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H Tabuchi, Y Furuichi and C Miyamoto

ABSTRACT

To investigate the nuclear signalling pathway induced by endothelin (ET) isopeptides, we have established permanent Chinese hamster ovary (CHO) cell lines, CHO-ETA/fos-lacZ and CHO-ETB/fos-lacZ, that produce both a c-fos-β-galactosidase fusion protein and either the type A or the type B human ET receptor. These cell lines permitted a colorimetric measurement of c-fos expression, which was induced by the signal transduction system with ET receptors and ET isopeptides. We found that the ET-1-dependent c-fos expression was so efficient that it could respond to low concentrations (even a physiological concentration) of ET-1. For example, CHO-ETA/fos-lacZ and CHO-ETB/fos-lacZ responded to ET concentrations of 5×10−9 m and 5×10−13 m respectively. Using this highly sensitive system, the H-7 sensitive protein kinase was found to be involved in signal transduction mediated by ETA, and also partly in the ETB-mediated pathway. These lines of evidence suggest that c-fos expression occurs through at least two different pathways, depending on the concentration of ET in plasma.

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Q Wu, XF Lin, XF Ye, B Zhang, Z Xie and WJ Su

Retinoic acid receptor alpha (RARalpha) plays an important role in mediating all-trans retinoic acid (ATRA) signals. In this study, we found that ATRA up-regulated RARalpha mRNA and protein expression in gastric cancer BGC-823 cells. However, in breast cancer MCF-7 cells it down-regulated RARalpha protein expression with no effect on its RARalpha mRNA. Immunoprecipitation/Western blot analysis showed that, although sumoylated and ubiquitinated RARalpha existed simultaneously in both cancer cell lines, ATRA exerted different regulatory effects on sumoylation and ubiquitination of RARalpha. In MCF-7 cells, ATRA treatment enhanced the ubiquitination of RARalpha and the subsequent degradation of RARalpha through the ubiquitin/proteasome pathway. This resulted in a reduction in the DNA binding activity of RARalpha/retinoid X receptor alpha (RXRalpha) heterodimer, the separation of RXRalpha from RARalpha and the translocation of RXRalpha from the nucleus to the cytoplasm. By contrast, in BGC-823 cells, ATRA augmented sumoylation, not ubiquitination, of RARalpha. The stability of sumoylated RARalpha was significantly stronger than in non-sumoylated RARalpha. These results also showed an increase in the DNA binding activity of the RARalpha/RXRalpha heterodimer and the stability of nuclear localization of this heterodimer, which normally facilitates the ATRA signal transduction. In conclusion, our results reveal a novel mechanism for the regulation of RARalpha-dependent signal transduction through the ubiquitin/proteasome pathway in breast cancer cells and the sumoylation pathway in gastric cancer cells.

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CJ Rhodes

Certain nutrients, pharmacological agents and growth factors can stimulate pancreatic beta-cell proliferation; however, mitogenic signal transduction pathways in beta-cells have not been particularly well characterized. As a model system we have focussed on characterizing the signal transduction pathways immediately downstream of the IGF-I and GH receptors in beta-cells. The original idea was to gain an idea of important elements in mitogenic signaling pathways which might then be exploited to generate a marked increase in beta-cell proliferation. Such an approach could eventually reveal a means to increase the number of human pancreatic endocrine cells in vitro, in order to obtain an abundant source of beta-cells for routine transplantation therapy of type-I diabetes. However, in the course of our studies, we have also unveiled an unexpected insight into the pathogenesis of obesity-linked type-II diabetes. It has been observed that free fatty acids inhibit glucose- and glucose-dependent IGF-I/GH-induced beta-cell proliferation. We hypothesize that a gradual accumulation of intracellular fat in beta-cells during obesity can eventually lead to an inhibition of beta-cell mass expansion and hence failure to compensate for peripheral insulin resistance, so that type-II diabetes ensues.

