眞嶋 浩聡

膵臓の3大疾患とも言うべき急性膵炎、慢性膵炎、ならびに膵臓癌は依然難治性疾患であり、未だこれら疾患の画期的な予防法および治療法は開発されていない。一方、これら疾患の病態を解明し新たな治療法を開発すべく多くの研究者が膵疾患基礎研究に日夜尽力してきた結果、急性膵炎発症機転や慢性膵炎の発症・進展に関して新たな知見が次々と得られている。急性膵炎におけるオートファジー機構の関与や慢性膵炎の発症・進展における膵星細胞の役割がその代表的なものである。本稿ではこれら膵疾患の病態に関する知見を概説する。(著者抄録)

I型インターフェロンは抗ウイルス作用にとどまらず、免疫調整作用や抗腫瘍効果などの多彩な生物学的活性を有している。インターフェロン制御因子(IRF)はインターフェロン誘導遺伝子の転写を制御する因子として発見されたが、免疫関連遺伝子の転写調節の他に、細胞増殖・アポトーシスの制御、癌化などに関与する。膵臓においてIRF2は調節性外分泌にかかわっており、IRF2ノックアウトマウスの膵臓は急性膵炎の発症初期像を呈する。また、膵癌においてIRF1は癌抑制遺伝子、IRF2は癌遺伝子としての機能を有していることが明らかとなった。生物学的悪性度が高く、浸潤傾向が強い膵癌に対しては、多方面からの治療が不可欠である。インターフェロンシグナルの膵癌の発癌、進展に及ぼす影響やIRFの分子メカニズムの解明を進めることによって、膵癌の新たな治療法に結びつく可能性がある。(著者抄録)

膵臓は消化・吸収のための消化酵素を高率に産生するがゆえに、小胞体ストレスや酸化ストレスに曝されやすい。急性膵炎の発症にはトリプシンの異所性活性化やNF-κBの活性化、細胞内ストレス、オートファジーなどが関与している。オートファジーは老廃物の分解やリサイクルを通して細胞の恒常性の維持に寄与するが、膵炎発症時にも細胞内環境を維持するために動員されると考えられる。しかし、機能不全に陥って膵炎の発症に関与し、p62/SQSTM1(p62)の蓄積や転写因子Nrf2の活性化が細胞内で生じる。p62はオートファジーの基質であるだけでなく、シグナル伝達、細胞増殖、アポトーシス、炎症などをつなぐハブとしての機能を有している。オートファジーと膵炎発症の関係が注目されており、p62やKeap1-Nrf2を介するシグナルが重要な役割を果たしている可能性がある。(著者抄録)

Primary enterolith is a rare condition that can induce ileus and intestinal perforation. We report the first case of a true primary enterolith treated by balloon-assisted enteroscopy. The patient presented with a small intestinal ileus. After its improvement following the insertion of an ileus tube, radiography with amidotrizoate sodium meglumine detected a round, movable defect in the ileum measuring 42 mm diameter. The patient was diagnosed with a primary enterolith based on her past history. The enterolith was fractured and removed using balloon-assisted enteroscopy. This case suggests that balloon-assisted enteroscopy may be an effective non-invasive treatment option for enteroliths.

Objective: Pancreatic cancer is one of the most malignant diseases worldwide. Interferon regulatory factor (IRF) 1 and IRF2 function as a tumor suppressor and oncoprotein, respectively, in several types of cancers. We investigated whether IRF1 and IRF2 are involved in the progression of pancreatic cancer. Methods: We examined the expressions of IRF1 and IRF2 in pancreatic cancer specimens and analyzed the association with clinicopathologic features. We evaluated the biological effects of IRF1 and IRF2 using a pancreatic cancer cell line. Results: The expression levels of IRF1 and IRF2 were decreased and increased, respectively, in the pancreatic cancer cells compared with those observed in the paired normal areas. A higher expression of IRF1 was associated with better features of tumor differentiation, infiltration depth, tumor size, and survival, whereas that of IRF2 was associated with a worse feature of tumor infiltration depth. Interferon regulatory factor 2-overexpressing PANC-1 cells exhibited an increase in cell growth, less apoptotic features, and chemoresistance to gemcitabine treatment. In contrast, IRF1-overexpressing cells exhibited the opposite characteristics. Conclusions: Interferon regulatory factors 1 and 2 may regulate the progression of pancreatic cancer by functioning as an antioncoprotein and oncoprotein, respectively. These molecules may serve as potential targets of therapy.

