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M. C. Slootweg, S. T. van Genesen, A. P. Otte, S. A. Duursma and W. Kruijer

ABSTRACT

Addition of human GH (hGH) to primary mouse osteoblasts resulted in rapid and transient induction of the c-fos and c-myc proto-oncogenes and preceded hGH-induced mitogenesis. Human GH-induced c-fos expression was maximal after 30 min, resulting in a 10- to 15-fold increase over unstimulated cells, and returned to prestimulation levels within 60 min of the addition of hGH. Induction of the c-fos gene by hGH was dose dependent and also occurred in the absence of protein synthesis, resulting in superinduction of the c-fos gene. The induction of the c-fos gene by hGH was mediated by a somatotrophic (GH) rather than a lactogenic (prolactin) receptor on primary mouse osteoblasts, as indicated by a 10- to 100-fold greater potency of hGH compared with ovine prolactin in stimulating the expression of the c-fos gene. Primary mouse osteoblasts also induced the c-fos gene in response to epidermal growth factor, insulin-like growth factor-I and several agents, including phorbol 12-myristate 13-acetate (TPA), forskolin and A23187, that are known to activate signal transduction pathways involved in the action of growth factors.

Addition of hGH to primary mouse osteoblasts did not result in increased phosphoinositide breakdown, while selective deactivation of the diacylglycerol—protein kinase C and inositol 1,4,5—trisphosphate—Ca2+ pathways by long-term TPA pretreatment or depleting intracellular Ca2+ stores had no effect on hGH-induced c-fos expression. Human GH did not alter basal cyclic AMP levels in mouse osteoblasts. The immediate consequences of GH—receptor interaction as well as the mechanism of signal transduction leading to induction of the c-fos gene remain, therefore, unresolved.

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A. R. Solano, L. Dada and E. J. Podesta

ABSTRACT

Aldosterone secretion from adrenal glomerulosa cells can be stimulated by angiotensin II (AII), extracellular potassium and ACTH. Mitochondria from these cells respond to intracellular factors generated by AII (cyclic AMP (cAMP)-independent steroidogenesis) and ACTH (cAMP-dependent steroidogenesis), suggesting that the two signal-transduction mechanisms are linked by a common intermediate. We have evaluated this hypothesis by stimulating mitochondria from the unstimulated zona glomerulosa with a subcellular post-mitochondrial fraction (PMF) obtained from the zona glomerulosa after stimulation with AII or from the fasciculata gland after stimulation with ACTH; the subcellular fractions were also tested on mitochondria from fasciculata cells. PMFs obtained after incubation of adrenal zona glomerulosa with or without AII (0·1 μm) or ACTH (0·1 nm) were able to increase net progesterone synthesis 4·5-fold in mitochondria isolated from unstimulated rat zona glomerulosa. AII-pre-treated PMFs from the zona glomerulosa also stimulated steroidogenesis by mitochondria from zona fasciculata cells.

Separate experiments showed that inhibitors of arachidonic acid release and metabolism (bromophenacyl bromide, nordihydroguaiaretic acid, caffeic acid or esculetin) blocked corticosterone production in fasciculata cells stimulated with ACTH, suggesting that arachidonic acid could be the common intermediate in the actions of AII and ACTH on steroid synthesis. Evidence to support this concept was obtained from experiments in which the formation of an activated PMF by treatment of zona fasciculata with ACTH was blocked by the presence of the same inhibitors. Moreover, the inhibitory effects of these substances on PMF activation by ACTH were overcome by exogenous arachidonic acid and, in addition, arachidonic acid release was stimulated by ACTH.

We suggest that the mechanisms of action of ACTH and AII involve an increase in the release of arachidonic acid and conversion of arachidonic acid into lipoxygenase products. Both ACTH and AII may have a common intermediate, in spite of different membrane receptors and different signal-transduction mechanisms.

