Somatostatin receptor gene expression in neuroblastoma

Somatostatin receptor expression is a favorable prognostic factor in human neuroblastoma. Somatostatin receptors have been demonstrated in vitro by pharmacologic analysis of tumor tissue and in vivo by diagnostic radioreceptor scintigraphy. However, which receptor subtypes (sst(1), sst(2), sst(3), sst(4), and sst(5)) are expressed in these tumors has not yet been delineated. We used RT-PCR to analyze expression of the five somatostatin receptor genes in 32 neuroblastoma tumor specimens. All 32 tumor specimens expressed mRNA for c-abl and sst(1); sst(2) mRNA was detected in 27/32 samples and somatostatin mRNA was detected in 30/32 tumor specimens. The remaining receptor subtypes, sst(3), sst(4), and sst(5) were variably expressed. Receptor protein for sst(1) and sst(2) was visualized in tumor neuroblasts as well as in endothelial cells of tumor vessels using immunostaining with specific anti-receptor antibodies. The effect of high expression of somatostatin receptors on cell proliferation was examined in SKNSH neuroblastoma cells transfected with sst(1) and sst(2). SS(14) binding to wild-type SKNSH cells was undetectable; but the native peptide bound with high affinity to the SKNSH/sst(1) and SKNSH/sst(2) neuroblastoma cell lines. Pharmacologic analysis of binding with two long-acting analogues, CH275 and octreotide, confirmed selective expression of sst(1) and sst(2) in stably transfected SKNSH cells. Formation of neuroblastoma xenograft tumors in nude mice was significantly delayed for both SKNSH/sst(1) (P<0.001) and SKNSH/sst(2) (P<0.05) cells compared to wild-type SKNSH. We conclude that: (1) Somatostatin receptors, sst(1) and sst(2), are expressed in the majority of neuroblastomas at diagnosis; and (2) upregulation of functional sst(1) or sst(2) in neuroblastoma cell lines suppresses tumorigenicity in a xenograft model. These observations suggest that somatostatin receptors may be a useful therapeutic target in neuroblastoma.     

The Somatostatin 2A Receptor Is Enriched in Migrating Neurons during Rat and Human Brain Development and Stimulates Migration and Axonal Outgrowth

The neuropeptide somatostatin has been suggested to play an important role during neuronal development in addition to its established modulatory impact on neuroendocrine, motor and cognitive functions in adults. Although six somatostatin G protein-coupled receptors have been discovered, little is known about their distribution and function in the developing mammalian brain. In this study, we have first characterized the developmental expression of the somatostatin receptor sst2A, the subtype found most prominently in the adult rat and human nervous system. In the rat, the sst2A receptor expression appears as early as E12 and is restricted to post-mitotic neuronal populations leaving the ventricular zone. From E12 on, migrating neuronal populations immunopositive for the receptor were observed in numerous developing regions including the cerebral cortex, hippocampus and ganglionic eminences. Intense but transient immunoreactive signals were detected in the deep part of the external granular layer of the cerebellum, the rostral migratory stream and in tyrosine hydroxylase- and serotonin- positive neurons and axons. Activation of the sst2A receptor in vitro in rat cerebellar microexplants and primary hippocampal neurons revealed stimulatory effects on neuronal migration and axonal growth, respectively. In the human cortex, receptor immunoreactivity was located in the preplate at early development stages (8 gestational weeks) and was enriched to the outer part of the germinal zone at later stages. In the cerebellum, the deep part of the external granular layer was strongly immunoreactive at 19 gestational weeks, similar to the finding in rodents. In addition, migrating granule cells in the internal granular layer were also receptor-positive. Together, theses results strongly suggest that the somatostatin sst2A receptor participates in the development and maturation of specific neuronal populations during rat and human brain ontogenesis.     

