induced dimerization of NK1. These data show that the activity of NK1 and NK2 can be modulated by heparin and other related glycosaminoglycans to induce proliferation in cells expressing c-Met.

Safety and antitumor activity of recombinant soluble Apo2 ligand.

TNF and Fas ligand induce apoptosis in tumor cells; however, their severe toxicity toward normal tissues hampers their application to cancer therapy. Apo2 ligand (Apo2L, or TRAIL) is a related molecule that triggers tumor cell apoptosis. Apo2L mRNA is expressed in many tissues, suggesting that the ligand may be nontoxic to normal cells. To investigate Apo2L's therapeutic potential, we generated in bacteria a potently active soluble version of the native human protein. Several normal cell types were resistant in vitro to apoptosis induction by Apo2L. Repeated intravenous injections of Apo2L in nonhuman primates did not cause detectable toxicity to tissues and organs examined. Apo2L exerted cytostatic or cytotoxic effects in vitro on 32 of 39 cell lines from colon, lung, breast, kidney, brain, and skin cancer. Treatment of athymic mice with Apo2L shortly after tumor xenograft injection markedly reduced tumor incidence. Apo2L treatment of mice bearing solid tumors induced tumor cell apoptosis, suppressed tumor progression, and improved survival. Apo2L cooperated synergistically with the chemotherapeutic drugs 5-fluorouracil or CPT-11, causing substantial tumor regression or complete tumor ablation. Thus, Apo2L may have potent anticancer activity without significant toxicity toward normal tissues.

Overexpression of the retinoic acid-responsive gene Stra6 in human cancers and its synergistic induction by Wnt-1 and retinoic acid.

Genetic defects in the Wnt-1 signaling pathway contribute to human tumor progression and are especially prevalent in colorectal cancer. We screened mouse C57MG cells to isolate mRNAs induced by Wnt-1 and identified Stra6, an mRNA known to be up-regulated by retinoic acid. Up-regulation of Stra6 mRNA was also observed in hyperplastic mammary tissue and mammary gland tumors from transgenic mice expressing Wnt-1 and in human tumors that frequently harbor defects in Wnt-1 signaling. Stimulation of C57MG cells with retinoic acid plus Wnt-1 resulted in expression of Stra6 transcript to levels greatly exceeding that observed with either stimulus alone. This synergy could be explained in part by the up-regulation of retinoic acid receptor-gamma that was observed in response to Wnt-1 signaling. Accordingly, treatment of human colorectal cancer cell lines with retinoic acid resulted in the up-regulation of Stra6 mRNA and accumulation of Stra6 protein at the cell membrane. The data support a model in which Wnt-1 signaling synergizes with retinoids to activate retinoic acid receptor-gamma-responsive genes in human cancers.

Purification and characterization of recombinant human activin B.

Recombinant human activin B has been isolated to more than 95% purity from a mammalian kidney cell line. Activin B is a covalently-linked homodimer with an apparent molecular mass of 25.9 kDa (unreduced) and 15.2 kDa (reduced) as determined by SDS-polyacrylamide-gel electrophoresis. On gel filtration in 6 M guanidine hydrochloride, activin B chromatographs with an apparent molecular mass of 11 kDa, whether reduced or not. The amino-terminal sequence of the purified protein is consistent with the expected sequence derived from the beta subunit of inhibin B. The amino acid composition of the purified molecule agrees with the expected theoretical composition of the beta subunit of inhibin B. Activin B has an apparent pI of 4.6 as determined by isoelectric focusing in 6 M urea and 4.7 as determined by chromatofocusing in 6 M urea. The extinction coefficient is 1.8.

Recombinant expression and characterization of human activin A.

