Epidermal Growth Factor Receptor Transactivation Mediates Substance P-induced Mitogenic Responses in U-373 MG Cells

Abstract

Ligand-induced activation of G protein-coupled receptors is emerging as an important pathway leading to the activation of certain receptors with intrinsic tyrosine kinase activity, such as the epidermal growth factor receptor (EGFR). Substance P (SP) exerts many effects via activation of its G protein-coupled receptor (neurokinin-1, NK-1). SP participates in acute inflammation and activates key proteins involved in mitogenic pathways, such mitogen-activated protein kinases (MAPKs), stimulating DNA synthesis. We tested the hypothesis that SP-induced MAPK activation and DNA synthesis require activation of the EGFR. In U-373 MG cells, which express functional NK-1, SP induced tyrosine phosphorylation of several proteins including EGFR. SP induced formation of an activated EGFR complex containing the adapter proteins SHC and Grb2, but not c-Src. SP activated the MAPK pathway as shown by increased Erk2 kinase activity. SP induced Erk2 activation, and DNA synthesis was inhibited in cells transfected with a dominant negative EGFR plasmid lacking kinase activity, as well as in cells treated with a specific EGFR inhibitor. In addition, pertussis toxin, an inhibitor of G␣ protein subunits, prevented SP-induced EGFR transactivation and subsequent DNA synthesis. Our results implicate EGFR as an essential regulator in SP/NK-1-induced activation of the MAPK pathway and cell proliferation in U-373 MG cells, and these events are mediated by a pertussis toxin-sensitive G␣ protein. We suggest that this mechanism by which SP controls cell proliferation is an important pathway in tissue restoration and healing.

Mitogenic effects of gastrin-releasing peptide in head and neck squamous cancer cells are mediated by activation of the epidermal growth factor receptor

“…MAPK activation has been previously implicated in mediating GPCR/EGFR signaling pathways in fibroblasts. It is generally believed that MAPK activation mediated through GPCR/EGFR drives cellular proliferation (Sharif et al, 1997;Castagliuolo et al, 2000). However, Santiskulvong et al showed that an EGFRdependent, but MAPK-independent pathway could also mediate the mitogenic effects of GPCR activation (Santiskulvong et al, 2001).…”

“…However, little is known about the contribution of GPCR-mediated EGFR transactivation to carcinogenesis and tumor growth (Gschwind et al, 2001). EGFR transactivation by GPCRs has been observed in ovarian cancer cells SK-OV-3 by interleukin-8 (Venkatakrishnan et al, 2000), astroglioma cells U-3T3 MG by substance P (Castagliuolo et al, 2000) and PC-3 prostate cancer cells by bombesin (Prenzel et al, 1999).…”

Substance P mediates antiapoptotic responses in human colonocytes by Akt activation

“…Moreover, prostaglandin E 2 induces the association of a prostaglandin E receptor 4/␤-arrestin 1/c-Src signaling complex, resulting in the transactivation of the EGFR and downstream Akt signaling (41). SP binding to NK-1R causes transactivation of the EGFR and MAPK phosphorylation linked to cell proliferation (9,14) and protection from colitis (15). Several groups also showed an EGFR-Akt-MAPK cross-talk in several cell types (42,43).…”

“…NK-1R-deficient mice are protected during the acute phase of colitis, but they have an enhanced inflammatory response at a later stage chronic colitis (15). Improved mucosal healing in the chronic phase of colitis in NK-1R-deficient mice was shown to involve metalloproteinasedependent EGFR transactivation, leading to cell proliferation in colonic fibroblasts and human colonocytes (9,14,15). In this study we report that in colonocytes, SP, by NK-1R activation, stimulates phosphorylation of the antiapoptotic molecule Akt and reduces colonocyte cell death and apoptosis both in vitro and in vivo.…”

“…Pharmacological antagonism of NK-1R (7,11,12) or genetic deletion of this receptor results in reduced intestinal inflammation in the acute model of colitis induced by Clostridium difficile toxin A (13). Interestingly, studies with NK-1R-deficient mice showed that this receptor plays a protective role in prolonged experimental colitis induced by 2,4,6-trinitrobenzensulfonic acid and dextran sulfate (DSS) (14). The mechanisms involved in this protective effect are activation of epidermal growth factor receptor (EGFR)-dependent mitogen-activated protein kinase (MAPK) phosphorylation, and cell proliferation of colonic fibroblasts (15) and colonic epithelial cells (9).…”