Requirement for active glycogen synthase kinase-3β in TGF-β1 upregulation of connective tissue growth factor (CCN2/CTGF) levels in human gingival fibroblasts

Connective tissue growth factor (CCN2/CTGF) mediates transforming growth factor-beta (TGF-beta)-induced fibrosis. Drug-induced gingival overgrowth is tissue specific. Here the role of the phosphoinositol 3-kinase (PI3K) pathway in mediating TGF-beta(1)-stimulated CCN2/CTGF expression in primary human adult gingival fibroblasts and human adult lung fibroblasts was compared. Data indicate that PI3K inhibitors attenuate upregulation of TGF-beta(1)-induced CCN2/CTGF expression in human gingival fibroblasts independent of reducing JNK MAP kinase activation. Pharmacologic inhibitors and small interfering (si)RNA-mediated knockdown studies indicate that calcium-dependent isoforms and an atypical isoform of protein kinase C (PKC-delta) do not mediate TGF-beta(1)-stimulated CCN2/ CTGF expression in gingival fibroblasts. As glycogen synthase kinase-3 beta (GSK-3 beta) can undergo phosphorylation by the PI3K/pathway, the effects of GSK-3 beta inhibitor kenpaullone and siRNA knockdown were investigated. Data in gingival fibroblasts indicate that kenpaullone attenuates TGF-beta(1)-mediated CCN2/CTGF expression. Activation of the Wnt canonical pathways with Wnt3a, which inhibits GSK-3 beta, similarly inhibits TGF-beta(1)-stimulated CCN2/CTGF expression. In contrast, inhibition of GSK-3 beta by Wnt3a does not inhibit, but modestly stimulates, CCN2/CTGF levels in primary human adult lung fibroblasts and is beta-catenin dependent, consistent with previous studies performed in other cell models. These data identify a novel pathway in gingival fibroblasts in which inhibition of GSK-3 beta attenuates CCN2/CTGF expression. In adult lung fibroblasts inhibition of GSK-3 beta modestly stimulates TGF-beta(1)-regulated CCN2/CTGF expression. These studies have potential clinical relevance to the tissue specificity of drug-induced gingival overgrowth.