膵臓は外分泌機能を担う膵腺房および導管、内分泌機能を担うランゲルハンス氏島、ならびに膵星細胞などによって構成される臓器である。とくにランゲルハンス氏島(ラ氏島)の各種内分泌細胞にて産生・分泌される膵ホルモンは膵腺房細胞の生理的機能を制御していると考えられている。さらに最近では、生理的条件下ならびに各種病態において膵臓においてTGF-betaスーパーファミリーに属するサイトカインであるactivin Aが発現し、膵の発生、分化、ならびに線維化などにおいて大きな役割を果たしていることが明らかになってきている。本稿では、膵内分泌相関として知られる膵ホルモンの膵外分泌機構への作用とその分子機序、ならびにactivin Aのさまざまな膵生理機能ならびに膵疾患病態に対する作用を中心に紹介する。(著者抄録)

We report a case of a 64-year-old woman with Stage IV breast cancer who responded well to chemotherapy containing bevacizumab. She noticed a left breast tumor with acute progression and was diagnosed as having Stage IV, estrogen receptor (ER) (-), progesterone receptor (PgR) (-), human epidermal growth factor receptor 2 (HER2) (-) breast cancer (T4cN3cM1 [lymph nodes]). She received 5 courses of adriamycin (60 mg/m2) plus cyclophosphamide (600mg/m2) (AC therapy) and 4 courses of weekly paclitaxel (PTX 90 mg/m2) plus bevacizumab (AVA 10 mg/m2) as systemic therapy. Computed tomography (CT) and magnetic resonance imaging (MRI) revealed a complete response (CR). After local resection of the breast tumor and radiation to the breast and regional lymph nodes, capecitabine therapy was initiated. Currently, at 5 months after surgery, no new lesion has been detected.

We describe the case of a 74-year-old female with a mesenteric lymph node abscess caused by a Yersinia enterocolitica infection. She had been administered an immunosuppressive drug and was admitted to the hospital due to a high fever, right lower abdominal pain and advanced leukocytosis. We initially diagnosed her with lymphadenitis based on the symptoms and the imaging studies. However, conservative treatment with antibiotics did not yield any improvement, and abscess formation was suspected. Surgical treatment was performed, and the culture from the drainage fluid grew Y. enterocolitica. The histological findings suggested that an ulcerative lesion of the terminal ileum was the entry port of Y. enterocolitica. The pathogen infected the mesenteric lymph nodes and spread along the ileocecal lymphatic vessels, resulting in the formation of an abscess. We also provide a review of the previously published literature on lymph node abscesses due to Y. enterocolitica infections. © Springer 2014.

異時性に内分泌細胞癌小細胞型と扁平上皮癌の多発を認めた稀な食道癌の1例を経験したので報告する．症例は62歳，男性．嚥下時つかえ感を主訴に施行した内視鏡検査で胸部食道に2型腫瘍を認め，生検で内分泌細胞癌小細胞型と診断された．CTでは気管膜様部浸潤，リンパ節転移などを認め，cStage IVa（T4N3M0）と診断され，肺小細胞癌に準じてCDDP，CPT-11による化学療法と放射線療法を併用した．化学療法計11コース終了後，CT，内視鏡検査では腫瘍，リンパ節腫脹が消失し，完全奏効であったが，腹部食道に表面隆起型扁平上皮癌を認め，内視鏡的粘膜下層剥離術を施行した（moderately to poorly differentiated squamous cell carcinoma，pT1a（MM），ly0，v0，pHM0，pVM0）．内分泌細胞癌の診断から現在まで，3.5年間無再発生存中である．

Insulin-like peptide 5 (INSL5) is a member of the insulin superfamily, and is a potent agonist for RXFP4. We have shown that INSL5 is expressed in enteroendocrine cells (EECs) along the colorectum with a gradient increase toward the rectum. RXFP4 is ubiquitously expressed along the digestive tract. INSL5-positive EECs have little immunoreactivity to chromogranin A (CgA) and might be a unique marker of colorectal EECs. CgA-positive EECs were distributed normally along the colorectum in INSL5 null mice, suggesting that INSL5 is not required for the development of CgA-positive EECs. Exogenous INSL5 did not affect the proliferation of human colon cancer cell lines, and chemically-induced colitis in INSL5 null mice did not show any significant changes in inflammation or mucosa] healing compared to wild-type mice. In contrast, all of the rectal neuroendocrine tumors examined co-expressed INSL5 and RXFP4. INSL5 may be a unique marker of colorectal EECs, and INSL5 RXFP4 signaling might play a role in an autocrine/ paracrine fashion in the colorectal epithelium and rectal neuroendocrine tumors. (C) 2013 Elsevier Inc. All rights reserved.