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CE Wrede, LM Dickson, MK Lingohr, I Briaud and CJ Rhodes

It is possible that activation of protein kinase C (PKC) isoforms by free fatty acids (FFA) plays a role in the failure of pancreatic beta-cell mass expansion to compensate for peripheral insulin resistance in the pathogenesis of type-2 diabetes. The effect of lipid moieties on activation of conventional (PKC-alpha and -beta1), novel (PKC-delta) and atypical (PKC-zeta) PKC isoforms was evaluated in an in vitro assay, using biotinylated neurogranin as a substrate. Oleoyl-Coenzyme A (CoA) and palmitoyl-CoA, but not unesterified FFA, significantly increased the activity of all PKC isoforms (P< or =0.05), particularly that for PKC-delta. It was found that FFA (0.4 mM oleate/complexed to 0.5% bovine serum albumin) inhibited IGF-I-induced activation of protein kinase B (PKB) in the pancreatic beta-cell line (INS-1), but this was alleviated in the presence of the general PKC inhibitor (Go6850; 1 microM). To further investigate whether conventional or novel PKC isoforms adversely affect beta-cell proliferation, the effect of phorbol ester (phorbol 12-myristate 13-acetate; PMA)-mediated activation of these PKC isoforms on glucose/IGF-I-induced INS-1 cell mitogenesis, and insulin receptor substrate (IRS)-mediated signal transduction was investigated. PMA-mediated activation of PKC (100 nM; 4 h) reduced glucose/IGF-I mediated beta-cell mitogenesis (>50%; P< or =0.05), which was reversible by the general PKC inhibitor Go6850 (1 microM), indicating an effect of PKC and not due to a non-specific PMA toxicity. PMA inhibited IGF-I-induced activation of PKB, correlating with inhibition of IGF-I-induced association of IRS-2 with the p85 regulatory subunit of phosphatidylinositol-3 kinase. However, in contrast, PMA activated the mitogen-activated protein kinases, Erk1/2. Titration inhibition analysis using PKC isoform inhibitors indicated that these PMA-induced effects were via novel PKC isoforms. Thus, FFA/PMA-induced activation of novel PKC isoforms can inhibit glucose/IGF-I-mediated beta-cell mitogenesis, in part by decreasing PKB activation, despite an upregulation of Erk1/2. Thus, activation of novel PKC isoforms by long-chain acyl-CoA may well contribute to decreasing beta-cell mass in the pathogenesis of type-2 diabetes, similar to their inhibition of insulin signal transduction which causes insulin resistance.

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A G Aprikian, K Han, S Chevalier, M Bazinet and J Viallet

ABSTRACT

Bombesin and gastrin-releasing peptide (GRP) are potent neuropeptides expressed by prostate cancer neuroendocrine cells and are related to the progression of this malignancy. This study characterizes bombesin receptors in human prostate cancer cell lines (PC-3, DU-145, LNCaP) and assesses the in vitro effect of bombesin on signal transduction and cell proliferation. [125I]Tyr4-bombesin binding assays (37 °C) and Scatchard analyses revealed the presence of a single class of high-affinity receptors with similar K d values (1·5, 1·1 and 3·6 × 10−10 m in PC-3, DU-145 and LNCaP cells respectively) but with significant differences in the number of binding sites per cell (47·6, 1·5 and 0·1 × 103 in PC-3, DU-145 and LNCaP cells respectively). Molecular characterization of the binding sites performed in PC-3 cells by cross-linking experiments and SDS/PAGE revealed a single radioactive band of 85 kDa. To determine which of the three known bombesin receptor subtypes (GRP receptor (GRP-R), neuromedin B receptor, bombesin receptor subtype-3) were expressed in the cell lines, reverse transcription/PCR analysis of cellular RNA followed by hybridization with receptor-specific cDNA was performed. This revealed the presence of GRP-R transcript in all cell lines, while neither of the other two receptor transcripts were expressed. When intracellular calcium mobilization was measured by Fura-2/AM cell labeling and spectrofluorometric monitoring, bombesin (100 nm) induced rapid calcium mobilization in both PC-3 (>200% of baseline) and DU-145 (>100% of baseline) cells, but not in LNCaP cells. However, as measured by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay and [3H]thymidine incorporation, no growth modulation was observed with bombesin or bombesin receptor antagonist at various concentrations (0-500 nm). Our data indicate that bombesin is a potent inducer of signal transduction via GRP-R receptors in androgen-insensitive PC-3 and DU-145 prostate cancer cells. This suggests that the bombesin/GRP family of neuropeptides may play a regulatory role in the biology of androgen-independent prostate cancer.

