Atsushi Masamune

Rho kinase inhibitors block activation of pancreatic stellate cells

In response to pancreatic injury and in cell culture, pancreatic stellate cells (PSCs) are transformed (‘activated’) into highly proliferative myofibroblast‐like cells, which express α‐smooth muscle actin (α‐SMA), and produce type I collagen and other extracellular matrix components. There is accumulating evidence that activated PSCs play important roles in pancreatic fibrosis and inflammation. The small GTP‐binding protein Rho has emerged as an important regulator of the actin cytoskeleton and cell morphology through the downstream effector Rho kinase (ROCK). But, the roles of Rho‐ROCK pathway in PSCs are unknown. Here, we examined the effects of (+)‐(R)‐trans‐4‐(1‐aminoethyl)‐N‐(4‐pyridyl) cyclohexanecarboxamide (Y‐27632) and HA‐1077 (fasudil), specific inhibitors of ROCK, on the activation of PSCs. PSCs were isolated from the pancreas of male Wistar rats after perfusion with collagenase P. The actin cytoskeleton was analyzed by phalloidin staining. Expression of RhoA and ROCK was examined by immunostaining and Western blotting. Effects of Y‐27632 and HA‐1077 on α‐SMA expression, platelet‐derived growth factor‐induced proliferation and chemotaxis, and collagen production were assessed. Culture‐activated PSCs developed a well‐spread cell shape, with extended stress fiber formation. PSCs expressed RhoA, ROCK‐1, and ROCK‐2. Y‐27632 caused disassembly of stress fibers. Y‐27632 and HA‐1077 inhibited α‐SMA expression, proliferation, chemotaxis, and type I collagen production in culture‐activated PSCs. In addition, Y‐27632 and HA‐1077 inhibited spontaneous activation of freshly isolated PSCs in culture on plastic. These findings suggest a role of Rho‐ROCK pathway in the activation process of PSCs by regulating the actin cytoskeleton, and a potential application of Rho‐ROCK pathway inhibitors for the treatment of pancreatic inflammation and fibrosis.

Activated rat pancreatic stellate cells express intercellular adhesion molecule-1 (ICAM-1) in vitro.

IntroductionActivated pancreatic stellate cells (PSCs) have recently been implicated in the pathogenesis of pancreatic fibrosis.AimsTo examine the role of PSCs in pancreatic inflammation by determining whether activated PSCs express intercellular adhesion molecule-1 (ICAM-1).MethodologyCulture-activ

Hereditary pancreatitis as the premalignant disease: a Japanese case of pancreatic cancer involving the SPINK1 gene mutation N34S.

Abstract: Mutations in the cationic trypsinogen gene are acknowledged as a risk factor for pancreatic cancer in patients with hereditary pancreatitis. However, whether patients with mutations in other genes, such as the serine protease inhibitor Kazal type 1 (SPINK1) gene, are also at a higher risk of pancreatic cancer remains unknown. We report a case of pancreatic cancer associated with chronic calcifying pancreatitis in a patient with a homozygous N34S mutation in the SPINK1 gene. A 44-year-old woman was hospitalized due to obstructive jaundice. Preoperative examination showed a tumor in the head of the pancreas and multiple pancreatic stones; pancreatoduodenectomy revealed a solid tumor, 3.0 × 2.5 cm in size, in the head of the pancreas, and numerous pancreatic stones throughout the pancreas. Pathologic studies revealed moderately differentiated tubular adenocarcinoma. Mutational analyses of the SPINK1 and PRSS1 genes in members of the patient’s family were carried out. The homozygous N34S mutation in the SPINK1 gene was found in the patient and her older sister, who was previously diagnosed with chronic calcific pancreatitis and had undergone the Frey operation. The patient’s parents and brother were unaffected carriers of the N34S heterozygous mutation. No family members had any mutations in the cationic trypsinogen gene. To our knowledge, this is the first reported case of chronic pancreatitis accompanied by pancreatic cancer in a patient with the SPINK1 N34S mutation. Although this case does not meet the classic criteria of hereditary pancreatitis, it does suggest that the SPINK1 N34S mutation may be associated with cancer development in patients with hereditary pancreatitis. Further prospective, multicenter trials investigating secondary screening for pancreatic cancer in hereditary pancreatitis are necessary to clarify the role of SPINK1 mutations in the development of pancreatic cancer.

