眞嶋 浩聡

Calcium plays a critical part in the regulation of cell growth, and growth factors stimulate calcium entry into cells through calcium-permeable channels. However, the molecular nature and regulation of calcium-permeable channels are still unclear at present. Here we report the molecular characterization of a calcium-permeable cation channel that is regulated by insulin-like growth factor-I (IGF-I). This channel, which we name growth-factor-regulated channel (GRC), belongs to the TRP-channel family and localizes mainly to intracellular poets under basal conditions. Upon stimulation of cells by IGF-I, GRC translocates to the plasma membrane. Thus, IGF-I augments calcium entry through GRC by regulating trafficking of the channel.

dPancreatic AR42J cells have the feature of pluripotency of the common precursor cells of the pancreas. Dexamethasone (Dx) converts them to exocrine cells, whereas activin A (Act) converts them into endocrine cells expressing pancreatic polypeptide. A combination of Act and betacellulin (BTC) converts them further into insulin-secreting cells, The present study identifies some of the genes involved in the process of differentiation that is induced by these factors, using the mRNA differential display and screening of the cDNA expression array. The expression levels of 7 genes were increased by Act alone, and a combination of Act and ETC increased the expression of 25 more genes. Of these, 16 represented known genes or homologues of genes characterized previously, Nine of the identified genes were unrelated to any other sequences in the database. An inhibitor of the mitogen-activated protein kinase pathway, PD098059, which blocks the differentiation into insulin-secreting cells, inhibited the expression of 18 of the 25 genes, suggesting that the proteins encoded by these genes are associated with the differentiation into insulin-producing cells, These include known genes encoding extracellular signaling molecules, such as parathyroid hormone-related peptide, cyloskeletal proteins, and intracellular signaling molecules. Identification and characterization of these differentially expressed genes should help to clarify the molecular mechanism of differentiation of pancreatic cells and the; gene products that enable the beta-cells to produce insulin.

Genes play an important role in the development of diabetes mellitus, a group of metabolic diseases characterized by hyperglycemia resulting from defects in insulin secretion, insulin action or both. Some forms of diabetes such as the condition called maturity-onset diabetes of the young (MODY) result from mutations in a single gene. Others such as type 1 and type 2 diabetes are multifactorial in origin with different combinations of several genes together with nongenetic factors (e.g., lifestyle and environmental factors) contributing to the development of hyperglycemia. MODY is a type of diabetes that phenotypically resembles type 2 diabetes. However, in contrast to the latter, it is characterized by an autosomal dominant pattern of inheritance and by onset or recognition at a young age, i.e., in childhood, adolescence or young adult life, usually under 25 years of age. MODY has been a valuable model for genetic and physiological studies of diabetes. Genetic studies have shown that mutations in the genes encoding the glycolytic enzyme glucokinase, and the transcription factors hepatocyte nuclear factor-1 alpha, -1 beta and -4 alpha and insulin promoter factor-1 can cause MODY. Physiological studies have shown that heterozygous mutations in these genes affect normal pancreatic beta cell functions, thereby resulting in diabetes. Molecular genetic and clinical investigative studies of patients with different forms of MODY have provided a better understanding of the pathophysiology of diabetes and a foundation for clinical studies of pancreatic beta cell function in other forms of diabetes.

