Calmodulin-Kinase-II: A Key Regulator of CTGF/CCN2 Expression in Gingival Fibroblasts

Introduction: Connective Tissue Growth Factor (CTGF/CCN2) expression is induced by TGFbeta in fibroblastic cells and independently stimulates the production and/or deposition of extracellular matrix components. CTGF/CCN2 is found to be overexpressed in most fibrotic conditions, including phenytoin-induced gingival overgrowth. Cyclosporine-A also induces gingival overgrowth and specifically inhibits calcineurin (protein-phosphatase-2B/PP2B) activity. The inhibition of calcineurin inactivates the transcription factor NFAT. Since the CTGF/CCN2 promoter contains a putative NFAT-binding sequence, the inhibition of calcineurin and NFAT by cyclosporine-A may contribute to the downregulation of the TGFbeta-stimulated expression of CTGF/CCN2 in gingival fibroblasts. Objective: Identify the role(s) of CaMKII-mediated signaling that regulates the TGFbeta-induced expression of CTGF/CCN2 and calcineurin activity in primary human gingival fibroblasts. Methods: Primary human gingival fibroblast cultures pretreated with specific peptide or pharmacologic inhibitors of CaMK-II, or with cyclosporine-A, prior to stimulation with 5ng/ml TGFbeta. Cell layer protein extracts were analyzed by Western blot. The phosphorylation of CaMK-II and CTGF/CCN2 protein levels were analyzed by Western blot and calcineurin activity was assessed using a free-phosphate assay. Results: TGFbeta-induced CTGF/CCN2 expression is reduced 75% in the presence of CaMKII inhibition and 50% by cyclosporine-A. The inhibition of CaMK-II results in a significant reduction in calcineurin activity suggesting that CaMK-II activity is required for calcineurin/NFAT activation. Conclusions: CaMKII activity is required for calcineurin activity in gingival fibroblasts. The reduction in TGFbeta-stimulated CTGF/CCN2 expression in response to cyclosporine-A is due, in part, to interference with calcineurin activity and implicates NFAT as a critical regulator of CTGF/CCN2 expression. The greater reduction of CTGF/CCN2 in response to the inhibition of CaMKII versus calcineurin suggests that CaMKII also mediates other pathways required for the TGFbeta-stimulated expression of CTGF/CCN2 in human gingival fibroblasts. Determining the signaling mechanisms of CaMKII on regulating CTGF/CCN2 expression may lead to novel methods for treating gingival overgrowth. K08-DE16609, RO1-DE11004, MO1-RR00533.