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

Abstract

Connective tissue growth factor (CCN2/CTGF) mediates transforming growth factor-β (TGF-β)-induced fibrosis. Drug-induced gingival overgrowth is tissue specific. Here the role of the phosphoinositol 3-kinase (PI3K) pathway in mediating TGF-β1-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-β1-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-δ) do not mediate TGF-β1-stimulated CCN2/CTGF expression in gingival fibroblasts. As glycogen synthase kinase-3β (GSK-3β) can undergo phosphorylation by the PI3K/pathway, the effects of GSK-3β inhibitor kenpaullone and siRNA knockdown were investigated. Data in gingival fibroblasts indicate that kenpaullone attenuates TGF-β1-mediated CCN2/CTGF expression. Activation of the Wnt canonical pathways with Wnt3a, which inhibits GSK-3β, similarly inhibits TGF-β1-stimulated CCN2/CTGF expression. In contrast, inhibition of GSK-3β by Wnt3a does not inhibit, but modestly stimulates, CCN2/CTGF levels in primary human adult lung fibroblasts and is β-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-3β attenuates CCN2/CTGF expression. In adult lung fibroblasts inhibition of GSK-3β modestly stimulates TGF-β1-regulated CCN2/CTGF expression. These studies have potential clinical relevance to the tissue specificity of drug-induced gingival overgrowth.

Molecular and Clinical Aspects of Drug-induced Gingival Overgrowth

“…3A). Therefore, inhibition of GSK-3β would attenuate CCN2 expression as was observed (Bahammam et al 2013). By contrast, lung fibroblasts would be expected to have GSK3β.…”

“…2 for a summary). Moreover, an unexpected inhibition of TGF-β1-stimulated CCN2 levels by the canonical WNT signaling pathway, and by inhibition of glycogen synthase kinase 3-beta (GSK3β) activity, was uncovered, which is opposite that which occurs in other biological systems (Bahammam et al 2013). Relative levels of PKA and GSK-3β expression have been postulated to differ between gingival fibroblasts and lung cells based on sequential and repressing and activating phosphorylations of CREB by PKA and GSK-3β, respectively.…”

“…The observed low cAMP response of gingival fibroblasts to PGE 2 is consistent with this hypothesis because PKA mediates many cellular responses to cAMP (Black et al 2007). A low level of PKA-dependent phosphorylation of CREB at Ser133 in gingival fibroblasts is predicted to repress CCN2 expression because non-phosphorylated CREB and initially phosphorylated CREB on Ser133 would fail to activate and repress, respectively, CCN2 expression (Bahammam et al 2013). A subsequent GSK-3β-dependent phosphorylation of PSer133-CREB on Ser129 would relieve this repression (see Fig.…”

“…By contrast, lung fibroblasts would be expected to have GSK3β. The proposed model may account for the unique sensitivity of (A) human gingival fibroblast expression of TGF-β-stimulated CCN2 to GSK3β inhibitors compared with (B) lung fibroblasts (Bahammam et al 2013). …”

Targeted inhibition of disheveled PDZ domain via NSC668036 depresses fibrotic process

“…Upon activation, β-catenin and Lef1/Tcf, which are downstream components of the Wnt signaling cascade, form a complex with Smad4, an essential mediator of signals initiated by TGF-β, then translocate to the nucleus to participate in transcription [23]. An active glycogen synthase kinase-3β, the important component of Wnt signaling, is requisite in TGF-β1 upregulation of connective tissue growth factor levels [24]. However, the effect of TGF-β on myofibroblast differentiation after inhibition of Wnt signaling remains unclear.…”