Somatostatin Gene Research Articles

Immunosuppressant agent FK506 stimulates growth hormone (GH) secretion and gene expression of hypothalamic GH-releasing hormone in the rat.

Immunosuppressant agent FK506 has been reported to stimulate ACTH release from pituitary cells. We examined the effects of FK506 on GH release from the rat anterior pituitary cells and the effects of FK506 on hypothalamic GH- releasing hormone (GRH) and somatostatin (SS) gene expression in conscious male rats. In vitro experiments, the monolayer pituitary culture and reverse hemolytic plaque assay were employed to examine the GH release from the rat anterior pituitary cells. In in vivo experiments, the FK506 was administered for 7 days and then sequential blood sampling was performed every 20 min during 6 h in conscious rats. The hypothalamus was removed, and total RNA was extracted for Northern blot analysis. The FK506 significantly stimulated GH release from the rat anterior pituitary cells in a dose-dependent manner in vitro. In in vivo experiments, the area under the curve of GH surges was significantly increased in FK506-treated rats, although the peak height and the trough level of GH surges were not altered. Pituitary GH messenger RNA (mRNA) levels were significantly increased by the FK506 treatment. Hypothalamic GRH mRNA levels were significantly increased in FK506- treated rats, whereas hypothalamic SS mRNA levels were not altered. These findings indicate that FK506 stimulates GH secretion and gene expression of hypothalamic GRH in the rat.

Expression of the somatostatin gene in human astrocytoma cell lines.

Somatostatin (somatotropin release-inhibiting hormone; SRIH) has been demonstrated in neurons of the central nervous system (CNS) as well as in endocrine cells of the pancreas and gastrointestinal tract and can suppress various immune functions including lymphocyte proliferation, immunoglobulin synthesis, and cytokine production. Since astrocytes possess antigen-presenting activity and can secrete a wide array of immunoregulatory and inflammatory cytokines, we studied SRIH gene expression in both astrocyte cell lines and mitogen-stimulated peripheral blood mononuclear leukocytes from healthy donors. We now report by means of a complementary DNA-based reverse transcription PCR that differential levels of SRIH mRNA were expressed in 9 of 11 human astrocytoma cell lines tested but were undetectable in activated peripheral blood mononuclear leukocytes as well as in a variety of human lymphocyte and monocyte cell lines. The synthesis and secretion of SRIH protein by astrocytoma cells that expressed SRIH transcripts were confirmed by specific radioimmunoassay of cell culture fluids. These findings support the notion that SRIH gene expression occurs in human astrocytoma cells but not in mature lymphoid cells of the immune system.

Gene expression of hypothalamic somatostatin, growth hormone releasing factor, and their pituitary receptors in hypothyroidism.

Thyroid hormones are important to growth in mammals and have been shown to rapidly stimulate the rate of GH gene transcription. In this study, we investigated whether thyroid hormones modulate GH secretion through their effects on the gene expression of GRF, somatostatin (SS), GRF receptor, and receptor subtype 2 for SS (SSTR2). Male adult Sprague-Dawley rats were rendered hypothyroid with a single injection of propylthiouracil followed by methimazole in drinking water (0.02%) for 1 day to 12 weeks. Total RNA extracted from the anterior pituitary and hypothalamus was analyzed by Northern hybridization. GH messenger RNA (mRNA) level in the anterior pituitary was significantly reduced in the hypothyroid animals (P < 0.0001 vs. controls for all treatment duration > or = 1 week). An increase in hypothalamic GRF mRNA level, by 2- and 4-fold, respectively, was seen after 3 and 12 weeks of antithyroid treatment (both P < 0.001 vs. controls). Hypothalamic GRF content, studied in 12-week hypothyroid rats only, was decreased compared with controls (P < 0.05). A reduction in pituitary GRF receptor mRNA level was observed after 1 week of antithyroid treatment (P < 0.01 after 1 week, P < 0.001 after 3 weeks). Total hypothalamic SS content and SS mRNA level in hypothalamic fragments consisting predominantly of the paraventricular and periventricular nuclei became significantly decreased (P < 0.05 and P < 0.005 respectively) after 12-weeks of antithyroid treatment. The reduction in SS gene expression in the periventricular nuclei was confirmed by in situ hybridization. No significant change in the mRNA level of pituitary SSTR2 was observed up to 12 weeks of antithyroid treatment. In conclusion, we have demonstrated a reduction in the gene expression of GRF receptor and SS in the hypothyroid rat. Our results suggest that the changes in hypothalamic GRF and SS gene expression in hypothyroid rats may be compensatory in nature and are likely to be secondary to the reduction in GH synthesis and secretion in these animals. The reduction in basal and GRF-stimulated GH secretion in hypothyroidism can be explained by the observed reduction in GH and GRF receptor gene expression.

