Multiple signaling pathways convey central and peripheral signals to regulate pituitary function: Lessons from human and non-human primate models.

The anterior pituitary gland is a key organ involved in the control of multiple physiological functions including growth, reproduction, metabolism and stress. These functions are controlled by five distinct hormone-producing pituitary cell types that produce growth hormone (somatotropes), prolactin (lactotropes), adrenocorticotropin (corticotropes), thyrotropin (thyrotropes) and follicle stimulating hormone/luteinizing hormone (gonadotropes). Classically, the synthesis and release of pituitary hormones was thought to be primarily regulated by central (neuroendocrine) signals. However, it is now becoming apparent that factors produced by pituitary hormone targets (endocrine and non-endocrine organs) can feedback directly to the pituitary to adjust pituitary hormone synthesis and release. Therefore, pituitary cells serve as sensors to integrate central and peripheral signals in order to fine-tune whole-body homeostasis, although it is clear that pituitary cell regulation is species-, age- and sex-dependent. The purpose of this review is to provide a comprehensive, general overview of our current knowledge of both central and peripheral regulators of pituitary cell function and associated intracellular mechanisms, focusing on human and non-human primates.

Somatostatin dramatically stimulates growth hormone release from primate somatotrophs acting at low doses via somatostatin receptor 5 and cyclic AMP.

Somatostatin and cortistatin have been shown to act directly on pituitary somatotrophs to inhibit growth hormone (GH) release. However, previous results from nonprimate species indicate that these peptides can also directly stimulate GH secretion, at low concentrations. The relevance of this phenomenon in a nonhuman primate model was investigated in the present study by testing the impact of somatostatin/cortistatin on GH release in primary pituitary cell cultures from baboons. High doses (> 10(-10) m) of somatostatin/cortistatin did not alter basal GH secretion but blocked GH-releasing hormone (GHRH)- and ghrelin-induced GH release. However, at low concentrations (10(-17)-10(-13) m), somatostatin/cortistatin dramatically stimulated GH release to levels comparable to those evoked by GHRH or ghrelin. Use of somatostatin receptor (sst) specific agonists/antagonists, and signal transduction blockers indicated that sst2 and sst1 activation via intact adenylate cylcase and mitogen-activated protein kinase systems mediated the inhibitory actions of high-concentration somatostatin. By contrast, the stimulatory actions of low-dose somatostatin on GH release were mediated by sst5 signalling through adenylate cylcase/cAMP/protein kinase A and intracellular Ca(2+) pathways, and were additive with ghrelin (not GHRH). Notably, low-concentrations of somatostatin, similar to sst5-agonists, inhibited prolactin release. These results clearly demonstrate that the ultimate impact of somatostatin/cortistatin on hormone release is dose-dependent, cell type-selective and receptor-specific, where the stimulatory effects of low-concentration somatostatin/cortistatin on GH release extend to primates, thereby supporting the notion that this action is relevant in regulating GH secretion in humans.

Cortistatin mimics somatostatin by inducing a dual, dose-dependent stimulatory and inhibitory effect on growth hormone secretion in somatotropes.

Cortistatin is a recently discovered neuropeptide that is structurally related to somatostatin, the classic inhibitor of growth hormone (GH) release. Cortistatin binds with high affinity to all five somatostatin receptors (sst1-5), and, like somatostatin, cortistatin inhibits in vivo GH release in man and rats. In this report, we compared the in vitro actions of cortistatin and somatostatin using primary pig pituitary cell cultures. In this species, we have previously reported that somatostatin not only inhibits GH-releasing hormone (GHRH)-stimulated GH release at high doses, but also stimulates basal GH release at low (pM) doses, a dual response that is markedly dependent on the subpopulation of pituitary somatotropes examined. Results reported herein demonstrate that cortistatin closely mimics the dose-dependent inhibitory and stimulatory effects of somatostatin on GH secretion. As cortistatin, unlike somatostatin, binds to the human receptor for ghrelin/GH secretagogs (GHS-R), we also investigated whether cortistatin stimulates GH release through this receptor by using a synthetic, short form of cortistatin, cortistatin-8 (CST8), which lacks the sst-binding capacity of full-length cortistatin but retains its GHS-R-binding capacity. Interestingly, CST8 stimulated GH release only at low doses (10(-15) M), and did not reduce GH secretion stimulated by GHRH, ghrelin, or low-dose, full-length cortistatin, yet it counteracted that induced by a nonpeptidyl GHS, L-163 255. Taken together, our results indicate that the dual, inhibitory and stimulatory effects of cortistatin on GH release closely parallel those of somatostatin and are probably mediated by the same receptor(s) and signaling pathway(s) for both peptides. Furthermore, they suggest that the pathway(s) activated by cortistatin (and somatostatin) to stimulate GH release are not initiated by GHS-R activation.

Differential responses of the growth hormone axis in two rat models of streptozotocin-induced insulinopenic diabetes.

