Bacterial DNA Promotes Proliferation of Rat Pancreatic Stellate Cells Thorough Toll-Like Receptor 9

Nakamura, Taichi; Ito, Tetsuhide; Oono, Takamasa; Igarashi, Hisato; Fujimori, Nao; Uchida, Masahiko; Niina, Yusuke; Yasuda, Masami; Suzuki, Koichi; Takayanagi, Ryoichi

doi:10.1097/mpa.0b013e318224a501

Cited by 13 publications

(22 citation statements)

References 54 publications

(33 reference statements)

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“…9 Purity of PSCs of 490% was confirmed by the typical star-like configuration of the cells and by presence of vitamin A autofluorescence. The cells were maintained in complete Dulbecco's modified Eagle medium (DMEM)/F-12, which is a mixture of DMEM and Ham's F-12 nutrient mixture (Wako Pure Chemicals, Osaka, Japan) supplemented with 10% fetal bovine serum, 50 units/ml of penicillin, and 50 mg/ml of streptomycin (Invitrogen, Carlsbad, CA, USA).…”

Section: Isolation Of Pscs and Cell Culturementioning

confidence: 97%

“…9 PSCs were serum starved and incubated for 24 h at 37 1C and fixed in 4% paraformaldehyde. After blocking with 2% normal bovine serum albumin, cells were incubated with rabbit anti-rat CX3CL1, MMP-2, and ADAM17 antibody at 1:100 dilution and with mouse anti-rat a-SMA antibody at 1:400 dilution overnight at 4 1C.…”

Section: Immunofluorescence Stainingmentioning

confidence: 99%

“…9 Cells were lysed in RIPA buffer (Nacalai Tesque, Kyoto, Japan) and cellular proteins (approximately 50 mg) were fractionated by electrophoresis on a 10% sodium dodecyl sulfate polyacrylamide gel (Bio-Rad, Hercules, CA, USA). The proteins were transferred to a nitrocellulose membrane (Bio-Rad), and the membrane was incubated for 2 to 6 h with primary antibodies at 1:1000 dilutions.…”

Section: Western Blottingmentioning

confidence: 99%

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ERK pathway and sheddases play an essential role in ethanol-induced CX3CL1 release in pancreatic stellate cells

Uchida

Ito

Nakamura

et al. 2013

Laboratory Investigation

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The clinical course of chronic pancreatitis (CP) worsens with drinking, and pancreatic stellate cells (PSCs) have an important role in the pathogenesis of alcoholic CP. Chemokines recruit inflammatory cells, resulting in chronic pancreatic inflammation. Although serum levels of fractalkine (CX3CL1) are significantly elevated in patients with alcoholic CP, the mechanism of this elevation remains unclear. This study aims to determine the effects of cytokines, pathogen-associated molecular patterns (PAMPs), and ethanol and its metabolites on CX3CL1 secretion by PSCs. Male Wistar/Bonn Kobori (WBN/Kob) rats aged 15 to 20 weeks were used as rodent models of CP in vivo. PSCs were isolated from 6-week-old male Wistar rats. The effects of cytokines, PAMPs, and ethanol and its metabolites on chemokine production and activation of signaling pathways in PSCs in vitro were examined by real-time reverse transcription-polymerase chain reaction (RT-PCR), western blotting, and enzyme-linked immunosorbent assay. Expression of CX3CL1 and matrix metalloprotease (MMP)-2 was increased in the pancreas of WBN/Kob rats. The rat PSCs expressed CX3CL1, MMP-2, and a disintegrin and metalloprotease domain (ADAM) 17. Cytokines and PAMPs induced CX3CL1 release and activated extracellular signal-regulated kinase (ERK), MMP-9, and ADAM17. CX3CL1 release was suppressed by specific inhibitors of ERK, MMP, and ADAM, and ERK was associated with CX3CL1 transcription. Ethanol and phorbol myristate acetate synergistically increased CX3CL1 release. Real-time PCR and western blotting confirmed the synergistic activation of ERK and ADAM17. Ethanol synergistically increased CX3CL1 release via ERK and ADAM17 activation in PSCs. In conclusion, we demonstrated for the first time that ethanol synergistically increased CX3CL1 release from PSCs at least in part through activation of ERK mitogen-activated protein kinase and ADAM17. This might be one of the mechanisms of serum CX3CL1 elevation and disease progression in patients with alcoholic CP.

