Phosphorylation-independent desensitization of the luteinizing hormone/chorionic gonadotropin receptor in porcine follicular membranes.

Phosphorylation of several G protein-coupled receptors mediates desensitization. This study determined whether LH/CG receptor was phosphorylated under conditions that promoted human CG (hCG)-induced desensitization. Cell-free desensitization of LH/CG receptor-mediated adenylylcyclase activity in porcine follicular membranes occurred in the presence of GTP and was time- and hCG dose-dependent, reaching 36-52% upon preincubation at 30 C for 40 min with 1.0 micrograms/ml hCG. However, under conditions that promoted GTP-dependent desensitization, there was no apparent phosphorylation of LH/CG receptor (obtained via immunoprecipitation) by endogenous membrane-associated protein kinases using [gamma-32P]GTP or [gamma-32P]ATP as phosphate donor. On the other hand, LH/CG receptor (88-90 kilodaltons) from both control and hCG-incubated membranes was phosphorylated in vitro by the catalytic subunit of cAMP-dependent protein kinase (protein kinase A). However, protein kinase A (in the absence of exogenous GTP) did not promote LH/CG receptor desensitization. These data demonstrate that, unlike with other G protein-linked receptors, LH/CG receptor phosphorylation by endogenous follicular membrane-associated protein kinase(s) does not mediate desensitization.

A proliferative effect of transforming growth factor-beta1 on a human prostate cancer cell line, TSU-Pr1.

Transforming growth factor -beta (TGF-beta) is growth inhibitory to many malignant cells, including prostate cancer cells. The present study reports an unusual observation in that TGF-beta is growth stimulatory to a human prostate cancer cell line, TSU-Pr1. The TSU-Pr1 line is highly aggressive and exhibits a rapid rate of proliferation in culture. These cells underwent further proliferation in response to TGF-beta1. Both type I and II receptors to TGF-beta (TPR-I, TPR-II) are expressed in TSU-Pr1 cells. Activation of a luciferase reporter gene, which contains a TGF-beta response element, confirmed that the TGF-beta receptors in TSU-Pr1 cells were functional. RT-PCR analysis and an ELISA assay determined that TSU-Pr1 cells secreted TGF-beta. In conclusion, TSU-Pr1 cells contain functional TGF-beta receptors but instead of the usual growth inhibition by TGF-beta1, these cells undergo proliferation. The present observation provides a proliferative role of TGF-beta in TSU-Pr1 cells, which may play a part in the aggressive phenotype of these cells and, perhaps other prostate cancer cells.

Epidermal growth factor-induced heterologous desensitization of the luteinizing hormone/choriogonadotropin receptor in a cell-free membrane preparation is associated with the tyrosine phosphorylation of the epidermal growth factor receptor.

Epidermal growth factor (EGF) attenuated hCG-stimulated adenylyl cyclase activity in rat luteal and follicular membranes. H7, an equipotent serine/threonine protein kinase inhibitor of cAMP-dependent protein kinases, cGMP-dependent protein kinases, and lipid-dependent protein kinase C, did not effect the ability of EGF to decrease hCG-responsive adenylyl cyclase activity, suggesting that a serine/threonine phosphorylation event catalyzed by these kinases was not critically involved in EGF-induced desensitization. Likewise, pertussis toxin-catalyzed ADP-ribosylation of a 40-kDa luteal membrane protein, which exhibited immunoreactivity with an antibody against Gi alpha, did not hinder the ability of EGF to attenuate hCG-stimulated adenylyl cyclase activity, indicating that Gi did not mediate EGF-induced desensitization. Rather, EGF-induced heterologous desensitization of LH/CG receptor in ovarian membranes was closely associated with the specific and prominent tyrosine phosphorylation of the 170-kDa EGF receptor. Both EGF-stimulated autophosphorylation of EGF receptor and EGF-induced LH/CG receptor desensitization were attenuated by genistein, a tyrosine kinase inhibitor. These results suggest that tyrosine phosphorylation of the 170-kDa EGF receptor is a necessary component of the signaling pathway in EGF-induced heterologous desensitization of the LH/CG receptor.

Transforming growth factor-beta1 inhibits membrane association of protein kinase C alpha in a human prostate cancer cell line, PC3.

