Induction of CTGF by TGF-beta1 in normal and radiation enteritis human smooth muscle cells: Smad/Rho balance and therapeutic perspectives.
OBJECTIVE
Transforming Growth Factor beta1 (TGF-beta1) and its downstream effector Connective Tissue Growth Factor (CTGF/CCN2), are well known fibrogenic activators and we previously showed that the Rho/ROCK pathway controls CTGF expression in intestinal smooth muscle cells isolated from patients with delayed radiation enteritis. The aim of the present work was to investigate the balance between Smad and Rho signalling pathways in the TGF-beta1 CTGF induction and modulation of radiation-induced fibrogenic differentiation after addition of pravastatin, an inhibitor of Rho isoprenylation.


METHODS
Primary human smooth muscle cells isolated from normal (N-SMC) or radiation enteritis (RE-SMC) biopsies were incubated with TGF-beta1 (10 ng/ml). Induction of CTGF, as well as nucleo-cytoplasmic distribution of phospho-Smad2/3, Smad2/3 and Smad4 were analysed by Western blot and immunocytochemistry. Smad DNA binding was assessed by EMSA and Rho activation was measured by pull-down assay.


RESULTS
After TGF-beta1 addition, Smads were translocated to the nucleus in both cell types. Nuclear accumulation of Smad as well as their DNA-binding activity were higher in N-SMC than in RE-SMC, whereas the opposite was observed for Rho activation, suggesting a main involvement of Rho pathway in sustained fibrogenic differentiation. This hypothesis was further supported by the antifibrotic effect observed in vitro after cell treatment with pravastatin (i.e. decreased expression of CTGF, TGF-beta1 and Collagen Ialpha2).


CONCLUSIONS
Our results suggest that TGF-beta1-induced CTGF transactivation mainly depends on the Smad pathway in N-SMC, whereas in RE-SMC, Smad and Rho pathways are involved. Inhibition of Rho activity by pravastatin alters fibrogenic differentiation in vitro which opens up new therapeutic perspectives.
Haydont
Valérie
V
UPRES EA 27-10 'Radiosensibilité des tumeurs et tissus sains', Institut Gustave Roussy/Institut de Radioprotection et de Sûreté Nucléaire, Villejuif, France.
Mathé
Denis
D
Bourgier
Céline
C
Abdelali
Jalil
J
Aigueperse
Jocelyne
J
Bourhis
Jean
J
Vozenin-Brotons
Marie-Catherine
MC
D002478:Cells, Cultured; D055513:Connective Tissue Growth Factor; D004751:Enteritis / Q000209:etiology / Q000378:metabolism* / Q000473:pathology; D006801:Humans; D017874:Immediate-Early Proteins / Q000378:metabolism*; D036341:Intercellular Signaling Peptides and Proteins / Q000378:metabolism*; D007421:Intestine, Small / Q000166:cytology; D047908:Intracellular Signaling Peptides and Proteins; D032389:Myocytes, Smooth Muscle / Q000378:metabolism*; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism*; D011832:Radiation Injuries / Q000378:metabolism*; D015398:Signal Transduction; D051785:Smad Proteins / Q000378:metabolism*; D027981:Symporters; D016212:Transforming Growth Factor beta / Q000494:pharmacology*; D053773:Transforming Growth Factor beta1; D054460:rho-Associated Kinases
C526317:CCN2 protein, human; C526316:CCN2 protein, rat; D017874:Immediate-Early Proteins; D036341:Intercellular Signaling Peptides and Proteins; D047908:Intracellular Signaling Peptides and Proteins; C089982:Slc6a18 protein, rat; D051785:Smad Proteins; D027981:Symporters; C508960:TGFB1 protein, human; C508962:Tgfb1 protein, rat; D016212:Transforming Growth Factor beta; D053773:Transforming Growth Factor beta1; D055513:Connective Tissue Growth Factor; D017346:Protein Serine-Threonine Kinases; D054460:rho-Associated Kinases