Autocrine growth hormone: effects on growth hormone receptor trafficking and signaling

GH and GH receptor are expressed in many extrapituitary tissues, permitting autocrine/paracrine activity. Autocrine GH has regulatory functions in embryonic development and cellular differentiation and proliferation and is reported to be involved in the development and metastasis of tumor cells. To understand the principles of transport and signaling of autocrine GH and GH receptor, we used a model system to express both proteins in the same cell. Our experiments show that GH binds the GH receptor immediately after synthesis in the endoplasmic reticulum and facilitates maturation of GH receptor. The hormone-receptor complexes arrive at the cell surface where exogenously added GH is unable to bind these receptors. Autocrine GH activates the GH receptors, but signal transduction occurs only after exiting the endoplasmic reticulum. This model study explains why autocrine GH-producing cells may be insensitive for GH (antagonist) treatment and clarifies autocrine signaling events.     

Sensitivity of rat forebrain neurons to growth hormone-releasing hormone

The effects of iontophoretically applied human pancreatic growth hormone-releasing factor (hpGRF), peptide histidine isoleucine (PHI-27), and somatostatin (SS) on the extracellular activity of single cells in the hypothalamus, thalamus, and cortex of the rat brain were studied in urethane-anesthetized, male rats. Neurons with membrane sensitivity to hpGRF, PHI-27, and SS were present in each brain region. Although neurons excited by these peptides were encountered in thalamus and hypothalamus, depression of neuronal firing was the predominant response observed. Overall, the neurons responding to hpGRF also possessed membrane sensitivity to PHI-27, whereas, the hpGRF sensitive neurons appeared to be more divided as to their ability to respond to SS. The results clearly demonstrate that hpGRF and PHI-27 are capable of affecting the membrane excitability of neurons in several brain regions. The distribution of neurons sensitive to hpGRF suggests that hypothalamic GRF, in addition to its well documented role in the regulation of pituitary growth hormone secretion, may subserve other physiological events in the rat central nervous system as a neurotransmitter and/or neuromodulator.     

Biological activities of two porcine growth hormone-releasing hormone receptor isoforms

Binding of growth hormone-releasing hormone (GHRH) to two isoforms (G3R and G5R) of the porcine GHRH receptor was studied. Both G3R- and G5R-cDNA were isolated from a porcine anterior pituitary cDNA library and have an identical primary structure from aa 1 to 418 and a different aa sequence from aa 419 to 423. In addition, the G5R isoform contains an extra C-terminal tail of 28 aa. The G3R and G5R mRNAs arise from alternative splicing of a single precursor mRNA for GHRH receptors. A mammalian cell expression vector containing either G3R or G5R cDNA under the regulation of a strong human cytomegalovirus promoter was constructed and used to transfect a human embryonic kidney 293 cell line. Two stable transfectants (293/G3R-4 and 293/G5R-12) were isolated on the basis of high expression of the receptor mRNAs. Both G3R and G5R mRNAs were expressed at similarly high levels in 293/G3R-4 and 293/G5R-12 cells; however, GHRH binding to 293/G3R-4 cells was much greater than that observed for 293/G5R-12 cells. Basal as well as GHRH-stimulated GTPase activity and intracellular cAMP concentration are also significantly greater in 293/G3R-4 cells as compared to 293/G5R-12 cells. We conclude that the modification of GHRH receptor at the C-terminal region hindered GHRH binding to the receptor and thus attenuates its biological activities.     

Cellular distribution of growth hormone-releasing hormone receptor in human reproductive system and breast and prostate cancers

Growth hormone releasing hormone receptor (GHRH-R) mRNA and protein was first localized to the anterior pituitary gland, consequent with the action of its ligand on GH synthesis and release. Subsequent studies found GHRH-R also expressed in the hypothalamus and in systemic tissues including those of the reproductive system. In the present work, we studied the distribution of GHRH-R in human reproductive system of males and females by immunohistochemical method. GHRH-R immunostaining was localized in male reproductive system: Leydig cells, Sertoli and basal germ cells of the seminiferous tubules and prostate secretory cells. GHRH-R immunostaining was also demonstrated in the ovary: oocytes, follicular cells, granulosa, thecal and corpus luteum cells. Endometrial glands, placenta and normal mammary glands also showed GHRH-R immunostaining. Our results demonstrate the localization of GHRH-R in the reproductive system, which may mediate the direct action of GHRH in these tissues. Moreover, GHRH-R was demonstrated in prostate and breast carcinomas, opening a variety of possibilities for the use of GHRH antagonists in the treatment of prostatic and mammary tumors.     

