Lysyl oxidase regulation, gingival overgrowth, and collagen accumulation in osteoblastic cultures

PLEASE NOTE: This work is protected by copyright. Downloading is restricted to the BU community: please click Download and log in with a valid BU account to access. If you are the author of this work and would like to make it publicly available, please contact open-help@bu.edu.Thesis (D.Sc.D.)--Boston University, Henry M. Goldman School of Dental Medicine, 1997 (Periodontology).Includes bibliographical references (leaves 69-91).Lysyl oxidase catalyzes the enzymatic step that is critical for collagen and elastin cross-linking. The relationship between drug-induced gingival overgrowth and lysyl oxidase is not understood. The fibrogenic drugs, cyclosporine-A (CsA), phenytoin (PHT) and nifedipine may directly stimulate lysyl oxidase production and cause increased insoluble collagen accumulation. Alternatively, altered cytokine levels caused by these drugs may regulate extracellular matrix production. In addition, the temporal regulation of lysyl oxidase in mineralizing osteoblastic cultures has not been previously analyzed. Therefore, the goals of this research were to determine the direct effects of CsA on lysyl oxidase and collagen biosynthesis in cultured human gingival fibroblasts, to determine the effects of selected cytokines on lysyl oxidase and collagen biosynthesis in these cultured human fibroblasts, to determine by immunohistochemistry lysyl oxidase levels in tissue samples from patients suffering drug-induced gingival overgrowth, and to determine the temporal relationship between the regulation of lysyl oxidase and insoluble collagen accumulation in developing murine MC3T3-EI osteoblastic cells. In normal human gingival fibroblast cultures, 100 ng/ml CsA treatment did not consistently increase collagen, elastin or lysyl oxidase mRNA levels or lysyl oxidase activity after up to 48 hours of exposure. However, when these cultures were treated instead with 4 to 400 pM TGF-[Beta]1 for 4, 24 and 48 hours, 400 pM TGF-[Beta]1 maximally stimulated lysyl oxidase enzyme activity, lysyl oxidase and [alpha]-1-type 1 collagen mRNA levels at 48 hours. Lysyl oxidase enzyme activity was increased 2-3 fold, lysyl oxidase mRNA levels were increased by 1.9 fold, and [alpha]-1-type 1 collagen mRNA levels were increased by 3.8 fold after 48 hours of treatment. TGF-[Beta] 1 has been shown to be increased in gingival tissues from drug-induced gingival overgrowth patients, therefore increased lysyl oxidase and collagen synthesis mediated by TGF-[Beta]1 may be important in the etioIogy of gingival overgrowth. On the other hand, bFGF (0.02 to 5 nM) did not regulate lysyl oxidase enzyme activity. Interestingly, 1 nM bFGF decreased lysyl oxidase and [alpha]-1-type 1 collagen mRNA levels to 30%-50%, and to less than 30% of the control levels after 48 hours of treatment, respectively. IL-1, IL-6, INF-[alpha] and PDGF-BB did not significantly regulate collagen, elastin and lysyl oxidase in human gingival fibroblasts under the conditions tested. Immunohistochemical studies showed significantly increased numbers of lysyl oxidase positive cells in gingival tissues from PHT-treated and nifedipine-treated subjects compared to tissues from control subjects with similar levels of inflammation. Compared to control groups, increased numbers of both inflammatory cells and lysyl oxidase positive cells were found in CsA plus nifedipine-treated subect. These observations suggest inflammation itself may not directly up-regulate lysyl oxidase, though, inflammation may play an important role in the increasing of lysyl oxidase levels in drug-treated subjects. Finally, the results of developing MC3T3-E1 osteoblastic cultures indicated that up-regulation of lysyl oxidase activity in osteoblastic cells coincides with increased collagen accumulation but not with collagen mRNA levels. Lysyl oxidase activity appears to be regulated in proportion to increased lysyl oxidase mRNA levels through the second stage of osteoblast phenotype development, and post-translationally in the third stage