Activin, which stimulates the secretion of FSH from anterior pituitary cells, is a dimer of the beta-subunits of inhibin. Two species of activin (A and AB) have been purified from ovarian follicular fluid and characterized. We have been able to biosynthetically produce recombinant human activin A by constructing stable cell lines expressing the mRNA for the beta A subunit of human inhibin. These cell lines secreted a 24 kilodalton beta A dimer which stimulated FSH secretion in cultured pituitary cells. The ability of this protein to stimulate FSH secretion was sensitive to reduction of disulfide bonds, exhibited a slow onset of action, and was blocked by actinomycin D. In addition, recombinant activin A stimulated hemoglobin accumulation in K562 cells. These data show that recombinant activin A has the biochemical properties and biological activities that have been ascribed to native activin A. In addition, these results provide an independent confirmation, and thereby a final proof, of the structure and function of activin.

Activin B: precursor sequences, genomic structure and in vitro activities.

We report here the complete amino acid sequence of the human inhibin beta B-subunit as deduced from the sequence of cDNA and genomic clones. The primary translation product of the beta B mRNA predicts a protein of 407 amino acids, containing a prepro region of 292 amino acids separated by basic amino acids from the mature C-terminal 115 amino acids. Mammalian tissue culture cells transfected with a beta B-subunit expression plasmid secreted an activin B homodimer of approximately 22K mol wt. Coexpression of the beta A- and beta B-subunit mRNAs resulted in the secretion of the three forms of activin, A, AB, and B. Purified activin B was shown to elicit FSH release in an in vitro pituitary assay and trigger the accumulation of hemoglobin in K562 cells. The potency of activin B in both of these assays (ED50 approximately 2 ng/ml) was indistinguishable from that observed for activin A.

Localization of inhibin/activin subunit mRNAs within the primate ovary.

In order to gain further understanding of the physiology of inhibin and activin in the primate, the expression of inhibin/activin subunit mRNAs in the monkey ovary was examined by in situ hybridization. Granulosa cells of small antral follicles were found to express mRNA for the beta B subunit, which decreased to undetectable levels in dominant follicles. In contrast, expression of alpha and beta A subunit mRNAs was detected in granulosa cells of dominant follicles and in corpora lutea, but not in small antral follicles. These results indicate that the expression of the beta A and beta B subunits is differentially regulated during the growth and development of ovarian follicles in the monkey.

Retrovirus-mediated in vivo gene transfer in the replicating liver using recombinant hepatocyte growth factor without liver injury or partial hepatectomy.

Retrovirus-mediated gene delivery into hepatocytes in vivo provides long-term gene expression, which is of great importance for treating most genetic and metabolic disorders. However, clinical application has not been realized because of the requirement for prior 70% partial hepatectomy or chemical (toxic) liver injury to initiate hepatocyte replication at the time of retroviral gene transduction. In this paper, we describe a novel gene delivery system that uses recombinant hepatocyte growth factor (rHGF) prior to retrovirus-mediated in vivo gene transfer in the liver without partial hepatectomy or liver injury. A single retroviral infusion through the portal vein following five systemic injections (via the tail vein) of 100 microg/kg rHGF resulted in a 10.4% 5-bromo-2'-deoxyuridine (BrdU) labeling index (BLI) and 0.14% retroviral gene transduction efficiency (RGTE) in hepatocytes, which were 6.3- and 12.9-fold higher than those of controls, respectively. Modest additional increases in BLI and RGTE (13.4% and 0.22%, respectively) were seen after five systemic injections of 500 microg/kg rHGF. The correlation between BLI and RGTE was statistically confirmed regardless of treatment. When rats received multiple retroviral infusions through a cannulated portal vein following five portal injections of 100 microg/kg rHGF, RGTE was dramatically increased (1.3%) and in some areas of the liver exceeded more than 10%. There was no evidence of liver injury in any animal. This approach has great potential for clinical application in terms of avoiding invasive procedures or liver injury.

Inhibition of synthesis of luteinizing hormone (LH) receptors by a down-regulating dose of LH.