The impact of streptozotocin (STZ)-induced, insulinopenic diabetes on the GH axis of rats and mice differs from study to study, where this variation may be related to the induction scheme, severity of the diabetes and/or the genetic background of the animal model used. In order to begin differentiate between these possibilities, we compared the effects of two different STZ induction schemes on the GH axis of male Sprague-Dawley rats: (1) a single high-dose injection of STZ (HI STZ, 80 mg/kg, i.p.), which results in rapid chemical destruction of the pancreatic beta-cells, and (2) multiple low-dose injections of STZ (LO STZ, 20 mg/kg for 5 consecutive days, i.p.), which results in a gradual, autoimmune destruction of beta-cells. STZ-treated animals were killed after 3 weeks of hyperglycemia (>400 mg/dl), and in both paradigms circulating insulin levels were reduced to <40% of vehicle-treated controls. HI STZ-treated rats lost weight, while body weights of LO STZ-treated animals gradually increased over time, similar to vehicle-treated controls. As previously reported, HI STZ resulted in a decrease in circulating GH and IGF-I levels which was associated with a rise in hypothalamic neuropeptide Y (NPY) mRNA (355% of vehicle-treated controls) and a fall in GH-releasing hormone (GHRH) mRNA (45% of vehicle-treated controls) levels. Changes in hypothalamic neuropeptide expression were reflected by an increase in immunoreactive NPY within the arcuate and paraventricular nuclei and a decrease in GHRH immunoreactivity in the arcuate nucleus, as assessed by immunohistochemistry. Consistent with the decline in circulating GH and hypothalamic GHRH, pituitary GH mRNA levels of HI STZ-treated rats were 58% of controls. However, pituitary receptor mRNA levels for GHRH and ghrelin increased and those for somatostatin (sst2, sst3 and sst5) decreased following HI STZ treatment. The impact of LO STZ treatment on the GH axis differed from that observed following HI STZ treatment, despite comparable changes in circulating glucose and insulin. Specifically, LO STZ treatment did suppress circulating IGF-I levels to the same extent as HI STZ treatment; however, the impact on hypothalamic NPY mRNA levels was less dramatic (158% of vehicle-treated controls) where NPY immunoreactivity was increased only within the paraventricular nucleus. Also, there were no changes in circulating GH, hypothalamic GHRH or pituitary receptor expression following LO STZ treatment, with the exception that pituitary sst3 mRNA levels were suppressed compared with vehicle-treated controls. Taken together these results clearly demonstrate that insulinopenia, hyperglycemia and reduced circulating IGF-I levels are not the primary mediators of hypothalamic and pituitary changes in the GH axis of rats following HI STZ treatment. Changes in the GH axis of HI STZ-treated rats were accompanied by weight loss, and these changes are strikingly similar to those observed in the fasted rat, which suggests that factors associated with the catabolic state are critical in modifying the GH axis following STZ-induced diabetes.

New insights in the mechanism by which SRIF influences GH secretion.

Once thought to act only as a somatotropin release-inhibiting factor (SRIF), SRIF is currently viewed as a pleiotropic neuroendocrine factor controlling secretion, gene expression, apoptosis and signalling in many different targets. Actually, despite the numerous studies that have characterized SRIF action on somatotropes, new facets are continuously being discovered which help enlightening the biology of this cell type. As an example, ten years ago we demonstrated that SRIF exerts a dual, inhibitory/stimulatory effect on GH release from cultured pig somatotropes, which depends on the concentration of the peptide and on a divergent responsiveness of the two main cell subsets comprising the somatotrope population. Specifically, very low, picomolar doses of SRIF were found to stimulate GH release in vitro from intact cultures of dispersed pig pituitary cells and from purified somatotrope subpopulations. Conversely, higher (10(-7)M) SRIF concentrations inhibited, as expected, GHRH-induced GH release from intact pituitary cells and from one of the somatotrope subtypes; yet, at this same dose, it stimulated GH release from the other somatotrope subset. Analysis of second messenger pathways revealed that cAMP is the main signal conveying the stimulatory effects of low-dose SRIF. This peptide also exerts a distinct, dose-dependent regulation of the expression of three of its receptor subtypes (sst1, sst2 and sst5) at the pituitary. Indeed, acute in vitro treatment with a high SRIF dose increased mRNA levels of all three subtypes, whereas a low SRIF concentration only increased that of sst5. Interestingly, short term treatment with GHRH or ghrelin reduced the expression of sst5, and not that of sst1 and sst2. Hopefully, ongoing studies on cloning and individual characterization of porcine sst will help to unravel the complex and exciting response of somatotropes to SRIF.

Homologous and heterologous in vitro regulation of pig pituitary somatostatin receptor subtypes, sst1, sst2 and sst5 mRNA.

