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Inhibition of Rho kinase modulates radiation induced fibrogenic phenotype in intestinal smooth muscle cells through alteration of the cytoskeleton and connective tissue growth factor expression.

ABSTRACT: BACKGROUND:Late radiation enteritis in humans is associated with accumulation of extracellular matrix and increased connective tissue growth factor (CTGF) expression that may involve intestinal muscular layers. AIMS:We investigated the molecular pathways involved in maintenance of radiation induced fibrosis by gene profiling and postulated that alteration of the Rho pathway could be associated with radiation induced fibrogenic signals and CTGF sustained expression. PATIENTS AND METHODS:Ileal biopsies from individuals with and without radiation enteritis were analysed by cDNA array, and primary cultures of intestinal smooth muscle cells were established. Then, the effect of pharmacological inhibition of p160 Rho kinase, using Y-27632, was studied by real time reverse transcription-polymerase chain reaction, western blot, and electrophoretic mobility shift assay. RESULTS:Molecular profile analysis of late radiation enteritis showed alterations in expression of genes coding for the Rho proteins. To investigate further the involvement of the Rho pathway in intestinal radiation induced fibrosis, primary intestinal smooth muscle cells were isolated from radiation enteritis. They retained their fibrogenic differentiation in vitro, exhibited a typical cytoskeletal network, a high constitutive CTGF level, increased collagen secretory capacity, and altered expression of genes coding for the Rho family. Rho kinase blockade induced a simultaneous decrease in the number of actin stress fibres, alpha smooth muscle actin, and heat shock protein 27 levels. It also decreased CTGF levels, probably through nuclear factor kappaB inhibition, and caused decreased expression of the type I collagen gene. CONCLUSION:This study is the first showing involvement of the Rho/Rho kinase pathway in radiation fibrosis and intestinal smooth muscle cell fibrogenic differentiation. It suggests that specific inhibition of Rho kinase may be a promising approach for the development of antifibrotic therapies.

SUBMITTER: Bourgier C

PROVIDER: S-EPMC1774418 | biostudies-literature | 2005 Mar

REPOSITORIES: biostudies-literature

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Inhibition of Rho kinase modulates radiation induced fibrogenic phenotype in intestinal smooth muscle cells through alteration of the cytoskeleton and connective tissue growth factor expression.

Bourgier C C Haydont V V Milliat F F François A A Holler V V Lasser P P Bourhis J J Mathé D D Vozenin-Brotons M-C MC

Gut 20050301 3

<h4>Background</h4>Late radiation enteritis in humans is associated with accumulation of extracellular matrix and increased connective tissue growth factor (CTGF) expression that may involve intestinal muscular layers.<h4>Aims</h4>We investigated the molecular pathways involved in maintenance of radiation induced fibrosis by gene profiling and postulated that alteration of the Rho pathway could be associated with radiation induced fibrogenic signals and CTGF sustained expression.<h4>Patients and ...[more]

PMID: 15710979

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Project description:Intestinal crypt progenitor/stem (ICPS) cell apoptosis and vascular endothelial cell apoptosis are responsible for the initiation and development of ionizing radiation (IR)-evoked gastrointestinal syndrome. The signaling mechanisms underlying IR-induced ICPS cell apoptosis remain largely unclear. Our findings provide evidence that ?-arrestin-2 (?arr2)-mediated ICPS cell apoptosis is crucial for IR-stimulated intestinal injury. ?Arr2-deficient mice exhibited decreased ICPS cell and intestinal Lgr5(+) (leucine-rich repeat-containing G-protein-coupled receptor 5-positive) stem cell apoptosis, promoted crypt proliferation and reproduction, and protracted survival following lethal doses of radiation. Radioprotection in the ICPS cells isolated from ?arr2-deficient mice depended on prolonged nuclear factor-?B (NF-?B) activation via direct interaction of ?arr2 with I?B? and subsequent inhibition of p53-upregulated modulator of apoptosis (PUMA)-mediated mitochondrial dysfunction. Unexpectedly, ?arr2 deficiency had little effect on IR-induced intestinal vascular endothelial cell apoptosis in mice. Consistently, ?arr2 knockdown also provided significant radioresistance by manipulating NF-?B/PUMA signaling in Lgr5(+) cells in vitro. Collectively, these observations show that targeting the ?arr2/NF-?B/PUMA novel pathway is a potential radiomitigator for limiting the damaging effect of radiotherapy on the gastrointestinal system. Significance statement: acute injury to the intestinal mucosa is a major dose-limiting complication of abdominal radiotherapy. The issue of whether the critical factor for the initiation of radiation-induced intestinal injury is intestinal stem cell apoptosis or endothelial cell apoptosis remains unresolved. ?Arrs have recently been found to be multifunctional adaptor of apoptosis. Here, we found that ?-arrestin-2 (?arr2) deficiency was associated with decreased radiation-induced ICPS cell apoptosis, which prolonged survival in abdominally irradiated mice. Moreover, ?arr2 deficiency-mediated intestinal progenitor/stem cell radioprotection relied on protracted NF-?B activation and subsequent suppression of PUMA induction. Our results suggest that ICPS cell apoptosis is the factor involved in the initiation and development of radiation-induced gastrointestinal syndrome. ?Arr2 is a potential target for lessening radiation-induced ICPS cell apoptosis.