Gene expression of hypothalamic somatostatin, growth hormone releasing factor, and their pituitary receptors in hypothyroidism

Thyroid hormones are important to growth in mammals and have been shown to rapidly stimulate the rate of GH gene transcription. In this study, we investigated whether thyroid hormones modulate GH secretion through their effects on the gene expression of GRF, somatostatin (SS), GRF receptor, and receptor subtype 2 for SS (SSTR2). Male adult Sprague-Dawley rats were rendered hypothyroid with a single injection of propylthiouracil followed by methimazole in drinking water (0.02%) for 1 day to 12 weeks. Total RNA extracted from the anterior pituitary and hypothalamus was analyzed by Northern hybridization. GH messenger RNA (mRNA) level in the anterior pituitary was significantly reduced in the hypothyroid animals (P < 0.0001 vs. controls for all treatment duration > or = 1 week). An increase in hypothalamic GRF mRNA level, by 2- and 4-fold, respectively, was seen after 3 and 12 weeks of antithyroid treatment (both P < 0.001 vs. controls). Hypothalamic GRF content, studied in 12-week hypothyroid rats only, was decreased compared with controls (P < 0.05). A reduction in pituitary GRF receptor mRNA level was observed after 1 week of antithyroid treatment (P < 0.01 after 1 week, P < 0.001 after 3 weeks). Total hypothalamic SS content and SS mRNA level in hypothalamic fragments consisting predominantly of the paraventricular and periventricular nuclei became significantly decreased (P < 0.05 and P < 0.005 respectively) after 12-weeks of antithyroid treatment. The reduction in SS gene expression in the periventricular nuclei was confirmed by in situ hybridization. No significant change in the mRNA level of pituitary SSTR2 was observed up to 12 weeks of antithyroid treatment. In conclusion, we have demonstrated a reduction in the gene expression of GRF receptor and SS in the hypothyroid rat. Our results suggest that the changes in hypothalamic GRF and SS gene expression in hypothyroid rats may be compensatory in nature and are likely to be secondary to the reduction in GH synthesis and secretion in these animals. The reduction in basal and GRF-stimulated GH secretion in hypothyroidism can be explained by the observed reduction in GH and GRF receptor gene expression.

Coexpression of preprotachykinin-A, alpha-calcitonin gene-related peptide, somatostatin, and neurotrophin receptor family messenger RNAs in rat dorsal root ganglion neurons.

