Serine proteinase dependent collagen degradation

Temporomandibular joint disorders (TMD) are estimated to affect 10 million Americans every year. About 8% of TMD population undergoes destructive changes in the temporomandibular joint (TMJ), which is primarily composed of Type I collagen.;The purpose of the first part of this dissertation was to examine the ability of diseased human TMJ synovial fibroblasts to degrade Type I collagen and their expression metalloproteinases (MMPs) and the tissue inhibitors of MMPs (TIMPs), which regulate collagen cleavage. Reverse transcriptase-PCR indicated that these TMJ fibroblasts expressed mRNA for multiple MMPs and TIMPs. The collagen degradation assays showed that these fibroblasts were capable of digesting the collagen underneath there on collagen-coated plates at passages 3 to 8 and digesting all the collagen in the wells at passages 8 to 13. Along with the changes in their ability to degrade collagen, the inhibition of collagen cleavage was altered from complete inhibition by a MMP inhibitor to complete inhibition by a combination of a MMP inhibitor and a serine proteinase inhibitor. The data suggest that these TMJ cells utilize MMP dependent and MMP independent pathways to degrade Type I collagen.;The MMP independent pathway was further characterized by incubating the conditioned media from the cell-mediated collagen degradation assays with Type I collagen at pH 7.5 with or without different inhibitors. The MMP inhibitor or the serine proteinase inhibitors partially inhibited the collagen cleavage. The cysteine proteinase inhibitors had no effect. The combination of a MMP inhibitor and a serine proteinase inhibitor was the most effective in inhibiting the collagen cleavage. Zymography identified a proteinase at approximate 22.5 kDa in the conditioned media from cells at passage 8 to 13, which was effectively inhibited by the serine proteinase inhibitors. Reverse transcript-PCR and real-time PCR results demonstrated that these TMJ cells did not express trypsinogen-2 mRNA, a serine proteinases capable of cleaving Type I collagen.;These studies demonstrated that these TMJ fibroblasts utilize both MMP dependent and independent pathways to digest Type I collagen. The MMP independent pathway is the principal pathway utilized by these TMJ fibroblasts and involves a serine proteinase, yet to be identified.