Although LH is known to cause down-regulation of the LH/human CG (hCG) receptor, the mechanisms underlying this process are not well understood. Here we have analyzed the effects of LH on the turnover of LH receptors in cultured granulosa cells. Down-regulation was stimulated in FSH-primed granulosa cells by exposure to LH for 1 h. By 24 h after LH exposure, the LH/hCG receptor content, as measured using [125I]iodo-hCG was 70% less than controls. To determine whether this loss of LH/hCG receptors was due to accelerated receptor degradation, turnover measurements were made using tunicamycin to inhibit LH/hCG receptor synthesis. Under these conditions, there was a small (16%) increase in the rate of degradation of LH/hCG receptors in LH-treated cells compared to controls. By contrast, the rate of LH/hCG receptor synthesis, estimated mathematically, was reduced by 75% in the LH-treated cells. LH treatment had no effect on the incorporation of [35S]methionine into total cellular protein. Withdrawing FSH from the culture medium had a similar effect, slightly enhancing LH/hCG receptor degradation while markedly reducing its synthesis. The role of lysosomes in the degradation of LH/hCG receptors was also investigated. The lysosomotropic amine NH4Cl failed to cause LH/hCG receptors to accumulate, contrary to what would be expected if LH/hCG receptors were degraded lysosomally. In contrast, NH4Cl inhibited the degradation of receptor-bound [125I]iodo-hCG, suggesting that the ligand and the receptor may be handled differently by the granulosa cell. Our results suggest that in the granulosa cell, LH down-regulates its own receptor by inhibiting the synthesis of LH/hCG receptors, possibly by interfering with the ability of FSH to stimulate the synthesis of LH/hCG receptors. Furthermore, they suggest that the degradation of LH/hCG receptors may occur by nonlysosomal mechanisms.

In vivo comparison of the follicle-stimulating hormone-suppressing activity of follistatin and inhibin in ovariectomized rats.

The present study was performed to compare and contrast the effects of two gonadal polypeptides, inhibin and follistatin, on ovariectomy-induced hypersecretion of FSH and LH. Ovariectomies were performed 1 week before study. Follistatin was purified from porcine follicular fluid, and human inhibin A was produced by recombinant DNA technology. On the day of study, a blood sample was taken from intraatrial cannulae inserted on the previous day, the materials were injected, and additional blood samples were taken at various times thereafter. Serum FSH and LH levels were determined by RIA. Both follistatin and inhibin exhibited dose-dependent suppression of circulating FSH but not LH levels, with initial decreases in FSH levels by both materials observed between 2-4 h post injection. Maximal suppression of FSH by each polypeptide occurred between 4-6 h depending on dose. Based on the dose-response relationships, it was determined that inhibin was approximately five times as potent as follistatin in suppressing FSH release. However, despite the greater biopotency of inhibin than follistatin, the duration of action of even the highest dose of inhibin (50 micrograms) was between 4-9 h, whereas the duration of FSH-suppressing activity by the two highest doses of follistatin (40 and 80 micrograms) was between 10-21 h. Data obtained from a second experiment conducted to examine the effects of inhibin and follistatin on anterior pituitary gonadotropin responses to LHRH were consistent with in vitro data showing direct pituitary effects of the gonadal polypeptides. Collectively, these results demonstrate that both purified porcine follistatin and recombinant human inhibin A profoundly suppress serum FSH levels in a dose- and time-dependent manner, with inhibin being more potent in this regard. Whereas the onset of action is similar for the two polypeptides, the duration of action of follistatin is longer than that for inhibin, suggesting, among other factors, different metabolic clearance rates or different pretranscriptional mechanisms of action of follistatin and inhibin.

In vivo regulation of FSH synthesis by inhibin and activin.

