No abstract

Focal adhesion complexes are actin-rich, cytoskeletal structures that mediate cell adhesion to the substratum and also selectively regulate signal transduction pathways required for interleukin (IL)-1␤ signaling to the MAP kinase, ERK. IL-1-induced ERK activation is markedly diminished in fibroblasts deprived of focal adhesions whereas activation of p38 and JNK is unaffected. While IL-1 signaling is known to involve the activity of protein and lipid kinases including MAP kinases, FAK, and PI3K, little is known about the role of phosphatases in the regulation of IL-1 signal generation and attenuation. Here we demonstrate that SHP-2, a protein tyrosine phosphatase present in focal adhesions, modulates IL-1-induced ERK activation and the transient actin stress fiber disorganization that occurs following IL-1 treatment in human gingival fibroblasts. Using a combination of immunoblotting, immunoprecipitation, and immunostaining we show that SHP-2 is present in nascent focal adhesions and undergoes phosphorylation on tyrosine 542 in response to IL-1 stimulation. Blocking anti-SHP-2 antibodies, electoporated into the cytosol of fibroblasts, inhibited IL-1-induced ERK activation, actin filament assembly, and cell contraction, indicating a role for SHP-2 in these processes. In summary, our data indicate that SHP-2, a focal adhesion-associated protein, participates in IL-1-induced ERK activation likely via an adaptor function. Interleukin-1 (IL-1)1 is a potent monocyte/macrophage-derived cytokines (1) capable of stimulating multiple signaling cascades and inducing the expression of a variety of immune and inflammatory factors including c-Fos and c-Jun (2, 3), matrix metalloproteinases (4, 5), nitric-oxide synthetase (6, 7), prostaglandin E (8), and other cytokines (9, 10). Consequently, the unregulated production of IL-1 is associated with significant cellular and tissue damage observed in inflammatory diseases such as rheumatoid arthritis and periodontal diseases (11,12). Following IL-1 binding, the IL-1 receptor-associated protein is recruited to the type I IL-1 receptor thereby increasing the avidity of the receptor for its ligand (13-15). The adapter protein MyD88 then recruits Toll-interacting protein (16) and the IL-1 receptor-associated kinases (17-20), which initiate downstream signaling. Engagement of the IL-1 receptor initiates multiple signaling events implicated in the pathogenesis of chronic inflammatory diseases, including: (i) phosphorylation of multiple kinases and IL-1R 1 -associated proteins (21, 22), (ii) Ca 2ϩ flux (23), (iii) reorganization of the actin cytoskeleton (24), and (iv) activation of three members of the MAPK family, ERK, JNKs/SAPKs, and p38 MAP kinases (25)(26)(27).Gingival fibroblasts and chondrocytes are cells involved in inflammatory disorders that require focal adhesion complex (FAC) formation in vitro for IL-1-induced ERK activation (23,28). FAC are adhesive domains that comprise the termini of actin stress fibers in physical association with a number of actin-binding proteins includi...