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W Ge, M Tanaka, M Yoshikuni, Y Eto and Y Nagahama

ABSTRACT

We have cloned a full length cDNA coding for the activin type IIB receptor (GactRIIB) from the goldfish ovary. GactRIIB shares 73 and 70% amino acid identity in the extracellular domain, and 78 and 80% identity in the intracellular domain with the type IIB receptors of the mouse and Xenopus respectively. The intracellular domain of GactRIIB contains two serine kinase consensus sequences, DFKSRN and GTRRYMAPE, in agreement with the reports in other vertebrates that serine/threonine phosphorylation is involved in activin signal transduction. The identity of GactRIIB was confirmed by transient expression in the COS cells followed by activin binding. Iodinated human activin A bound to the GactRIIB-transfected cells and the binding could be completely inhibited by unlabeled activin. Affinity labeling revealed a band of about 85 kDa, which is in agreement with the reported type II receptors in other vertebrates. Together with the fact that activin is expressed in the goldfish ovary, the cloning of activin receptors from the ovary suggests paracrine and autocrine roles for activin in the goldfish ovarian functions.

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A J Notini, R A Davey, J F McManus, K L Bate and J D Zajac

Androgens mediate their effects in target cells via the androgen receptor (AR), which acts predominantly as a ligand-dependent transcription factor. In addition, androgens induce rapid activation of second messenger signal transduction cascades, and this is thought to occur via non-genomic mechanisms. We have used the Cre/loxP system to generate an AR knockout (ARKO) mouse targeting exon 3, which encodes the second zinc finger of the DNA-binding domain. To generate universal ARKO mice, floxed AR mice were mated with CMV-Cre mice, which express Cre recombinase ubiquitously. Deletion of the floxed allele in our mice does not disrupt the reading frame, and has been designed so that the mutant AR can bind ligand but not target genes. ARKO males displayed a complete androgen insensitivity phenotype, with female external genitalia and a reduction in body weight compared with wild-type males (P < 0.001). Testes of ARKO males were smaller than control males (P < 0.0001) and were located intra-abdominally. We have demonstrated that genotypically XY mice lacking the second zinc finger of the AR have a female phenotype, and we conclude that the genomic actions of the AR (mediated by DNA binding) are indispensable for normal male sexual differentiation.

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P. N. Monk and P. Banks

ABSTRACT

The signal transduction mechanisms involved in complement fragment C5a-induced recruitment of actin to the cytoskeleton have been investigated using U-937 cells differentiated by exposure to dibutyryl cyclic AMP. Two parameters of cytoskeletal activation were compared: F-actin formation and nucleation of polymerization of pyrenyl-actin in whole cell lysates. The dose dependency of these responses to C5a was clearly different to that observed for [3H]inositol phosphate formation and also markedly different from that observed for the production of reactive oxygen intermediates (ROI). Further evidence to dissociate inositol lipid hydrolysis from these cytoskeletal responses was obtained by treating cells with neomycin, phorbol myristate acetate and pertussis toxin and by modulating the levels of intracellular Ca2+ using quin 2. Inhibition of [3H]inositol phosphate and ROI production was not correlated with effects on actin recruitment or nucleation. In addition, these agents had differing effects on F-actin formation and nucleation activity. The results show that the production of inositol phosphates is not required for stimulating either F-actin formation or nucleation activity and also that ligand-induced polymerization of actin depends primarily upon an increase in the availability of G-actin rather than nucleation sites. These cytoskeletal responses are apparently controlled by different signalling pathways which diverge at an early stage.

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M Tena-Sempere, L Pinilla and E Aguilar

ABSTRACT

In the present work in vitro GH pituitary responsiveness to GHRH in short-term (STO) and long-term orchidectomized (LTO) male rats was compared. In agreement with previous data obtained in vivo, pituitaries from STO rats showed reduced GH release after GHRH stimulation while LTO male pituitaries presented responses similar to those from control animals after maximal GHRH (10-6 m) stimulation. This suggests that compensatory mechanisms have taken place, probably at the pituitary level, in order to restore GH pituitary responsiveness to high doses of GHRH. However, LTO male rats showed a reduced sensitivity to GHRH relative to intact males, as indicated by a higher EC50 vs controls (40·82 ± 12·03 nm vs 0·35 ± 0·09 nm in intact males). We aimed to investigate further the events involved in the compensatory mechanisms that take place in LTO rats. For this purpose, we compared in vitro GH secretion by pituitaries from intact and LTO male rats after stimulation with specific activators of the signal transduction pathways related to GH release. Forskolin and dibutyryl cyclic-adenosine 3′,5′-monophosphate were more effective in eliciting GH secretion (expressed in terms of percent increment over basal GH release) in LTO males, whereas phorbol 12-myristate 13-acetate was completely ineffective in stimulating GH release in this group. Thus, our results clearly showed that long-term orchidectomy enhances the effectiveness of the cAMP pathway in inducing GH release while it completely blunts that of the protein kinase C pathway. In conclusion, orchidectomy decreased the effectiveness of GHRH in eliciting GH release in vitro. However, long-term orchidectomy activated compensatory mechanisms that restored complete GH pituitary responsiveness to maximal GHRH stimulation. These mechanisms seem not to operate in STO rats. An increased effectiveness of the cAMP pathway in eliciting GH release in LTO rats is probably involved in the aforementioned compensatory mechanisms.