Developmental Expression and Functional Characterization of the 4C5 Antigen in the Postnatal Cerebellar Cortex

Abstract: The monoclonal antibody 4C5 recognizes a neuron-specific surface antigen (4C5 antigen) in the CNS and PNS of the rat. In the present study we investigated the expression of 4C5 antigen in the developing cerebellum of the rat and the functional role of this molecule during cerebellar morphogenesis. Immunoblotting and immunohistochemistry in sections of cerebellar cortex revealed an age-dependent decrease in the expression of the 4C5 antigen. In cerebellar primary cell cultures, 4C5 immunoreactivity was detected both on granule and on Purkinje neurons. Granule cell migration was inhibited in cerebellar explants derived from 8-day-old rats and cultured for 2 days in the presence of antibodies against the 4C5 antigen. Electron microscope immunocytochemistry revealed that in 8-day-old rat cerebellum, 4C5 immunoreactivity was localized on the cell bodies of granule neurons in the external and internal granular layers and on parallel fibers in the developing molecular layer as well as at contact sites between these cellular elements. It was not detected on Bergmann glia. These results suggest strongly that the 4C5 antigen is involved in granule cell migration during cerebellar development, possibly via neuron-neuron interactions.     

Distribution and characterisation of somatostatin receptor mRNA and binding sites in the brain and periphery.

The distribution and nature of (somatostatin) SRIF receptors and receptor mRNAs was studied in the brain and periphery of various laboratory animals using in situ hybridisation, autoradiography and radioligand binding. The messenger RNA (mRNA) expression of SRIF receptors msst1, msst2, msst3, msst4 and msst5 was studied in the adult mouse brain by in situ hybridisation histochemistry using specific oligonucleotide probes and compared to that of adult rats. As observed in rat brain, sst3 receptor mRNA is prominently expressed across the mouse brain, although equivalent binding has not yet been identified in situ. Sst1 and sst2 receptor mRNA expression, was prominent and again comparable to that observed in rat brain, whereas sst4 and especially sst5 receptor mRNA show comparatively low levels, although the former appears to be widely distributed while the latter could only be identified in a few nuclei. Altogether, the data are compatible with current knowledge, i.e. sst1 and sst2 receptor mRNA is prominent (both receptors have been functionally identified in the brain and for sst2 in the periphery), sst3 mRNA is highly expressed but in the absence of any functional correlate remains elusive. The expression of sst4 mRNA is comparatively low (especially when compared to what is seen in the lung, where high densities of sst4 receptors are present) and it remains to be seen whether sst5 receptor mRNA, which is confined to a few nuclei, will play a role in the brain, keeping in mind that high levels are found in the pituitary. Radioligand binding studies were performed in CCL39 cells expressing the five human recombinant receptors and compared to binding in membranes of rat cerebral cortex with [125I]Tyr11-SRIF14 which in the presence of 120 mM labels primarily sst1 receptor as suggested by the better correlation hsst1 and similar rank order of potency. The profile of [125I]Tyr3-octreotide labelled sites in rat cortex correlates better with recombinant sst2 than sst3 or sst5 binding profiles. Finally, [125I]LTT-SRIF28-labelled sites in rat lung express a sst4 receptor profile in agreement with previous findings. SRIF receptor autoradiography was performed in the brain and peripheral tissue of rat and/or guinea-pig using a number of ligands known to label recombinant SRIF receptors: [125I]LTT-SRIF28, [125I]CGP 23996, [125I]Tyr10-CST, or [125I]Tyr3-octreotide. Although, [125I]Tyr10-CST has been shown to label all five recombinant SRIF receptors, it is apparent that this radioligand is not useful for autoradiographic studies. By contrast, the other three ligands show good signal to noise ratios in rat or guinea-pig brain, rat lung, rat pancreas, or guinea-pig ileum. In most tissues, [125I]Tyr3-octreotide represents a prominent part of the binding (when compared to [125I]LTT-SRIF28 and [125I]CGP 23996), suggesting that sst2 receptors are strongly expressed in most tissues; it is only in rat lung that [125I]LTT-SRIF28 and [125I]CGP 23996 show marked binding, whereas [125I]Tyr3-octreotide does apparently label no sites, in agreement with the sole presence of sst4 receptors in this tissue.     

