RICERCA ITALIANA     

Somatostatin and related peptides: distribution, biological effects, and characterization of transgenic models

Università degli Studi di Torino

Abstract

This project derives from the collaboration of four research groups each possessing specific expertise/background in cellular and structural morphology, electrophysiology, pathology and histopathology, pharmacology and endocrinology. By such a mutifaceted approach this program will aim to clarify some cellular and molecular mechanisms that undierlie the biological effects of somatostatin and two related peptides (cortistatin and ghrelin) in different experimental models in vitro and in vivo, and in human tissues. These biologically active peptides display pleiotropic functions, many of which are of potential applicative interest. Therefore we will primarily address:
- the comparative distribution in the neuroendocrine system on KO mice, human fetal and adult material, tumors of nervous and/or neuroendocrine origin;
- the regulatory effects on proliferation and apoptosis in normal and pathological nerve cells, endocrine cells or hematological cells;
- the analysis of the endocrine-metabolic profile and evaluation of the physio-pathological and pharmacological responses in phlogistic conditions and somatic and visceral hyperalgesia in single/double KO mice;
- the short-term modulation of excitatory glutamatergic neurotransmission within the superficial laminae of the spinal dorsal horn with particular emphasis on pain neurotrasmission in acute and chronic conditions;
- the short and long-term modulation of inhibitory GABAergic neurotrasmission within the superficial laminae of the spinal dorsal horn with particular emphasis on the regulation of the inhibitory tone of substantia gelatinosa neurons.
To do so we will use histology, light and electron immunocytochemistry, electrophysiology, pharmacology, and molecular biology, possibly in combination to each other by taking advantage of the interactions among our groups. <<<

Principal Investigator

Adalberto Merighi Università degli Studi di TORINO

Research Objectives

The main goal of this program will be to clarify some cellular and molecular mechanisms that undierlie the biological effects of somatostatin and two related peptides (cortistatin and ghrelin) by using a multidisciplinary approach in different experimental models in vitro and in vivo.
The four units collaborating to the project will liaise with each others in term of exchange of technical expertise and cultural background. Each group will primarily address one or more of the following major issues of somatostatin biology:
- Comparative distribution in the neuroendocrine system on KO mice, human fetal and adult material, tumors of nervous and/or neuroendocrine origin;
- Regulatory effects on proliferation and apoptosis in normal and pathological nerve cells, endocrine cells or hematological cells;
- Analysis of the endocrine-metabolic profile and evaluation of the physio-pathological and pharmacological responses in phlogistic conditions and somatic and visceral hyperalgesia in single/double KO mice;
- Short-term modulation of excitatory glutamatergic neurotransmission within the superficial laminae of the spinal dorsal horn with particular emphasis on pain neurotrasmission in acute and chronic conditions;
- Short and long-term modulation of inhibitory GABAergic neurotrasmission within the superficial laminae of the spinal dorsal horn with particular emphasis on the regulation of the inhibitory tone of substantia gelatinosa neurons.

To do so we will use histology, light and electron immunocytochemistry, electrophysiology, pharmacology, and molecular biology, possibly in combination to each other by taking advantage of the interactions among units.
The animal models used by each group have already been characterized in previous studies and appear particularly suitable to our purposes. In particular in vitro models and KO animals will be useful to clarify some of the biological effects of somatostatin and related peptides, offering a controlled and simplified experimental paradigm to the study of different types of CNS and endocrine disorders/pathologies. Finally we believe that the comparison of these models will be fundamental to the comprehension of the pleiotropic functions of these biologically active peptides.

