Transforming Growth Factor-β1 (TGFβ1) Stimulates Connective Tissue Growth Factor (CCN2/CTGF) Expression in Human Gingival Fibroblasts through a RhoA-independent, Rac1/Cdc42-dependent Mechanism

Abstract

Regulation of connective tissue growth factor (CCN2/CTGF) in gingival fibroblasts is unique and may provide therapeutic opportunities to treat oral fibrotic diseases. RhoA was previously implicated in mediating the expression of CCN2/CTGF. We now present evidence that Rho family GTPases Rac1 and Cdc42 are the principal mediators of the transforming growth factor-␤1 (TGF␤1)-stimulated expression of CCN2/CTGF in primary human gingival fibroblasts. TGF␤1 does not stimulate RhoA activation in gingival fibroblasts, and the overexpression of dominant-negative RhoA does not reduce CCN2/CTGF expression in response to TGF␤1. In contrast, the overexpression of dominant-negative forms of Cdc42 or Rac1 results in a dramatic reduction of CCN2/CTGF protein levels. Lovastatin and a geranylgeranyltransferase inhibitor reduce the TGF␤1-stimulated levels of CCN2/CTGF protein by ϳ75 and 100%, respectively. We previously demonstrated that JNK1 phosphorylation by TGF␤1 is also critical for TGF␤1-induced CCN2/CTGF expression, and forskolin partially reduces levels of phosphorylated JNK1. Inhibition of geranylgeranyltransferase has no effect on levels of JNK phosphorylation in response to TGF␤1 suggesting RhoGTPases act independently of JNK1. The combination of lovastatin and forskolin results in a greater inhibitory effect than each agent alone and reduces CCN2/CTGF mRNA and protein expression by greater than 90%. This novel combination has additive inhibitory effects on the TGF␤1-stimulated expression of CCN2/ CTGF in human gingival fibroblasts through the simultaneous disruption of Rho-and JNK1-mediated pathways, respectively. This combination of available therapeutic compounds may therefore be useful in designing treatment strategies for oral fibrotic conditions in which gingival CCN2/CTGF is elevated.

Molecular and Clinical Aspects of Drug-induced Gingival Overgrowth

“…As noted above, active RAC1 and CDC42 are uniquely required for TGF-β-stimulated CCN2 levels in gingival fibroblasts [Fig. 2;Black and Trackman (2008)]. Statins are widely prescribed for treating hypercholesterolemia and, as approved drugs, are of particular interest as a potential therapeutic option for addressing gingival overgrowth.…”

“…One example is the combination of lovastatin plus foskolin. Lovastatin inhibits the activation of the small G-proteins RAC1 and CDC42, which contribute to TGF-β-stimulated CCN2 expression (Black and Trackman 2008). In addition, lovastatin has been reported to inhibit GSK-3β activation, which could result in elevated repression of CCN2 as proposed in Figure 3 (Lee et al 2012).…”

“…3). Indeed, data analysis showed that the combination of lovastatin and forskolin inhibits CCN2 levels in an additive manner (Black et al 2007;Black and Trackman 2008). It is clear that additional detailed mechanistic studies are needed to confirm and extend this understanding to utilize these findings to develop new clinical strategies to address gingival overgrowth.…”

“…In addition, the cAMP response to PGE 2 was found to be weak and delayed in gingival fibroblasts (Black et al 2007). Subsequent studies of signaling in gingival and lung cells identified CDC42 and RAC1 but not RHOA as mediators of TGF-β1 up-regulation of CCN2 (Black and Trackman 2008) (see Fig. 2 for a summary).…”

Epithelial to Mesenchymal Transition in Gingival Overgrowth

“…2 Patients who suffer from gingival overgrowth do not have fibrosis elsewhere, indicating that this condition is tissue specific, some features of which are due to unique signal transduction pathways in gingival fibroblasts. 3,4 It is interesting that a classic feature of gingival overgrowth is that epithelial rete pegs extend deep into the connective tissue compared with normal tissues, and all forms of gingival overgrowth lesions contain fibrotic or expanded connective tissues with characteristic levels of inflammation. 5 Moreover, we have observed elevated expressions of CCN2/ CTGF in both epithelial and connective tissue cells in phenytoin-induced gingival overgrowth tissues.…”