眞嶋 浩聡

Pancreatic AR42J cells have the feature of pluripotency of the precursor cells of the gut endoderm. Betacellulin (BTC) and activin A (Act) convert them into insulin-secreting cells. Using mRNA differential display techniques, we have identified a novel mitochondrial transporter, which is highly expressed during the course of differentiation, and have designated it citrate transporter protein-like protein (CTPL). Recently sideroflexin 1 (Sfxn1) was shown to be a susceptible gene of flexed-tail (f/f) mice, and CTPL has turned out to be, a rat orthologous protein of Sfxn3, a member of sideroflexin family. CTPL/Sfxn3 was targeted to mitochondrial membrane like Sfxn1; The expression levels of CTPL/Sfxn3, Sfxn2, and Sfxn5 were upregulated in the early phase of differentiation into insulin-secreting cells but the expression levels of Sfxn1 and Sfxn3 did not change. All Sfxn family members were expressed in rat pancreatic islet. The expression levels of CTPL/Sfxn3, Sfxn2, and Sfxn5 were also upregulated in islets of streptozotocin-induced diabetic rats. The downregulation of CTPL/Sfxn3 in a rat insulinoma cell line, INS-1, With the antisense oligonucleotide did not affect the insulin secretion. Taken together, CTPL/Sfxn3 and some other family members might be important in the differentiation of pancreatic beta-cells as a channel or a carrier molecule and be related to the regeneration of pancreatic endocrine cells.

The WD40 repeats containing zinc finger protein 106 (ZFP106) is a conserved mammalian protein of unknown function. However, its cDNA shares an extended region of identity with the scr homology domain 3 binding protein 3 (Sh3bp3) cDNA encoding a protein implicated in the insulin signaling pathway. Asking, whether Zfp106 and Sh3bp3 are products of the same gene, we characterized the structures and transcriptional regulation of Zfp106 and its human homologue, ZFP106. A TATA-less, CpG island associated promoter (PI), was mapped by 5'-RACE to a region 19 kb upstream of the ZFP106 translation start site. P1 is active throughout development and at low levels in all adult tissues examined. A conserved cis-element in the proximal PI region showed specific binding to nuclear respiratory factor-1 (NRF-1). Mutagenesis of this site and transfection of a dominant-negative NRF-1 both revealed the crucial role of NRF-1 in activation of P1. The broad tissue expression of PI was in contrast to the high level of ZFP106 mRNA observed in striated muscle. This prompted additional Y-RACE experiments that established a second, TATA box-containing promoter (P2) upstream of the third coding exon. P1 and P2 transcripts encode proteins with distinct N-terminal sequences, with Sh3hp3 corresponding to a rare, alternatively spliced P2 transcript. P2 initiated transcripts are specifically expressed in striated muscle and their level is strongly upregulated during myogenic, but not adipogenic differentiation. By deletion analysis, the region between nucleotides -296 to +96 was sufficient for robust P2 responsiveness to myogenic differentiation. This response is mediated by the additive effect of binding of myogenin to three critical E boxes within this region. In addition, transcriptional enhancer factor-1 family factors contribute to both basal and myogenesis induced P2 activity. In situ hybridization of mouse embryos confirmed predominant expression of Zfp106 in tissues with high developmental expression of either NRF-1 (brown fat and developing brain) or myogenin (striated muscle). Our results suggest distinct roles of tissue-specific ZFP106 isoforms in growth related metabolism and provide the foundation for further studies into the regulation and function of ZFP106. (C) 2004 Elsevier B.V. All rights reserved.

Membrane-type I matrix metalloproteinase (MT1-MMP) localized on the plasma membrane plays a central role in various normal biological responses including tissue remodeling, wound heeling, and angiogenesis and in cancer cell invasion and metastasis, by functioning as a collagenase and activating other matrix metalloproteinases. In order to elucidate the molecular mechanism of the MT1-MMP targeted localization on the plasma membrane, we examined the participation of syntaxin proteins in MT1-MMP intracellular transport to the plasma membrane in human gastric epithelial AGS cells. Western blotting showed that syntaxin 3 and 4 proteins, which are known to function in intracellular transport towards the plasma membrane, were expressed in AGS cells. Immunocytochemistry revealed that transient transfection of AGS cells with dominant-negative mutant syntaxin 4 decreased plasma membrane MT1-MMP expression. In contrast, transient transfection with either dominant-negative mutant syntaxin 3 or 7 did not affect MT1-MMP localization on the plasma membrane. Cell surface biotinylation assay and Matrigel chamber assay demonstrated that stable transfection with dominant-negative mutant syntaxin 4 decreased the amount of MT1-MMP on the plasma membranes and inhibited the cell invasiveness. We suggest that syntaxin 4 is involved in the intracellular transport of MT1-MMP toward the plasma membrane. (C) 2004 Elsevier Inc. All rights reserved.