The effect of a single sc injection of the gonadal peptide, recombinant human activin A (rhActivin A), on gonadotropin synthesis and secretion was examined in adult and immature male and female rats and the effect of recombinant human inhibin A (rhInhibin A) was examined in adult male rats. Pituitary FSH beta, LH beta and alpha messenger RNA (mRNA) levels were determined by blot hybridization. Trunk blood was collected to measure serum FSH levels. Treatment with rhInhibin A (100 micrograms/kg) resulted in a decrease in FSH beta mRNA to 2% of controls levels 6 h after injection. FSH beta mRNA levels started to rebound at 10 h, but were still significantly lower than vehicle-treated controls. Serum FSH levels were significantly reduced at 2 h and were reduced further at 6 and 10 h. There were no significant changes in alpha and LH beta mRNA levels. RhActivin A, at the highest dose (500 micrograms/kg), in immature male rats had only a modest effect (1.2- and 1.3-fold increase) on FSH beta mRNA levels and FSH secretion, respectively, at 2 h. No increase in FSH synthesis and FSH secretion was observed in adult male rats. In contrast, both immature and adult-ovariectomized E2 implanted females showed a robust response to rhActivin A. In immature females, 2 h after rhActivin A (100 and 500 micrograms/kg) administration, FSH beta mRNA levels were elevated 2.0- and 2.2-fold. At this time serum FSH was also elevated. At 6 and 10 h rhActivin A significantly reduced FSH beta mRNA levels from vehicle-treated controls. In contrast, FSH secretion was elevated at 6 h and returned to baseline at 10 h. Administration of rhActivin A (500 micrograms/kg) to adult, ovariectomized-E2 females resulted in a significant increase in FSH beta mRNA levels and FSH secretion at 2 and 6 h. There were no significant changes in alpha and LH beta mRNA levels in either males or females. Thus, these in vivo studies have shown that rhInhibin A can inhibit FSH beta mRNA levels and FSH secretion in the adult male rat. RhActivin A stimulates FSH synthesis and secretion in the immature and adult ovariectomized-E2 females, but has little or no effect in immature and adult males. Hence, there is a sexual dimorphic response to rhActivin A in vivo in the rat.

Reduction of alpha-naphthylisothiocyanate-induced hepatotoxicity by recombinant human hepatocyte growth factor.

Hepatocyte growth factor (HGF) is a potent stimulator of DNA synthesis in cultured hepatocytes. To determine whether HGF has any activity in vivo, we have tested HGF in rats in which intrahepatic cholestasis was induced by acute administration of alpha-naphthylisothiocyanate (ANIT). The hepatotoxic effects of a single injection of ANIT were manifested 48 h later as large increases in serum bilirubin, alanine aminotransferase, aspartate aminotransferase, and alkaline phosphatase. These biochemical changes were accompanied by widespread periportal edema, hypertrophy of bile duct epithelium, and randomly scattered areas of liquifaction necrosis in the hepatic parenchyma. The increases in bilirubin, alanine aminotransferase, aspartate aminotransferase and alkaline phosphatase were markedly attenuated when HGF was administered 30 min before ANIT and again at 6, 12, 24, 30, and 36 h after ANIT. In addition, this HGF dosing regimen completely prevented the occurrence of parenchymal lesions, although it had no effect on periportal histopathology. The effect of ANIT was dose dependent; a maximal response was observed at 320 micrograms/kg per injection, with an intermediate response at 105 micrograms/kg. Delaying the administration of HGF until 12 h after ANIT was as effective as when administration was begun 30 min before ANIT. Taken together these results show that HGF can prevent some aspects of ANIT hepatotoxicity.

Hepatocyte growth factor and vitamin D cooperatively inhibit androgen-unresponsive prostate cancer cell lines.

Expression of MET, the receptor for hepatocyte growth factor (HGF), has been associated with androgen-insensitive prostate cancer. In this study we evaluated MET activation by HGF and HGF action in prostate cancer cell lines. HGF causes phosphorylation (activation) of the MET receptor in three androgen-unresponsive cell lines (DU 145, PC-3, and ALVA-31) together with morphological change. Although HGF is known to stimulate the growth of normal epithelial cells, including those from prostate, we found that HGF inhibited ALVA-31 and DU 145 (hormone-refractory) cell lines. Moreover, HGF and vitamin D additively inhibited growth in each androgen-unresponsive cell line, with the greatest growth inhibition in ALVA-31 cells. Further studies in ALVA-31 cells revealed distinct cooperative actions of HGF and vitamin D. In contrast to the accumulation of cells in G1 seen during vitamin D inhibition of androgen-responsive cells (LNCaP), growth inhibition of the androgen-unresponsive ALVA-31 cell line with the HGF and vitamin D combination decreased, rather than increased, the fraction of cells in G1, with a corresponding increase in the later cell cycle phases. This cell cycle redistribution suggests that in androgen-unresponsive prostate cancer cells, HGF and vitamin D act together to slow cell cycle progression via control at sites beyond the G1/S checkpoint, the major regulatory locus of growth control in androgen-sensitive prostate cells.