Induction of galanin receptor-1 (GalR1) expression in external granule cell layer of post-natal mouse cerebellum

Galanin is a modulator of fast transmission in adult brain and recent evidence suggests that it also acts as a trophic factor during neurogenesis and neural injury and repair. Previous studies in our laboratory have identified galanin mRNA in Purkinje cells of adult and developing rat (but not adult mouse) cerebellum; and galanin-binding sites in adult mouse (but not rat) cerebellum. The post-natal development of the cerebellum provides a unique and convenient model for the investigation of developmental processes and to learn more about putative cerebellar galanin systems, the current study examined the presence and distribution of galanin-like-immunoreactivity (- LI), [(125)I]-galanin binding sites and galanin receptor-1 (GalR1) mRNA in post-natal mouse cerebellum. Using autoradiography and in situ hybridization, [(125)I]-galanin binding sites and GalR1 mRNA were first detected on post-natal day 10 (P10) in the external germinal layer of all lobes and high levels were maintained until P14. Quantitative real-time PCR assays detected GalR1 mRNA in whole cerebellum across the post-natal period, with a strong induction and peak of expression at P10. Assessment of galanin levels in whole cerebellum by radioimmunoassay revealed relatively similar concentrations from P5 to P20 and in adult mice (80-170 pg/mg protein), with a significantly higher concentration (250 pg/mg, p < 0.01) detected at P3. These concentrations were some four- to six-fold lower than those in adult forebrain samples. Using immunohistochemistry, galanin-like-immuno-reactivity was observed in prominent fibrous elements within the white matter tracts of the cerebellum at P3-5 and in more punctate elements in the internal granule cell layer and associated with the Purkinje cell layer at P12 and P20. Increased levels of GalR1 mRNA and galanin binding (attributed to GalR1) in the external granule cell layer at P10-12/(14) coincide with granule cell migration from the external to the inner granule cell layer and together with demonstrated effects of other neuropeptide-receptor systems suggest a role for GalR1 signalling in regulating this or related developmental processes.     

The Elusive Nature of Cerebellar Somatostatin Receptors: Studies in Rat,Monkey and Human Cerebellum

Abstract

The pharmacological profile and localization of somatostatin (SRIF) receptors were determined in rat, monkey and human cerebellum. In rat cerebellar cortex, low ss₁/sst₄, intermediate sst₂ and very high sst₃ receptor mRNA levels were found, sst₁ mRNA was also expressed in the deep cerebellar nuclei. [¹²⁵I]Tyr³-octreotide binding sites in cerebellar membranes correlated with recombinant sst₂, but not with sst₅ or sst₃ receptors and were found in the molecular layer of the cerebellum. [¹²⁵I]CGP 23996 (in Na⁺-buffer) binding in rat cerebellum correlated with sst₁ or sst₄, but not with sst₂, sst₃ or sst₅ receptor binding. Similar data were obtained in rhesus monkey cerebellum. mRNAs for all five receptors were found in the granule cell layer of the human cerebellum and/or in the dentate nucleus. [¹²⁵I]Tyr³-octreotide binding was strong in the molecular layer and correlated with that of recombinant sst₂ receptors, but not with sst₃ or sst₅ receptors. [¹²⁵I]CGP 23996 (in Mg⁺⁺-buffer) binding was heterogeneous (about 75%. to sst₂ and 25% to sst₁ and/or sst₄ receptors). The molecular and granular layers were equally and the dentate nucleus strongly labeled. Thus. SRIF receptors of the sst₂, sst₁ and/or sst₄ subtype are present in the rat, monkey and human cerebellum. In the latter two species, the sst₂ type appears to be predominant. Surprisingly, the high expression of sst₃ receptor mRNA is not supported by radioligand binding data in any of the species studied. The reason for this discrepancy remains to be elucidated.     

Glycosylation affects agonist binding and signal transduction of the rat somatostatin receptor subtype 3.