Syntheses of substance P, somatostatin, and calcitonin gene-related peptide in sensory neurons have been suggested to be regulated by neurotrophic factors retrogradely transported from target tissues. In this study, we re-examined this idea by investigating the coexpression of neurotrophin receptor (trk family proto-oncogene) messenger RNAs, and preprotachykinin-A (a precursor peptide of substance P), alpha-calcitonin gene-related peptide and somatostatin messenger RNAs in lumbar dorsal root ganglion neurons by means of in situ hybridization histochemistry in rats. Approximately 35-40%, 5% and 15-20% of sensory neurons displayed signals for trkA, trkB, and trkC messenger RNAs, respectively. Approximately 28% of dorsal root ganglion neurons were positive for preprotachykinin-A messenger RNA, and were divided into two groups; those labeled strongly and those labeled weakly by in situ hybridization. All the strongly-labeled neurons (78% of preprotachykinin-A-positive cells) expressed trkA messenger RNA at the same time, while the weakly-labeled neurons did not. Thirty-seven per cent of dorsal root ganglion neurons expressed alpha-calcitonin gene-related peptide messenger RNA, and most of these neurons (84%) also expressed trkA messenger RNA. No or few preprotachykinin-A messenger RNA- and/or alpha-calcitonin gene-related peptide messenger RNA-expressing neurons were also positive for trkB or trkC messenger RNAs. Nine per cent of dorsal root ganglion neurons expressed somatostatin messenger RNA, and these neurons lacked all three trk messenger RNAs. Furthermore, most of these neurons (about 90%) showed positive, albeit weak, signals for preprotachykinin-A and alpha-calcitonin gene-related peptide messenger RNAs. The results suggest that expression of preprotachykinin-A and alpha-calcitonin gene-related peptide messenger RNAs is mediated by nerve growth factor via trkA receptor but not by brain-derived neurotrophic factor or neurotrophin-3, and that somatostatin gene transcription is not regulated by any member of the neurotrophin family in rat sensory neurons.

Regulation of Somatostatin Gene Transcription by cAMP

A number of hormones and growth factors stimulate target cells through receptors which are coupled to second messenger pathways. The second messenger cAMP, for example, mediates a wide variety of cellular responses to hormonal signals, including changes in intermediary metabolism, cellular proliferation and cellular motility. In mammalian cells, all of these biological responses are triggered by the activation of the cAMP-dependent protein kinase A, a heterotetramer consisting of paired catalytic and regulatory subunits. Upon hormonal stimulation, cAMP binds tightly to the regulatory subunits, thereby liberating catalytic subunits and promoting the phosphorylation of cellular substrates. In the liver, cAMP functions as a starvation state signal, mediating hormonal cues from the pancreas and adrenal gland to stimulate glucose production. cAMP stimulates glucose production, in part, by regulating transcription of the gene for phosphoenolpyruvate carboxykinase (PEPCK), a rate-limiting enzyme in gluconeogenesis. Following hormonal stimulation, cAMP induces PEPCK gene expression 10-fold within 20-30 min. This induction appears to be independent of new protein synthesis.

Regulation of somatostatin gene expression by veratridine-induced depolarization in cultured fetal cerebrocortical cells

The stimulatory effect of veratridine (VTD) depolarization upon somatostatin mRNA (SS mRNA) levels in primary cultures of fetal cerebrocortical cells was analyzed. Depolarizing stimuli, such as 100 microM VTD exposure for 30 min, elicited an increase in immunoreactive somatostatin (IR-SS) release to the media without affecting SS mRNA levels. These levels increased when exposure to depolarization stimuli was prolonged up to 3 or more hours. At this time, veratridine acted as a secretagogue, stimulating somatostatin secretion, but was also effective in stimulating somatostatin mRNA levels. These changes were blunted by the Na+ channel blockade tetrodotoxin (TTX), and partially abolished by the Ca2+ channel antagonist, verapamil (VPM). To study whether VTD may affect mRNA stability we determine the rate of disappearance of SS mRNA after inhibition of transcription by actinomycin D and demonstrated that VTD stimulation did not stabilize the SS mRNA. These results indicate that the induction of SS mRNA expression by VTD involves the modulation of Ca2+ and Na+ channels. The time course study confirmed that the VTD-induced SS mRNA accumulation is time-dependent, and requires a prolonged exposure to stimulate SS gene expression. VTD stimulation does not modify the SS mRNA rate of degradation.

Diminution of preprosomatostatin-mRNA in cerebral cortex of the aged rat.