Evidence for an autocrine role of activin B within rat anterior pituitary cultures.

Activins, dimers of inhibin beta subunits, are potent stimulators of FSH secretion in vivo and in vitro and of FSH beta mRNA expression in rat anterior pituitary cultures. In this study, we investigated the possibility that locally secreted activin B (beta B beta B) may function as an autocrine modulator of basal FSH secretion and expression based on the previous observation that beta B is expressed within gonadotropes. The incubation of cultured rat anterior pituitary cells with a m mouse monoclonal antibody specific for the activin B homodimer (MAb-activin B) significantly attenuated the basal secretion of FSH in a concentration- and time-dependent manner, without influencing LH secretion. Moreover, MAb-activin B selectively inhibited FSH beta mRNA accumulation without affecting either LH beta or alpha subunit mRNAs. The MAb-activin B completely blocked the stimulation of FSH secretion by exogenous activin B, but not by activin A, confirming its specificity. As previously shown, inhibin A and follistatin significantly suppressed basal FSH secretion in these cultures. This inhibitory effect, albeit of lower magnitude, was still evident even in the presence of the MAb-activin B which by itself suppressed basal FSH secretion. These data suggest that the secretion of activin B by the gonadotropes of the anterior pituitary may serve as an autocrine signal in the selective modulation of FSH expression and secretion. Furthermore, the inhibitory actions of inhibins and follistatins on gonadotropes may, in part, be explained by their ability to interfere with the actions of endogenous activin B.

The effects of activin on follicle-stimulating hormone secretion and biosynthesis in human glycoprotein hormone-producing pituitary adenomas.

The effects of activin on pituitary FSH biosynthesis have been previously characterized using primary rat pituitary cultures; however, little is known of the effects of activin on FSH biosynthesis and secretion in human pituitary tissue. Production of intact glycoprotein hormones and free subunits is increasingly recognized in pituitary tumors; however, the regulation of gonadotropins in such tumors has not been addressed. We have investigated the effects of human recombinant activin on glycoprotein hormone biosynthesis and secretion in primary cultures of 12 human glycoprotein hormone-producing pituitary adenomas and compared this with the effects of activin in normal rat anterior pituitary cells. In 33% of the human pituitary tumors studied, significant (P less than 0.05) increases in FSH beta secretion occurred in response to incubation with 20 ng/mL activin for 24 h (19-287% stimulation), without changes in the production of intact FSH. A Northern analysis performed on cells derived from one tumor indicated that FSH beta mRNA levels increased 350% after activin treatments; however, FSH secretion did not parallel the mRNA changes. None of the human glycoprotein hormone-producing tumors significantly increased FSH secretion in response to activin. To validate the biological activity of recombinant human activin-A and to confirm time and dose conditions for the human tumor cultures, we also examined its ability to stimulate FSH production in rat pituitary cultures. Activin (20 ng/mL) added to the culture medium significantly increased FSH secretion and steady state levels of FSH beta mRNA after 24 h. These data indicate that some glycoprotein hormone-producing pituitary tumors treated with purified activin have discordant responses of intact gonadotropins and free subunit responses. In contrast to responses in normal rat gonadotrophs, FSH beta biosynthetic pathways may be uncoupled from intact FSH secretion in a subset of glycoprotein hormone-producing pituitary adenomas.

Effect of recombinant activin on androgen synthesis in cultured human thecal cells.