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K Haraguchi, T Saito, M Kaneshige, T Endo and T Onaya

ABSTRACT

To understand the functional significance of the carboxy-terminal half of the intracellular region of the human TSH receptor (hTSHR), a mutant hTSHR lacking amino acids from the carboxyterminal to His726 was constructed.

Wild type hTSHR cDNA and truncated hTSHR cDNA were subcloned into a eukaryotic expression vector, pRc/CMV, and transfected into Chinese hamster ovary cells to obtain cell lines which stably expressed hTSHRs at high levels. This allowed us to observe highly efficient coupling of hTSHR and adenylyl cyclase as well as desensitization and internalization of hTSHR.

Despite the differences in potential phosphorylation sites and internalization signals, dose-dependent stimulation of adenylyl cyclase by TSH, TSH-dependent desensitization and the rate of hTSHR internalization were similar for wild type and truncated hTSHRs. We conclude that the carboxy-terminal half of the intracellular region of hTSHR does not have a major functional role in TSH-dependent signal transduction.

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S Mani

The current and expanded view of transcriptional regulation by the steroid/thyroid superfamily of nuclear transcription factors integrates not only the hormone-dependent but also the hormone-independent cellular signaling mechanisms in physiology and reproduction. This effort has vastly been aided by the identification of steroid hormone receptors as transcriptional mediators of a variety of ligands, whose transcriptional response is dependent upon cross-talk with distinct signal transduction pathways, their recruitment of coregulators, alteration of chromatin structure and identification of specific interactive motifs within the receptors themselves. This review will provide a framework for the current concepts in the field of steroid hormone action as exemplified by our studies on progesterone receptor in female sexual behavior.

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P E Clayton, R N Day, C M Silva, P Hellmann, K H Day and M O Thorner

ABSTRACT

GH induces hepatic IGF-I synthesis by increasing transcription of its gene. IGF-I is synthesized, however, in many other tissues where the effect of GH on its gene expression is less well characterized. IGF-I and GH are produced by human lymphocytes and may function as autocrine regulators of lymphoproliferation. We have therefore used the human IM9 lymphocyte cell line to (A) define the IGF-I gene transcripts expressed and (B) investigate the effect of GH on early (protein tyrosine phosphorylation) and late (changes in IGF-I mRNA levels) events in intracellular signal transduction. Multiple IGF-I mRNA species, ranging in size from 0·9 to 5·8 kb, were detected by Northern hybridization of poly(A)+ mRNA from IM9 cells. The human IGF-I gene contains at least six exons and alternative splicing produces a number of transcripts. Solution hybridization with exon-specific riboprobes and amplification by PCR using exon-specific primers revealed that multiple transcripts were expressed in IM9 cells, and that exon 2 was the dominant leader exon.

Treatment of IM9 cells with 200 ng recombinant human (rh)GH/ml led to the specific tyrosine phosphorylation of three intracellular proteins (93, 120 and 134 kDa), which are involved in the initial signalling of the GH transduction pathway. However a solution hybridization assay using the IGF-IA specific riboprobe on IM9 cell RNA from similar experiments revealed that GH treatment did not change IGF-I gene expression.

This study has demonstrated (A) that the IGF-I gene is expressed in human IM9 lymphocytes, (B) that in contrast to other human tissue, exon 2 is the major leader exon, and (C) that rhGH induces tyrosine phosphorylation of 93, 120 and 134 kDa proteins but does not alter IGF-I gene expression. The IM9 cell may form an important model to investigate a GH transduction pathway not coupled to the IGF-I gene.

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C Sen Gupta and RR Dighe

The strategy of translationally fusing the two subunits of human chorionic gonadotropin (hCG) has been used to produce recombinant single chain hCG in which the C-terminus of the alpha subunit is fused to the N-terminus beta without any linker using Pichia pastoris expression system. The Pichia-expressed hCGalphabeta (phCGalphabeta) attained an overall conformation similar to that of hCG, and could bind to the receptor and elicit biological response, suggesting that receptor binding and signal transduction can take place even with a molecule having blocked the C-terminus of the alpha subunit. The carboxyl terminal of the alpha subunit has been shown to be involved in hormone binding and signal transduction of all the heterodimeric glycoprotein hormones. However, deletion of five amino acids from the C-terminus of the alpha subunit in the single chain hCG did not alter the overall conformation of the fusion molecule and its receptor binding ability, but led to a significant reduction in its ability to elicit biological response. These data show that these five amino acids at the C-terminus of the alpha subunit in the single chain hCG are not absolutely essential for attaining a conformation required for receptor binding, but are essential for obtaining a full biological response.