We herein describe the case of a 51-year-old man with a duodenocolic fistula (DCF) caused by a stomal ulcer. The patient complained of watery diarrhea, dysgeusia and malnutrition. His medical history included distal gastrectomy with Billroth I reconstruction for duodenal ulcer perforation. A combination study using endoscopy and contrast imaging confirmed the presence of DCF. Laparotomic fistulectomy was performed, which resulted in the patient's recovery from diarrhea and malnutrition. The histological findings suggested that the fistula had originated from a stomal ulcer. In patients with chronic watery diarrhea of obscure origin following gastrectomy, DCF is a possible cause of the diarrhea.

胃がん検診は、X線検査のみがいくつかの症例対照研究によって死亡率減少効果を示したことにより対策型検診として推奨されている。一方で近年、血清検査を用いた効率的な胃がん検診法が提唱されている。しかし、内視鏡検診も含めて有効性の評価がなされていないのが現状である。そこで、新しい胃がん検診システムの評価をおこなうことを目的として、「X線検査・精査内視鏡検査群」(バリウム検診群)と「抗H.pylori抗体+ペプシノゲン測定・内視鏡検査群」(血清胃癌リスク検診群)の無作為化比較対照試験をスタートさせた。(著者抄録)

Pancreatic acinar cells are used to study regulated exocytosis. We investigated the role of interferon regulatory factor-2 (IRF2) in exocytosis in pancreatic acinar cells.<br /> Pancreas tissues from Irf2⁺/⁺, Irf2⁺/⁻), and Irf2⁻/⁻ mice were examined by microscopy, immunohistochemical, and immunoblot analyses; amylase secretion was quantified. We also compared salivary glands and pancreatic islets of Irf2⁻/⁻ mice with those of Irf2⁺/⁻ mice. To examine the effects of increased signaling by type I interferons, we studied pancreatic acini from Irf2⁻/⁻Ifnar1⁻/⁻ mice. The effect of IRF2 on amylase secretion was studied using an acinar cell line and a retroviral system. We studied expression of IRF2 in wild-type mice with cerulein-induced pancreatitis and changes in pancreatic tissue of Irf2⁻/⁻ mice, compared with those of Irf2⁺/⁻ mice.<br /> Irf2⁻/⁻ pancreas was white and opaque; numerous and wide-spread zymogen granules were observed throughout the cytoplasm, along with lack of fusion between zymogen granules and the apical membrane, lack of secretagogue-stimulated amylase secretion, and low serum levels of amylase and elastase-1, indicating altered regulatio

BACKGROUND & AIMS: Pancreatic acinar cells are used to study regulated exocytosis. We investigated the role of interferon regulatory factor-2 (IRF2) in exocytosis in pancreatic acinar cells. METHODS: Pancreas tissues from Irf2(+/+), Irf2(+/-) and Irf2(-/-) mice were examined by microscopy, immunohistochemical, and immunoblot analyses; amylase secretion was quantified. We also compared salivary glands and pancreatic islets of Irf2(-/-) mice with those of Irf2(+/-) mice. To examine the effects of increased signaling by type I interferons, we studied pancreatic acini from Irf2(-/-) Ifnar1(-/-) mice. The effect of IRF2 on amylase secretion was studied using an acinar cell line and a retroviral system. We studied expression of IRF2 in wild-type mice with cerulein-induced pancreatitis and changes in pancreatic tissue of Irf2(-/-) mice, compared with those of Irf2(+/-) mice. RESULTS: Irf2(-/-) pancreas was white and opaque; numerous and wide-spread zymogen granules were observed throughout the cytoplasm, along with lack of fusion between zymogen granules and the apical membrane, lack of secretagogue-stimulated amylase secretion, and low serum levels of amylase and elastase- 1, indicating altered regulation of exocytosis. The expression pattern of soluble N-ethylmaleimide-sensitive factor attachment protein receptors changed significantly, specifically in pancreatic acini, and was not rescued by disruption of type I interferon signaling. Down-regulation of IRF2 decreased amylase secretion in an acinar cell line. In mice with pancreatitis, levels of IRF2 were reduced. Irf2(-/-) acini were partially resistant to induction of pancreatitis. CONCLUSIONS: IRF2 regulates exocytosis in pancreatic acinar cells; defects in this process might be involved in the early phases of acute pancreatitis.

