Activin A is an autocrine activator of rat pancreatic stellate cells: potential therapeutic role of follistatin for pancreatic fibrosis.

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.

Smad7 is induced by norepinephrine and protects rat hepatocytes from activin A-induced growth inhibition.

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-kappa B (NF-kappa B). The amount of active NF-kappa B complex rapidly increased after NE treatment. Preincubation of the cells with an NF-kappa B pathway inhibitor N-tosyl-l-phenylalanine chloromethyl ketone or infection of the cells with an adenovirus expressing an I kappa B super-repressor (Ad5I kappa B) 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.

Scale up of fuel ethanol production from sugar beet juice using loofa sponge immobilized bioreactor.

Production of fuel ethanol from sugar beet juice, using cells immobilized on loofa sponge was investigated. Based on ethanol productivity and ease of cell immobilization, a flocculating yeast strain, Saccharomyces cerevisiae IR2 was selected for ethanol production from sugar beet juice. It was found that raw sugar beet juice was an optimal substrate for ethanol production, requiring neither pH adjustment nor nitrogen source supplement. When compared with a 2 l bubble column bioreactor, mixing was not sufficient in an 8 l bioreactor containing a bed of sliced loofa sponges and consequently, the immobilized cells were not uniformly distributed within the bed. Most of the cells were immobilized in the lower part of the bed and this resulted in decreased ethanol productivity. By using an external loop bioreactor, constructing the fixed bed with cylindrical loofa sponges, dividing the bed into upper, middle and lower sections with approximately 1 cm spaces between them and circulating the broth through the loop during the immobilization, uniform cell distribution within the bed was achieved. Using this method, the system was scaled up to 50 l and when compared with the 2 l bubble column bioreactor, there were no significant differences (P > 0.05) in ethanol productivity and yield. By using external loop bioreactor to immobilize the cells uniformly on the loofa sponge beds, efficient large scale ethanol production systems can be constructed.

Changes in the expression of transcription factors in pancreatic AR42J cells during differentiation into insulin-producing cells.

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.

Dynamin is involved in human epithelial cell vacuolation caused by the Helicobacter pylori-produced cytotoxin VacA.

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.

Genetic variation in the gene encoding calpain-10 is associated with type 2 diabetes mellitus.

Type 2 or non-insulin-dependent diabetes mellitus (NIDDM) is the most common form of diabetes worldwide, affecting approximately 4% of the world's adult population. It is multifactorial in origin with both genetic and environmental factors contributing to its development. A genome-wide screen for type 2 diabetes genes carried out in Mexican Americans localized a susceptibility gene, designated NIDDM1, to chromosome 2. Here we describe the positional cloning of a gene located in the NIDDM1 region that shows association with type 2 diabetes in Mexican Americans and a Northern European population from the Botnia region of Finland. This putative diabetes-susceptibility gene encodes a ubiquitously expressed member of the calpain-like cysteine protease family, calpain-10 (CAPN10). This finding suggests a novel pathway that may contribute to the development of type 2 diabetes.

Kinesin is involved in regulation of rat pancreatic amylase secretion.

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.

Molecular cloning of a novel brain-type Na(+)-dependent inorganic phosphate cotransporter.

We have isolated a human cDNA encoding a protein, designated DNPI, that shows 82% amino acid identity and 92% similarity to the human brain-specific Na(+)-dependent inorganic phosphate (Na(+)/P(i)) cotransporter (BNPI), which is localized exclusively to neuron-rich regions. Expression of DNPI mRNA in Xenopus oocytes resulted in a significant increase in Na(+)-dependent P(i) transport, indicating that DNPI is a novel Na(+)/P(i) cotransporter. Northern blot analysis shows that DNPI mRNA is expressed predominantly in brain, where the highest levels are observed in medulla, substantia nigra, subthalamic nucleus, and thalamus, all of which express BNPI mRNA at low levels. In contrast, DNPI mRNA is expressed at low levels in cerebellum and hippocampus, where BNPI mRNA is expressed at high levels. No hybridizing signal for DNPI mRNA is observed in the glia-rich region of corpus callosum. In other regions examined, both mRNAs are moderately or highly expressed. These results indicate that BNPI and DNPI, which coordinate Na(+)-dependent P(i) transport in the neuron-rich regions of the brain, may form a new class within the Na(+)/P(i) cotransporter family.

Translocation of a calcium-permeable cation channel induced by insulin-like growth factor-I.

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 pools 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.

Genes expressed during the differentiation of pancreatic AR42J cells into insulin-secreting cells.

Pancreatic 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 BTC 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, cytoskeletal 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.

Assessment of the role of activin A and transforming growth factor beta in the regulation of AML12 cell growth.

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. We 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. Follistatin 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.

Formation of insulin-producing cells from pancreatic acinar AR42J cells by hepatocyte growth factor.

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 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.

Characterization of the activin receptor in cultured rat hepatocytes.

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 125I-activin A binding. Scatchard analysis of 125I-activin A binding indicated the existence of two classes of binding sites with apparent Kd 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 125I-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 125I-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.

Betacellulin and activin A coordinately convert amylase-secreting pancreatic AR42J cells into insulin-secreting cells.

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, approximately 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.

Norepinephrine reverses the effects of activin A on DNA synthesis and apoptosis in cultured rat hepatocytes.

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 [3H] 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 A in human intestinal epithelial cell line.

Production of activin was studied in four cell lines of epithelial cells: FRTL-5, JCT-12, GH4C1, 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 f1p4e 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 G1 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 [125I]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.

Conversion of amylase-secreting rat pancreatic AR42J cells to neuronlike cells by activin A.

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.

Derangements in the activin-follistatin system in hepatoma cells.

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 125I-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 125I-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.

The effect of lansoprazole treatment on the healing and relapse of peptic ulcer and serum levels of IgG anti-Helicobacter pylori antibody.

In the present study, the effects of short-term treatment with lansoprazole on healing and recurrence of peptic ulcer were investigated. Complete healing (change to the S2 stage) after 3 or 4 weeks of treatment with lansoprazole was observed in 9.9% of gastric ulcers and 38.3% of duodenal ulcers. Complete healing was observed after 3 or 4 weeks of treatment with lansoprazole in gastric ulcers and was significantly related to the serum titer of IgG anti-Helicobacter pylori antibodies. This was not the case in duodenal ulcers. Complete healing after 6 or 8 weeks of treatment with lansoprazole was not dependent on serum levels of IgG anti-H. pylori antibodies. Short-term treatment by lansoprazole did not affect serum levels of IgG anti-H. pylori antibodies immediately after the end of lansoprazole treatment, but significantly reduced serum levels of IgG anti-H. pylori antibodies were found 9 months after treatment.