The new truncated somatostatin receptor variant sst5TMD4 is associated to poor prognosis in breast cancer and increases malignancy in MCF-7 cells.

Somatostatin receptors (sst1-5) are present in different types of tumors, where they inhibit key cellular processes such as proliferation and invasion. Although ssts are densely expressed in breast cancer, especially sst2, their role and therapeutic potential remain uncertain. Recently, we identified a new truncated sst5 variant, sst5TMD4, which is related to the abnormal response of certain pituitary tumors to treatment with somatostatin analogs. Here, we investigated the possible role of sst5TMD4 in breast cancer. This study revealed that sst5TMD4 is absent in normal mammary gland, but is abundant in a subset of poorly differentiated human breast tumors, where its expression correlated to that of sst2. Moreover, in the MCF-7 breast cancer model cell, sst5TMD4 expression increased malignancy features such as invasion and proliferation abilities (both in cell cultures and nude mice). This was likely mediated by sst5TMD4-induced increase in phosphorylated extracellular signal-regulated kinases 1 and 2 and p-Akt levels, and cyclin D3 and Arp2/3 complex expression, which also led to mesenchymal-like phenotype. Interestingly, sst5TMD4 interacts physically with sst2 and thereby alters its signaling, enabling disruption of sst2 inhibitory feedback and providing a plausible basis for our findings. These results suggest that sst5TMD4 could be involved in the pathophysiology of certain types of breast tumors.

Identification and characterization of new functional truncated variants of somatostatin receptor subtype 5 in rodents.

Somatostatin and cortistatin exert multiple biological actions through five receptors (sst1-5); however, not all their effects can be explained by activation of sst1-5. Indeed, we recently identified novel truncated but functional human sst5-variants, present in normal and tumoral tissues. In this study, we identified and characterized three novel truncated sst5 variants in mice and one in rats displaying different numbers of transmembrane-domains [TMD; sst5TMD4, sst5TMD2, sst5TMD1 (mouse-variants) and sst5TMD1 (rat-variant)]. These sst5 variants: (1) are functional to mediate ligand-selective-induced variations in [Ca(2+)]i and cAMP despite being truncated; (2) display preferential intracellular distribution; (3) mostly share full-length sst5 tissue distribution, but exhibit unique differences; (4) are differentially regulated by changes in hormonal/metabolic environment in a tissue- (e.g., central vs. systemic) and ligand-dependent manner. Altogether, our results demonstrate the existence of new truncated sst5-variants with unique ligand-selective signaling properties, which could contribute to further understanding the complex, distinct pathophysiological roles of somatostatin and cortistatin.

Identification of novel genes involved in the plasticity of pituitary melanotropes in amphibians.

Melanotrope cells from the amphibian intermediate lobe are composed of two subpopulations that exhibit opposite secretory behavior: hypersecretory and hormone-storage hyposecretory melanotropes. Isolation of these subpopulations allowed a comparison of their gene expression profiles by differential display, leading to the identification of a number of genes differentially expressed in hypersecretory or hyposecretory melanotropes. Among them, we chose two (preferentially expressed in hyposecretory cells) of unknown function but structurally related to proteins involved in the secretory process: Rab18 and KIAA0555. We demonstrate that, upon activation of the regulated secretory pathway, Rab18 associates with secretory granules, inhibits their mobilization, and, consequently, reduces the secretory capacity of neuroendocrine cells. The other gene, KIAA0555, was predicted by in silico analysis to encode a protein with a long coiled-coil domain, a structural feature also shared by different proteins related to intracellular membrane traffic (i.e., golgins), and a hydrophobic C-terminal domain that could function as a transmembrane domain. A database search unveiled the existence of a KIAA0555 paralogue, KIAA4091, displaying a long coiled-coil region highly similar to that of KIAA0555 and an identical C-terminal transmembrane domain. Both KIAA0555 and KIAA4091 were found to be predominantly expressed in tissues containing cells with regulated secretory pathway, that is, endocrine and neural tissues. Moreover, when exogenously expressed in HEK293 cells, both proteins showed a yuxtanuclear distribution, which partially overlaps with that of a Golgi complex marker, thus suggesting a possible role of these two proteins in the control of the secretory process.