Pancreatic stellate cells (PSCs) play a major role in promoting pancreatic fibrosis. Transforming growth factor-beta(1) (TGF-beta(1)) regulates PSC activation and proliferation in an autocrine manner. The intracellular signaling pathways of the regulation were examined in this study. Immunoprecipitation and immunocytochemistry revealed that Smad2, Smad3, and Smad4 were functionally expressed in PSCs. Adenovirus-mediated expression of Smad2, Smad3, or dominant-negative Smad2/3 did not alter TGF-beta(1) mRNA expression level or the amount of autocrine TGF-beta(1) peptide. However, expression of dominant-negative Smad2/3 inhibited PSC activation and enhanced their proliferation. Co-expression of Smad2 with dominant-negative Smad2/3 restored PSC activation inhibited by dominant-negative Smad2/3 expression without changing their proliferation. By contrast, co-expression of Smad3 with dominant- negative Smad2/3 attenuated PSC proliferation enhanced by dominant- negative Smad2/3 expression without altering their activation. Exogenous TGF-beta(1) increased TGFbeta(1) mRNA expression in PSCs. However, PD98059, a specific inhibitor of mitogen-activated protein kinase kinase (MEK1), inhibited ERK activation by TGF-beta(1), and consequently attenuated TGF-beta(1) enhancement of its own mRNA expression in PSCs. We propose that TGF-beta(1) differentially regulates PSC activation, proliferation, and TGF-beta(1) mRNA expression through Smad2-, Smad3-, and ERK-dependent pathways, respectively.

Although angiotensin II (Ang II) is known to participate in pancreatic fibrosis, little is known as to the mechanism by which Ang II promotes pancreatic fibrosis. To elucidate the mechanism, we examined the action of Ang II on the proliferation of rat pancreatic stellate cells (PSCs) that play central roles in pancreatic fibrosis. Immunocytochemistry and Western blotting demonstrated that both Ang II type 1 and type 2 receptors were expressed in PSCs. [H-3]Thymidine incorporation assay revealed that Ang II enhanced DNA synthesis in PSCs, which was blocked by Ang II type 1 receptor antagonist losartan. Western blotting using anti-phospho-epidermal growth factor (EGF) receptor and anti-phospho-extracellular signal regulated kinase (ERK) antibodies showed that Ang II-activated EGF receptor and ERK. Both EGF receptor kinase inhibitor AG1478 and MEK1 inhibitor PD98059 attenuated ERK activation and DNA synthesis enhanced by Ang II. These results indicate that Ang II stimulates PSC proliferation through EGF receptor transactivation-ERK activation pathway. (C) 2004 Elsevier Inc. All rights reserved.

MAGI-1 and CASK are membrane-associated guanylate kinases of epithelial junctions. MAGI-1 is localized at tight junctions in polarized epithelial cells, whereas CASK is localized along the lateral membranes. We obtained the KIAA0769 gene product through the yeast two-hybrid screening using MAGI-1 as a bait and named it Carom. Carom has a coiled-coil domain in the middle region, and two src homology 3 domains and a PSD-95/Dlg-A/ZO-1 (PDZ)-binding motif in the C-terminal region. Carom binds to the fifth PDZ domain of MAGI-1 and the calmodulin kinase domain of CASK in vitro. MAGI-1 and CASK bind to the distinct sequences in the C-terminal region of Carom, but still compete with each other for Carom binding. The study using a stable transformant of Madine Darby canine kidney (MDCK) cells expressing GFP-Carom revealed that Carom was partially overlapped by MAGI-1 in MDCK cells, which have not yet established mature cell junctions, but became separated from MAGI-1 and colocalized with CASK in polarized cells. Carom was highly resistant to Triton X-100 extractions and recruited CASK to the Triton X-100-insoluble structures. Carom is a binding partner of CASK, which interacts with CASK in polarized epithelial cells and may link it to the cytoskeleton.

Background and aim: The present study was conducted to examine the effect of activin A on activation of rat pancreatic stellate cells (PSCs). Methods: PSCs were prepared from rat pancreas using collagenase digestion and centrifugation with Nycodenz gradient. Activation of PSCs was examined by determining smooth muscle actin expression with western blotting. The presence of activin A receptors in PSCs was investigated by reverse transcription-polymerase chain reaction (RT-PCR), western blotting, and immunocytochemistry. Expression of activin A and transforming growth factor beta (TGF-beta) mRNA was examined by RT-PCR. Activin A and TGF-beta peptide concentrations were examined with ELISA. Existence of activin A peptide in PSCs was investigated by immunocytochemistry. Collagen secretion was determined by Sirius red dye binding. Results: Activin A receptors I and IIa were present in PSCs. PSCs expressed activin A mRNA and secreted activin A. Activin A enhanced PSC activation and collagen secretion in a dose dependent manner. TGF-beta and activin A increased each other's secretion and mRNA expression of PSCs. Follistatin decreased TGF-beta mRNA expression and TGF-beta secretion of PSCs, and inhibited both PSC activation and collagen secretion. Conclusion: Activin A is an autocrine activator of PSCs. Follistatin can inhibit PSC activation and collagen secretion by blocking autocrined activin A and decreasing TGF-beta expression and secretion of PSCs.