The postreceptor signaling pathway(s) that mediates the effects of transforming growth factor-beta1 (TGF-beta1) is incompletely understood. The present study investigated the involvement of protein kinase C (PKC) in the growth-inhibitory action of TGF-beta1 in PC3, a human prostate cancer cell line. PKC alpha, the only conventional PKC isoform detected in PC3 cells, appeared to be constitutively active based on its presence in both Triton-soluble membrane fraction and cytosol. However, levels of membrane-associated PKC alpha were decreased by a growth-inhibitory dose of TGF-beta1. The response to TGF-beta1 was rapid (within 5 min), time dependent, isoform specific, and occurred without apparent changes in levels of total PKC alpha protein. TGF-beta1 also decreased the levels of membrane-associated PKC activity coincident with its inhibitory effect on PKC alpha's membrane association. Inhibition of PKC activity appeared to be associated with growth inhibition in PC3 cells, because chelerythrine (a specific PKC inhibitor) likewise decreased cell proliferation. Taken together, our data suggest that inhibition of PKC activity, at least in part due to inactivation of PKC alpha, is an early event associated with TGF-beta1 postreceptor signaling that might mediate suppression of cell proliferation.

Transforming growth factor-beta1-induced proliferation of the prostate cancer cell line, TSU-Pr1: the role of platelet-derived growth factor.

The results of our previous study revealed that transforming growth factor-beta1 (TGFbeta1) stimulated proliferation of the prostate cancer cell line, TSU-Pr1. This observation is unexpected, for TGFbeta usually inhibits proliferation in prostate cancer cells. The present study examines possible mechanisms through which TGFbeta1 induces this proliferation. We postulate that TGFbeta1 action is mediated through an indirect mechanism by inducing the expression of platelet-derived growth factor (PDGF), which, in turn, stimulates proliferation. The TGFbeta1-induced proliferation can be abrogated by treatment with a PDGF-neutralizing antibody. Treatment with exogenous PDGF significantly increased TSU-Pr1 proliferation. Finally, treatment of TSU-Pr1 cells with TGFbeta1 resulted in an increase in PDGF secretion. These results indicate that TGFbeta1-induced proliferation in TSU-Pr1 cells is at least mediated through an increased secretion of PDGF.

The effect of protein kinases on desensitization of the porcine follicular membrane luteinizing hormone/chorionic gonadotropin-sensitive adenylyl cyclase.

Because the acute homologous phase of desensitization of the LH/CG-sensitive adenylyl cyclase in porcine follicles is readily demonstrated in a cell-free membrane preparation, it follows that any enzyme(s) required to achieve desensitization must be present in the membranes and must be activated upon LH/CG receptor activation. The purpose of the following studies was to determine whether modulation of endogenous membrane protein kinases, with activators or inhibitors, or addition of exogenous protein kinases affected desensitization of the LH/CG-sensitive adenylyl cyclase. The effects of these potential modulators were evaluated in both the presence and absence of ligand (hCG)-stimulated receptor activation. To this end, membranes were incubated in the presence or absence of hCG (stage 1) and then assayed for adenylyl cyclase activity in the presence or absence of hCG (stage 2). The results showed that although porcine follicular membranes rich in LH/CG-sensitive adenylyl cyclase activity also exhibited cAMP-dependent [protein kinase-A (PKA)], cGMP-dependent (PKG), lipid-dependent (PKC), Ca2+/calmodulin, and casein kinase-I and -II activities, only full hCG-stimulated adenylyl cyclase activity (measured with BSA in stage 1 and hCG in stage 2) was reduced upon addition of exogenous PKC (to the stage 1 incubation). hCG-dependent desensitization of cAMP synthesis (measured with hCG in stages 1 and 2) was unaffected by activators or inhibitors of endogenous PKA, PKC, or PKG, by an inhibitor of casein kinases and kinases in the beta-adrenergic receptor kinase family, or by the addition of exogenous active PKA, PKC, or rhodopsin kinase to the stage 1 incubation. These results suggest that the acute homologous phase of hCG-dependent desensitization of adenylyl cyclase activity in follicular membranes is not regulated by PKA, PKC, PKG, or messenger-independent heparin-sensitive protein kinases.

Luteinizing hormone/choriogonadotropin receptor-mediated activation of heterotrimeric guanine nucleotide binding proteins in ovarian follicular membranes.

