Connective tissue growth factor in human gingiva, and studies of lysyl oxidase processing proteinases

Abstract

The first part of our studies is focused on gingival overgrowth. Gingival overgrowth is characterized by excess extracellular matrix (ECM) accumulation. TGF-J31 regulation of connective tissue growth factor (CTGF) was assessed for the first time in human gingival cells and tissues. CTGF protein is strongly induced by TGF-J31 in human gingival fibroblasts. Exogenous addition of CTGF to gingival fibroblasts stimulates production of lysyl oxidase enzyme activity up to 1.5-fold after 48 hours, and 50 ng/ml CTGF stimulated insoluble collagen accumulation by only 1.5 to 2.0 fold after 4 to18 days of treatment. Thus, although CTGF itself contributes to increased insoluble collagenous extracellular matrix accumulation, CTGF does not mediate more potent effects of TGF-J31. Gingival overgrowth samples obtained from patients undergoing therapy with phenytoin, nifedipine, and cyclosporin A and control tissues from systemically healthy donors were subjected to immunohistochemistry by staining with CTGF, and TGF-J3 l antibodies. The results indicate for the first time increased levels of CTGF protein in overgrown gingival tissues, particularly in phenytoin induced gingival overgrowth. The second part of our studies is focused on the Bmpl-related genes. The Bmpl gene encodes bone morphogenetic protein 1 (BMP-1) and mammalian Tolloid-like (mTLD), both of which have procollagen C-proteinase activity. Two BMP-1/mTLD­related protease genes named mammalian Tolloid-like 1 and 2 (Till and Tll2) have recently been described. The present study determines and compares the abilities of recombinant BMP-1, mTLD, mTLL-1, and mTLL-2 to process a recombinant pro-lysyl oxidase fusion protein in vitro. Results suggest that recombinant BMP-1, mTLD, and mTLL-1 productively cleave pro-lysyl oxidase at the correct physiological site in vitro, and that BMP-1 itself appears to be the most efficient lysyl oxidase processing activity while mTLL-2 was shown to process pro-lysyl oxidase slowly in vitro. These studies suggest that pro-lysyl oxidase processing may depend principally on the presence of Bmpl gene products, and that mTLL-1, and mTLL-2 may contribute to pro-lysyl oxidase processing.