In chronic pancreatitis, pancreatic stellate cells (PSCs) play a central role in tissue fibrogenesis. Transforming growth factor beta(1) (TGF-beta(1)) and the Th2 lymphokines such as interleukin (IL)-13 are major profibrogenic cytokines in many organs. Activated PSCs produce various inflammatory cytokines including TGF-beta(1). In this study, we investigated whether IL-13 affects pancreatic fibrogenesis by modulating the functions of PSCs. IL-13 promoted PSCs proliferation without activation through the suppression of autocrine TGF-beta(1). IL-13 enhanced Stat6 phosphorylation in PSCs but Stat6 was not involved in the suppression of TGF-beta(1). IL-13 inhibited the transcriptional activity of NF-kappa B, and the expression of mutant I-kappa B reproduced the suppression of autocrine TGF-beta(1) and promoted PSCs proliferation. Taken together, we demonstrated that IL-13 promotes PSCs proliferation through the suppression of the transcriptional activity of NF-kappa B, resulting in the decrease of autocrine TGF-beta(1). This finding provides an unequivocal evidence of IL-13 participation in pancreatic fibrosis, illustrating a new strategy for chronic pancreatitis. (C) 2010 Elsevier Inc. All rights reserved.

Although hepatic stellate cells, endothelial cells, glomerular podocytes and plateles were reported to be a source of ADAMTS13, it is not clarified which source is involved in the regulation of plasma ADAMTS13 activity. It was demonstrated previously that selective hepatic stellate cell damage in rats caused decreased plasma ADAMTS13 activity. To further elucidate the potential contribution of hepatic stellate cells to the regulation of plasma ADAMTS13 activity, this study examined plasma ADAMTS13 activity when hepatic stellate cells proliferate during the process of liver fibrosis by employing rat models of liver fibrosis due to cholestasis, bile duct ligation, and steatohepatitis, a choline-deficient L-amino acid-defined-diet. ADAMTS13 expression was increased with co-localisation with smooth muscle alpha-actin, a marker of hepatic stellate cells, in bile duct-ligated livers up to four weeks, in which a close correlation between ADAMTS13 and smooth muscle alpha-actin mRNA expressions was determined. Plasma ADAMTS13 activity, measured by a sandwich ELISA involving a specific substrate to ADAMTS I 3,was increased in bile duct-ligated rats with a significant correlation with ADAMTS13 mRNA expression levels in the liver. Furthermore, ADAMTS13 mRNA expression was increased with enhanced mRNA expression in smooth muscle alpha-actin in the livers of rats fed a choline-deficient L-amino acid-defined-diet for 16 weeks, in which increased plasma ADAMTS13 activity was determined. Thus, increased plasma ADAMTS13 activity in cholestasis and steatohepatitis in rats may be due, at least in part,to enhanced ADAMTS13 production in the liver, suggesting a significant role of hepatic stellate cells in the regulation of plasma ADAMTS13 activity.

Pancreatic AR42J cells demonstrate the pluripotency in precursor cells of the gut endoderm and also provide an excellent model system to study the differentiation of the pancreas. Using the mRNA differential display technique, we identified junctional adhesion molecule-1 (JAM-1), a component of the tight junction, was highly up-regulated during the differentiation of AR42J cells, although junctions were not formed. The expression level of JAM-1 showed an up-regulation in the mRNA level after 3 hours and in the protein level after 24 hours in [activin A + betacellulin]-treated AR42J cells. The expressions of its signaling molecules, PAR-3 and atypical PKC lambda, also increased after the addition of activin A + betacellulin. When JAM-1 was over-expressed in [activin A + betacellulin]-treated AR42J cells, tagged-JAM-1 was observed in cytoplasm as vesicular structures and JAM-1 was colocalized with Rab3B and Rab13, members of the Rab family expressed at tight junctions. In streptozotocin-induced regenerating islets, the expression of JAM-1 was also up-regulated in the mRNA level and the protein level. JAM-1 might therefore play an important role in the differentiation of AR42J cells and the regeneration of pancreatic islets.