In vivo biological potency of rat and human growth hormone-releasing factor and fragments of human growth hormone-releasing factor

The biological potency of the synthetic replicates of three peptides isolated from a human pancreatic tumor with growth hormone releasing activity and rat hypothalamic growth hormone-releasing factor was evaluated in conscious freely-moving rats and anesthetized rats. All 4 peptides are equipotent on a molar basis in their ability to stimulate GH secretion. Studies with synthetic fragments of the human derived material indicated that the amino-terminal amino acid is required for activity. Deletion of as many as 13 amino acids from the carboxy-terminal failed to decrease GH-releasing activity; however, deletion of 16 amino acids resulted in a significant decrease and deletion of 20 amino acids resulted in complete loss of bioactivity.     

Growth hormone-releasing hormone antagonists inhibit growth of human ovarian cancer

Epithelial ovarian carcinoma is the leading cause of cancer-related deaths among women with gynecologic malignancies. Antagonists of the growth hormone-releasing hormone (GHRH) have been shown to inhibit growth of various cancers through endocrine, autocrine, and paracrine mechanisms. In this study, we have investigated the effects of GHRH antagonists (GHRHa) in ES-2 human clear cell ovarian cancer and in UCI-107 human serous ovarian cancer in vitro and in vivo. We evaluated the expression of mRNA for GHRH receptor, the binding to GHRH receptors, in specimens of ES-2 ovarian cancer. We evaluated also the in vitro effects of GHRHa on ES-2 cells and the in vivo effect of 2 different GHRHa on ES-2 and UCI-107 tumors. Nude mice bearing xenografts on ES-2 and UCI-107 ovarian cancer were treated with JMR-132 and MZ-J-7-118, respectively. Tumor growth was compared to control. ES-2 cells expressed mRNA for the functional splice variant SV1 of the GHRH receptor. JMR-132 inhibited cell proliferation in vitro by 42% and 18% at 10 and 1 μM concentration, respectively. Specific high affinity receptors for GHRH were detected in ES-2 cancer samples. In vivo daily subcutaneous injections of GHRHa significantly reduced tumor growth compared to a control group in both animal models. Our results indicate that GHRHa such as JMR-132 and MZ-J-7-118 can inhibit the growth of human ovarian cancer. The efficacy of GHRHa in ovarian cancer should be assessed in clinical trials.     

Growth hormone-releasing hormone induced transactivation of epidermal growth factor receptor in human triple-negative breast cancer cells

Triple-negative breast cancer (TNBC) is a subset of breast cancers which is negative for expression of estrogen and progesterone receptors and human epidermal growth factor receptor-2 (HER2). Chemotherapy is currently the only form of treatment for women with TNBC. Growth hormone-releasing hormone (GHRH) and epidermal growth factor (EGF) are autocrine/paracrine growth factors in breast cancer and a substantial proportion of TNBC expresses receptors for GHRH and EGF. The aim of this study was to evaluate the interrelationship between both these signaling pathways in MDA-MB-468 human TNBC cells. We evaluated by Western blot assays the effect of GHRH on transactivation of EGF receptor (EGFR) as well as the elements implicated. We assessed the effect of GHRH on migration capability of MDA-MB-468 cells as well as the involvement of EGFR in this process by means of wound-healing assays. Our findings demonstrate that in MDA-MB-468 cells the stimulatory activity of GHRH on tyrosine phosphorylation of EGFR is exerted by two different molecular mechanisms: i) through GHRH receptors, GHRH stimulates a ligand-independent activation of EGFR involving at least cAMP/PKA and Src family signaling pathways; ii) GHRH also stimulates a ligand-dependent activation of EGFR implicating an extracellular pathway with an important role for metalloproteinases. The cross-talk between EGFR and GHRHR may be impeded by combining drugs acting upon GHRH receptors and EGFR family members. This combination of GHRH receptors antagonists with inhibitors of EGFR signalling could enhance the efficacy of both types of agents as well as reduce their doses increasing therapeutic benefits in management of human breast cancer.     

Molecular cloning and expression of a pituitary-specific receptor for growth hormone-releasing hormone.

A novel cDNA was isolated from rat pituitary mRNA using the polymerase chain reaction to amplify sequences encoding G protein-coupled receptors. The human homolog of this cDNA was isolated and expressed in human kidney 293 cells, and membrane fractions from these cells were found to bind human GH-releasing hormone (GHRH) with high affinity and specificity. GHRH also stimulates intracellular cAMP production in these transfected cells. The encoded receptor protein contains seven potential membrane-spanning domains, a hallmark of G protein-coupled receptors, and is homologous to previously identified receptors for secretin and vasoactive intestinal peptide, ligands that are related to GHRH. The rat GHRH receptor mRNA is expressed predominantly, if not exclusively, in the anterior pituitary gland, the major target for GHRH action. These results define a mechanism for cellular signaling by GHRH and provide the opportunity to examine the role of the GHRH receptor in growth abnormalities that involve the GH axis.     

