FAK mediates HLA-II-induced signaling in gingival fibroblasts

Objectives: We previously reported that HLA-II molecules on gingival fibroblast (GF) do not present antigens, but transduce signals into the cells by making complex with antigenic peptide-T-cell receptor, resulting in the expression of several cytokines such as IL-6, MCP-1, and RANTES (Ohyama H et al., Cytokine, 2002). However, HLA-II-induced signaling cascades are not fully understood yet. We recently reported that stimulation of HLA-II on gingival fibroblasts by anti-HLA-II antibody resulted in the phosphorylation of focal adhesion kinase (FAK) (Yoshizawa S et al., 53rd JADR, 2005). Thus, in this study, we aimed to see if 1) FAK structurally associates with HLA-II molecules, and, if so, 2) FAK mediates HLA-II-induced signals into the cells. Methods: Human GF was obtained from periodontally healthy donors during the extraction of impacted third molar. Recombinant human interferon-γ was used to up-regulate HLA-II expression on cell surface. To see if FAK directly associates with HLA-II molecules, the cell lysates were immunoprecipiteted with anti-HLA-DR antibodies (L243, Leinco), and FAK was detected by immunoblotting. To see FAK is phosphorylated upon stimulation with anti-HLA-II molecules, the cells were first stimulated with L243, and then immunoprecipitataed. FAK and phosphorylated form of FAK were detected by immunoblotting. To see FAK actually mediates HLA-II-induced signals, the cells were stimulated with L243 in the presence or absence of luteolin (Extrasynthese), an inhibitor for FAK, and the cytokine production into culture supernatants was examined by ELISA. Results: FAK was co-immunoprecipitated with HLA-II molecules by L243. Stimulation of GF by anti-HLA-II antibody resulted in the enhanced phosphorylation of FAK. Luteolin suppressed the phosphorylation of FAK, and inhibited the secretion of IL-6, MCP-1 and RANTES in a dose dependent manner in GF when stimulated with anti-HLA-II antibody. Conclusions: The results suggested that FAK directly associated with HLA-II molecules, and actually mediated HLA-II-induced signals in GF.