Indian hedgehog (Ihh) is a member of hedgehog peptides family that exerts diverse effects on multiple cellular functions. Since Ihh expression is elevated in the pancreas of chronic pancreatitis patients, Ihh has been assumed to participate in the chronic pancreatic injury, especially in pancreatic fibrosis. However, its function in pancreatic fibrosis is still unknown. We thus examined Ihh effects on rat activated pancreatic stellate cells (PSCs) that play a central role in pancreatic fibrosis. Activated PSCs express both patched-I and smoothened that are essential components of hedgehog receptor system. Ihh did not alter the PSC expression of collagen-I or alpha-smooth muscle actin, a parameter of PSC transformation, or did not change PSC proliferation. However, Ihh enhanced PSC migration in both chemotactic and chemokinetic manners. Furthermore, Inn increased the amount of membrane-type I matrix metalloproteinase (MTI-MMP) and altered its localization on the plasma membrane, which plays a stimulatory role in cellular migration. In addition, tissue inhibitor of metalloproteinase-2 (TIMP-2) attenuated lhh-stimulated PSC migration. Since most hedgehog intracellular signals are mediated by Gli-I transcription factor, we investigated its contribution to lhh-enhancement of PSC migration. Ihh induced Gli-I nuclear accumulation in PSCs, indicating that Ihh stimulates Gli-I-dependent signaling pathway in PSCs. Unexpectedly, however, adenovirus-mediated Gli-I overexpression blocked the Inn enhancement of both MTI-MMP localization on the plasma membrane and PSC migration. Furthermore, reduction of Gli-I expression with RNA interference augmented lhh-stimulated PSC migration. These data indicate that lhh promotes PSC migration by enhancing MTI-MMP localization on the plasma membrane but is negatively regulated by Gli-I.

Helicobacter pylori-produced cytotoxin VacA induces intracellular vacuolation. The VacA-induced vacuole is assumed to represent the pathological status of intracellular trafficking. The fusion mechanism of the endosomes requires the formation of a tight complex between the Q-SNAREs and the R-SNAREs. We recently reported that syntaxin 7, a family member of the Q-SNARE protein, is involved in VacA-induced vacuole formation. In order to further elucidate the molecular mechanism, we identified the participation of vesicle-associated membrane protein 7 (VAMP7) as a partner of syntaxin 7. Immunocytochemistry revealed endogenous VAMP7 to be localized to the vacuoles induced by VacA. A Northern blotting study demonstrated that VacA intoxication increased VAMP7 mRNA in a time-dependent manner. VAMP7 was coimmunoprecipitated with syntaxin 7, and the amounts of endogenous VAMP7 and syntaxin 7 bound to syntaxin 7 and VAMP7, respectively, increased in response to VacA. The down-regulation of VAMP7 using small interfering RNA inhibited VacA-induced vacuolation, and the transient transfection of dominant-negative mutant VAMP7, the N-terminal domain of VAMP7, also inhibited the vacuolation. We therefore conclude that R-SNARE VAMP7 plays an important role in VacA-induced vacuolation as a partner of Q-SNARE syntaxin 7.

Bile acids, which have been implicated in gastrointestinal-tract cell carcinogenesis, share properties with tumor promoters in that both affect signal transduction pathways responsible for cell proliferation and apoptosis. In the present study, we demonstrate that EGFR-ERK1/2 is activated following treatment of AGS human gastric carcinoma cells with bile acids. EGFR phosphoactivation is ligand-dependent, since treatment of cells with HB-EGF antisera or CM 197 (a selective inhibitor of HB-EGF) markedly inhibits deoxycholate (DC)-promoted activation. Membrane-type bile acid receptor (M-BAR)/TGR5 is a recently identified G-protein-coupled receptor (GPCR). In AGS cells, siRNAs that target M-BAR suppress DC-induced phosphorylation of EGFR. Furthermore, introduction of siRNAs targeting ADAM17 transcripts resulted in suppression of DC-induced activation of EGFR and ERK1/2. These results suggest that in AGS cells, DC transactivates EGFR through M-BAR- and ADAM/HB-EGF-dependent mechanisms. (c) 2006 Elsevier Inc. All rights reserved.