Somatostatin (SRIF) is commonly regarded as an inhibitor of GH release in rodents and humans. However, in pigs, SRIF can stimulate the release of GH at low (picomolar) doses, while inhibiting GHRH-stimulated GH release at high (nanomolar) doses in primary pituitary cell cultures. One possible mechanism by which pig cells respond differently to the actions of SRIF is by differential expression and regulation of SRIF receptor subtypes. As no information is available on the homologous regulation of SRIF receptors in pigs, we examined the acute (4 h) in vitro effects of SRIF on mRNA levels of SRIF receptors sst1, sst2 and sst5 by multiplex RT-PCR. These particular sst subtypes were selected because all three have been implicated in the inhibitory effects of SRIF on GH release in both rodents and humans. At a high dose (10(-7) M), SRIF stimulated the expression of sst1, sst2 and sst5 in pig pituitary cell cultures. At a low dose (10(-13) M), SRIF markedly increased sst1, without affecting sst2 or sst5. Given that our laboratory has shown SRIF at high and low doses stimulates cAMP production in a subpopulation of pig somatotropes, we sought to determine if this signaling pathway may be responsible for the stimulatory effect of SRIF on its own receptor expression. The receptor-independent cAMP activator forskolin elevated sst1 and sst2 mRNA levels but did not affect sst5 expression, suggesting the stimulatory actions of high- and low-dose SRIF on sst1 and high-dose SRIF on sst2 mRNA levels can be mediated by activation of cAMP, whereas the stimulatory effect of high-dose SRIF on sst5 mRNA is elicited by a cAMP-independent pathway. Interestingly, both GHRH (10(-8) M) and ghrelin (10(-6) M), which release GH in pig pituitary cell cultures via cAMP-dependent mechanisms, decreased sst5 without altering sst1 or sst2 mRNA levels. Since the actions of GHRH and ghrelin on sst expression markedly contrasted with that observed for SRIF and forskolin these results clearly indicate GHRH and ghrelin are regulating sst5 mRNA levels by a cAMP-independent signaling pathway. Taken together, our results demonstrate that expression of pig SRIF receptors is under a complex, receptor subtype-selective regulation, wherein the concerted actions of key regulators of somatotrope function would play divergent and dose-dependent effects.

Growth hormone-releasing hormone and pituitary somatotrope proliferation.

Growth hormone-releasing hormone (GHRH) is a hypothalamic hormone that is essential for normal expansion of the somatotrope lineage during pituitary development. Decreased GHRH secretion and/or action leads to impairment of this process and somatotrope hypoplasia in both humans and experimental animals. Excessive GHRH secretion and/or action result in dysregulated somatotrope proliferation, leading to hyperplasia and neoplastic transformation. Our understanding of the molecular and morphologic bases for these effects from both animal and clinical studies has greatly increased during the past decade. However, many features of the cellular pathways remain to be defined, including the interaction of other genes in the multistep process of somatotrope tumorigenesis.

The effect of GHRH on somatotrope hyperplasia and tumor formation in the presence and absence of GH signaling.

Excessive GHRH stimulation leads to somatotrope hyperplasia and, ultimately, pituitary adenoma formation in the metallothionein promoter-driven human GHRH (hGHRH) transgenic mouse. This pituitary phenotype is similar to that observed in humans with ectopic production of GHRH. In both mice and man, GHRH hyperstimulation also results in dramatic increases in circulating GH and IGF-I. To determine whether GH/IGF-I modulates the development and growth rate of GHRH-induced pituitary tumors, pituitary growth and histology were evaluated in mice generated from cross-breeding metallothionein promoter-driven hGHRH transgenic mice with GH receptor binding protein (GHR) gene disrupted mice (GHR(-/-)). Expression of the hGHRH transgene in 2-month-old GHR intact (GHR(+)) mice resulted in the doubling of pituitary weight that was largely attributed to an increase in the number of GH-immunopositive cells. Pituitary weight of GHR(+) hGHRH mice did not significantly change between 2 and 6 months of age, whereas at 12 months, weights increased up to 100-fold those of GHR(+) pituitaries, and 70% of the glands contained grossly visible adenomas. All adenomas stained positively for GH, whereas some showed scattered PRL staining. Pituitaries of GHR(-/-) mice were half the size of those of GHR(+) mice. Although reduced in size, the histological features of GHR(-/-) mouse pituitaries were suggestive of somatotrope hyperplasia. Despite evidence of somatotrope hyperplasia, pituitaries from GHR(-/-) mice as old as 28 months of age were similar in size to those of 2-month-old mice and did not show signs of adenoma formation. Expression of the hGHRH transgene in GHR(-/-) mice did not significantly increase pituitary size between 2 and 6 months of age. However, at 12 months the majority of GHR(-/-), hGHRH pituitaries developed adenomas with mean pituitary weight and histological features similar to those of GHR(+), hGHRH mice. These observations demonstrate that intact GH signaling is not required for GHRH tumor formation. Although the majority of GHR(+), hGHRH and GHR(-/-), hGHRH pituitaries developed tumors by 12 months of age, a small subset remained morphologically indistinct from those at 2 months of age. These observations taken together with the fact that overt tumor formation is preceded by a static pituitary growth phase between 2 and 6 months, indicates that protective mechanisms are in place to maintain pituitary mass despite hGHRH hyperstimulation.

The growth hormone (GH)-axis of GH receptor/binding protein gene-disrupted and metallothionein-human GH-releasing hormone transgenic mice: hypothalamic neuropeptide and pituitary receptor expression in the absence and presence of GH feedback.