The present study was conducted to determine the role of two autocrine factors, activin A and transforming growth factor beta (TGF-beta), in the growth regulation of AML12 hepatocytes, me overexpressed truncated type II activin and/or TGF-beta receptors in AML12 cells, In AML12 cells overexpressing truncated type II activin receptors (AML-tAR cells), the inhibitory effect of activin A on DNA synthesis was completely blocked, AML-tAR cells proliferated faster than parental cells, both in the presence and absence of epidermal growth factor (EGF). However, AML-tAR cells could not grow in soft agar. Fol listatin augmented EGF-induced DNA synthesis in AML12 cells, whereas it was ineffective in AML-tAR cells, In AML12 cells overexpressing truncated type II TGF-beta receptor (AML-tTR cells), the inhibitory effect of TGF-beta on DNA synthesis was blocked. AML-tTR cells proliferated faster than parental cells, both in the presence and absence of EGF, but at a slower rate than that of AML-tAR cells, AML-tTR cells did not grow in soft agar. The growth rate of cells overexpressing both types of truncated receptors was identical to that of AML-tAR cells, and these cells did not grow in soft agar, These results indicate that both activin A and TGF-beta act as autocrine inhibitors of DNA synthesis in AML12 cells, and that the blocking of the actions of two factors does not lead to transformation. Activin A is a predominant autocrine factor in these cells.

Pancreatic AR42J cells are derived from acinar cells and express both exocrine and neuroendocrine properties. We have recently shown that these cells convert into insulin-producing cells in vitro after treatment with activin A and betacellulin. Here, we investigated the effect of hepatocyte growth factor (HGF) in those cells. When AR42J cells were incubated with HGF, DNA synthesis was attenuated, and the amylase content was reduced in a concentration-dependent manner. HGF-treated cells extended processes, but bundle formation was not observed using an antibody against tubulin. Reverse transcription-PCR analysis revealed that messenger RNAs for both insulin and pancreatic polypeptide (PP) were expressed in HGF-treated, but not naive, AR42J cells. Immunocytochemical analysis indicated that approximately 3% of the HGF-treated cells were stained with antiinsulin antibody, and some were also stained with anti-PP antibody. When AR42J cells were exposed to a combination of activin A and HGF, cells extended longer processes, and over 10% of them were stained with antiinsulin antibody. In these cells, messenger RNAs for insulin, PP, glucose transporter 2, and glucokinase, but not those for glucagon or somatostatin, were expressed. A subclone of AR42J cells, AR42J-B13, was obtained. Most of the AR42J-B13 cells converted to insulin-producing cells after the incubation with activin A and HGF. Insulin secretion was augmented by tolbutamide, depolarizing concentrations of potassium, carbachol, and glucagon-like peptide-1 in these cells. These results indicate that HGF reduces the acinar cell-like property of AR42J cells and converts them into insulin-producing cells. The effect of HGF was markedly enhanced by activin A.

Activin A is an autocrine inhibitor of initiation of DNA synthesis in rat hepatocytes. The present study was conducted to characterize the cell-surface receptors for activin A in cultured rat hepatocytes by measuring I-125-activin A binding, Scatchard analysis of I-125-activin A binding indicated the existence of two classes of binding sites with apparent Ma values of 3 x 10(-10) mol/L and 3.5 x 10(-9) mol/L. Pretreatment of the cells with heparitinase reduced the number of low-affinity binding sites, whereas pretreatment with excess exogenous follistatin increased the number of low-affinity binding sites. Affinity cross-linking of I-125-activin A to hepatocytes-revealed distinct protein complexes with molecular weights of approximately 48, 65, and 85 kd, which may represent cross-linked cell-bound follistatin, type I and type II activin receptors, respectively, Another band with a molecular weight of 180 kd was also found, which may represent the type III activin receptor. When hepatocytes were cultured with epidermal growth factor (EGF), both high- and low-affinity binding sites increased at 12 hours without altering their affinities, At 60 hours of the incubation with EGF, the high-affinity binding sites decreased while the number of low-affinity binding sites increased slightly. These results indicate that two classes of I-125-activin A binding sites exist in cultured hepatocytes: the high-affinity binding site may represent oligomeric complex of the type I and type II receptors, and at least part of the low-affinity binding site may represent cell-bound follistatin. The number of activin receptors in hepatocytes is increased after the stimulation with EGF.