The somatostatin receptor subtypes, sst1-sst5, bind their natural ligands, somatostatin-14, somatostatin-28 and cortistatin-17, with high affinity but do not much discriminate between them. Detailed understanding of the interactions between these receptors and their peptide ligands may facilitate the development of selective compounds which are needed to identify the biological functions of individual receptor subtypes. The influence of the amino-terminal domain and of the two putative N-linked glycosylation sites located in this region of rat sst3 was analysed. Biochemical studies in transfected cell lines suggested that the amino-terminus of sst3 is glycosylated at both sites. Mutation of the N-linked glycosylation site, Asn18Thr, had only a small effect on binding properties and inhibition of adenylyl cyclase. The double mutant Asn18Thr/Asn31Thr lacking both glycosylation sites showed a significant reduction in high affinity binding and inhibition of adenylyl cyclase while peptide selectivity was not affected. Truncation of the amino-terminal region by 32 amino acid residues including the two glycosylation sites caused similar but much stronger effects. Immunocytochemical analysis of receptor localisation revealed that the amino-terminal domain but not the carbohydrates appear to be involved in the transport of the receptor polypeptide to the cell surface.     

Glutamate receptor subunit expression in primary neuronal and secondary glial cultures

We report on the expression of ionotropic glutamate receptor subunits in primary neuronal cultures from rat cortex, hippocampus and cerebellum and of metabotropic glutamate (mGlu) receptor subtypes in these neuronal cultures as well as in cortical astroglial cultures. We found that the NMDA receptor (NR) subunits NR1, NR2A and NR2B were expressed in all three cultures. Each of the three cultures showed also expression of the four AMPA receptor subunits. Although RT-PCR detected mRNA of all kainate (KA) subunits in the three cultures, western blot showed only expression of Glu6 and KA2 receptor subunits. The expression analysis of mGlu receptors indicated the presence of all mGlu receptor subtype mRNAs in the three neuronal cultures, except for mGlu2 receptor mRNA, which was not detected in the cortical and cerebellar culture. mGlu1a/alpha, -2/3 and -5 receptor proteins were present in all three cultures, whereas mGlu4a and mGlu8a receptor proteins were not detected. Astroglial cultures were grown in either serum-containing or chemically defined medium. Only mGlu5 receptor protein was found in astroglial cultures grown in serum-containing medium. When astrocytes were cultured in chemically defined medium, mGlu3, -5 and -8 receptor mRNAs were detected, but at the protein level, still only mGlu5 receptor was found.     

Expression of Cytochrome P450 Side-Chain Cleavage Enzyme and 3β-Hydroxysteroid Dehydrogenase in the Rat Central Nervous System: A Study by Polymerase Chain Reaction and In Situ Hybridization

Abstract: In examining steroid synthesis in the CNS, expression of the mRNAs encoding for cytochrome P450 side-chain cleavage enzyme (P450_SCC) and 3β-hydroxysteroid dehydrogenase/Δ⁵-Δ⁴ isomerase (3β-HSD) has been studied in the rat brain. P450_SCC transforms cholesterol into pregnenolone and 3β-HSD transforms pregnenolone into progesterone. PCR was used to amplify cDNA sequences from total RNA extracts. Classical steroidogenic tissues, like adrenal and testis, as well as the non-steroidogenic tissue lung have been used as controls. The expression of P450_SCC and 3β-HSD have been demonstrated by PCR in cortex, cerebellum, and spinal cord. In addition, primary cultures of rat cerebellar glial cells and rat cerebellar granule cells were found to express P450_SCC and 3β-HSD at comparable levels. Furthermore, three of the four known isoenzymes of 3β-HSD were identified, as determined using selective PCR primers coupled with discriminative restriction enzymes and sequencing analysis of the amplified brain products. Using RNA probes, in situ hybridization indicated that P450_SCC and 3β-HSD are expressed throughout the brain at a low level and mainly in white matter. Enrichment of glial cell cultures in oligodendrocytes, however, does not increase the relative abundance of P450_SCC and 3β-HSD mRNA detected by PCR. This discrepancy suggests that the developmental state of cultured cells and their intercellular environment may be critical for regulating the expression of these enzymes. These findings support the proposal that the brain apparently has the capacity to synthesize progesterone from cholesterol, through pregnenolone, but that the expression of these enzymes appears to be quite low. Furthermore, the identification of these messages in cerebellar granule cell cultures implies that certain neurons, in addition to glial cells, may express these steroidogenic enzymes.     