Elevation of circulating GH acts to feed back at the level of the hypothalamus to decrease GH-releasing hormone (GHRH) and increase somatostatin (SRIF) production. In the rat, GH-induced changes in GHRH and SRIF expression are associated with changes in pituitary GHRH receptor (GHRH-R), GH secretagogue receptor (GHS-R), and SRIF receptor subtype messenger RNA (mRNA) levels. These observations suggest that GH regulates its own synthesis and release not only by altering expression of key hypothalamic neuropeptides but also by modulating the sensitivity of the pituitary to hypothalamic input, by regulating pituitary receptor synthesis. To further explore this possibility, we examined the relationship between the expression of hypothalamic neuropeptides [GHRH, SRIF, and neuropeptide Y (NPY)] and pituitary receptors [GHRH-R, GHS-R, and SRIF receptor subtypes (sst2 and sst5)] in two mouse strains with alterations in the GH-axis; the GH receptor/binding protein gene-disrupted mouse (GHR/BP-/-) and the metallothionein promoter driven human GHRH (MT-hGHRH) transgenic mouse. In GHR/BP-/- mice, serum insulin-like growth factor I levels are low, and circulating GH is elevated because of the lack of GH negative feedback. Hypothalamic GHRH mRNA levels in GHR/BP-/- mice were 232 +/- 20% of GHR/BP+/+ littermates (P < 0.01), whereas SRIF and NPY mRNA levels were reduced to 86 +/- 2% and 52 +/- 3% of controls, respectively (P < 0.05; ribonuclease protection assay). Pituitary GHRH-R and GHS-R mRNA levels of GHR/BP-/- mice were elevated to 275 +/- 55% and 319 +/- 68% of GHR/BP+/+ values (P < 0.05, respectively), whereas the sst2 and sst5 mRNA levels did not differ from GHR/BP intact controls as determined by multiplex RT-PCR. Therefore, in the absence of GH negative feedback, both hypothalamic and pituitary expression is altered to favor stimulation of GH synthesis and release. In MT-hGHRH mice, ectopic hGHRH transgene expression elevates circulating GH and insulin-like growth factor I. In this model of GH excess, endogenous (mouse) hypothalamic GHRH mRNA levels were reduced to 69 +/- 6% of nontransgenic controls, whereas SRIF mRNA levels were increased to 128 +/- 6% (P < 0.01). NPY mRNA levels were not significantly affected by hGHRH transgene expression. Also, MT-hGHRH pituitary GHRH-R and GHS-R mRNA levels did not differ from controls. However, sst2 and sst5 mRNA levels in MT-hGHRH mice were increased to 147 +/- 18% and 143 +/- 16% of normal values, respectively (P < 0.05). Therefore, in the presence of GH negative feedback, both hypothalamic and pituitary expression is altered to favor suppression of GH synthesis and release.

Modulation of pituitary somatostatin receptor subtype (sst1-5) messenger ribonucleic acid levels by changes in the growth hormone axis.

The role of individual components of the hypothalamic-pituitary-GH axis in the modulation of pituitary somatostatin (SRIF) receptor subtype (sst1-5) synthesis was assessed using multiplex RT-PCR to measure receptor messenger RNA (mRNA) levels in normal rats and spontaneous dwarf rats (SDRs). In SDRs, a strain with no immunodetectable GH, pituitary sst1 and sst2 mRNA levels were elevated, sst5 mRNA levels were reduced, and sst3 and sst4 mRNA levels did not significantly differ from those in normal controls. Treatment of SDRs with GH (72 h), but not insulin-like growth factor I, significantly decreased sst2 mRNA levels and increased sst4 and sst5 mRNA levels above vehicle-treated control levels. To test whether more rapid changes in circulating GH levels could alter SRIF receptor subtype expression, normal rats were infused (iv) with GH-releasing hormone (GHRH) for 4 h in the presence or absence of SRIF antiserum. GHRH infusion increased pituitary sst1 and sst2 and decreased sst5, but had no effect on sst3 and sst4 mRNA levels. Immunoneutralization of SRIF, which produced a rise in circulating GH levels, did not alter basal or GHRH-mediated SRIF receptor subtype expression. These observations indicate that acute suppression of SRIF tone does not regulate pituitary SRIF receptor subtype mRNA levels in vivo. The possibility that elevated circulating GH concentrations induced by GHRH infusion were responsible for the observed changes in SRIF receptor subtype mRNA levels was examined by infusing SDRs with GHRH for 4 h. GHRH did not increase sst1 mRNA levels in SDRs above their already elevated value. However, GHRH infusion produced an increase in sst2 and a decrease in sst5 mRNA levels similar to those observed in normal rats, indicating that the acute effects of GHRH on SRIF receptor subtype expression are independent of circulating GH levels. Primary rat pituitary cell cultures were incubated with GHRH (10 nM) or forskolin (10 microM) for 4 h to determine whether GHRH could directly mediate SRIF receptor subtype mRNA. GHRH treatment increased sst1 and sst2 mRNA levels and decreased sst5 mRNA levels, but had no effect on sst3 and sst4, similar to the results in vivo. The effect of forskolin mimicked that of GHRH on sst1, sst2, and sst5 mRNA, suggesting that GHRH acts through cAMP to directly mediate gene transcription or mRNA stability of these SRIF receptor subtypes. In addition, forskolin reduced sst3 and sst4 expression. These results strongly suggest that rat pituitary sst1, sst2, and sst5 mRNA levels are regulated both in vivo and in vitro by GHRH. The stimulatory action of GHRH on sst1 and sst2 and the inhibitory action on sst5 indicate that these receptor subtypes have independent and unique roles in the modulation of pituitary GH release.