Rat pancreatic AR42J cells possess exocrine and neuroendocrine properties, Activin A induces morphological changes and converts them into neuron-like cells. In activin-treated cells, mRNA for pancreatic polypeptide (PP) but not that for either insulin or glucagon was detected by reverse transcription-PCR. About 25% of the cells were stained by anti-PP antibody. When AR42J cells were incubated with betacellulin, a small portion of the cells were stained positively with antiinsulin and anti-PP antibodies, The effect of betacellulin was dose dependent, being maximal at 2 nM, Approximately 4% of the cells became insulin positive at this concentration, and mRNAs for insulin and PP were detected. When AR42J cells were incubated with a combination of betacellulin and activin A, similar to 10% of the cells became insulin positive, Morphologically, the insulin-positive cells were composed of two types of cells: neuron-like and round-shaped cells, Immunoreactive PP was found in the latter type of cells, The mRNAs for insulin, PP, glucose transporter 2, and glucokinase, but not glucagon, were detected, Depolarizing concentration of potassium, tolbutamide, carbachol, and glucagon-like peptide-1 stimulated the release of immunoreactive insulin, These results indicate that betacellulin and activin A convert amylase-secreting AR42J cells into cells secreting insulin. AR42J cells provide a model system to study the formation of pancreatic endocrine cells.

Activin A, an autocrine factor produced by hepatocytes, inhibits mitogen-stimulated DNA synthesis and induces apoptotic death of cultured rat hepatocytes. Several lines of evidence indicate that norepinephrine (NE), as a comitogenic growth factor, alters the balance between growth stimulation and inhibition and acts as a trigger for the initiation of hepatocyte proliferation. In the present study, we examined whether NE modulated the effects of activin A on rat hepatocytes in primary culture. Activin A, at a concentration of 10(-9) mol/L, blocked the effect of epidermal growth factor (EGF) on DNA synthesis, that was assessed by measuring [H-3] thymidine incorporation and nuclear labeling, almost completely, and NE reversed the inhibitory effect of activin A on DNA synthesis. This effect of NE was dose-dependent, being significant at concentrations of 10(-6) mol/L and above, but was overcome by higher concentrations of activin A, and was attenuated by prazosin, but not by yohimbine or propranolol. NE exerted its effect during the first 24 hours of culture, but was ineffective when added after 24 hours. EGF augmented the release of follistatin, an activin-binding protein known to block the action of activin A, by hepatocytes and NE did not affect the amount of follistatin they released In addition to inhibiting DNA synthesis by hepatocytes cultured with EGF, activin A induced death of hepatocytes cultured in the absence of EGF. The nuclear morphology of cells cultured with activin A alone was strikingly changed compared with untreated control cells and marked identation of the nuclear membranes and moderate chromatin condensation were observed. Fragmentation of DNA was also observed, suggesting that activin A induced apoptosis, and activin-mediated cell death was prevented significantly by NE. These results indicate that NE, acting on alpha(1)-adrenergic receptors, attenuates the effects of activin A on DNA synthesis by and apoptosis of cultured rat hepatocytes.

Production of activin was studied in four cell lines of epithelial cells: FRTL-5, JCT-12, GH(4)C(1), and FHs74Int cells. Bioactivity of activin was detected in conditioned media of FRTL-5, JCT-12 and FHs74Int cells. Among these three cell lines, FHs74Int cells, which were derived from human embryonic intestine, released a relatively large amount of bioactive activin. In these cells, serum and epidermal growth factor (EGF), which were capable of stimulating DNA synthesis, augmented release of bioactive activin in middle to late G(1) phase. In addition, basic FGF (bFGF), which had no effect on DNA synthesis in these cells, also increased release of activin. In bFGF-treated FHs74Int cells, bioactive activin was released within 4 hr of the addition of bFGF. The reverse-transcription polymerase chain reaction reveals that mRNA for only the beta(A) subunit of activin is expressed in these cells. Immunoblotting of lysate from serum-treated cells using anti-human activin A antibody indicated the existence of a 12.5-kDa protein under a reducing condition. FHs74Int cells did not express binding site for [I-125]activin A and exogenous activin A did not affect DNA synthesis in these cells. These results indicate that FHs74Int cells derived from human embryonic intestine synthesize and release activin A. Activin A released from intestinal epithelial cells might be a modulatory factor in cells in intestinal mucosa.