The LH/CG receptor signals to adenylyl cyclase via the stimulatory heterotrimeric GTP binding regulatory protein, Gs, and to phospholipase C and potentially to other effectors, such as ion channels, via a G protein or proteins that have not been identified in gonadal cells. To identify G proteins activated in a physiological membrane environment upon LH/CG receptor activation, we used the ability of activated G proteins to bind GTP and incubated ovarian follicular membranes with the photoaffinity GTP analog, P3-(4-azidoanilido)-P1-5'-GTP ([32P]AAGTP). Results showed that human CG (hCG) stimulated the binding of [32P]AAGTP to a 45-kDa protein(s) in follicular membranes that comigrated with immunoreactive G alphas, G alphaq/11, and G alpha13. When G alpha proteins were immunoprecipitated from Triton X-100 solubilized membrane extracts after photoaffinity labeling with [32P]AAGTP, a time-dependent increase in hCG-dependent [32P]AAGTP binding to G alphas, G alphaq/11, and G alphai was detected. hCG-dependent [32P]AAGTP binding to G alpha13 was also detected. These results demonstrate that agonist-dependent LH/CG receptor activation promotes the activation of Gs, Gi, Gq/11, and G13 in porcine ovarian follicular membranes. These results further suggest that G alphas remains coupled to the agonist-bound LH/CG receptor during at least the initial 10 min after agonist-dependent receptor activation.

Monoclonal antibodies specific for rat relaxin. II. Passive immunization with monoclonal antibodies throughout the second half of pregnancy disrupts birth in intact rats.

The purpose of this investigation was to use an approach targeted specifically on endogenous relaxin to determine the influence of relaxin on birth in the rat. To that end, a monoclonal antibody specific for rat relaxin, designated MCA1, was used to passively neutralize endogenous relaxin in intact pregnant rats. MCA1 was administered iv to intact rats daily from days 12-22 of pregnancy. Animals were observed for birth continuously from 2100 h on day 22 until 1200 h on day 24. MCA1-treated rats exhibited significantly prolonged durations of straining and litter delivery as well as reduced incidence of live pups compared with controls. Whereas approximately 20-25% of fetuses and placentae were retained in utero at 1200 h on day 24 in MCA1-treated rats, neither fetuses nor placentae were retained in control rats. Passive immunization with MCA1 throughout the second half of pregnancy had no apparent influence on normal ovarian function. The time of occurrence of the antepartum decline in serum progesterone levels (functional luteolysis) as well as the time interval between the attainment of basal progesterone levels and the onset of litter delivery in MCA1-treated and control rats that delivered on day 23 were in close agreement with previous reports. In conclusion, the prolonged durations of straining and litter delivery, reduced incidence of live pups, and increased incidence of retained fetuses and placentae after passive immunization with MCA1 establish the physiological need for endogenous relaxin to attain normal delivery of the young in the rat.

Monoclonal antibodies specific for rat relaxin. I. Production and characterization of monoclonal antibodies that neutralize rat relaxin's bioactivity in vivo.

The physiological role of relaxin during pregnancy and at parturition in the rat is not absolutely established. There are limitations to the experimental approach used in the few studies that examined the influence of relaxin in the pregnant rat. These studies were unphysiological, since they involved administration of porcine relaxin as well as progesterone and estrogen to ovariectomized pregnant rats. A more physiological approach is to use antibodies to neutralize the biological actions of endogenous relaxin in the intact pregnant rat. The purpose of the present study was to produce and characterize monoclonal antibodies suitable for this approach. Six stable and rapidly growing hybridoma clones which produced monoclonal antibodies specific for rat relaxin (MCA-rR) were obtained after the fusion of NSO mouse myeloma cells with lymphocytes from the spleen of a BALB/c mouse immunized with rat relaxin. Five MCA-rR (MCA1-5; all immunoglobulin G1 kappa) inhibited the ability of exogenously administered rat relaxin to increase the interpubic ligament length in estrogen-primed mice. Of the five MCA-rR that neutralized rat relaxin's bioactivity in vivo, MCA1 exhibited the highest relative affinity for rat relaxin. MCA1 was also highly specific for rat relaxin. MCA1 demonstrated no cross-reactivity with rat insulin, rat insulin-like growth factor I and II, or porcine relaxin-proteins that are structurally related to rat relaxin. In view of its high affinity and high specificity for rat relaxin as well as its ability to neutralize rat relaxin's bioactivity in vivo, MCA1 was selected for use in subsequent studies aimed at the neutralization of endogenous relaxin in intact pregnant rats.

Hormonal regulation of PKC-delta protein and mRNA levels in the rabbit corpus luteum.