Secretagogues and the somatotrope: signaling and proliferation.

Somatotrope function requires consideration of both growth hormone (GH) secretion and cellular proliferation. The regulation of these processes is, to a large extent, controlled by three hypothalamic hormones: GH-releasing hormone (GHRH), somatostatin (SRIF), and an as-yet-unidentified GH secretagogue (GHS). Each binds to G protein-linked membrane receptors through which signaling occurs. Our laboratory has used a series of genetic and transgenic models with perturbations of individual components of the GH regulatory system to study both somatotrope signaling and proliferation. Impaired GHRH signaling is present in the lit mouse, which has a GHRH receptor (R) mutation, and the dw rat, which has a post-receptor signaling defect. Both models also have impaired responses to a GHS, implying an interaction between the two signaling systems. The spontaneous dwarf rat (SDR), in which a mutation of the GH gene results in total absence of the hormone, shows characteristic changes in the hypothalamic regulatory hormones due to an absence of GH feedback and alterations in the expression of each of their pituitary receptors. Treatment of SDRs with GHRH and a GHS has allowed demonstration of a stimulatory effect of GHRH on GHRH-R, GHS-R, and SRIF type 2 receptor (SSTR-2) expression and an inhibitory effect on SSTR-5 expression. GH also modifies the expression of these receptors, though its effects are seen at later time periods and appear to be indirect. Overall, the results indicate a complex regulation of GH secretion in which somatotrope receptor, as well as ligand expression, exerts an important physiological role. Both the SDR and the GH-R knockout (ko) mouse have small pituitaries and decreased somatotropes, despite elevated GHRH secretion and intact GHRH-R signaling. Introduction of the hGHRH transgene into GH-R ko mice confirmed that the proliferative effects of GHRH require GH/insulin-like growth factor-I (IGF-I) action. The results offer new insights into factors participating in somatotrope proliferation.

Glucocorticoids regulate pituitary growth hormone secretagogue receptor gene expression.

Glucocorticoids regulate growth hormone (GH) secretion by modulating both hypothalamic and pituitary function. At the level of the pituitary, glucocorticoids increase GH and GH-releasing hormone receptor (GHRH-R) gene expression. To test if glucocorticoids might also regulate the pituitary expression of the recently identified GH secretagogue (GHS) receptor, GHS-R; adult male rats were adrenalectomized or sham operated, and treated with the synthetic glucocorticoid (dexamethasone, 200 microg/day) or vehicle for 8 days. Pituitary GHS-R mRNA levels were assessed by reverse transcriptase polymerase chain reaction (RT-PCR). Adrenalectomy decreased pituitary GHS-R mRNA to 45% of vehicle-treated, sham-operated rats (P < 0.05). Administration of dexamethasone increased GHS-R mRNA levels in sham-operated as well as in adrenalectomized rats (199 +/- 24% (P < 0.05) and 369 +/- 48% (P < 0.01) of vehicle-treated controls). Addition of dexamethasone to primary rat pituitary cell cultures increased GHS-R mRNA levels in a dose- and time-dependent manner while the transcriptional inhibitor, actinomycin D, completely blocked the stimulatory action of dexamethasone. Taken together, these results suggest glucocorticoids directly increase pituitary GHS-R mRNA levels by stimulating GHS-R gene transcription.

p27Kip1-deficient mice exhibit accelerated growth hormone-releasing hormone (GHRH)-induced somatotrope proliferation and adenoma formation.

p27Kip1 (p27) controls cell cycle progression by binding to and inhibiting the activity of cyclin dependent kinases. Disruption of the p27 gene in mice (p27-/-) results in increased body growth with a disproportionate enlargement of the spleen, thymus, testis, ovary and pituitary. The increase in pituitary size is due to selective hyperplasia of the intermediate lobe (IL) while the anterior lobe (AL) is not overtly affected. p27 heterozygous mice (p27+/-), as well as p27-/- mice, are hypersensitive to radiation- and chemical-induced tumors compared to wildtype (p27+/+) littermates. Therefore, unlike classical tumor suppressors, only a reduction in p27 levels is necessary to predispose tissues to secondary tumor promoters. Consistent with these studies is the fact that the p27 gene sequence and mRNA levels appear normal in human pituitary adenomas while p27 protein levels are decreased. Therefore, a reduction in p27 levels could be sufficient to sensitize pituitary cells to tumorigenic factors. To test this hypothesis, metallothionein promoter-driven, human growth hormone-releasing hormone (MT-hGHRH) transgenic mice, that exhibit somatotrope hyperplasia before 9 months of age and subsequent adenoma formation with 30 - 40% penetrance, were crossbred with p27+/- mice for two successive generations to produce p27+/+, p27+/- and p27-/- mice that expressed the hGHRH transgene. At 10 - 12 weeks of age, p27-/- and p27+/+, hGHRH mice were larger than their p27+/+ littermates and displayed characteristic hyperplasia of the IL and AL, respectively. Expression of the hGHRH transgene in both p27+/- and p27-/- mice selectively expanded the population of somatotropes within the AL, where pituitaries of p27+/-, hGHRH and p27-/-, hGHRH mice were two- and fivefold larger than p27+/+, hGHRH pituitaries, respectively. There was also a synergistic effect of hGHRH transgene expression and p27-deficiency on liver, spleen and ovarian growth. At 6 - 8 months of age, 83% of p27+/-, hGHRH mice displayed macroscopic AL adenomas (>100 mg), while all pituitaries from p27+/+, hGHRH mice remained hyperplastic (<20 mg). In contrast to the dramatic effects of p27-deficiency on hGHRH-induced organ growth, elimination of p53, by crossbreeding MT-hGHRH mice to p53-deficient mice, did not augment the hyperplastic/tumorigenic effects of hGHRH transgene expression. Taken together these results demonstrate that a reduction in p27 expression is sufficient to sensitize somatotropes to the proliferative actions of excess GHRH, resulting in the earlier appearance and increased penetrance of hGHRH-induced pituitary tumors.

