The Protein Tyrosine Phosphatase SHP-2 Regulates Interleukin-1-induced ERK Activation in Fibroblasts

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...

SHP-2 Modulates Interleukin-1-induced Ca2+ Flux and ERK Activation via Phosphorylation of Phospholipase Cγ1

“…All media contained 10% fetal bovine serum and antibiotics (0.17% penicillin V, 0.1% gentamicin sulfate, and 0.01% amphotericin). Cells were used between the 5th and 12th passages as described previously (20). Murine embryonic fibroblasts (SHP-2 (⌬46 -110) and the same cells reconstituted with wild-type murine SHP-2) were generated and grown in high glucose Dulbecco's modified Eagle's medium as described previously (21).…”

“…2ϩ Release, and ERK Activation-A general role for protein tyrosine phosphorylation in the regulation of Ca 2ϩ fluxes has been reported (27,28), and we demonstrated earlier that the protein tyrosine phosphatase SHP-2 is enriched in the focal adhesions of fibroblasts and can modulate IL-1-induced ERK activation (20). Although several protein tyrosine phosphatases, including protein tyrosine phosphatase-␣ and low molecular weight protein tyrosine phosphatase, are known to associate with focal adhesions (20, 29 -31), we focused on SHP-2, a non-receptor type of protein tyrosine phosphatase that has been implicated in the regulation of cytokine, growth factor, and integrin signaling (15,20).…”

Identification of CD245 as myosin 18A, a receptor for surfactant A: A novel pathway for activating human NK lymphocytes

“…27 Of note, SHP-2 has previously been shown to negatively regulate NK cell function, 31 although in other settings, it mediated a positive proliferation signal 34 and activated the IL-1/Erk-mediated cytoskeleton reorganization. 35 Our novel molecular and functional data on a newly described NK cell co-activating receptor have broad potential applications. The unconventional myosin 18A is a member of the myosin superfamily of motor enzymes.…”

Regulation of smooth muscle by inducible nitric oxide synthase and NADPH oxidase in vascular proliferative diseases

“…This study confirmed an earlier report in gingival fibroblasts which also provided evidence supporting a requirement for PLCγ in IL-1β-dependent activation of ERK1/2 [158]. Not only is there evidence for the involvement of PLCγ in IL-1β-dependent activation of ERK1/2 in gingival fibroblasts, a series of studies by McCulloch et al also report the involvement of the traditional mediators of IL-1β signaling such as IRAK along with the non-receptor tyrosine kinase focal adhesion kinase (FAK) [104] [108] and the tyrosine phosphatase SHP2 [107]. While little is known concerning the IL-1 receptor and its adaptor molecules in VSM cells, it is tempting to speculate that the mechanism by which IL-1β stimulation results in a PLCγ-and PKCδ-dependent activation of ERK1/2 and subsequent increases in iNOS expression in VSM cells is similar.…”