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S Gahr, M Merger, LC Bollheimer, CG Hammerschmied, J Scholmerich and SR Hugl

We investigated the role of hepatocyte growth factor (HGF) in beta-cell growth and its complex intracellular signal transduction pathways. Cell proliferation was measured in the beta-cell line INS-1 using [3H]thymidine incorporation. Activation of mitogenic signaling proteins was assessed using co-immunoprecipitation, immunoblot analysis and specific protein activity inhibitors in proliferation assays. HGF (1 x 375 nM) increased INS-1 cell proliferation in the presence of 3-24 mM glucose up to 45-fold vs unstimulated controls. HGF exceeded the effect of glucose alone (2 x 2-fold at 3 mM glucose and 1 x 7-fold in the presence of 15 mM glucose). The HGF-induced INS-1 cell proliferation was further increased by addition of IGF-I or GH. Stimulation with HGF activated the JAK-2/STAT-5 pathway with a subsequent activation of phosphatidylinositol-3'-kinase (PI3'K). PI3'K activation was necessary for HGF- and glucose-stimulated INS-1 cell proliferation. The effect of PI3'K was mediated via 70 kDa S6 kinase and protein kinase B, which showed maximum activation in the presence of 3-6 mM glucose. Protein kinase C was essential for HGF-induced INS-1 cell proliferation. The HGF effect was also mediated at low glucose concentrations via insulin receptor substrate 4 (IRS-4) whereas other IRS proteins did not show any activation. High glucose concentrations also showed an increased IRS-4/PI3'K binding and therefore activation. In conclusion, beta-cell proliferation is mediated via complex interacting signal transduction pathways. HGF, in contrast to other growth factors, seems to be of importance particularly in the presence of low glucose concentrations and therefore takes a special role in this complex concert.

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P R Riley, A P F Flint, D R E Abayasekara and H J Stewart

ABSTRACT

A sheep endometrial oxytocin receptor (OTR) cDNA (1·5 kb) was isolated from a λ-ZAP library using a reverse transcription-PCR product probe generated from oestrous endometrial mRNA. The sheep OTR cDNA shared an overall similarity of 82% with human OTR cDNA, 85% with pig OTR cDNA and 76% with rat OTR cDNA. The encoded receptor was a 391 amino acid polypeptide 94% similar to human OTR, 94% similar to pig OTR and 93% similar to rat OTR. The sheep OTR contained two additional amino acids compared with human OTR which were located in the highly GC-rich third intracytoplasmic loop. This region is thought to be associated with G protein coupling and signal transduction. Expression of the cDNA in Cos-7 cells and measurement of oxytocin-induced phosphoinositide turnover confirmed that it coded for a functional product. The affinity of the expressed receptor was comparable with that observed for the in vivo receptor.

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J.-P. Weniger, R. L. Cate and A. Zeis

ABSTRACT

To determine whether mammalian Müllerian-inhibiting substance (MIS) is active in birds, Müllerian ducts from 7- to 8-day-old male or female chick embryos were cultured in the presence of human recombinant MIS at concentrations between 2.5 and 12.5μg/ml. None of 20 ducts regressed at any concentration. In contrast, at concentrations of 2.5–5 μg/ml, all 12 Müllerian ducts from 13-day-old male mouse embryos and 13 out of 14 female ducts were inhibited to varying degrees. It is concluded that avian Müllerian ducts are unresponsive to mammalian MIS. There may be a difference in structure between the MIS of birds and mammals, or the signal-transduction system may be different.