Increase in mRNA concentrations of pituitary receptors for growth hormone-releasing hormone and growth hormone secretagogues after neonatal monosodium glutamate treatment.

Previous studies have demonstrated that neonatal monosodium glutamate (MSG) treatment destroys growth hormone releasing-hormone (GHRH) neurones within the hypothalamic arcuate nucleus, decreases serum GH and insulin-like growth factor (IGF-I) concentrations, and retards linear growth. In the present study we investigated whether expression of pituitary GH, GHRH receptors (GHRH-R), growth hormone secretagogue receptors (GHS-R) and liver IGF-I is altered in this model of GHRH deficiency. In addition, we investigated if treatment of MSG-lesioned rats with the GHRH agonist, JI-38, would 'normalise' the GH-axis. Serum GH and IGF-I concentrations were determined by RIA, GH mRNA levels were evaluated by Northern blotting, and GHRH-R, GHS-R and IGF-I mRNA levels were measured by semiquantitative RT-PCR. In accord with previous reports, neonatal MSG treatment caused 50% and 76% decreases in serum GH and IGF-I concentrations, respectively, at 8 weeks of age. The decline in circulating GH was accompanied by a 56% reduction in total pituitary GH content, which was a reflection of the decrease in total pituitary protein. However, GH concentration (per mg protein) was unaltered. Despite the maintenance of a normal GH concentration, GH mRNA concentration (per microg total RNA) was suppressed by 42%, compared to saline-treated controls (P<0.05). These data indicate that a post-transcriptional mechanism, such as a reduction in the GH secretory rate, acts to maintain intracellular GH concentrations. The fall in circulating concentrations of GH leads to a 42% decrease in liver IGF-IB mRNA levels, while liver IGF-IA transcripts showed only a 27% suppression. In contrast, pituitary GHRH-R and GHS-R mRNA levels (per microg total RNA) were increased in MSG-lesioned rats by 96% and 180% of normal values (P<0.01), respectively. Twice daily treatment of MSG-lesioned rats (for 2 weeks) with the GHRH agonist, JI-38, increased serum GH and IGF-I levels, as measured 20 h after the last agonist injection. However, GH, IGF-I, GHRH-R and GHS-R mRNA levels were not altered at this time. These results demonstrate that intermittent GHRH agonist treatment stimulates pituitary GH secretion and GH in turn stimulates hepatic IGF-I but that effects on gene expression are not sustained. Collectively, our observations demonstrate a complex interplay between transcriptional, translational and post-translational mechanisms within each level of the GH-axis following destruction of GHRH neurones by neonatal MSG treatment.

Isolated familial somatotropinomas: establishment of linkage to chromosome 11q13.1-11q13.3 and evidence for a potential second locus at chromosome 2p16-12.

The majority of somatotropinomas are sporadic, although a small number occur with a familial aggregation, either as a component of an endocrine neoplasia complex that includes multiple endocrine neoplasia type 1 (MEN-1) and Carney complex (CNC) or as isolated familial somatotropinomas (IFS). IFS is defined as the occurrence of at least two cases of acromegaly or gigantism in a family that does not exhibit MEN-1 or CNC. This rare disease is associated with loss of heterozygosity (LOH) on chromosome 11q13, the locus of the MEN-1 gene, although the MEN-1 sequence and expression appear normal. These data suggest the presence of another tumor suppressor gene located at 11q13 that is important in the control of somatotrope proliferation. To establish linkage of IFS to 11q13 and to define the candidate interval of the IFS gene, we performed haplotype and allelotype analyses on two families with IFS. Collectively, allelic retention in one tumor and a recombinant haplotype in an affected individual mapped the tumor suppressor gene involved in the pathogenesis of IFS to a region of 8.6 cM between polymorphic microsatellite markers D11S1335 and INT-2 located at chromosome 11q13.1-13.3. Maximum two-point LOD scores for five markers within this region were 3.0 or more at theta = 0.0. As somatotropinomas are the predominant pituitary tumor subtype associated with CNC and arise before 30 yr of age, which is strikingly similar to the age at diagnosis for IFS, we explored the possibility that the putative CNC genes might also contribute to the pathogenesis of IFS. Although the genetic defect responsible for the complex is unknown, CNC has been mapped by linkage analysis to chromosomes 2p15-16 and 17q23-24 in different kindreds. Two-point LOD scores less than -2.0 were obtained using marker D17S949 from chromosome 17q23-24, excluding linkage. However, LOD scores of 2.5 were obtained for markers within 2p16-12; therefore, linkage of IFS to chromosome 2p cannot be excluded. This report establishes linkage of the tumor suppressor gene involved in the pathogenesis of IFS to chromosome 11q13.1-13.3 and identifies a potential second locus at chromosome 2p16-12.

