July 30-August 1, 2000
6th International Workshop on Scleroderma Research, Oxford, UK.

Control of CTGF Expression by Fibroblasts: Role of IFNgamma, TNF-alpha and the Proteasome. A Leask, X Shi-wen, S Sa, Y Xu, GR Martin, AM Holmes, DJ Abraham and CM Black.
Centre for Rheumatology, Royal Free and University College Medical School, Rowland Hill Street, London NW3 2PF, U.K. FibroGen Inc. 255 Gateway Blvd, South San Francisco, CA, U.S.A.

Objective: In scleroderma connective tissue fibrosis results in the disruption of the normal architecture of affected organs and ultimately leads to their dysfunction and failure. The TGFbeta/Smad pathway and the activation of CTGF are key effector pathways likely to be responsible for the persistent activation of the genes encoding extracellular matrix proteins such as type I collagen, that underlie the fibrotic process. Understanding of the mode of action of these growth factors in particular the pathways they activate will allow the development of modulatory therapeutic strategies. We have studied the influence of the anti-fibrotic cytokines TNF-alpha and IFNgamma and inhibition of 26S proteasome function on TGFbeta induction of CTGF and type I collagen.

Methods: We have used primary dermal fibroblast cell cultures from normal individuals, ELISA, western blotting and transient transfection using reporter gene constructs to examine the influence of TNF-alpha and IFNgamma on the induction of fibroblast CTGF and type I collagen by TGFbeta, and the effect of inhibiting 26S proteasome on matrix gene expression.

Results: We found that TNF-alpha potently repressed TGFbeta induction of CTGF and type I collagen expression. A role for the transcription factor NFkappaB in this down-regulation was suggested by the ability of an inhibitor of the NFkappaB pathway to block the effects of TNF-alpha, and this was further supported by the ability of a dominant-negative mutant of IkappaBalpha to blocked the inhibition by TNF-alpha. The presence of IFNgamma a known inducer of the inhibitory Smad, Smad7, was also shown to cause marked down-regulation of TGFbeta-dependent type I collagen expression. Furthermore, we showed that addition of two inhibitors of the 26S proteasome, MG-132 or lactacystin, which inhibit degradation of ubiquitinated proteins prevented TNF-alpha inhibition of TGFbeta-dependent CTGF and type I collagen expression.

Conclusion: TNF-alpha appears to be able to regulate CTGF and type I collagen expression via the NFkappaB pathway, whereas IFNgamma may modulate TGFbeta-dependent type I collagen expression via the induction of Smad7. In addition, the ubiquitin-dependent degradation of signalling molecules relevant to the TGFbeta/Smad and the NFkappaB pathway is also likely to be key regulatory events in controlling the expression of pro-fibrotic growth factors and matrix production.