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.
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.


OBJECTIVE
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.


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.


CONCLUSIONS
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.
Bourgier
C
C
Laboratoire UPRES EA 27-10, "Radiosensibilité des tumeurs et tissus sains", PR1, 39, Rue Camille Desmoulins, 94805 Villejuif Cedex, France.
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
D000199:Actins / Q000378:metabolism; D000328:Adult; D000368:Aged; D000369:Aged, 80 and over; D000577:Amides / Q000494:pharmacology; D002454:Cell Differentiation; D002478:Cells, Cultured; D055513:Connective Tissue Growth Factor; D003599:Cytoskeleton / Q000378:metabolism / Q000528:radiation effects; D004268:DNA-Binding Proteins / Q000378:metabolism; D004751:Enteritis / Q000201:enzymology* / Q000209:etiology / Q000473:pathology; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005260:Female; D005355:Fibrosis / Q000209:etiology / Q000473:pathology; D020869:Gene Expression Profiling / Q000379:methods; D006801:Humans; D007082:Ileum / Q000473:pathology; D017874:Immediate-Early Proteins / Q000378:metabolism*; D036341:Intercellular Signaling Peptides and Proteins / Q000378:metabolism*; D047908:Intracellular Signaling Peptides and Proteins; D008297:Male; D008875:Middle Aged; D009130:Muscle, Smooth / Q000378:metabolism / Q000473:pathology / Q000528:radiation effects*; D016328:NF-kappa B / Q000494:pharmacology; D017346:Protein Serine-Threonine Kinases / Q000037:antagonists & inhibitors* / Q000502:physiology; D011725:Pyridines / Q000494:pharmacology; D011832:Radiation Injuries / Q000201:enzymology* / Q000209:etiology / Q000473:pathology; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods; D015398:Signal Transduction; D020741:rho GTP-Binding Proteins / Q000502:physiology; D054460:rho-Associated Kinases
D000199:Actins; D000577:Amides; C526317:CCN2 protein, human; D004268:DNA-Binding Proteins; D004791:Enzyme Inhibitors; D017874:Immediate-Early Proteins; D036341:Intercellular Signaling Peptides and Proteins; D047908:Intracellular Signaling Peptides and Proteins; D016328:NF-kappa B; D011725:Pyridines; C108830:Y 27632; D055513:Connective Tissue Growth Factor; D017346:Protein Serine-Threonine Kinases; D054460:rho-Associated Kinases; D020741:rho GTP-Binding Proteins