Identification and characterization of two novel truncated but functional isoforms of the somatostatin receptor subtype 5 differentially present in pituitary tumors.

CONTEXT: Somatostatin and its related peptide cortistatin exert multiple actions on normal and tumoral tissue targets through a family of receptors termed somatostatin receptor (sst)1-5. Despite the considerable advances in the knowledge on these receptors and their (patho)physiological roles, there is still evidence that additional receptors for these peptides should exist to fully explain their actions. OBJECTIVE: The growing number of spliced variants found in similar receptor families, often present in tumors, and results from our group obtained on sst5 from other species (pig) led us to explore the existence of new human sst5 isoforms. DESIGN AND RESULTS: A rapid amplification of cDNA ends PCR approach on samples from a human pituitary tumor and a cell line enabled identification of two novel alternatively spliced sst5 receptor variants. The sequences obtained encode putative proteins that correspond to truncated isoforms of five and four transmembrane domains (TMDs), accordingly named sst5TMD5 and sst5TMD4, respectively. Both novel receptors show a differential expression pattern in normal tissues and are also present in pituitary tumors of diverse etiology including nonfunctioning adenomas, corticotropinomas, somatotropinomas, and a prolactinoma. In contrast to the predominant plasma membrane localization of full-length sst5, both sst5TMD5 and sst5TMD4 show a preferentially intracellular localization. Despite their truncated nature, both receptors are functional, as shown by their ability to mediate selective, ligand-induced rises in free cytosolic calcium concentration. Specifically, whereas sst5TMD5 is selectivity activated by somatostatin compared with cortistatin, cells transfected with sst5TMD4 almost exclusively respond to cortistatin and not to somatostatin. CONCLUSIONS: Our results demonstrate the existence of two previously unidentified sst5 spliced variants with distinct distribution in normal tissues and pituitary tumors, unique ligand-selective signaling properties, and subcellular distribution, which could contribute to somatostatin and cortistatin signaling in normal and tumoral cells.

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 expression and processing of chromogranin A and secretogranin II in relation to the secretory status of endocrine cells.

Chromogranin A (CgA) and secretogranin II (SgII) are neuroendocrine secretory proteins that participate in regulation of the secretory pathway and also serve as precursors of biologically active peptides. To investigate whether there is a relationship between the expression, distribution, and processing of CgA and SgII and the degree of secretory activity, we employed two melanotrope subpopulations of the pituitary intermediate lobe that exhibit opposite secretory phenotypes. Thus, although one of the melanotrope subtypes shows high secretory activity, the other exhibits characteristics of a hormone storage phenotype. Our data show that SgII expression levels were higher in secretory melanotropes, whereas CgA expression showed similar rates in both cell subsets. The use of various antibodies revealed the presence of the unprocessed proteins as well as three CgA-derived peptides (67, 45, and 30 kDa) and six SgII-derived peptides (81, 66, 55, 37, 32, and 30 kDa) in both subpopulations. However, the smallest molecular forms of both granins predominated in secretory melanotropes, whereas the largest SgII- and CgA-immunoreactive peptides were more abundant in storage melanotropes, which is suggestive of a more extensive processing of granins in the secretory subset. Confocal microscopy studies showed that CgA immunoreactivity was higher in storage cells, but SgII immunoreactivity was higher in secretory melanotropes. Taken together, our results indicate that SgII and CgA are differentially regulated in melanotrope subpopulations. Thus, SgII expression is strongly related to the secretory activity of melanotrope cells, whereas CgA expression may not be related to secretory rate, but, rather, to hormone storage in this endocrine cell type.

Analysis of Rab18 and a new golgin in the secretory pathway.