When AR42J cells, an amylase-secreting pancreatic exocrine cell line, were treated with activin A, cells extended neuritelike processes, and, concomitantly, amylase-containing vesicles disappeared, Immunofluorescence and immunoelectron microscopy revealed that these processes had neurite-specific cytoskeletal architectures: neurofilaments and microtubule bundles with cross-bridges of microtubule-associated protein 2, In addition to such morphological changes, activin-treated cells exhibited a marked increase in cytoplasmic free calcium concentration in response to depolarizing concentration of potassium, Moreover, activin-treated AR42J cells expressed mRNA for alpha(1) subunit of the neuroendocrine/beta cell-type voltage-dependent calcium channel, In naive AR42J cells, a sulfonylurea compound, tolbutamide, did not affect free calcium concentration, while it induced a marked elevation of free calcium in activin-treated cells, Single channel recording of the membrane patch revealed the existence of ATP-sensitive potassium channel in activin-treated cells, These results indicate that activin A converts amylase-secreting AR42J cells to neuronlike cells, Given that pancreatic endocrine cells possess neuronlike properties and express ATP-sensitive potassium channel as well as neuroendocrine/beta cell-type voltage-dependent calcium channel, activin treatment of AR42J cells may provide an in vitro model system to study the conversion of pancreatic exocrine cells to endocrine cells in islets.

Background/Aims: The growth of normal hepatocytes is regulated by the activin-follistatin system. The aim of this study was to investigate the activin-follistatin system in hepatoma cells. Methods: The production and action of activin and follistatin in human hepatoma cell lines were examined. Activin A and follistatin were measured by bioassay and protein-binding assay, respectively. Results: Activin A inhibited cell growth in HepG2 cells but not in either PLC/PRF/5 or HLE cells. However, the effect of activin A in HepG2 cells was attenuated at high cell density. In HepG2 cells, two classes of activin-binding sites were expressed, and affinity cross-linking showed that I-125-activin A bound specifically to three proteins with molecular weights of 48, 67, and 94 kilodaltons. In PLC/PRF/5 cells, a single class of binding site was observed, and the binding capacity was approximately 60% of the capacity in HepG2 cells. Virtually no I-125-activin A binding was detected in HLE cells. Bioactivity and messenger RNA for activin A were undetectable in three cell lines. In contrast, follistatin was released from three cell lines. Conclusions: Multiple alterations in the activin-follistatin system were found in three hepatoma cell lines. The accelerated growth observed in hepatoma cells may be caused, at least partly, by the attenuation of the action of activin A.

Activin A, a member of the transforming growth factor-p supergene family, exists in secretory granules of non-B-cells of rat pancreatic islet (H. Yasuda, K. Inoue, H. Shibata, T. Takeuchi, Y. Eto, Y. Hasegawa, N. Sekine, Y. Totsuka, T. Mine, E. Ogata, and I. Kojima. Endocrinology 133: 624-630, 1993). Because functions of exocrine pancreas are influenced by hormones in pancreatic islet, it is possible that activin A affects the function of pancreatic acinar cells. To examine this possibility, we studied the effects of activin A on amylase secretion and DNA synthesis in AR42J cells. In these cells, dexamethasone !Dr) induces increases in secretory organelles and secretion of amylase (C. D. Logsdon, J. Moessner, J. A. Williams, and I. D. Goldfine. J. Cell Biol. 100: 1200-1208 1985). Activin A did not change the rate of amylase release by itself nor affect the cholecystokinin-stimulated amylase release from Dr-treated differentiated AR42J cells. However, when activin A was added together with Dx, activin A inhibited Dx-induced increase in amylase content in a dose-dependent manner. In the presence of 1 nM activin A, the effect of Dx was abolished. In the absence of Dx, amylase content of the cells was also reduced by activin A in a dose-dependent manner. The maximum inhibitory effect was obtained by 10 nM activin A, and at this concentration amylase content became undetectable. In addition, activin A potently inhibited DNA synthesis as assessed by [H-3]thymidine incorporation. The maximal inhibitory effect was obtained by 10 nM activin A, and [H-3]thymidine incorporation was similar to 39 of the control value. Electron microscopic analysis revealed that in activin A-treated cells, crinophagy-like structures were observed. Rough endoplasmic reticulum disappeared, and only free ribosomes were observed. These results indicate that activin A inhibits both proliferation and amylase production in AR42J cells.