<<<

Timescale

24 months

National and international background

Somatostatin as a pleiotropic peptide
Bioactive peptides, cytokines and growth factors represent the principal types of chemical messengers. Classically, the term bioactive peptides designate a group of hormones and neuromediators of petidic nature. Cytokines are mediators of the cell communication in the immune system whereas, growth factors represent signal molecules controlling cell division and/or differentiation notably during embryonic development. Distinction between these three families of polypeptides/glycoproteins is more historical and didactic than functional. Indeed, there is often no clear distinction between the three families and, more importantly, the same peptide might functionally belong to all three families, being released by a multitude of cell types and thus displaying pleiotropic effects [1].
Concerning the relevant receptors, there are slight differences in the way the ligand triggers the initial
receptor activation upon binding, but the final cellular response is elaborated according to a common, general principle consisting in activation of different types of protein kinases. In the case of bioactive peptides, receptors are made of a single polypeptide chain spanning the plasma membrane in seven transmembrane domains and are coupled to G-proteins (G-protein-coupled receptors or GPCRs). In all cases, protein kinase-driven phosphorylation results in functional alteration of down-stream effectors (ion channels, enzymes, transcription factors) and adequate cellular responses.
The somatostatin [somatotroph (growth hormone = GH) release inhibiting factor, SRIF] neuropeptide family comprises few peptides originating from different post-translational processing of the prepro-SRIF precursor . Only two biologically active SRIF isoforms have been identified so far: the tetradecapeptide SRIF-14 and the amino-terminally extended octacosapeptide SRIF-28 (the entire SRIF-14 sequence is present in the C-terminus of SRIF-28). Both SRIF-14 and SRIF-28 are found in non-nervous tissues and in peripheral and central nervous system (CNS), but the predominant isoform is SRIF-14. The relative proportions of the two isoforms differ among various SRIF-producing tissues. However, SRIF-14 and SRIF-28 display overlapping functions.
These include the inhibition of a wide range of endocrine and exocrine secretions. Thus, SRIF inhibits the secretions of the pituitary (GH, prolactin and thyrotropin), gastrointestinal tract (cholecystokinin, gastric inhibitory peptide, gastrin, motilin, neurotensin and secretin) and pancreas (glucagon, insulin and pancreatic polypeptide). Inhibition of various exocrine functions by SRIF is well documented for the secretion of amylase, by salivary glands; hydrochloric acid and pepsinogen, by the gastrointestinal mucosa; pancreatic enzymes and bicarbonate, by pancreatic acini; and bile, by the liver. SRIF also regulates the intestinal absorption of nutrients and gastro-intestinal motility.
In the CNS, SRIF acts itself as a neurotransmitter in distinct, anatomically defined pathways, and as
a neuromodulator since it has the capacity to modulate the release of other neurotransmitters (among which are serotonin, acetylcholine, glutamate) and neurohormones (e.g. GH-releasing hormone).
In addition, SRIF has potent immunomodulatory actions on the secretion activity of immune cells such
as immunoglobulin (Ig) production by activated B-lymphocytes, and cytokine production by activated T-lymphocytes and macrophages. The proliferative response to antigens/mitogens of different types of immune cells is also modulated by SRIF [2].
Peptides related to SRIF
Another peptide, which does not belong strictu senso to the SRIF peptide family but shares 11 amino acids with SRIF, has recently been characterized and named cortistatin (CST). The biologically active form of CST is the tetradecapeptide (CST-14) in rodents and the heptadecapeptide (CST-17) in humans, corresponding to rodent CST-14 amino-terminally extended by 3 amino acids. In contrast to SRIF, CST appears predominantly to be confined to the CNS and, in particular, to inhibitory inter-neurons of the cerebral cortex and hippocampus. The biological actions of CST involve the regulation of sleep-phase transitions, consolidation of short- and long-term memory and locomotor activity. Among human peripheral tissues studied, the highest CST expression has been reported in kidney and testis[3], but CST has also been found in pancreas [4] and hepatocellular carcinoma cells [5]. In the immune system, CST is expressed in human T- and B-lymphocytes [6] as well as in monocytes
and monocyte-derived cells such as macrophages and dendritic cells [7].