The effect of activin-A on ovarian androgen synthesis was tested in vitro using serum-free monolayer cultures of human thecal cells. Maximal rates of androgen (androstenedione and dehydroepiandrosterone) production were induced by treating the cells for 4 days with LH (10 ng/mL) in the presence of insulin-like growth factor-I (greater than or equal to 30 ng/mL). The additional presence of recombinant activin-A (1-100 ng/mL) in culture medium caused dose-dependent suppression of thecal cell androgen production, with 50% maximal inhibition occurring at an activin-A concentration of about 10 ng/mL. Progesterone production was only suppressed by high dose (100 ng/mL) activin-A, and inhibition of steroid production occurred without inhibition of DNA synthesis (tritiated thymidine uptake). These results reveal a potent and selective inhibitory action of activin-A on thecal cell androgen synthesis, consistent with a paracrine function for activin(s) in modulating follicular androgen biosynthesis in the human ovary.

Effect of recombinant inhibin on androgen synthesis in cultured human thecal cells.

Effects of inhibin (recombinant human inhibin-A) on ovarian androgen synthesis were tested in vitro using serum-free monolayer cultures of human thecal cells. Treatment for 4 days with inhibin alone at doses between 10 and 100 ng/ml caused modest (approximately 2-fold) increases in production of androgen (androstenedione and dehydroepiandrosterone): similar to the maximal level of stimulation caused by luteinizing hormone (LH) (10 ng/ml) alone but only about one-third of that caused by insulin-like growth factor I (IGF-I) (30 ng/ml) alone. Combined treatment with LH and inhibin elicited additive effects on androgen production whereas LH and IGF-I were synergistic, giving rise to androgen production rates at least 40 times greater than control. Additional presence of inhibin caused up to 10-fold augmentation of the response to LH + IGF-I. Activin (recombinant human activin-A) was previously shown to inhibit LH + IGF-I-induced androgen synthesis in this human thecal cell culture system. In the present study we found that the additional presence of inhibin (greater than 1 ng/ml) completely neutralized this inhibitory action of activin (10 ng/ml). These effects of inhibin were dose-dependent (ED50 1-10 ng/ml) and maximal at approximately 100 ng/ml. Inhibin stimulation of androgen synthesis occurred in the absence of measurable effects on progesterone production, and cell numbers in cultured cell monolayers were unaltered by the protein. It is concluded that inhibin exerts potent and selective stimulation of human thecal cell androgen synthesis in vitro. These results a paracrine role for inhibin(s) in modulating follicular androgen biosynthesis in the human ovary.

A central role for cyclic AMP, but not progesterone, in luteinizing hormone receptor down-regulation in the granulosa cell.

Although luteinizing hormone (LH) is known to down-regulate its own receptor in several gonadal cell types, the mechanisms underlying this process are poorly understood. To elucidate these mechanisms we have examined the role of cAMP and progesterone in LH-stimulated down-regulation of the LH receptor, using cultured granulosa cells as a model. LH receptors were induced by culturing the cells with follicle-stimulating hormone for 2 days, and once induced, could be down-regulated by a brief exposure to LH. Down-regulation also occurred when cells were cultured with activators of adenylate cyclase, inhibitors of phosphodiesterase, or analogues of cAMP. Cholera toxin and N6,O2'-dibutyryladenosine 3':5'-cyclic monophosphate, like LH, decreased the number of LH receptors, without affecting affinity for 125I-human chorionic gonadotropin (hCG). The extent of receptor loss after treatment with LH plus cholera toxin was no greater than that caused by LH alone. LH, hCG, and deglycosylated hCG, which binds to the LH receptor but has little bioactivity, caused down-regulation, and their relative capacity to cause down-regulation was highly correlated with their relative capacity to stimulate cAMP production. Indirect evidence suggested that maximal down-regulation requires activation of adenylate cyclase for at least 3 h. Consistent with this idea, a 3-h exposure to dibutyryl cAMP caused near-maximal down-regulation. Progesterone secretion was enhanced by all agents that caused down-regulation of the LH receptor; however, there was little correlation between progesterone secretion and down-regulation. Furthermore, maximal down-regulation occurred when progesterone secretion was inhibited greater than 99% with cyanoketone. These data indicate that cAMP, but not progesterone, plays a central role in LH receptor down-regulation in the granulosa cell and that elevation of intracellular cAMP levels for 3 h is both necessary and sufficient to trigger maximal down-regulation.