We have previously reported that rabbit corpora lutea exhibit a prominent phosphorylated substrate protein at 76 kDa which corresponds to the autophosphorylated form of protein kinase C (PKC) delta and that the expression of PKC-delta protein is increased in rabbit corpora lutea of pseudopregnancy at least 2-fold when serum estrogen levels are raised by the presence of an estrogen implant inserted at the time of human chorionic gonadotropin (hCG)-induced ovulation. The purpose of the experiments described herein was to evaluate further the hormonal regulation of PKC-delta in the rabbit corpus luteum. Results demonstrate that luteal PKC-delta protein and mRNA are concomitantly induced some 5-fold within 48 h in response to an ovulatory surge of hCG; that, as in corpora lutea of pseudopregnancy, luteal PKC-delta expression is relatively constant during the life span of the corpus luteum following a fertile mating; that exogenous estrogen does not modulate the induction of luteal PKC-delta during luteinization but promotes an additional two-fold increase in steady state PKC-delta mRNA (and protein) levels in corpora lutea by day 10 of pseudopregnancy; and that luteal PKC-delta expression can be abruptly and reversibly modulated upon withdrawal and subsequent replacement of an estrogen implant to pseudopregnant rabbits. These results demonstrate that an ovulatory surge of luteinizing hormone induces the expression of PKC-delta mRNA and protein in rabbit corpora lutea, and that once the corpus luteum becomes estrogen responsive, estrogen then regulates expression of PKC-delta mRNA and protein.

Reversal of the desensitized state of pig ovarian follicular human choriogonadotropin-sensitive adenylylcyclase by guanosine 5'-O-(2-thiodiphosphate).

We investigated the stability of the desensitized state of the human choriogonadotropin (hCG)-sensitive adenylylcyclase of the pig ovarian follicle. A 20,000 x g membrane preparation of pig follicular membranes was incubated under conditions which resulted in the hormone-induced desensitization of the hCG-responsive adenylylcyclase. The desensitized state was maintained upon subsequent incubation of the membranes with GTP, GDP, GMP, ATP, ADP, AMP, CTP, UTP, adenyl-5'-yl imidodiphosphate (AMP-P(NH)P), and adenyl (beta, gamma-methylene)-diphosphonate (AMP-P(CH2)P); however, the desensitized state was reverted to a fully active state upon incubation with guanosine 5'-O-(2-thiodiphosphate) (GDP beta S) and guanosine 5'-O-(3-thiotriphosphate) (GTP gamma S). The reversal effect of GDP beta S on hCG-responsive adenylylcyclase activity was time- and temperature-dependent, and showed a selectivity for GDP beta S over adenosine 5'-O-(2-thiodiphosphate) (ADP beta S) (half-maximal effective dose of 12 microM versus 260 microM, respectively). GDP beta S had no effect on the binding affinity or apparent number of luteinizing hormone (LH)/CG receptors or on the dissociation rate of 125I-hCG from the receptor. GDP beta S promoted an hCG- and time-dependent release of guanine nucleotides from the membranes. A model is proposed which accounts for the unique characteristics of LH/CG-sensitive adenylylcyclase desensitization and subsequent reactivation by GDP beta S.

Isozymes of cAMP-dependent protein kinase present in the rat corpus luteum.

Regulatory (R) subunits and their association with catalytic subunits to form cAMP-dependent protein kinase holoenzymes were investigated in corpora lutea of pregnant rats. Following separation by DEAE-cellulose chromatography, R subunits were identified by labeling with 8-N3[32P]cAMP and autophosphorylation on one and two-dimensional gel electrophoresis and by reactivity with antisera. DEAE-cellulose elution of R subunits with catalytic subunits as holoenzymes or without catalytic subunits was determined by sedimentation characteristics on sucrose density gradient centrifugation and by cAMP-stimulated kinase activation characteristics on Eadie-Scatchard analysis. We identified the presence of a type I holoenzyme containing RI alpha (Mr 47,000) subunits, a prominent type II holoenzyme containing RII beta (Mr 52,000) subunits, and a second more acidic type II holoenzyme peak containing both RII beta and RII alpha (Mr 54,000) subunits. However, the majority of total R subunit activity was associated with a catalytic subunit-free peak of RI alpha protein which on elution from DEAE-cellulose was associated with cAMP. This report establishes the more basic elution position from DEAE-cellulose of the prominent rat luteal RII beta holoenzyme in very close proximity to free RI alpha and presents one of the few reports of a normal tissue containing a large percentage of catalytic subunit-free RI alpha.

Mesenchymal factor bone morphogenetic protein 4 restricts ductal budding and branching morphogenesis in the developing prostate.