The aim of the present study was to examine the effect of normal aging on somatostatin neurotransmission. Somatostatin gene expression was analysed in several regions of the cerebral cortex and hippocampus in 3, 7 and 21 month-old Sprague-Dawley rats using quantitative in situ hybridization with a 48mer oligodeoxynucleotide antisense probe. Furthermore the distribution of 125I-Tyr11 somatostatin receptor binding sites was studied using quantitative receptor autoradiography. The results demonstrated a significant reduction of preprosomatostatin-mRNA in the frontal cortex of the aged (21 month) group compared with the young (3 month) and the middle-aged (7 month) groups. The receptor binding densities of the aged (21 month) group tended to be lower, compared to the other groups although no significant region-specific changes were evident. These results indicate neurochemical changes in somatostatin-containing neurons in the frontal cortex during aging.

Diminution of preprosomatostatin-mRNA in cerebral cortex of the aged rat

The aim of the present study was to examine the effect of normal aging on somatostatin neurotransmission. Somatostatin gene expression was analysed in several regions of the cerebral cortex and hippocampus in 3, 7 and 21 month-old Sprague-Dawley rats using quantitative in situ hybridization with a 48mer oligodeoxynucleotide antisense probe. Furthermore the distribution of 125I-Tyr 11 somatostatin receptor binding sites was studied using quantitative receptor autoradiography. The results demonstrated a significant reduction of preprosomatostatin-mRNA in the frontal cortex of the aged (21 month) group compared with the young (3 month) and the middle-aged (7 month) groups. The receptor binding densities of the aged (21 month) group tended to be lower, compared to the other groups although no significant region-specific changes were evident. These results indicate neurochemical changes in somatostatin-containing neurons in the frontal cortex during aging.

Pancreatic somatostatin-14 and somatostatin-25 release in rainbow trout is stimulated by glucose and arginine

Previous work suggested that teleost fish possess two somatostatin (SS) genes. Regulation of differential secretion of SS gene I (SS-25) and SS gene II products (SS-14) by nutrients was studied using rainbow trout. Hemi-islets were cultured in medium containing different concentrations of glucose and arginine. SS-14 and insulin were determined by radioimmunoassay; SS-25 was determined by a novel enzyme-linked immunosorbent assay. Glucose stimulated SS-25 release in a concentration-related manner, with 10 mM being the most effective concentration; at higher (25 mM) glucose, release decreased. Insulin levels in the medium also were found to be more elevated from islets incubated in 10 mM glucose than those incubated in 25 mM glucose. Maximum SS-14 release was observed at lower (1-5 mM) glucose concentrations. Arginine stimulated SS release so that maximum release of both SS forms occurred at the lowest glucose concentrations (1 mM). A concentration-related effect of arginine was observed on SS-25 release. The results of this study indicate that glucose and arginine regulate secretion of both pancreatic SS gene I and gene II products from rainbow trout islets, suggesting that two different pancreatic SS interact in nutrient homeostasis in this species.

Characterization of Insulin, Glucagon, and Somatostatin from the River Lamprey, Lampetra fluviatilis

Insulin has been isolated from an extract of the pancreas of an Agnathan, the river lamprey Lampetra fluviatilis. The primary structure of the peptide (A-chain: GIVEQ CCHRK CSIYD MENYC N; B-chain: SALTG AGGTH LCGSH LVEAL YVVCG DRGFF YTPSK T) is the same as that in insulin from the sea lamprey Petromyzon marinus . In contrast, Lamperta glucagon (HAQGS FTSDY SKYLD SKQAK DFVIW LMNT), isolated from an extract of intestine, is structurally more similar to human glucagon (five amino acid substitutions) than to Petromyzon glucagon (six substitutions). Similarly, the primary structure of somatostatin (AAAAP GAAGG AQLPL GNRER KAGCK NFFWK TFSSC), isolated from Lampetra pancreas, contains eight amino acid substitutions and an additional residue compared with Petromyzon somatostatin. Somatostatin, isolated from Lampetra brain, has an identical structure to mammalian somatostatin-14 (AGCKN FFWKT FTSC). indicative of the same tissue specific expression of different somatostatin genes that was previously observed in Petromyzon . In contrast to the reduced binding affinity of other fish insulins, lamprey insulin was equipotent with porcine insulin in inhibiting the binding of [3-[125I]iodotyrosine-A14] human insulin to the human insulin receptor.

Light-induced CREB phosphorylation and gene expression in rat retinal cells.

