Mutations in the Heparin Binding Domain of Fibronectin in Cooperation with the V Region Induce Decreases in pp125 Levels Plus Proteoglycan-mediated Apoptosis via Caspases

Abstract

Intact fibronectin (FN) protects cells from apoptosis.When FN is fragmented, specific domains induce proteinase expression in fibroblasts. However, it is not known whether specific domains of FN can also regulate apoptosis. We exposed fibroblasts to four recombinant FN fragments and then assayed for apoptosis using criteria of cellular shape change, condensed nuclear morphology, and DNA fragmentation. The fragments extended from the RGD-containing repeat III10 to III15; they included (V FAK levels and regulate apoptosis through a chondroitin sulfate proteoglycan-and possibly ␣4 integrin-mediated pathway, which triggers a caspase cascade. The extracellular matrix molecule fibronectin (FN)1 is composed of several domains that mediate multiple cell functions through cell surface integrin and proteoglycan receptors. When isolated, specific domains of FN display activities not exhibited by the intact molecule. For example, the central cell binding domain of FN (FN 120) induces rabbit synovial fibroblasts (1) and human fibroblasts (2) to express elevated levels of matrix metalloproteinases, whereas fragments from the amino-terminal and gelatin binding domains induce chondrolysis in vitro, the latter effect presumably through matrix metalloproteinase and serine proteinase induction (3, 4). These FN fragments are also associated with chronic inflammatory states in vivo, since high levels of such fragments have been found in synovial fluids from arthritic patients (5-7) and in gingival crevicular fluid from patients with periodontitis (8, 9).Another function recently attributed to FN is protection against programmed cell death, or apoptosis. Apoptosis is generally characterized by cell rounding, nuclear condensation, and DNA fragmentation and by signaling pathways that activate a cascade of cell death proteases. Intact FN as a substrate for cell adhesion can rescue cells from apoptosis, although it is not known whether specific domains of FN regulate this function. The mechanism underlying the protective effect of FN seems to involve integrin-mediated signaling and activation of the focal adhesion kinase (pp125 FAK ) (10 -13) and/or activation of the Bcl-2 cell survival pathway, which is independent of pp125 FAK (14). This difference in utilization of pp125 FAK as the signaling molecule may depend on cell type.We tested the hypothesis that specific domains of FN are important in protecting fibroblasts from undergoing apoptosis. Specifically, we tested the roles of the high affinity heparinbinding domain and the alternatively spliced V region of FN in this process. EXPERIMENTAL PROCEDURESFibroblast Cell Culture-Primary cultures of human periodontal ligament fibroblasts were routinely obtained from human teeth extracted from patients undergoing therapeutic removal of third molars or orthodontic treatment. To obtain cells, the periodontal ligament (PDL) was scraped from the midroot section of extracted teeth with a scalpel blade using standard protocols (2). The tissue was placed under a coverslip and kept in culture medium...

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