Project description:Vascular smooth muscle cell (SMC) switching between differentiated and dedifferentiated phenotypes is reversible and accompanied by morphological and functional alterations that require reconfiguration of cell-cell and cell-matrix adhesion networks. Studies attempting to explore changes in overall composition of the adhesion nexus during SMC phenotype transition are lacking. We have previously demonstrated that T-cadherin knockdown enforces SMC differentiation, whereas T-cadherin upregulation promotes SMC dedifferentiation. This study used human aortic SMCs ectopically modified with respect to T-cadherin expression to characterize phenotype-associated cell-matrix adhesion molecule expression, focal adhesions configuration and migration modes. Compared with dedifferentiated/migratory SMCs (expressing T-cadherin), the differentiated/contractile SMCs (T-cadherin-deficient) exhibited increased adhesion to several extracellular matrix substrata, decreased expression of several integrins, matrix metalloproteinases and collagens, and also distinct focal adhesion, adherens junction and intracellular tension network configurations. Differentiated and dedifferentiated phenotypes displayed distinct migrational velocity and directional persistence. The restricted migration efficiency of the differentiated phenotype was fully overcome by reducing actin polymerization with ROCK inhibitor Y-27632 whereas myosin II inhibitor blebbistatin was less effective. Migration efficiency of the dedifferentiated phenotype was diminished by promoting actin polymerization with lysophosphatidic acid. These findings held true in both 2D-monolayer and 3D-spheroid migration models. Thus, our data suggest that despite global differences in the cell adhesion nexus of the differentiated and dedifferentiated phenotypes, structural actin cytoskeleton characteristics per se play a crucial role in permissive regulation of cell-matrix adhesive interactions and cell migration behavior during T-cadherin-induced SMC phenotype transition.

Project description:BackgroundIntestinal fibrosis is a serious complication of inflammatory bowel disease, including Crohn's disease and ulcerative colitis. There is no specific treatment for intestinal fibrosis. Studies have indicated that peroxisome proliferator-activated receptor- ? (PPAR-?) agonists have anti-fibrogenic properties in organs besides the gut; however, their effects on human intestinal fibrosis are poorly understood. This study investigated the anti-fibrogenic properties and mechanisms of PPAR-? agonists on human primary intestinal myofibroblasts (HIFs).MethodsHIFs were isolated from normal colonic tissue of patients undergoing resection due to colorectal cancer. HIFs were treated with TGF-?1 and co-incubated with or without one of two synthetic PPAR-? agonists, troglitazone or rosiglitazone. mRNA and protein expression of procollagen1A1, fibronectin, and ?-smooth muscle actin were determined by semiquantitative reverse transcription-polymerase chain reaction and Western blot. LY294002 (Akt inhibitor) was used to examine whether Akt phosphorylation was a downstream mechanism of TGF-?1 induced expression of procollagen1A1, fibronectin, and ?-smooth muscle actin in HIFs. The irreversible PPAR-? antagonist GW9662 was used to investigate whether the effect of PPAR-? agonists was PPAR-? dependent.ResultsBoth PPAR-? agonists reduced the TGF-?1-induced expression of ?-smooth muscle actin which was integrated into stress fibers in HIFs, as determined by actin microfilaments fluorescent staining and ?-smooth muscle actin-specific immunocytochemistry. PPAR-? agonists also inhibited TGF-?1-induced mRNA and protein expressions of procollagen1A1, fibronectin, and ?-smooth muscle actin. TGF-?1 stimulation increased phosphorylation of downstream signaling molecules Smad2, Akt, and ERK. TGF-?1 induced synthesis of procollagen1A1, fibronectin, and ?-smooth muscle actin through a phosphatidylinositol 3-kinase/Akt-dependent mechanism. PPAR-? agonists down regulated fibrogenesis, as shown by inhibition of Akt and Smad2 phosphorylation. This anti-fibrogenic effect was PPAR-? independent.ConclusionsTroglitazone and rosiglitazone suppress TGF-?1-induced synthesis of procollagen1A1, fibronectin, and ?-smooth muscle actin in HIFs and may be useful in treating intestinal fibrosis.

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Inhibition of Rho kinase modulates radiation induced fibrogenic phenotype in intestinal smooth muscle cells through alteration of the cytoskeleton and connective tissue growth factor expression.

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Inhibition of Rho kinase modulates radiation induced fibrogenic phenotype in intestinal smooth muscle cells through alteration of the cytoskeleton and connective tissue growth factor expression.

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