The signal pathway for light-induced expression of c-fos and the neuropeptide somatostatin (SS) in rat retinal cells was investigated. Flashing light induced c-fos and SS mRNA in the inner nuclear layer and the ganglion cell layer. As both c-fos and SS genes have a cyclic AMP response element (CRE) in their promoters, CRE-binding protein (CREB) phosphorylation in retinal cells was examined with a phospho-CREB-specific antibody. Both flashing light and administration of the L-type Ca2+ channel activator Bay K 8644 induced phosphorylation of CREB in the nuclei of the amacrine cells and the ganglion cells where c-fos/SS mRNAs were expressed. These cells could be double-stained with anti-calmodulin kinase II (anti-CaM kinase II) monoclonal antibody and phospho-CREB-specific polyclonal antiserum after Bay K 8644 administration, indicating the colocalization of phosphorylated CREB at Ser133 and CaM kinase II in the neural retina.

Chloride channel 2 gene (Clc2) maps to chromosome 16 of the mouse, extending a region of conserved synteny with human chromosome 3q

The Clc2 gene of the mouse codes for the ubiquitously expressed chloride channel ClC-2, a member of a family of at least seven voltage gated chloride channels, some of which are implicated in hereditary diseases. Using a mouse interspecies back-cross panel, we have mapped Clc2 to Chr 16, proximal to the somatostatin gene Smst, extending a region of documented conserved synteny to human Chr 3q.

Differential stimulation of somatostatin but not neuropeptide Y gene expression by quinolinic acid in cultured cortical neurons.

Somatostatin (SS) and neuropeptide Y (NPY) are coproduced in a subpopulation of neurons that are selectively resistant to NMDA neurotoxicity. We have previously reported that quinolinic acid (QUIN), an NMDA receptor agonist, augments SS mRNA in cultured fetal rat cortical neurons. This study examines coregulation of SS and NPY by QUIN and NMDA in cultured cortical neurons and compares the effects of these agents with those of forskolin and phorbol 12-myristate 13-acetate (PMA), known to activate SS and NPY gene transcription by protein kinase A- and protein kinase C-dependent mechanisms. In addition, transcriptional regulation of the SS gene was investigated by acute transfection of cortical cultures with an SS promoter-chloramphenicol acetyltransferase (CAT) construct. QUIN and NMDA displayed dose-dependent fourfold augmentation of levels of mRNA for SS but not for NPY. In contrast, forskolin and PMA increased both SS and NPY mRNA levels. QUIN- and NMDA-mediated induction of SS mRNA was blocked by the NMDA receptor antagonist (-)-2-amino-5-phosphonovaleric acid and displayed regional brain specificity because it was not observed in fetal hypothalamic cell cultures. In time course studies, the effects of QUIN/NMDA on SS mRNA occurred after a latency of 8 h, indicating a delayed effect. Cortical cells transfected with pSS-750 CAT showed three- to fourfold stimulation of CAT activity with forskolin but not by QUIN or NMDA. These data reveal a dose-dependent, tissue-specific, NMDA receptor-mediated stimulation of SS but not NPY mRNA.(ABSTRACT TRUNCATED AT 250 WORDS)

Combined non-radioactive detection of peptide hormones and their mRNAs in stomach somatostatin cells

Non-radioactive in situ hybridization (ISH) and immunocytochemistry (ICC) have been used to detect somatostatin (SS) messenger RNA (mRNA) and peptide in antropyloric mucosa of the stomach in the rats. We have applied a method of non-radioactive in situ hybridization histochemistry using digoxigenin labelled oligonucleotide probes to detect somatostatin gene expression in the stomach. In prehybridization stage we used proteinase K (PK) in various concentrations (from 1 to 10 μg/ml) and periods (from 10 min to 1 h) but we maintained high background. However it was possible to detect the somatostatin mRNAs in the stomach mucosa making use of either background preventing solutions during the prehybridization, or of levamisole (20 μl/mg) added into the hybridization buffer or of pepsin. Somatostatin mRNA and peptide signals were scattered all through the mucosa especially localized particularly at the base of the pyloric glands. SS peptide shown by ICC and SS mRNA shown by ISH were observed in different cells.