Ghrelin is another recently discovered peptide related to SRIF. It was originally purified from the rat stomach [8]. The name ghrelin is based on “ghre,” a word root in Proto-Indo-European languages for “grow,” in reference to its ability to stimulate GH release. Ghrelin is a 28-amino acid peptide, in which the serine-3 (Ser3) is n-octanoylated, and this modification is essential for ghrelin’s activity. Rat and human ghrelins differ in only two amino acid residues [9]. In rat stomach, a second type of ghrelin peptide has been purified and identified as des-Gln14-ghrelin [8]. Except for the deletion of Gln14, des-Gln14-ghrelin is identical to ghrelin, even retaining the n-octanoic acid modification. Des-Gln14-ghrelin has the same potency of activities as that of ghrelin.
In all vertebrate species, ghrelin is mainly produced in the stomach [10], where ghrelin-containing cells are more abundant in the fundus than in the pylorus [11-13]. In situ hybridization and immunohistochemical analyses indicate that ghrelin-containing cells are a distinct endocrine cell type found in gastric mucosa . Ghrelin-immunoreactive cells are also found in the duodenum, jejunum, ileum, and colon [11,14,15], their concentration gradually decreasing from the duodenum to the colon. As in the stomach, the main molecular forms of intestinal ghrelin are n-octanoyl ghrelin and des-acyl ghrelin [11]. The pancreas is also a ghrelin-producing organ. Analyses combining HPLC and ghrelin-RIA revealed that ghrelin and des-acyl ghrelin both exist in the rat pancreas [16]. However, the cell type that produces ghrelin in the pancreatic islets remains controversial [16-19].
Ghrelin has been found in the hypothalamic arcuate nucleus, an important region for controlling appetite [9,20]. In addition, a recent study has reported the presence of ghrelin in previously uncharacterized hypothalamic neurons adjacent to the third ventricle between the dorsal,
ventral, paraventricular, and arcuate hypothalamic nuclei [21]. These ghrelin-containing neurons send efferent fibers to neurons that contain neuropeptide Y (NPY) and agouti-related protein (AgRP) and may stimulate the release of these orexigenic peptides. These localization patterns of ghrelin suggest a role in controlling food intake.
Recently, by a combined physiological and histochemical approach it was demonstrated that: i. ghrelin, administered either systemically or centrally, exerts potent, time-dependent GH-releasing activity under physiological conditions; ii. ghrelin is a functional antagonist of SRIF, but its GH-releasing activity at the pituitary level is not dependent on inhibiting endogenous SRIF release; iii. SRIF antagonizes the action of ghrelin at the level of the pituitary gland; and iv. the GH response to ghrelin in vivo requires an intact endogenous GHRH system [22].
SRIF and SRIF-related peptide receptors
Molecular characterization of rodent and human SRIF receptors indicated that five different receptors underlie the biological actions of the peptide. They are encoded by five different genes and are highly conserved between species and within the same species. Their expression products correspond to monomers composed of 391 (sst1), 369 (sst2), 418 (sst3), 388 (sst4) and 383 (sst5) amino acids. All five SRIF receptors have been identified throughout the CNS, endocrine and exocrine glands. The expression of mRNA for the five cloned receptors is overlapping but the combination (profile) of expression is tissue- and cell type-specific. In more detail, rodent CNS mRNAs for all five types of SRIF receptors have been seen in the cerebral cortex, striatum, hippocampus, amygdala, olfactory bulb and preoptic area [23,24]. Among different types of receptors, sst1 and sst2 are the most abundantly expressed. Transcripts for all five receptor types have been visualized in rat but not in human pituitaries where sst4 mRNA is not expressed. Rat and human pancreas, stomach, duodenum, jejunum and ileum contain the transcripts for sst1–sst5 receptors. Other peripheral organs and tissues express SRIF receptor transcripts more selectively. For example, rat adrenals and testes contain sst1–
sst3 transcripts. Elevated expressions of sst3 in liver and spleen and of sst4 in lung, heart and placenta
have been seen. The expression of sst2A and sst3 receptor mRNAs has been reported in cells of the immune system such as activated macrophages, and T- and B lymphocytes [2,25,26].
Both SRIF-14 and SRIF-28 recognize the five cloned receptors with similar affinities. Two subfamilies of SRIF receptors are distinguished based on their affinity for routinely used analogs [27]. All five cloned SRIF receptors (sst1–sst5) belong to the superfamily of G-protein-coupled receptors with seven transmembrane domains. The activation of G-proteins upon SRIF binding is, however, coupled to multiple signaling pathways.