Establishment and characterization of a rat pancreatic stellate cell line by spontaneous immortalization

AIM Activated pancreatic stellate cells (PSCs) have been implicated in the pathogenesis of pancreatic fibrosis and inflammation. Primary PSCs can be subcultured only several times because of their limited growth potential. A continuous cell line may therefore be valuable in studying molecular mechanisms of these pancreatic disorders. The aim of this study was to establish a cell line of rat PSCs by spontaneous immortalization. METHODS PSCs were isolated from the pancreas of male Wistar rats, and conventional subcultivation was performed repeatedly. Telomerase activity was measured using the telomere repeat amplification protocol. Activation of transcription factors was assessed by electrophoretic mobility shift assay. Activation of mitogen-activated protein (MAP) kinases was examined by Western blotting using anti-phosphospecific antibodies. Expression of cytokine-induced neutrophil chemoattractant-1 was determined by enzyme immunoassay. RESULTS Conventional subcultivation yielded actively growing cells. One clone was obtained after limiting dilution, and designated as SIPS. This cell line has been passaged repeatedly more than 2 years, and is thus likely immortalized. SIPS cells retained morphological characteristics of primary, culture-activated PSCs. SIPS expressed alpha-smooth muscle actin, glial acidic fibrillary protein, vimentin, desmin, type I collagen, fibronectin, and prolyl hydroxylases. Telomerase activity and p53 expression were negative. Proliferation of SIPS cells was serum-dependent, and stimulated with platelet-derived growth factor-BB through the activation of extracellular signal-regulated kinase. Interleukin-1beta activated nuclear factor-kappaB, activator protein-1, and MAP kinases. Interleukin-1beta induced cytokine-induced neutrophil chemoattractant-1 expression through the activation of nuclear factor-kappaB and MAP kinases. CONCLUSION SIPS cells can be useful for in vitro studies of cell biology and signal transduction of PSCs.

Activated rat pancreatic stellate cells express intercellular adhesion molecule-1 (ICAM-1) in vitro

Introduction Activated pancreatic stellate cells (PSCs) have recently been implicated in the pathogenesis of pancreatic fibrosis. Aims To examine the role of PSCs in pancreatic inflammation by determining whether activated PSCs express intercellular adhesion molecule-1 (ICAM-1). Methodology Culture-activated rat PSCs were treated with interleukin-1&bgr; (IL-1&bgr;), tumor necrosis factor-&agr; (TNF-&agr;), ethanol, or acetaldehyde. ICAM-1 expression was analyzed by flow cytometry. The induction of mRNA was assessed by Northern blot analysis. The binding activity of transcription factors was examined by electrophoretic mobility shift assay. The activation of mitogen-activated protein kinases was assessed by Western blotting with use of antiphosphospecific antibodies. The adhesion of MOLT-4 cells to activated PSCs was also assessed. Results Culture-activated PSCs expressed ICAM-1. The expression was increased in response to IL-1&bgr; and TNF-&agr; but not to alcohol, with comparable mRNA induction. IL-1&bgr; and TNF-&agr; increased the nuclear factor-&kgr;B (NF-&kgr;B)–specific and activator protein–1-specific DNA binding activity, whereas the NF-IL6 activity was not altered. Alcohol did not increase NF-&kgr;B binding activity. IL-1&bgr; and TNF-&agr; activated extracellular signal–regulated kinase 1/2, c-Jun N-terminal kinase/stress-activated protein kinase, and p38 mitogen–activated protein kinase. Inducible ICAM-1 expression was blocked by pyrrolidine dithiocarbamate, a specific inhibitor of NF-&kgr;B activation, but not by inhibitors of mitogen-activated protein kinase pathways. IL-1&bgr; and TNF-&agr; increased the ICAM-1-mediated binding of MOLT-4 cells to activated PSCs, indicating a role of ICAM-1 in the adhesion of leukocytes to PSCs. Conclusion Our results suggest that activated PSCs express ICAM-1 mainly through the activation of NF-&kgr;B, thus playing a role in the pathogenesis of pancreatic inflammation.