Characterization of a growth hormone-releasing hormone binding site in the rat renal medulla

Receptor binding analysis was performed in the renal medulla from 2-month-old rats, an extrapituitary tissue containing the highest level of GHRH receptor mRNA. At 4 degrees C, in the presence of a cocktail of protease inhibitors, binding of [125I-Tyr(10)]hGHRH (1-44)NH(2) to medullary homogenates was specific, time-dependent, reversible and saturable (K(d): 28 nM; B(max): 30 fmol/mgprot.). In these experimental conditions, no change of binding parameters could be detected in the course of aging. The structure-affinity profile was different in the two tissues and chemical cross-linking revealed the presence of 65-, 55- and 38-kDa 125I-GHRH-labeled complexes in the renal medulla compared to 65-, 47- and 28-kDa radioactive complexes in the anterior pituitary. It is suggested that GHRH binding sites, and possibly the receptor, may be different in the two tissues.     

Response of growth hormone release to human growth hormone-releasing factor and its analogs in the bovine

Responses of growth hormone (GH) release to synthetic human growth hormone-releasing factor (hGRF)-44-NH2 analogs were determined, and the GH-releasing potency based on dose per kg of body weight (bw) was compared with that of hGRF-44-NH2 in female dairy calves. Four- and 12-month-old calves were injected intravenously with 0.25 microgram of hGRF-44-NH2 or its analogs per kg of bw. Blood samples were collected before, and during 180 min after each injection, and plasma GH concentrations were measured by radioimmunoassay. Areas under the GH response curves for 180 min after injection of hGRF-44-NH2 and its analogs were used as an index of the GH-releasing potency of each peptide. The GH-releasing potency of hGRF(1-26)-NH2 was significantly lower than that of hGRF-44-NH2 (P less than 0.05). On the other hand, hGRF(1-29)-NH2 possessed similar potency to hGRF-44-NH2. [D-Tyr1]-hGRF-44-NH2 showed prolonged GH-releasing activity, though its potency was similar to that of hGRF-44-NH2. Also, [D-Ala2]-hGRF(1-29)-NH2 exhibited prolonged GH-releasing activity, and its potency was 2.5 (P less than 0.05) and twice (P less than 0.05) as great as that of hGRF-44-NH2 and hGRF(1-29)-NH2, respectively. These results demonstrate that the N-terminal 29 amino acid residues of hGRF possess the activity site required for full GH release in vivo, and [D-Ala2]-hGRF(1-29)-NH2 has longer and greater activity, on a dose basis, than hGRF-44-NH2 in the calves.     

Characterization of a growth hormone-releasing hormone binding site in the rat renal medulla

Receptor binding analysis was performed in the renal medulla from 2-month-old rats, an extrapituitary tissue containing the highest level of GHRH receptor mRNA. At 4 degrees C, in the presence of a cocktail of protease inhibitors, binding of [125I-Tyr(10)]hGHRH NH(2) to medullary homogenates was specific, time-dependent, reversible and saturable (K(d): 28 nM; B(max): 30 fmol/mg prot.). In these experimental conditions, no change of binding parameters could be detected in the course of aging. The structure-affinity profile was different in the two tissues and chemical cross-linking revealed the presence of 65-, 55- and 38-kDa 125I-GHRH-labeled complexes in the renal medulla compared to 65-, 47- and 28-kDa radioactive complexes in the anterior pituitary. It is suggested that GHRH binding sites, and possibly the receptor, may be different in the two tissues.     

Internalization and sequestration of the human prostacyclin receptor

Prostacyclin (PGI(2)), the major product of cyclooxygenase in macrovascular endothelium, mediates its biological effects through its cell surface G protein-coupled receptor, the IP. PKC-mediated phosphorylation of human (h) IP is a critical determinant of agonist-induced desensitization (Smyth, E. M., Hong Li, W., and FitzGerald, G. A. (1998) J. Biol. Chem. 273, 23258-23266). The regulatory events that follow desensitization are unclear. We have examined agonist-induced sequestration of hIP. Human IP, tagged at the N terminus with hemagglutinin (HA) and fused at the C terminus to the green fluorescent protein (GFP), was coupled to increased cAMP (EC(50) = 0.39 +/- 0.09 nm) and inositol phosphate (EC(50) = 86. 6 +/- 18.3 nm) generation when overexpressed in HEK 293 cells. Iloprost-induced sequestration of HAhIP-GFP, followed in real time by confocal microscopy, was partially colocalized to clathrin-coated vesicles. Iloprost induced a time- and concentration-dependent loss of cell surface HA, indicating receptor internalization, which was prevented by inhibitors of clathrin-mediated trafficking and partially reduced by cotransfection of cells with a dynamin dominant negative mutant. Sequestration (EC(50) = 27.6 +/- 5.7 nm) was evident at those concentrations of iloprost that induce PKC-dependent desensitization. Neither the PKC inhibitor GF109203X nor mutation of Ser-328, the site for PKC phosphorylation, altered receptor sequestration indicating that, unlike desensitization, internalization is PKC-independent. Deletion of the C terminus prevented iloprost-induced internalization, demonstrating the critical nature of this region for sequestration. Internalization was unaltered by cotransfection of cells with G protein-coupled receptor kinases (GRK)-2, -3, -5, -6, arrestin-2, or an arrestin-2 dominant negative mutant, indicating that GRKs and arrestins do not play a role in hIP trafficking. The hIP is sequestered in response to agonist activation via a PKC-independent pathway that is distinct from desensitization. Trafficking is dependent on determinants located in the C terminus, is GRK/arrestin-independent, and proceeds in part via a dynamin-dependent clathrin-coated vesicular endocytotic pathway although other dynamin-independent pathways may also be involved.     