Growth hormone (GH)-releasing hormone (GHRH) and the GH secretagogue (GHS), L692,585, differentially modulate rat pituitary GHS receptor and GHRH receptor messenger ribonucleic acid levels.

The ability of synthetic GH secretagogues (GHSs) to elicit a maximal release of GH in vivo is dependent on an intact GH-releasing hormone (GHRH) signaling system. The role of GHRH in GHS-induced GH release has been attributed primarily to the ability of GHS to release GHRH from hypothalamic neurons. However, GHS also releases GH directly at the pituitary level. Several lines of evidence suggest that GHRH is necessary to maintain pituitary responsiveness to GHS by stimulating GHS receptor (GHS-R) synthesis. To test this hypothesis, male rats (250-290 g) were anesthetized with ketamine/xylazine (which does not alter pulsatile GH secretion) and infused i.v. with a GHRH analog ([des-NH2Tyr1,D-Ala15]hGRF-(1-29)-NH2; 10 microg/h) or saline for 4 h. Serum was analyzed for GH, pituitaries were collected, and GHS-R and GHRH receptor (GHRH-R) messenger RNA (mRNA) levels were determined by RT-PCR. GHRH infusion resulted in a 10-fold increase in circulating GH concentrations that were accompanied by an increase in GHS-R mRNA levels to 200% of those in saline-treated controls (P < 0.01). In contrast, GHRH reduced GHRH-R mRNA levels slightly, but not significantly (P < 0.07). The stimulatory effect of GHRH on GHS-R mRNA levels was independent of somatostatin tone, as pretreatment with somatostatin antiserum did not alter the effectiveness of GHRH infusion. In contrast, blockade of somatostatin actions up-regulated GHRH-R mRNA levels under basal conditions and unmasked the inhibitory effects GHRH on its own receptor mRNA. These observations suggest GHRH-R mRNA is tonically suppressed by somatostatin. The stimulatory effect of GHRH on GHS-R mRNA levels was independent of circulating GH, as GHRH infusion in spontaneous dwarf rats, which do not have immunodetectable GH, increased GHS-R mRNA levels to 150% of those in saline-treated controls (P < 0.05). To determine whether this effect occurred by a direct action on the pituitary, primary cell cultures from normal rat pituitaries were incubated with GHRH (0.01-10 nM) or forskolin (10 microM) for 4 h. These GH secretagogues did not alter GHS-R mRNA levels in vitro. However, GHRH and forskolin reduced GHRH-R mRNA levels by 40% (P < 0.05). To determine whether the synthesis of the GHS-R, like that of the GHRH-R, is negatively mediated by its own ligand, anesthetized rats were infused with the nonpeptidyl secretagogue, L-692,585 (100 microg/h) for 4 h. Neither circulating GH (at 4 h) nor GHRH-R mRNA levels were significantly altered by L-692,585, whereas GHS-R mRNA levels were reduced by 50% (P < 0.05). Taken together, these results indicate that GHRH-induced up-regulation of pituitary GHS-R synthesis in vivo is indirect and independent of both somatostatin and GH. They also demonstrate that GHS-R synthesis, like that of GHRH-R, can be rapidly down-regulated by its own ligand.

Growth hormone-releasing hormone receptor (GHRH-R) and growth hormone secretagogue receptor (GHS-R) mRNA levels during postnatal development in male and female rats.

