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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 - PubMed

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

C Bourgier et al. Gut. 2005 Mar.

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.

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Figures

Figure 1
Figure 1

(A) Intestinal smooth muscle cells exhibited fibrogenic differentiation in vivo. In the muscularis propria, Sirius red staining showed collagen infiltration within smooth muscle bundles in radiation enteritis (II, ×200) versus normal bowel (I, ×200) that colocalised with vimentin positive cells (IV, ×200). Connective tissue growth factor (CTGF) immunostaining was negative in normal muscularis propria (V, ×200) whereas strong staining was observed in radiation enteritis (VI, ×200). (B) Gene array analysis revealed induction of genes coding for the Rho family and for actin polymerisation control in radiation enteritis samples (n = 6) compared with normal bowel samples (n = 6).

Figure 2
Figure 2

(A) Immunofluorescence experiments showed that normal smooth muscle cells (N SMC) exhibited typical intestinal differentiation markers in vitro. They were positive for vimentin and tropomyosin whereas only a few cells expressed α smooth muscle actin (α-sm actin). (B) Smooth muscle cells derived from radiation enteritis (RE SMC) showed greater densities of stress fibres and a strong constitutive α-sm actin protein expression. RE SMC (two cell lines) exhibited a prosecretory phenotype with a higher constitutive connective tissue growth factor (CTGF) mRNA level (experiments done in triplicate; C) and procollagen type I (PIP) secretion (experiments done in duplicate; D) than their normal counterpart (three cell lines). *p<0.05 compared with untreated N SMC.

Figure 3
Figure 3

Gene array analysis revealed induction in the expression profile of genes coding for the Rho pathway in radiation enteritis smooth muscle cells (RE SMC) compared with normal smooth muscle cells (N SMC). Hybridisation intensities were obtained using the Atlas Image 1.5 software, converted into ratios, and adjusted for background and housekeeping gene expression. A “normal composite array” was established by averaging the three arrays obtained from the three N SMC lines and compared with the two RE SMC lines.

Figure 4
Figure 4

Alteration of actin stress fibre network by Rho kinase inhibition. F-actin was determined by FITC-phalloidin staining after Y-27632 incubation in normal smooth muscle cells (N SMC) (A) and radiation enteritis smooth muscle cells (RE SMC) (B). Rho kinase inhibition decreased heat shock protein (HSP)27 and α smooth muscle actin (α-sm actin) protein expression. (C) HSP27 and α-sm actin protein levels were assessed by western blot. Values were normalised to glyceraldehyde-3-phosphate dehydrogenase (GAPDH) protein levels. The blot is representative of three independent experiments.

Figure 5
Figure 5

(A, B) Regulation of connective tissue growth factor (CTGF) expression by Rho kinase inhibition. (A) CTGF mRNA level was assessed by real time reverse transcription-polymerase chain reaction (RT-PCR). Values are mean (SEM); n = 4. **p<0.01 compared with untreated normal smooth muscle cells (N SMC); ††p<0.01 compared with untreated radiation enteritis smooth muscle cells (RE SMC). (B) CTGF protein level was assessed by western blot in RE SMC. The blot is representative of three independent experiments. GAPDH, glyceraldehyde-3-phosphate dehydrogenase. (C) Regulation of type I collagen alpha 1 (COL1A1) expression by Rho kinase inhibition. COL1A1 mRNA levels were assessed by real time RT-PCR. Values are mean (SEM); n = 4. **p<0.01 compared with untreated N SMC; ††p<0.01 compared with untreated RE SMC.

Figure 6
Figure 6

(A) Results of electrophoretic mobility shift assay (EMSA) protein binding to the nuclear factor κB (NFκB) consensus site after incubation with 10 µM Y-27632 (Y) for 30 minutes and 120 minutes, and with 25 mM sodium salicylate (S) for 45 minutes. Non-radioactive NFκB oligo (CPX10) blocked NFκB DNA binding. The blot is representative of two independent experiments. (B) Western blot analysis of IκBα and p65 in cytoplasmic extract of normal smooth muscle cells (N SMC) and radiation enteritis smooth muscle cells (RE SMC) incubated with Y-27632 (Y) and sodium salicylate (S). The blot is representative of two independent experiments.

Figure 7
Figure 7

Chronic activation of the Rho/Rho kinase (ROCK) cascade in radiation enteritis is involved in intestinal smooth muscle cell differentiation towards an immature phenotype with altered prosecretory and contractile properties. CTGF, connective tissue growth factor; α/γ-sm actin, α/γ smooth muscle actin; HSP, heat shock protein; NFκB, nuclear factor κB.

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