Two new amphibian genes have been isolated and characterized from frog melanotropes, and the level of expression of these genes is related to the secretory status of the cells. Both genes, Rab18 and a novel member of the golgin family of proteins, are ubiquitously expressed in endocrine and nonendocrine tissues, and their corresponding proteins appear to show intracellular distributions associated with discrete vesicular and tubular structures, respectively, suggesting that they may play relevant roles in the regulation of the secretory pathway.

Ghrelin induces growth hormone (GH) secretion via nitric oxide (NO)/cGMP signaling.

Ghrelin, a recently discovered 28-aa peptide, stimulates GH release through a mechanism involving PLC- and cAMP-related signaling pathways. Recently, nitric oxide (NO) and its mediator, cGMP, have been shown to be required for the response of somatotropes to various regulators (GHRH, somatostatin, leptin). Here, we explore the possible role of the NO synthase (NOS)/NO/guanylate cyclase (GC)/cGMP signaling pathway in ghrelin-induced GH release from cultured pig somatotropes using blockers or activators of this route.

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.

Research progress in the stimulatory inputs regulating growth hormone (GH) secretion.

A review is presented on progress in the research of stimulatory inputs that regulate growth hormone secretion, including recent results on the action of the hypothalamic peptides growth-hormone releasing factor (GHRH) and pituitary adenylate cyclase-activating polypeptide (PACAP), as well as that of both peptidic (growth hormone-releasing hexapeptide; GHRP-6) and non-peptidyl (L-163,255) synthetic GHSs on somatotrope cell function.

Pituitary adenylate cyclase-activating polypeptide and its receptors in amphibians.

Pituitary adenylate cyclase-activating polypeptide (PACAP), a novel peptide of the secretin/glucagon/vasoactive intestinal polypeptide superfamily, has been initially characterized in mammals in 1989 and, only 2 years later, its counterpart has been isolated in amphibians. A number of studies conducted in the frog Rana ridibunda have demonstrated that PACAP is widely distributed in the central nervous system (particularly in the hypothalamus and the median eminence) and in peripheral organs including the adrenal gland. The cDNAs encoding the PACAP precursor and 3 types of PACAP receptors have been cloned in amphibians and their distribution has been determined by in situ hybridization histochemistry. Ontogenetic studies have revealed that PACAP is expressed early in the brain of tadpoles, soon after hatching. In the frog Rana ridibunda, PACAP exerts a large array of biological effects in the brain, pituitary, adrenal gland, and ovary, suggesting that, in amphibians as in mammals, PACAP may act as neurotrophic factor, a neurotransmitter and a neurohormone.

Melanotrope cell plasticity: a key mechanism for the physiological adaptation to background color changes.

The intermediate lobe of the pituitary secretes the melanotropic hormone alpha-MSH, which in amphibians plays a crucial role in skin color adaptation. It has been previously demonstrated that, in the frog Rana ridibunda, the intermediate lobe is composed of two distinct subpopulations of melanotrope cells that can be separated in vitro by using Percoll density gradients. These two melanotrope cell subsets, referred to as high-density (HD) and low-density (LD) cells, differ in their ultrastructural characteristics as well as in their biosynthetic and secretory activity. However, the specific, physiological role of the heterogeneity displayed by melanotrope cells remains elusive. In the present study, we investigated the effects of background color adaptation on melanotrope cell subpopulations. We found that adaptation of frogs to dark or white environment did not modify either the overall number of cells per intermediate lobe or the apoptotic and proliferation rates of melanotrope cells. On the other hand, adaptation of the animals to a white background significantly increased the proportion of hormone-storage HD cells and caused a concomitant decrease in that of LD cells (which exhibit higher levels of alpha-MSH release and POMC messenger RNA than HD cells). Conversely, after black-background adaptation the proportion of LD cells was markedly increased, suggesting that interconversion of HD cells to LD cells occurs during physiological activation of the intermediate lobe. In addition, black-background adaptation also enhanced alpha-MSH release by both cell subpopulations and increased inositol phosphate production in LD cells. These data indicate that, in frog, the proportions of the two melanotrope cell subsets undergo marked modifications during skin color adaptation, likely reflecting the occurrence of a secretory cell cycle whose dynamics are highly correlated to the hormonal demand imposed by the environment.