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M Zhang, Y Tao, B Zhou, H Xie, F Wang, L Lei, L Huo, Q Sun and G Xia

Atrial natriuretic peptide (ANP) as well as its receptors is found in mammalian ovary and follicular cells and its function in oocyte meiotic maturation has also been reported in Xenopus, hamster and rat. But the results are controversial and the physiological mechanism of ANP on oocyte maturation is not clear, especially the relationship between gonadotrophin and ANP as well as the signal transduction, and these need further study. The present study conducted experiments to examine these questions by using drug treatment and Western blot analysis and focused on pig oocyte meiotic maturation and cumulus expansion in vitro. The results revealed that ANP could inhibited FSH-induced pig oocyte maturation and cumulus expansion and prevent the full phosphorylation of mitogen-activated protein kinase in both oocytes and cumulus cells, and that these inhibitory effects could be mimicked by 8-Br-cyclic guanosine 5′-monophosphate (8-Br-cGMP), but blocked by a protein kinase G (PKG) inhibitor KT5823. Zaprinast, a cGMP-specific phosphodiesterase inhibitor, could enhance the inhibitory effect of ANP on oocyte maturation. A specific analogue of ANP, C-ANP-(4–23), which binds to the natriuretic peptide receptor-C (NPRC), had no effect in either FSH-induced or spontaneous oocyte maturation. Treatment with forskolin, a stimulator of adenylate cyclase, had a biphasic effect; 44 h treatment induced cumulus expansion but inhibited oocyte maturation while 2 h treatment induced maturation of cumulus-enclosed oocytes (CEOs). Both ANP and C-ANP-(4–23) could inhibit the effect of forskolin on CEO maturation, and these inhibitory effects of ANP/C-ANP-(4–23) could be blocked by preincubation with pertussis toxin (PT), consistent with mediation by a Gi protein(s) in the cumulus cells. All these results suggest that ANP is a multifunctional regulator of FSH and forskolin on pig CEO maturation by two signalling mechanisms: one is via a cGMP/PKG pathway, the other is via NPRC receptors in cumulus cells and the activation of the PT-sensitive Gi protein(s).

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E Dare, O Kifor, EM Brown and G Weber

The regulation of parathyroid hormone secretion by the chief cells of the parathyroid is mediated by a 7-transmembrane (7-TM) Ca2+-sensing receptor (CaR), which signals via activation of pertussis toxin-insensitive G proteins, causing stimulation of phosphatidylinositol-specific phospholipase C (PI-PLC). We have identified the PI-PLC isoforms expressed in two model systems utilized for studying CaR signal transduction, i.e. dispersed bovine parathyroid cells and a human embryonic kidney cell line (HEK 293) stably transfected with the human parathyroid CaR-cDNA. All of the eight PI-PLC isozymes examined in this study were found to be expressed to varying extents in the bovine parathyroid gland and in the CaR-transfected HEK cells as assessed by immunoblotting. We localized the expression of the more abundant isozymes (beta1, beta2, beta3, gamma1, gamma2, delta2) to the chief cells of the bovine parathyroid by immunocytochemistry, while the two less abundant isozymes (delta1, beta4) were not detectable in parathyroid sections. G proteins activated by 7-TM receptors are known to activate mainly PI-PLC of the beta class. Therefore, beta1, beta2, beta3 and beta4, all expressed in the bovine parathyroid, are candidate isozymes for coupling to the CaR. A comparison of the levels of expression of PI-PLC isozymes between CaR-transfected HEK cells and non-transfected HEK cells suggested that the expression of the CaR in this human cell line does not cause a significant up-regulation of any of the PLCbeta and PLCgamma isozymes. PLCdelta2, showing predominantly nuclear localization in the parathyroid, was the sole PI-PLC isozyme with higher levels of expression in CaR-transfected HEK cells.