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Section: Isolation Of Pscs and Cell Culturementioning

confidence: 97%

Section: Immunofluorescence Stainingmentioning

confidence: 99%

Section: Western Blottingmentioning

confidence: 99%

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ERK pathway and sheddases play an essential role in ethanol-induced CX3CL1 release in pancreatic stellate cells

Uchida

Ito

Nakamura

et al. 2013

Laboratory Investigation

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“…PSCs were isolated from the pancreas of normal male Wistar rats (weighing 180–200 g) by a density-gradient centrifugation method as previously described 56 . Briefly, purity of PSCs was more than 90% as evaluated by the typical star-like configuration and by detecting vitamin A autofluorescence.…”

Section: Methodsmentioning

confidence: 99%

Pancreatic Stellate Cells and CX3CR1

et al. 2014

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Objectives Numerous studies suggest important roles of the chemokine, fractalkine (CX3CL1) in acute/chronic pancreatitis, however the possible mechanisms of the effects are unclear. Pancreatic stellate cells (PSCs) can play important roles in pancreatitis, secreting inflammatory cytokines/chemokines, as well as proliferation. Therefore, we investigated CX3CL1 receptor (CX3CR1) occurrence in normal pancreas and pancreatitis (acute/chronic) tissues, and the effects of CX3CL1 on activated-PSCs. Methods CX3CR1 expression/localization in normal pancreas and pancreatitis (acute/chronic) tissues were evaluated with immunohistochemical analysis. CX3CR1 expression and effects of CX3CL1 on activated-PSCs were examined with realtime-PCR, BrdU assays and Western Blotting. Results In normal pancreas, acinar cells expressed CX3CR1 within granule-like-formations in the cytoplasm, whereas in acute/chronic pancreatitis, acinar, ductal and activated-PSCs expressed CX3CR1 on cell membranes. With activation of normal PSCs, CX3CR1 is increased. CX3CL1 activated multiple signaling cascades in PSCs. CX3CL1, did not induce inflammatory-genes expression in activated-PSCs, but induced proliferation. Conclusions CX3CR1s are expressed in normal pancreas. Expression is increased in acute/chronic pancreatitis and the CX3CR1s are activated. CX3CL1 induces proliferation of activated-PSCs without increasing release of inflammatory-mediators. These results suggest that CX3CR1 activation of PSCs could be important in their effects in pancreatitis, especially to PSCs proliferation in pancreatitis where CX3CL1 levels are elevated.

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“…Activated PSCs produce inflammatory mediators, including interleukin (IL) -1, IL-6, IL-8, IL-15, CTGF, TGFβ and RANTES which are all capable of activating other nearby quiescent PSCs via an autocrine loop. (Andoh et al , 2000, Shek et al , 2002 Activated PSC play key roles in the development of a fibrotic pancreatic stroma with the synthesis of extracellular matrix proteins such as collagen and enzymes responsible for matrix remodelling such as matrix metalloproteinases (MMPs) and their inhibitors, tissue inhibitor of metalloproteinases (TIMPs) (Nakamura et al , 2011, Phillips et al , 2003.…”

Section: Introductionmentioning

confidence: 99%

The role of exosomes in the pathogenesis of pancreatic ductal adenocarcinoma

Robinson

Fan

White

et al. 2016

The International Journal of Biochemistry & Cell Biology

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Bacterial DNA Promotes Proliferation of Rat Pancreatic Stellate Cells Thorough Toll-Like Receptor 9

Abstract: Our findings demonstrate that bacterial DNA promotes migration and proliferation of PSCs and suggest that bacterial DNA can initiate and sustain pancreatic inflammation and fibrosis by means of TLR9.

Cited by 13 publications

References 54 publications

ERK pathway and sheddases play an essential role in ethanol-induced CX3CL1 release in pancreatic stellate cells

ERK pathway and sheddases play an essential role in ethanol-induced CX3CL1 release in pancreatic stellate cells

Pancreatic Stellate Cells and CX3CR1

The role of exosomes in the pathogenesis of pancreatic ductal adenocarcinoma

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