Growth hormone (GH)-releasing factor differentially activates cyclic adenosine 3',5'-monophosphate- and inositol phosphate-dependent pathways to stimulate GH release in two porcine somatotrope subpopulations.

Somatotropes comprise two morphologically and functionally distinct subpopulations of low (LD) and high (HD) density cells. We recently reported that GRF induces different patterns of increase in the cytosolic free Ca2+ concentration in single porcine LD and HD somatotropes, which for LD cells required not only Ca2+ influx but also intracellular Ca2+ mobilization. This suggested that GRF may activate multiple signaling pathways in pig LD and HD somatotropes to stimulate GH secretion. To address this question, we first assessed the direct GRF effect on second messenger activation in cultures of LD and HD cells by measuring cAMP levels and [3H]myo-inositol incorporation. Secondly, to determine the relative importance of cAMP- and inositol phosphate (IP)-dependent pathways, and of intra- and extracellular Ca2+, GRF-induced GH release from cultured LD and HD somatotropes was measured in the presence of specific blockers. GRF increased cAMP levels in both subpopulations, whereas it only augmented IP turnover in LD cells. Accordingly, adenylate cyclase inhibition by MDL-12,330A abolished GRF-stimulated GH release in both subpopulations, whereas phospholipase C inhibition by U-73122 only reduced this effect partially in LD cells. Likewise, blockade of Ca2+ influx with Cl2Co reduced GRF-stimulated GH secretion in both LD and HD somatotropes, whereas depletion of thapsigargin-sensitive intracellular Ca2+ stores only decreased the secretory response to GRF in LD cells. These results demonstrate that GRF specifically and differentially activates multiple signaling pathways in two somatotrope subpopulations to stimulate GH release. Thus, although the prevailing signaling cascade employed by GRF in both subpopulations is adenylate cyclase/cAMP/extracellular Ca2+, the peptide also requires activation of the phospholipase C/IP/intracellular Ca2+ pathway to exert its full effect in porcine LD somatotropes.

Pituitary adenylate cyclase-activating polypeptide (PACAP) 38 and PACAP27 activate common and distinct intracellular signaling pathways to stimulate growth hormone secretion from porcine somatotropes.

We have recently shown that the two bioactive forms of pituitary adenylate cyclase-activating polypeptide, PACAP38 and PACAP27, stimulate GH release and GH messenger RNA (mRNA) accumulation in cultured porcine pituitary cells. However, dose- and time-related differences in the response to both peptides suggested that the signaling mechanisms activated by PACAP38 and PACAP27 in this cell type could differ. To test this hypothesis, we have evaluated hormone release and GH mRNA content after PACAP treatment in combination with selective activators and inhibitors of the adenylate cyclase/cAMP/protein kinase A and the phospholipase C/inositol phosphate (IP)/protein kinase C pathways, and with blockers of intra- and extracellular Ca2+. Our results show that activation of the adenylate cyclase/cAMP/protein kinase A system, and extracellular Ca2+ entry through L-type Ca2+-channels are prevailing and requisite signals for the transduction of the stimulatory effects of both PACAP38 and PACAP27 on GH release and transcription in porcine somatotropes. However, phospholipase C and intracellular Ca2+ also contribute, although partially, to PACAP38-induced, but not to PACAP27-induced increase in porcine GH secretion and mRNA levels. These findings demonstrate that in normal somatotropes, PACAP38 can activate multiple transduction pathways that differ from those employed by PACAP27. Moreover, these differences could account for the previously described divergences in the actions of either peptide in porcine somatotropes.

Growth hormone-releasing factor mobilizes cytosolic free calcium through different mechanisms in two somatotrope subpopulations from porcine pituitary.