In the present study, the role of endogenous prostanoid synthesis in gastric mucosa in the healing of chronic gastric ulcers was investigated. Nineteen patients were divided into two groups in accordance with healing state after one month of treatment with cimetidine only: ''healed group'' and ''unhealed group''. Biopsy specimens taken from the mucosa around the ulceration (damaged gastric mucosa) and at a distance from the ulceration (normal gastric mucosa) at endoscopy prior to treatment were homogenized, and the mucosal prostanoid synthesis was determined using [C-14]arachidonic acid. The mean value of prostaglandin E(2) synthesis in the normal gastric mucosa of the healed group was 60 % higher than in that of the unhealed group, but the difference was not significant. However, prostaglandin E(2) synthesis in the damaged gastric mucosa of the healed group was 117 % higher than in that of the unhealed group. The same tendency was observed for prostaglandin D-2 and 6-keto prostaglandin F-1 alpha synthesis as for prostaglandin E(2). In our study it was demonstrated that there is a good correlation of prostaglandin synthesis in the damaged mucosa with healing of chronic gastric ulceration. Furthermore, our study indicated that prostaglandin synthesis, especially in damaged mucosa, might be important in the healing of gastric ulceration.

Activin A, a member of the transforming growth factor-β supergene family, exists in secretory granules of non-B-cells of rat pancreatic islet (H. Yasuda, K. Inoue, H. Shibata, T. Takeuchi, Y. Eto, Y. Hasegawa, N. Sekine, Y. Totsuka, T. Mine, E. Ogata, and I. Kojima. Endocrinology 133:624-630, 1993). Because functions of exocrine pancreas are influenced by hormones in pancreatic islet, it is possible that activin A affects the function of pancreatic acinar cells. To examine this possibility, we studied the effects of activin A on amylase secretion and DNA synthesis in AR42J cells. In these cells, dexamethasone (Dx) induces increases in secretory organelles and secretion of amylase (C. D. Logsdon, J. Moessner, J. A. Williams, and I. D. Goldfine, J. Cell Biol. 100: 1200-1208 1985). Activin A did not change the rate of amylase release by itself nor affect the cholecystokinin-stimulated amylase release from Dx-treated differentiated AR42J cells. However, when activin A was added together with Dx, activin A inhibited Dx-induced increase in amylase content in a dose-dependent manner. In the presence of 1 nM activin A, the effect of Dx was abolished. In the absence of Dx, amylase content of the cells was also reduced by activin A in a dose-dependent manner. The maximum inhibitory effect was obtained by 10 nM activin A, and at this concentration amylase content became undetectable. In addition, activin A potently inhibited DNA synthesis as assessed by [3H]thymidine incorporation. The maximal inhibitory effect was obtained by 10 nM activin A, and [3H]thymidine incorporation was ~3% of the control value. Electron microscopic analysis revealed that in activin A-treated cells, crinophagy- like structures were observed. Rough endoplasmic reticulum disappeared, and only free ribosomes were observed. These results indicate that activin A inhibits both proliferation and amylase production in AR42J cells.