The budding of the urogenital sinus epithelium into the surrounding mesenchyme signals the onset of prostate morphogenesis. The epithelial and mesenchymal factors that regulate ductal budding and the ensuing process of ductal growth and branching are not fully known. We provide evidence that bone morphogenetic protein 4 (BMP4) is a mesenchymal factor that regulates ductal morphogenesis. The Bmp4 gene was most highly expressed in the male urogenital sinus from embryonic day 14 through birth, a period marked by formation of main prostatic ducts and initiation of ductal branching. From an initial wide distribution throughout the prostatic anlage of the urogenital sinus, Bmp4 expression became progressively restricted to the mesenchyme immediately surrounding the nascent prostatic ducts and branches. Exogenous BMP4 inhibited epithelial cell proliferation and exhibited a dose-dependent inhibition of ductal budding in urogenital sinus tissues cultured in vitro. Adult Bmp4 haploinsufficient mice exhibited an increased number of duct tips in both the ventral prostate and coagulating gland. Taken together, our data indicate that BMP4 is a urogenital sinus mesenchymal factor that restricts prostate ductal budding and branching morphogenesis.

Activation of the luteinizing hormone/choriogonadotropin hormone receptor promotes ADP ribosylation factor 6 activation in porcine ovarian follicular membranes.

Previously we demonstrated in a cell-free ovarian follicular plasma membrane model that agonist-dependent desensitization of the luteinizing hormone/choriogonadotropin receptor (LH/CG R) is GTP-dependent, mimicked by the addition of ADP-ribosylation factor (ARF) nucleotide binding site opener, which acts as a guanine nucleotide exchange factor for ARFs 1 and 6, and selectively inhibited by synthetic N-terminal ARF6 peptides. We therefore sought direct evidence that activation of the LH/CG R promotes activation of ARF1 and/or ARF6. Using a classic ARF activation assay, the cholera toxin-catalyzed ADP-ribosylation of G alpha(s), results show that LH/CG R activation stimulates an ARF protein by a brefeldin A-independent mechanism. Synthetic N-terminal inhibitory ARF6 but not ARF1 peptide blocks LH/CG R-stimulated ARF activity. LH/CG R activation also promotes the binding of a photoaffinity GTP analog to a protein that migrates on one- and two-dimensional polyacrylamide gel electrophoresis with ARF6. These results suggest that ARF6 is the predominant ARF activated by the LH/CG R. To activate ARF6, the LH/CG R does not appear to signal through the C-terminal regions of G alpha(i) or G alpha(q) or through the second or third intracellular loops or the N terminus of the cytoplasmic tail of the LH/CG R. Although exogenous recombinant ARNO promotes only a small increase in ARF6 activation in the presence of activated LH/CG R, hCG-stimulated ARF6 activation is reduced to basal levels by catalytically inactive ARF nucleotide binding-site opener. These results provide direct evidence that LH/CG R activation leads to the activation of membrane-delimited ARF6.

The physiological effects of relaxin during pregnancy: studies in rats and pigs.

For 50 years after its discovery in 1926, there was a general lack of interest in relaxin among both reproductive biologists and clinicians. A key reason for this lack of interest was the lack of information concerning relaxin's physiological importance during pregnancy in any species. Research conducted since the early 1980s has established that the hormone relaxin is essential during pregnancy in at least two species--rats and pigs. Two vital roles for relaxin during pregnancy have been identified. Relaxin promotes growth and softening of the uterine cervix and thereby enables rapid and safe delivery in both rats and pigs. Relaxin also promotes growth and development of the mammary apparatus in both species. Interestingly, the major effects of relaxin on mammary growth and development are targeted on the nipple in the rat, whereas they are targeted on the glandular parenchyma in the pig. Relaxin-dependent growth of the nipple in rats is required for normal lactational performance. Although likely, it remains to be established that relaxin's profound effects upon mammary gland development in pigs are required for normal lactational performance. The fact that relaxin has effects upon cervical and mammary gland development during pregnancy in both rats and pigs encourages the view that relaxin may have similar effects during pregnancy in other species. Nevertheless, one must keep in mind that there is great diversity in the physiology of relaxin among species (reviewed by Sherwood 1988). This diversity includes not only relaxin's source, regulation of synthesis and secretion, and secretory profiles during pregnancy, but also its biological effects. It seems essentially certain that relaxin's effects during pregnancy differ among species. For example, transformation of the pubic joint cartilage to a flexible and elastic interpubic ligament occurs during pregnancy in several species, including guinea pigs, mice, and bats. This pelvic adaptation, which is nearly certainly relaxin dependent, does not occur in species such as rats and sheep. It is possible that relaxin may have little or no physiological significance during pregnancy in some species. Although considerable progress has been made toward an understanding of the physiological role(s) of relaxin in pregnant rats and pigs, many fundamental questions remain unanswered.(ABSTRACT TRUNCATED AT 400 WORDS)