Porcine somatotropes can be separated by Percoll density gradient centrifugation into low (LD) and high density (HD) subpopulations that differ ultrastructurally and functionally. Here, we report the effects of growth hormone-releasing factor (GRF) on the cytosolic free calcium concentration ([Ca2+]i) of single LD and HD somatotropes. Resting [Ca2+]i in LD somatotropes was 2-fold higher than in HD cells. GRF induced [Ca2+]i increases in a similar percentage of somatotropes from both subsets. However, amplitude and kinetics of the responses were markedly different. In all responsive LD somatotropes, GRF evoked a rapid initial peak followed by a sustained plateau (plateau-type response). Blockade of extracellular Ca2+ entry by 3 mM EDTA, 2 mM CoCl2, or 100 microM verapamil completely abolished the plateau phase without affecting the initial Ca2+ spike. Conversely, only the plateau phase was preserved in thapsigargin (TG)-treated LD cells. The vast majority of GRF-responsive HD somatotropes exhibited a transient [Ca2+]i peak that returned gradually to baseline (transient-type response). This response was completely blocked by removal of extracellular Ca2+, whereas TG treatment had no effect. Taken together, our results indicate that the response of LD somatotropes to GRF depends on mobilization of Ca2+ of both extra- and intracellular origin, whereas that of HD somatotropes seems to be exclusively dependent on extracellular Ca2+ entry through L-type voltage sensitive Ca2+ channels (VSCC). These findings are the first to demonstrate a differential effect of GRF on Ca2+ mobilization in two somatotrope subpopulations, and suggest the existence of differences in the GRF receptor(s) expressed in each subpopulation and/or in the intracellular signalling pathways activated upon GRF binding.

Pituitary adenylate cyclase-activating polypeptide (PACAP) 38 and PACAP27 differentially stimulate growth hormone release and mRNA accumulation in porcine somatotropes.

Pituitary adenylate cyclase-activating polypeptide (PACAP) has been suggested to regulate growth hormone (GH) secretion in several species. Here, we analyzed the in vitro effects of PACAP38 and PACAP27 on the secretory activity of porcine somatotropes. Cultures of porcine pituitary cells were treated with PACAP38 and PACAP27, and GH release, intracellular GH content, and GH mRNA levels were evaluated. Also, the time course of changes in the somatotrope content of GH and its mRNA in response to PACAPs were measured. Both PACAPs stimulated GH release from porcine somatotropes in a broad range of doses (10(-10)-10(-6) M), yet only PACAP27 elicited a dose-dependent response. GH cell content remained essentially unchanged after PACAP treatment. In contrast, both PACAPs induced significant and sustained increases in GH mRNA cell content, although the response to PACAP27 appeared faster (8 h) than to PACAP38 (16 h). These results demonstrate that PACAP stimulates GH production in porcine somatotropes. Furthermore, the differential responses induced by PACAP38 and PACAP27 suggest that distinct mechanisms mediate their effects on this cell type.

Somatostatin at low doses stimulates growth hormone release from intact cultures of porcine pituitary cells.

Somatostatin (SRIF) is the primary inhibitory factor in the control of growth hormone (GH) release from somatotropes. This concept emerged from studies based mainly on the rat and human model. However, recent data suggest that the role of SRIF in the regulation of pituitary GH release might be different in other species such as the pig. Thus, in previous studies, we have demonstrated a dual (stimulatory/inhibitory) effect of SRIF on GH secretion in vitro in two porcine somatotrope subpopulations. In the present study, we have investigated whether SRIF can act as a GH-releasing factor in intact cultures of porcine somatotropes. To this end, both dose-related effects of SRIF on basal GH release and its effects on GH-releasing factor (GRF-)stimulated GH secretion were evaluated in monolayer cultures of porcine pituitary cells. SRIF did not affect basal secretion at the highest doses tested (10(-5), 10(-7), and 10(-9) M), whereas it induced a significant increase in GH secretion when applied at low doses (10(-11), 10(-13), and 10(-15) M). High-dose (10(-7) M) SRIF significantly reduced GRF-induced GH secretion, an effect that was absent at the lowest dose (10(-15) M) of the peptide tested. These results confirm the dual role af SRIF on GH secretion from porcine somatotropes, and demonstrate that SRIF, at low doses, can act as a true GH-releasing factor.