The question concerning the selective involvement of different SRIF receptors in specific physiological responses to SRIF is still a matter of debate. However, some precise physiological functions could be attributed to each of the five known receptors. The majority of the relevant data came from the studies on knock-out (KO) mice in which gene encoding for a given SRIF receptor has been invalidated. The studies of sst2 gene KO mice indicated that this receptor displays specific central actions such as fine motor control [28], modulation of spatial learning [29], exploratory activity and emotional reactivity [30]. Among the peripheral actions of SRIF, sst2 receptor appears specifically involved in the inhibition of gastric acid secretion [31,32]. Analysis of the sst5 KO phenotype showed that this receptor mediates SRIF-induced inhibition of insulin secretion and glucose homeostasis [33] in an age-dependent manner [34].
Certain physiological roles of SRIF receptors that had initially been suggested based on histological bases have therefore been confirmed by analysis of the relevant receptor KO phenotype. This is the case for the explicit demonstration coming from KO mice concerning sst5 receptor involvement in the regulation of insulin. The phenomenon of the co-regulation of sst receptor expression particularly hampered the analysis of data obtained in KO models in relation to the involvement of a given receptor type in the physiological effect of SRIF known to implicate multiple receptors. For example, the involvement of sst1 receptor in the regulation of GH secretion that has been documented in sst1-null mice [35] should now be reconsidered in the light of the co-regulation of sst1 and sst2 receptor expression [36], especially because sst2 receptor has also been involved in the regulation of GH secretion in both rodent [37] and human [38] pituitaries.
The capacity of SRIF to negatively regulate cell proliferation through both indirect and direct mechanisms has repeatedly been reported. Indirect actions include the inhibition of secretion of growth-promoting hormones and growth factors stimulating the growth of various cell types. For example, the ability of SRIF to inhibit insulin-like growth factor-I expression and serum
levels represents the rationale for its use in an adjuvant therapy for tamoxifen in the treatment of breast cancer [39]. However, it should be stressed that the recent clinical trials (phase III) indicated that adding SRIF analogs to tamoxifen did not increase the therapeutic benefit of tamoxifen in the treatment of advanced breast carcinoma [40]. Besides, the capacity of SRIF to inhibit angiogenesis is of potential clinical relevance for the indirect control of tumor cell growth [43,44].
The ability of SRIF to control cell number by both inhibiting cell division and triggering programmed
cell death by apoptosis has been demonstrated in vitro. Thus, it has been shown that SRIF inhibits the proliferation of cell lines transfected to express sst1, sst2 or sst5 receptors [43,44].
Cortistatin is also capable of binding to all SRIF receptor subtypes with high affinity, and a role as potential endogenous ligand of the sst2 subtype in the human immune system has recently been
proposed [45,46]. Nonetheless, until now it has not been shown that cortistatin mediates SRIF receptor activation in vivo. Quite recently, the orphan receptor MrgX2 has been suggested to be a candidate cortistatin receptor [47]. A BLAST search using the human MrgX2 amino acid sequence as a query revealed only 26% identity to human sst5 as closest search result for SRIF receptors.
Two distinct ghrelin receptor (GHS-R) cDNAs have been isolated [48]. The first, GHS-R type 1a, encodes a GPCR with binding and functional properties consistent with its role as a ghrelin receptor. Type 1b is a COOH-terminal truncated form of the type 1a receptor and thus pharmacologically inactive. GHS-R type 1a is well conserved across all vertebrate species examined, including a number of mammals, chicken, and pufferfish (Fugu) [49-51].
GHS-R type 1a mRNA is prominently expressed in the arcuate (ARC) and ventromedial nuclei (VMN) and in the hippocampus [48,52,53]. It is also detected in multiple hypothalamic nuclei and in the pituitary, as well as the dentate gyrus, CA2, and CA3 regions of the hippocampus, the substantia nigra, the ventral tegmental area, and the dorsal and median raphe nuclei. The existence of ghrelin and its receptor in the hippocampus [52,54], a region that is associated with learning and memory, suggest the role of ghrelin in memory formation.
RT-PCR analyses demonstrated GHS-R type 1a mRNA expression in many peripheral organs, including heart, lung, liver, kidney, pancreas, stomach, small and large intestines, adipose tissue, and immune cells [52,55-57], indicating that ghrelin has multiple functions in these tissues [58]
The signal transduction pathway following ghrelin receptor activation was investigated using a hepatoma cell line that responds to ghrelin [59]. Ghrelin upregulates several activities that are also potentiated by insulin, including tyrosine phosphorylation of insulin receptor substrate-1 (IRS-1), association of the adaptor molecule growth factor receptor-bound protein 2 with IRS-1 and stimulates mitogen-activated protein kinase activity. <<<