Impaired Degradation of Matrix Collagen in Human Gingival Fibroblasts by the Antiepileptic Drug Phenytoin

Abstract

Gingival overgrowth (GO) is a serious adverse effect associated with the administration of phenytoin (PHT), with PHT-induced GO characterized by a massive accumulation of extracellular matrix components, especially collagen, in gingival connective tissues. However, the etiology of such collagen accumulation is still largely unknown. We examined the effects of PHT on the collagen degradation process leading to collagen accumulation in human gingival fibroblasts (HGF). HGFs were cultured with various concentrations of PHT and viable cell numbers and collagen amounts were determined. Gene and protein expressions of matrix metalloproteinases (MMP) and tissue inhibitors of MMPs (TIMP) were quantified with reverse transcription-polymerase chain reaction (RT-PCR) analyses and Western blotting, respectively. Cellular endocytosis of collagen was assayed using flow-cytometric analysis. The effects of PHT on extracellular signal-regulated kinase 1/2 (ERK1/2) and inhibitor kappaB-alpha (IkappaB-alpha) were assayed. The proliferation of HGFs was not affected by PHT, whereas it significantly increased collagen accumulation. Further, the expressions of MMP-1, -2, and -3 were markedly suppressed by PHT, whereas that of TIMP-1 was induced in a dose- and time-dependent manner. PHT also markedly prevented collagen endocytosis by HGFs, which was associated with the suppression of alpha2beta1-integrin expression. In addition, the phosphorylation of ERK1/2 and IkappaB-alpha degradation were suppressed by PHT. These results suggest that PHT causes an impaired degradation of collagen by suppression of enzymatic degradation with MMPs/TIMP-1 and alpha2beta1-integrin-mediated endocytosis. Those alterations are likely mediated through the cellular signaling pathways of ERK1/2 and nuclear factor kappaB. These synergistic effects may cause collagen accumulation, leading to GO.