The Helicobacter pylori-produced cytotoxin VacA induces intracellular vacuolation. The formed vacuole is assumed to be a hybrid of late endosome and lysosome. To elucidate the molecular mechanism of VacA-induced vacuolation, we examined the participation of syntaxin 7 in the human gastric epithelial cell line AGS. Immunocytochemistry revealed that endogenous syntaxin 7 was localized to vacuoles induced by VacA. Northern and Western blotting demonstrated that VacA intoxication increased syntaxin 7 mRNA and protein expression, respectively, in a time-dependent manner. Transient transfection of dominant-negative mutant syntaxin 7, which lacks a carboxyl-terminal transmembrane domain, inhibited VacA-induced vacuolation. In contrast, transient transfection of wild-type syntaxin 7, dominant-negative mutant syntaxin 1a, or dominant-negative mutant syntaxin 4 did not alter VacA-induced vacuolation. Furthermore, under VacA treatment, neutral red dye uptake, a parameter of VacA-induced vacuolation, was inhibited in cells stably transfected with mutant syntaxin 7 but not in cells stably transfected with wild-type syntaxin 7, mutant syntaxin 1a, or mutant syntaxin 4. Sequential immunocytochemical observation confirmed that expression of mutant syntaxin 7 did not affect VacA attachment to or internalization into AGS cells. We suggest that syntaxin 7 is involved in the intracellular vacuolation induced by VacA.

Pancreatic AR42J cells have the feature of pluripotency of the precursor cells of the gut endoderm. Dexamethasone converts them to exocrine cells or liver cells. Using mRNA differential display techniques, we have identified a novel Ca2+-dependent member of the mitochondrial solute carrier superfamily, which is expressed during the course of differentiation, and have designated it MCSC. The corresponding cDNA comprises an open reading frame of 1407 base pairs encoding a polypeptide of 469 amino acids. The carboxyl-terminal-half of MCSC has high similarity with other mitochondrial carriers, and the amino-terminal-half has three canonical elongation factor-hand motifs and has calcium binding capacity. The deduced amino acid sequence revealed 79.1% homology to the rabbit peroxisomal Ca2+-dependent member of the mitochondrial superfamily, but the subcellular localization of the protein was exclusively mitochondrial, not peroxisomal. Northern blot and Western blot analyses revealed its predominant expression in the liver and the skeletal muscle. In the liver, the expression level of MCSC was higher in the adult stage than in the fetal stage, and MCSC was highly up-regulated in dexamethasone-treated AR42J cells before the expression of albumin. Taken together, MCSC may play an important role in regulating the function of hepatocytes rather than in differentiation in vivo.

Variations in the calpain-10 gene (CAPN10) have been identified among Mexican-Americans, and an at-risk haplotype combination (112/121) defined by three polymorphisms, UCSNP-43, -19, and -63, confers increased risk of type 2 diabetes. Here we examine the three polymorphisms in 1,594 -Scandinavian subjects, including 409 type 2 diabetic patients, 200 glucose-tolerant control subjects, 322 young healthy subjects, 206 glucose-tolerant offspring of diabetic patients, and 457 glucose-tolerant 70-year-old men. The frequency of the 112/121 combination was not significantly different in 409 type 2 diabetic subjects compared with 200 glucose-tolerant control subjects (0.06 vs. 0.05; odds ratio 1.32 [95% CI 0.58-3.30]). In glucose-tolerant subjects, neither the single-nucleotide polymorphisms individually nor the 112/121 combination were associated with alterations in plasma glucose, serum insulin, or serum C-peptide levels at fasting or during an oral glucose tolerance test, estimates of insulin sensitivity, or glucose-induced insulin secretion. In conclusion, the frequency of the 112/121 at-risk haplotype of CAPN10 is low among Scandinavians and we were unable to demonstrate significant associations between the CAPN10 variants and type 2 diabetes, insulin resistance, or impaired insulin secretion.