Sonic Hedgehog (Shh), a member of hedgehog peptides family, is expressed in gastric gland epithelium. To elucidate Shh function to gastric mucosal cells, we examined the effect of Shh on the proliferation of a rat normal gastric mucosal cell line, RGM-1. RGM-1 cells express essential components of Shh receptor system, patched-1, and smoothened. Shh enhanced DNA synthesis in RGM-1 cells and elevated intracellular calcium concentration ([Ca2+](i)). In addition, Shh as well as calcium ionophore A32187 rapidly activated ERK. However, Shh failed to activate ERK under calcium-free culture condition. Pretreatment of cells with PD98059 attenuated the DNA synthesis promoted by Shh. Moreover, when cells were pretreated with cyclopamine, Shh could not elevate [Ca2+],, activate ERK or promote DNA synthesis. On the other hand, although Shh induced Gli-1 nuclear accumulation in RGM-1 cells, Shh activated ERK even in cells pretreated with actinomycin D. These results indicate that Shh promotes the proliferation of RGM-1 cells through an intracellular calcium- and ERK-dependent but transcription-independent pathway via Patched/Smoothened receptor system. (c) 2006 Elsevier Inc. All rights reserved.

Dieulafoy's ulcer is a rare cause of gastrointestinal bleeding. The lesion is usually located in the stomach, although it may occur anywhere in the gastrointestinal tract. A 44-year-old man was admitted to hospital due to cerebral infarction. On the 23rd day of hospitalization, he showed massive hematochezia. He underwent an urgent colonoscopy. There was a visible protuberant vessel without significant ulceration at the fundus of the rectum, consistent with a Dieulafoy's ulcer. It was treated by endoscopic hemoclipping. However, rebleeding occurred three times despite repeated hemoclipping. Finally, endoscopic hand ligation was successfully perk formed to achieve permanent hemostasis. Endoscopic hand ligation is an effective treatment for bleeding rectal Dieulafoy's ulcer.

Pancreatic stellate cells ( PSCs) are activated during pancreatitis and promote pancreatic fibrosis by producing and secreting ECMs such as collagen and fibronectin. IL-1 beta has been assumed to participate in pancreatic fibrosis by activating PSCs. Activated PSCs secrete various cytokines that regulate PSC function. In this study, we have examined IL-1 beta secretion from culture-activated PSCs as well as its regulatory mechanism. RT-PCR and ELISA have demonstrated that PSCs express IL-1 beta mRNA and secrete IL-1 beta peptide. Inhibition of TGF-beta(1) activity secreted from PSCs by TGF-beta(1)-neutralizing antibody attenuated IL-1 beta secretion from PSCs. Exogenous TGF-beta(1) increased IL-1 beta expression and secretion by PSCs in a dose-dependent manner. Adenovirus-mediated expression of dominant-negative ( dn) Smad2/3 expression reduced both basal and TGF-beta(1)-stimulated IL-1 beta expression and secretion by PSCs. Coexpression of Smad3 with dnSmad2/3 restored IL-1 beta expression and secretion by PSCs, which were attenuated by dnSmad2/3 expression. In contrast, coexpression of Smad2 with dnSmad2/3 did not alter them. Furthermore, inhibition of IL-1 beta activity secreted from PSCs by IL-1 beta-neutralizing antibody attenuated TGF-beta(1) secretion from PSCs. Exogenous IL-1 beta enhanced TGF-beta(1) expression and secretion by PSCs. IL-1 beta activated ERK, and PD-98059, a MEK1 inhibitor, blocked IL-1 beta enhancement of TGF-beta(1) expression and secretion by PSCs. We propose that an autocrine loop exists between TGF-beta(1) and IL-1 beta in activated PSCs through Smad3- and ERK-dependent pathways.

Activated pancreatic stellate cells (PSCs) play major roles in promoting pancreatic fibrosis. We previously reported that angiotensin II (Ang II) enhances activated PSC proliferation through EGF receptor transactivation. In the present study, we elucidated a novel intracellular mechanism by which Ang II stimulates cellular proliferation. TGF-beta(1) inhibits activated PSC proliferation via a Smad3 and Smad4-dependent pathway in an autocrine manner. We demonstrated that Ang II inhibited TGF-beta(1)-induced nuclear accumulation of Smad3 and Smad4. Furthermore, Ang II rapidly induced inhibitory Smad7 mRNA expression. Adenovirus-mediated Smad7 overexpression inhibited TGF-beta(1)-induced nuclear accumulation of Smad3 and Smad4, and potentiated activated PSC proliferation. PKC inhibitor Go6983 blocked the induction of Smad7 mRNA expression by Ang II. In addition, 12-O-tetradecanoyl-phorbol 13-acetate, a PKC activator, increased Smad7 mRNA expression. These results Suggest that Ang II enhances activated PSC proliferation by blocking autocrine TGF-beta(1)-mediated growth inhibition by inducing Smad7 expression via a PKC-dependent pathway. (c) 2005 Elsevier Inc. All rights reserved.

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.