The mRNAs encoding GABAA/benzodiazepine receptor subunits are localized in different cell populations of the bovine cerebellum

The expression of the mRNAs encoding the alpha and beta subunits of the GABAA/benzodiazepine receptor was examined in the bovine cerebellum by in situ hybridization histochemistry. The alpha subunit mRNA, which encodes the benzodiazepine binding site, was localized in all Purkinje and granule cells and in some cells of the molecular layer. The distribution of the beta subunit mRNA, which encodes the GABA binding site, only partially overlapped with that of the alpha subunit mRNA. While cells in the granule cell layer expressed the beta subunit mRNA, no message could be detected in other cell populations. These findings suggest that the subunit composition of the GABAA/benzodiazepine receptor is heterogeneous and that additional, as yet unidentified, beta subunits exist.     

Expression and Agonist-Induced Down-Regulation of mRNAs of m2- and m3-Muscarinic Acetylcholine Receptors in Cultured Cerebellar Granule Cells

The regulation and expression of muscarinic acetylcholine receptor (mAChR) mRNA was studied in cultured cerebellar granule cells using Northern blot hybridization, mRNA species for m2- and m3-mAChRs but not m1- and m4-mAChRs were detected in these cells. The expression of mRNAs of both m2- and m3-mAChRs reached a maximum on the tenth day in culture but their expression patterns differed. Treatment of cerebellar granule cells after 8 days in culture with 100 microM carbachol led to differential down-regulation of the mRNA species of both mAChR subtypes present. Muscarinic receptor antagonists, atropine (1 microM) and pirenzepine (10 microM), prevented carbachol-induced m3-mAChR mRNA down-regulation observed at 8 h. However, exposure to either atropine or pirenzepine alone for 8 h led to a significant up-regulation of m3-mAChR mRNA. Thus, the mRNA species for both m2- and m3-mAChR subtypes are differentially expressed in culture and down-regulated by agonist stimulation. The loss of these mRNA species may play a role in the down-regulation of mAChR binding sites that occurs after desensitization.     

RT-PCR and immunocytochemistry studies support the presence of somatostatin, cortistatin and somatostatin receptor subtypes in rat Kupffer cells

The present study investigated the presence of somatostatin receptor subtypes (ssts) and the endogenous peptides somatostatin and cortistatin in rat Kupffer cells, since modulation of these cells by somatostatin may be important for the beneficial effect of somatostatin analogues in a selected group of hepatocellular carcinoma patients. Kupffer cells were isolated from rat liver in agreement with national and EU guidelines. RT-PCR was employed to assess the expression of somatostatin, cortistatin and ssts in Kupffer cells. Western blot analysis and immunocytochemistry were employed to assess the expression and the localization of the receptors, respectively. Quiescent Kupffer cells were found to express sst(1-4) mRNA, while immunocytochemical studies supported the presence of only the sst(3) and sst(4) receptors, which were found to be internalized. However, sst1 and sst(2A) receptors were detected by western blotting. RT-PCR and RIA measurements support the presence of both somatostatin and cortistatin. Stimulation of the cells with LPS activated the expression of the sst(2), sst(3) and sst(4) receptors. The present data provide evidence to support the presence of ssts and the endogenous neuropeptides somatostatin and CST in rat Kupffer cells. Both peptides may act in an autocrine manner to regulate sst receptor distribution. Studies are in progress in order to further characterize the role of ssts in Kupffer cells and in hepatic therapeutics.     

Somatostatin increases mitogen-induced IL-2 secretion and proliferation of human jurkat T cells via sst3 receptor isotype

