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Rho/ROCK pathway as a molecular target for modulation of intestinal radiation-induced toxicity

UPRES EA 27-10 "Radiosensibilité des tumeurs et tissus sains". Institut de Radioprotection et de Sûreté Nucléaire/Institut Gustave Roussy, Villejuif and Laboratoire de Radiopathologie, SLBE/DRPH, Institut de Radioprotection et de Sûreté Nucléaire, Fantenay-aux-Roses, France

View larger version (45K): [in this window] [in a new window]   Figure 1. Mucosa was dissected from ileum biopsies with and without radiation enteropathy(RE) and incubated in 0.2% collagenase Type II, 0.1% soybean trypsin inhibitor solution at 37°C to isolate primary subepithelial myofibroblasts. Cells were subcultured in FGM (Cambrex, Verviers, Belgium) used between passage 3 and passage 4. (a,b) After fixation and permeabilization, subsequent immunodetection of the -smooth muscle (sm) actin was performed as already described (CB) [13] (anti--sm actin; Sigma). Immunofluorescence experiments showed that subepithelial myofibroblasts derived from RE showed greater densities of stress fibres and nearly all cells were -sm actin positive. (c) Real-time reverse transcriptase polymerase chain reaction (RT-PCR) showed an increased connective tissue growth factor (CTGF) mRNA level in subepithelial myofibroblasts derived from RE vs subepithelial myofibroblasts derived from normal ileum. Values were normalized to GAPDH (glyceraldehyde-3-PDH) mRNA level. (d) Procollagen Type I enzyme-linked immunosorbent assay (ELISA) [procollagen Type I C-peptide (PIP) enzyme immunoassay; Takara Biomedicals, Cambrex, Belgium] showed an increased secretion of procollagen Type I in conditioned medium produced by RE subepithelial myofibroblasts vs normal subepithelial myofibroblasts.

View larger version (22K): [in this window] [in a new window]   Figure 2. Overview of Rho signalling. Rho proteins are activated by cell adhesion, mechanical stress and various extracellular signals including reactive oxygen species(ROS), phospholipids (LPA, lysophosphatidic acid; S1P, sphingosin-1 phosphate), cytokine and growth factors, many of which signal through the G protein-coupled receptor (GPCR). These receptors activate Rho through guanine nucleotide exchange factor (GEF) protein, which catalyses the exchange of guanosine diphosphate (GDP) for guanosine triphosphate (GTP). The receptors may also activate two negative regulators of Rho activity: GTPase activation protein (GAP) and GDP dissociation inhibitor (GDI). Active GTP-bound Rho form signals through downstream effectors, including citron kinase, mDia, ROCK (Rho-associated kinase), PKN (protein kinase N-related kinase) and rhotekin, to regulate various cell functions including stress fibres and focal adhesion, migration, cell cycle, survival and transcriptional regulation.

View larger version (20K): [in this window] [in a new window]   Figure 3. Gene array analysis reveals common alterations in the expression profile of genes coding for the Rho pathway in smooth muscle cells and subepithelial myofibroblasts derived from radiation enteropathy. Differences of more than twofold are considered significant(red line). Names of the genes are followed by the accession number.

View larger version (39K): [in this window] [in a new window]   Figure 4. (a,b) Subepithelial myofibroblasts derived from normal ileum and from radiation enteropathy were incubated with Y-27632 for 24 h (10, 100 µM). Subsequent fluorescein isothiocyanate (FITC) phalloidin staining showed alteration of the actin stress fibre network. (c,d) Rho kinase inhibition decreased connective tissue growth factor (CTGF) and COL1A1 (collagen Type 1 alpha 1) mRNA level, as assessed by real-time reverse transcriptase polymerase chain reaction (RT-PCR). Values are normalized to the level of 18S RNA.

View larger version (18K): [in this window] [in a new window]   Figure 5. Rho signalling in radiation-induced intestinal toxicity. (a) The Rho/ROCK pathway controls the molecular signals involved in the development and maintenance of fibrosis including differentiation of smooth muscle cells and subepithelial myofibroblasts, connective tissue growth factor (CTGF) expression and extracellular matrix (ECM) synthesis. (b) In addition, the Rho/ROCK pathway controls acute radiation-induced pathogenic events including inflammation and vascular damage that might contribute to the initiation of late intestinal toxicity. Rho/ROCK pathway pharmacological inhibitors are available. By inhibiting HMG-CoA reductase, the statins inhibit cholesterol synthesis and prevent the formation of isoprenoid intermediates, thus inhibiting Rho translocation to the membrane and subsequent activation. Statins simultaneously inhibit pathways mediated by other G proteins (Ras, Rac). In contrast, ROCK inhibitors (Y-27632) selectively inhibit ROCK activity in a competitive manner with adenosine triphosphate (ATP).