Activin A induces growth arrest of rat hepatocytes in vitro and in vivo. The alpha (1)-adrenergic agonist, norepinephrine (NE), enhances epidermal growth factor-stimulated DNA synthesis and inhibits activin A-induced growth inhibition, but the mechanisms of these actions are unclear. Smad proteins have recently been identified as intracellular signaling mediators of transforming growth factor-beta family members. In the present study, we explored how NE modulates the Smad signaling pathway in rat cultured hepatocytes. We demonstrate that NE inhibits activin A-induced nuclear accumulation of Smad2/3 and that NE rapidly induces inhibitory Smad7 mRNA expression. Infection of Smad7 adenovirus into rat hepatocytes inhibited activin A-induced nuclear accumulation of Smad2/3, enhanced epidermal growth factor-stimulated DNA synthesis, and abolished the growth inhibitory effect of activin A. We also demonstrated that the induction of Smad7 by NE is dependent on nuclear factor-kappaB (NF-kappaB). The amount of active NF-kappaB complex rapidly increased after NE treatment. Preincubation of the cells with an NF-kappaB pathway inhibitor N-tosyl-L-phenylalanine chloromethyl ketone or infection of the cells with an adenovirus expressing an I kappaB super-repressor (Ad5I kappaB) abolished the NE-induced Smad7 expression. These results indicate a mechanism of transmodulation between the Smad and trimeric G protein signaling pathways in rat hepatocytes.

The Helicobacter pylori-produced cytotoxin VacA induces intracellular vacuolation. To elucidate the molecular mechanism of vacuole formation by VacA, we examined the participation of dynamin, a GTPase functioning in intracellular vesicle formation, in human HeLa cells. Immunocytochemistry revealed that endogenous dynamin was localized to vacuoles induced by VacA, In cells transiently transfected with a GTPase-defective (dominant-negative) dynamin mutant, VacA Failed to induce vacuolation. In contrast, VacA did induce vacuolation in cells transiently transfected with wild-type dynamin, Furthermore, under VacA treatment, neutral red dye uptake, a parameter of VacA-induced vacuolation, was inhibited in cells stably transfected with the dominant-negative dynamin mutant. In contrast, uptake was markedly enhanced in cells stably transfected with wild-type dynamin. Moreover, VacA cytopathic effects on the viability of HeLa cells were inhibited in cells stably transfected with dominant-negative dynamin-1. Sequential immunocytochemical observation confirmed that expression of dominant-negative dynamin did not affect VacA attachment to or internalization into HeLa cells. We suggest that dynamin is involved in the intracellular vacuolation induced by VacA.

Pancreatic AR42J cells possess both exocrine and neuroendocrine properties and convert to insulin-producing cells upon treatment with activin A and hepatocyte growth factor (HGF). We studied changes in the mRNA expression of various transcription factors during the course of differentiation. Among the transcription factors studied, expression levels of Pax4 and neurogenin3 changed significantly. These two factors were not detected in naive cells, whereas their mRNA levels were markedly increased after treatment with activin A and HGF. Thus, these two factors were induced by activin A. Transfection of Pax4 did not induce any changes in morphology or expression of pancreatic polypeptide (PP). Furthermore, introduction of antisense Pax4 did not affect the conversion into insulin-producing cells induced by activin A and HGF. In contrast, transfection of neurogenin3 induced morphological changes similar to those induced by activin A. In addition, transfection of neurogenin3 induced the expression of PP. Conversely, introduction of antisense neurogenin3 blocked the differentiation of AR42J cells induced by activin A and HGF. These results indicate that activin A regulates the expression of neurogenin3, which is critical for the differentiation of AR42J into endocrine cells.

Background & Aims: Kinesin has recently been localized to zymogen granules of pancreatic acini and is suggested to participate in exocytosis of exocrine pancreas, We examined the function of kinesin in regulated exocytosis of pancreatic acini in this study, Methods: Kinesin function in exocytosis was examined by introducing hexahistidine-tagged recombinant kinesin protein and antikinesin monoclonal antibody into streptolysin-O-permeabilized acini. Intracellular localization of introduced recombinant kinesin was investigated by immunohistochemistry. Interaction between recombinant kinesin and the microtubule network was confirmed by nocodazole pretreatment of acini, Kinesin regulation by secretagogues was investigated by examining their effect on adenosine triphosphatase (ATPase) activity of endogenous kinesin, Results: Recombinant kinesin enhanced calcium-stimulated amylase release from streptolysin-O-permeabilized acini, Introduced recombinant kinesin was localized to both the microtubule network and zymogen granule, Nocodazole pretreatment of acini abolished the enhancing effect of recombinant kinesin on calcium-stimulated amylase release. Antikinesin antibody inhibited amylase release stimulated by the combination of calcium and cyclic adenosine monophosphate (cAMP) but not that stimulated by calcium alone, Secretin and 8-bromo-cAMP increased ATPase activity of endogenous kinesin. Conclusions: Kinesin plays a stimulatory role in regulated exocytosis of pancreatic acini and is involved in stimulus-secretion coupling through a cAMP-dependent pathway.

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.