Subcellular trafficking of growth hormone receptor and Jak2 under ligand exposure.

In CHO cells, growth hormone stimulation induces a rapid degradation of mature and precursor forms of its receptor, but does not affect Jak2 concentration. Confocal analysis of the receptor and of specific markers for subcellular localization shows that ligand exposure induced the disappearance of cell surface receptors, while some receptors seem to be sequestered in the endoplasmic reticulum (ER) and in the Golgi apparatus. Using a tagged version of Jak2 (HA-Jak2) and double immuno-fluorescence analysis with anti-HA and anti-Stat5 antibodies, we demonstrate that ligand stimulation induces Stat5 nuclear accumulation while Jak2 remains localized in the cytoplasm. Immunoblots of nuclear extracts confirm the Jak2 nuclear exclusion.     

Growth hormone regulation of growth hormone-releasing hormone gene expression

Slot-blot hybridization technique was used to evaluate growth hormone-releasing hormone (GHRH) mRNA levels in the hypothalamus of long-term (14 days) hypophysectomized (HPX) rats treated or not with 125 micrograms hGH/rat, twice daily IP, since the first day postsurgery. In addition, mRNA levels were determined in the hypothalamus of short-term (4 days) GH-treated (250 micrograms hGH/rat, twice daily IP) intact rats. GHRH mRNA levels were increased in HPX rats, and GH treatment partially counteracted this rise. Short-term administration of GH decreased GHRH mRNA levels in intact rats. These results, evaluated together with previous findings showing decreased hypothalamic GHRH-like immunoreactivity in both HPX rats and intact rats given GH (6, 7, 9), indicate that GH exerts a negative feedback action on the synthesis and release of GHRH.     

Binding and internalization of gold-conjugated somatostatin and growth hormone-releasing hormone in cultured rat somatotropes

Summary The synthetic peptides somatostatin (SRIF) and growth hormone-releasing hormone (GRH) were coupled directly to colloidal gold of different particle sizes. Both conjugates were biologically active in displacing the corresponding radiolabeled hormones from high affinity binding sites in pituitary membranes. Release of growth hormone (GH) from cultured anterior pituitary cells was modulated by both conjugates alone or in combination. Ultrastructural studies were performed with cells incubated at 4° C (2 h) and 37° C (2 min-2 h) with one of the labeled peptides or their combination. Somatotropes were identified by immunostaining with anti-rGH followed by protein A-ferritin, thus obtaining a triple labeling. Both hormone conjugates were internalized in different vesicles in the beginning but accumulated during longer incubation times in the same compartment. The secretory vesicles and the nucleus were not labeled by any hormone conjugate. In contrast to SRIF-gold, the uptake of GRH-gold conjugate decreased with longer incubation times. This effect could be neutralized by simulatenous incubation of the somatotropes with both regulating hormones. Hence, whereas the binding and internalization of SRIF by somatotropes do not seem to be influenced by GRH, the corresponding processes for GRH are stimulated by the presence of SRIF.     

Estradiol modulation of growth hormone secretion in the ewe: no growth hormone-releasing hormone neurons and few somatotropes express estradiol receptor alpha

Evidence suggests that estrogen modulates growth hormone (GH) release and that GH plays an important role in follicular and ovulatory processes. How estradiol affects GH secretion is unclear. Having verified that there is a coincident surge of GH at the time of the preovulatory LH surge, immunocytochemical studies incorporating high-temperature antigen retrieval were used to determine whether GH-releasing hormone (GHRH) neurons, somatotropes, or both, expressed estrogen receptor alpha (ER), in the ewe. Although GHRH neurons were surrounded by many ER cells, they did not express immunocytochemically detectable ERs. In contrast to gonadotropes, in which the majority expressed ERs, few somatotropes were estrogen receptive. These data suggest that estrogen does not act directly on GHRH neurons to influence GH secretion, and any direct effect on pituitary GH release, through the ERalpha, may be small.