Localization of the somatostatin gene to bovine chromosome 1q23-q25 by in situ hydridization.

Localization of the somatostatin gene to bovine chromosome 1q23-q25 by in situ hydridization.

Repression of somatostatin gene transcription mediated by two promoter silencer elements

We report that expression of the somatostatin gene in pancreatic islet and in non-islet cells is negatively regulated by two proximal silencer elements, PS1 and PS2. Transient transfection assays showed that PS1 decreases somatostatin gene promoter activity stimulated by an upstream enhancer in the islet D-cell line RIN-1027-B2, but not in the islet B-cell line RIN-1046-38, whereas PS2 inhibits gene transcription in both B- and D-cell lines. In BHK fibroblasts, both PS1 and PS2 independently inhibit somatostatin gene promoter activity, suggesting that both elements are involved in preventing the expression of the somatostatin gene in non-islet cells. DNA-binding studies revealed that both PS1 and PS2 bind similar nuclear protein complexes in islet and non-islet cells (120 and 130 kDa). PS1 also binds a 100-kDa protein present in islet B- and D-cell lines. In addition, both PS1 and PS2 bind three D-cell-specific protiens (40, 43 and 45 kDa). These observations support a direct involvement of both positive and negative transcriptional control mechanisms in the regulation of the islet cell-specific expression of the somatostatin gene.

Coexpression of the gastrin and somatostatin genes in differentiating and neoplastic human cells.

Double immunofluorescence and in situ hybridizations performed on adjacent thin sections show that a population of normal antropyloric cells of the human stomach expresses both gastrin and somatostatin mRNA's and the corresponding peptides. Such cells were present in both adult and fetal antropyloric mucosa and were situated in the regenerative (isthmus) region of the antropyloric tubes. It is, hence, likely that these cells represent immature endocrine cells that yet have to be committed to either the gastrin or somatostatin lineage. Cells coexpressing gastrin and somatostatin were also detected in pancreatic endocrine tumours. The presence of gastrin-somatostatin cells during development and in tumours suggests that gastrin and somatostatin cells may differentiate from such multipotent precursor cells.

Vitamin D3 effects on basal and cAMP modulated expression of cholecystokinin and somatostatin genes in a rat medullary thyroid carcinoma cell line [CA-77

The regulation of cholecystokinin and somatostatin expression by vitamin D and cyclic AMP in the rat medullary thyroid carcinoma cell line CA-77 was investigated. Treatment with 100 nmol/l vitamin D did not affect cholecystokinin mRNA and peptide concentrations significantly; somatostatin mRNA level increased 6 times and the somatostatin peptide concentration increased 2-fold after 5 days of drug treatment. Under the same experimental conditions cyclic AMP increased cholecystokinin mRNA level 4.5 times and the cellular cholecystokinin-peptide concentration 2-fold; somatostatin mRNA and peptide concentrations were not significantly changed. Cyclic AMP stimulated peptide secretion from the cells were not affected by vitamin D, but cyclic AMP mediated increase in CCK peptide concentration was significantly inhibited by vitamin D (p < 0.05).

The LIM-homeodomain protein Isl-1 segregates with somatostatin but not with gastrin expression during differentiation of somatostatin/gastrin precursor cells

The gastric epithelium is renewed from stem cells in the isthmus of the gastric glands. We describe that the two neuroendocrine peptides gastrin and somatostatin are coexpressed by isthmic stem cells. Bromodeoxyuridine labeling indicates that the coexpressing cells divide and differentiate into gastrin and somatostatin cells, which remain in paracrine contact during most of their migration down into the gland. The coexpressing cells display nuclear immunoreactivity for the transcription factors Isl-1 and CREB, which have been implicated in somatostatin gene expression. Differentiated gastrin cells lack Isl-1 reactivity and show variable staining for CREB while differentiated somatostatin cells display Isl-1 and CREB reactivity.