Signal transduction pathways mediating the expression of connective tissue growth factor by TGF[beta]1 in human gingival fibroblasts: Regulation by prostaglandin-E2

Introduction. Connective tissue growth factor (CCN2/CTGF) is expressed by human fibroblasts in response to TGF01 and stimulates the synthesis of the extracellular matrix (ECM). The over-expression of ECM has been implicated in the onset and progression of fibrosis in multiple tissues including human gingiva in patients taking the anticonvulsive drug phenytoin. Phenytoin-induced gingival overgrowth tissues are more fibrotic and less cellular than other forms of drug-induced gingival overgrowth suggesting that the accumulation of ECM constituents is the primary etiology of this form of drug-induced gingival enlargement. Methods. Primary fibroblasts cells from healthy human donors obtained from gingiva, renal mesangium and skin. Primary fibroblasts and IMR90 fibroblasts (human lung) were cultured for comparative analyses on the regulation of CCN2/CTGF expression. Real-time PCR was conducted to determine mRNA expression of specific genes. Western blotting was used to assess changes in protein levels. ELISA was used to determine cAMP accumulation. Results. PGE2 blocks CCN2/CTGF expression by stimulating an increase in cAMP and the subsequent inhibition of multiple MAPK pathways in a tissue-specific manner. Gingival fibroblasts are unique in that these cells resist the PGE2-mediated inhibition on the expression of CCN2/CTGF since only JNK-MAPK activation is required for CCN2/CTGF expression and the net effect of PGE2 is to stimulate JNK activation. Rho-GTPases Cdc42 and Rac1, but not RhoA, play a central role in promoting the TGFR1-stimulated expression of CCN2/CTGF in primary human gingival fibroblasts. Gingival fibroblast pretreated with Lovastatin or PDE-IV inhibitor in combination with forskolin significantly reduced the TGFR1-induced expression of CCN2/CTGF by >80% and >90%, respectively. The combination of Lovastatin, PDE-IV inhibitor and forskolin reduced CCN2/CTGF expression by more than 99%.;Conclusions. Gingival fibroblasts are unique among fibroblastic cell types in their signaling requirements and resistance to the inhibitory effects of PGE2 on CCN2/CTGF expression. The combination of forskolin with either Lovastatin or PDE-IV inhibitors dramatically reduces CCN2/CTGF expression stimulated by TGFbeta1. Local clinical application of a cocktail composed of lovastatin, forskolin and PDE-IV-specific inhibitor as this combination resulted in the near complete elimination of the TGFR1-induced expression of CCN2/CTGF in cultured gingival fibroblasts.