The neuropeptide somatostatin (SRIF) modulates normal and leukemia T cell proliferation. However, neither molecular isotypes of receptors nor mechanisms involved in these somatostatin actions have been elucidated as yet. Here we show by using RT-PCR approach that mitogen-activated leukemia T cells (Jurkat) express mRNA for a single somatostatin receptor, sst3. This mRNA is apparently translated into protein since specific somatostatin binding sites (K_I1 = 78 ± 3 pM) were detected in semipurified plasma membrane preparations by using ¹²⁵I-Tyr¹-SRIF14 as a radioligand. Moreover, somatostatin inhibits adenylyl cyclase activity with similar efficiency (IC₅₀ = 23 ± 4 pM) thus strongly suggesting a functional coupling of sst3 receptor to this transduction pathway. The involvement of sst3 receptor in immuno-modulatory actions of somatostatin was assessed by analysis of neuropeptide effects on IL-2 secretion and on proliferation of mitogen-activated Jurkat cells. Our data show that in the concentrations comprised between 10 pM and 10 nM, somatostatin potentiates IL-2 secretion. This effect is correlated with somatostatin-dependent increase of Jurkat cell proliferation since the EC₅₀ concentrations for both actions were almost identical (EC₅₀ = 22 ± 9 pM and EC₅₀ = 12 ± 1 pM for IL-2 secretion and proliferation, respectively). Altogether, these data strongly suggest that in mitogen-activated Jurkat cells, somatostatin increases cell proliferation through the increase of IL-2 secretion via a functional sst3 receptor negatively coupled to the adenylyl cyclase pathway. J. Cell. Biochem. 68:62–73, 1998. © 1998 Wiley-Liss, Inc.     

Neurotrophins promote the survival and development of neurons in the cerebellum of hypothyroid rats in vivo

The development of cerebellar cortex is strongly impaired by thyroid hormone (T3) deficiency, leading to altered migration, differentiation, synaptogenesis, and survival of neurons. To determine whether alteration in the expression of neurotrophins and/or their receptors may contribute to these impairments, we first analyzed their expression using a sensitive RNAse protection assay and in situ hybridization; second, we administered the deficient neurotrophins to hypothyroid animals. We found that early hypothyroidism disrupted the developmental pattern of expression of the four neurotrophins, leading to relatively higher levels of NGF and neurotrophin 4/5 mRNAs and to a severe deficit in NT-3 and brain-derived neurotrophic factor (BDNF) mRNA expression, without alteration in the levels of the full-length tyrosine kinase (trk) B and trkC receptor mRNAs. Grafting of P3 hypothyroid rats with cell lines expressing high levels of neurotrophin 3 (NT-3) or BDNF prevented hypothyroidism-induced cell death in neurons of the internal granule cell layer at P15. In addition, we found that NT-3, but not BDNF, induced the differentiation and/or migration of neurons in the external granule cell layer, stimulated the elaboration of the dendritic tree by Purkinje cells, and promoted the formation of the mature pattern of synaptic afferents to Purkinje cell somas. Thus, our results indicate that both granule and Purkinje neurons require appropriate levels of NT-3 for normal development in vivo and suggest that T3 may regulate the levels of neurotrophins to promote the development of cerebellum.     

Spatiotemporal expression and functional implication of CXCL14 in the developing mice cerebellum

Cerebellar granule neurons migrate from the external granule cell layer (EGL) to the internal granule cell layer (IGL) during postnatal morphogenesis. This migration process through 4 different layers is a complex mechanism which is highly regulated by many secreted proteins. Although chemokines are well-known peptides that trigger cell migration, but with the exception of CXCL12, which is responsible for prenatal EGL formation, their functions have not been thoroughly studied in granule cell migration. In the present study, we examined cerebellar CXCL14 expression in neonatal and adult mice. CXCL14 mRNA was expressed at high levels in adult mouse cerebellum, but the protein was not detected. Nevertheless, Western blotting analysis revealed transient expression of CXCL14 in the cerebellum in early postnatal days (P1, P8), prior to the completion of granule cell migration. Looking at the distribution of CXCL14 by immunohistochemistry revealed a strong immune reactivity at the level of the Purkinje cell layer and molecular layer which was absent in the adult cerebellum. In functional assays, CXCL14 stimulated transwell migration of cultured granule cells and enhanced the spreading rate of neurons from EGL microexplants. Taken together, these results revealed the transient expression of CXCL14 by Purkinje cells in the developing cerebellum and demonstrate the ability of the chemokine to stimulate granule cell migration, suggesting that it must be involved in the postnatal maturation of the cerebellum.