Experimental evidence suggests that differential pituitary sensitivity to hypothalamic signals exerts a role in mediating both age and sex dependent patterns of growth hormone (GH) release and synthesis. One mechanism by which pituitary sensitivity to hypothalamic GH regulators could be modified is by the differential synthesis of their pituitary receptors. In the present report we therefore studied the age and sex dependency of the expression of receptors for two known stimulators of GH release, growth hormone-releasing hormone (GHRH) and the synthetic peptidyl and non-peptidyl GH secretagogues (GHSs). Pituitary GHRH receptor (GHRH-R) and GHS receptor (GHS-R) mRNA levels were measured by reverse transcriptase-polymerase chain reaction (RT-PCR) in male and female rats at postnatal day 1, 10, 30 and 75. We also examined the age- and sex-dependent expression of the GHS-R in whole hypothalamic extracts, since the GHS-R is also expressed in a variety of nuclei within the hypothalamus and has been linked to central regulation of the GH-axis. Pituitary GHRH-R mRNA concentrations were age-dependent; the highest levels were observed in d1 pituitaries and then declined with age, reaching a nadir by d30. These results are in concordance with the age-related decline in pituitary GHRH sensitivity. In contrast, the ontogenic pattern of GHS-R expression was bimodal; GHS-R mRNA concentrations in dl and d30 pituitaries were approximately twice those at d10 and d75. These results mirror the transient increase in GHS sensitivity observed around the onset of puberty, suggesting that gonadal steroids mediate GHS-R expression. GHRH-R mRNA levels were comparable in males and females within each age while GHS-R mRNA levels were gender dependent. At d30, male GHS-R mRNA levels were 30% greater than in their female counterparts. This was reversed at d75, when females had 89% more GHS-R mRNA per pituitary and 65% more per somatotrope than did age-matched males. These sexual differences further support a role for gonadal steroids in the modulation of pituitary GHS-R synthesis. The ontogenic and gender-specific pattern of hypothalamic GHS-R expression differed from that observed for the pituitary. Hypothalamic GHS-R mRNA levels increased with age but exhibited no significant sex difference at each age tested. Taken together, these data demonstrate that changes in the levels of pituitary GHS-R mRNA, but not GHRH-R mRNA, are associated with changes in the gonadal steroid environment, thereby implicating the GHS/GHS-R signalling system as a control point in the establishment and maintenance of sexually dimorphic patterns of GH secretion.

Expression of growth hormone-releasing hormone (GHRH) messenger ribonucleic acid and the presence of biologically active GHRH in human breast, endometrial, and ovarian cancers.

GHRH is produced in a variety of extrahypothalamic tissues, including some neoplasms. We have previously reported that GHRH antagonists can inhibit the growth of various human cancers xenografted into nude mice. These observations suggest that locally produced GHRH might directly affect tumor cell proliferation. To investigate this possibility, we have examined the local production of GHRH in human endometrial, ovarian, and breast cancers obtained after surgery or grown in nude mice as xenografts. We have also examined whether the GHRH produced in these tumors is biologically active. RT-PCR and Southern blotting showed expression of messenger ribonucleic acid for GHRH in 17 of 22 endometrial and 17 of 22 ovarian cancer specimens and in all of the human endometrial, ovarian, and breast cancer xenografts studied. Acid extracts of endometrial cancer specimens and breast cancer xenografts that expressed the GHRH gene contained immunoreactive GHRH peptide, as assessed by RIA for GHRH. The level of immunoreactive GHRH detected was equivalent to 2.7-6.4 ng GHRH-(1-29)/g tissue. Purified extract from one of these tumor samples induced a powerful stimulation of GH release from rat pituitary cells. The presence of biologically and immunologically active GHRH and messenger ribonucleic acid for GHRH in human breast, endometrial, and ovarian cancers supports the hypothesis that locally produced GHRH may play a role in the proliferation of these tumors.

Loss of heterozygosity on chromosome 11q13 in two families with acromegaly/gigantism is independent of mutations of the multiple endocrine neoplasia type I gene.

Familial acromegaly/gigantism occurring in the absence of multiple endocrine neoplasia type I (MEN-1) or the Carney complex has been reported in 18 families since the biochemical diagnosis of GH excess became available, and the genetic defect is unknown. In the present study we examined 2 unrelated families with isolated acromegaly/gigantism. In family A, 3 of 4 siblings were affected, with ages at diagnosis of 19, 21, and 23 yr. In family B, 5 of 13 siblings exhibited the phenotype and were diagnosed at 13, 15, 17, 17, and 24 yr of age. All 8 affected patients had elevated basal GH levels associated with high insulin-like growth factor I levels and/or nonsuppressible serum GH levels during an oral glucose tolerance test. GHRH levels were normal in affected members of family A. An invasive macroadenoma was found in 6 subjects, and a microadenoma was found in 1 subject from family B. The sequence of the GHRH receptor complementary DNA in 1 tumor from family A was normal. There was no history of consanguinity in either family, and the past medical history and laboratory results excluded MEN-1 and the Carney complex in all affected and unaffected screened subjects. Five of 8 subjects have undergone pituitary surgery to date, and paraffin-embedded pituitary blocks were available for analysis. Loss of heterozygosity on chromosome 11q13 was studied by comparing microsatellite polymorphisms of leukocyte and tumor DNA using PYGM (centromeric) and D11S527 (telomeric), markers closely linked to the MEN-1 tumor suppressor gene. All tumors exhibited a loss of heterozygosity at both markers. Sequencing of the MEN-1 gene revealed no germline mutations in either family, nor was a somatic mutation found in tumor DNA from one subject in family A. The integrity of the MEN-1 gene in this subject was further supported by demonstration of the presence of MEN-1 messenger ribonucleic acid, as assessed by RT-PCR. These data indicate that loss of heterozygosity in these affected family members appears independent of MEN-1 gene changes and suggest that a novel (tissue-specific?) tumor suppressor gene(s) linked to the PYGM marker and expressed in the pituitary is essential for regulation of somatotrope proliferation.