Abstract
The functional interaction ("cross-talk") of integrins with growth factor receptors has become increasingly clear as a basic mechanism in cell biology, defining cell growth, adhesion, and motility. However, no studies have addressed the microdomains in which such interaction takes place nor the effect of gangliosides and tetraspanins (TSPs) on such interaction. Growth of human embryonal WI38 fibroblasts is highly dependent on fibroblast growth factor (FGF) and its receptor (FGFR), stably associated with ganglioside GM3 and TSPs CD9 and CD81 in the ganglioside-enriched microdomain. Adhesion and motility of these cells are mediated by laminin-5 ((LN5) and fibronectin (FN) through alpha3beta1 and alpha5beta1 integrin receptors, respectively. When WI38 cells or its transformant VA13 cells were adhered to LN5 or FN, alpha3beta1 or alpha5beta1 were stimulated, giving rise to signaling to activate FGFR through tyrosine phosphorylation and inducing cell proliferation under serum-free conditions without FGF addition. Types and intensity of signaling during the time course differed significantly depending on the type of integrin stimulated (alpha3beta1 versus alpha5beta1), and on cell type (WI38 versus VA13). Such effect of cross-talk between integrins and FGFR was influenced strongly by the change of GM3 and TSPs. (i) GM3 depletion by P4 caused enhanced tyrosine phosphorylation of FGFR and Akt followed by MAPK activation, without significant change of ceramide level. GM3 depletion also caused enhanced co-immunoprecipitation of FGFR with alpha3/alpha5/beta1 and of these integrins with CD9/CD81. (ii) LN5- or FN-dependent proliferation of both WI38 and VA13 was strongly enhanced by GM3 depletion and by CD9/CD81 knockdown by siRNA. Thus, integrin-FGFR cross-talk is strongly influenced by GM3 and/or TSPs within the ganglioside-enriched microdomain.
Highlights
Cell growth and associated differentiation are controlled by growth factors, and their receptors that have tyrosine kinases at the cytoplasmic domain [1,2,3]
Epidermal Growth factor receptors (GFRs) (EGFR) and platelet-derived GFR are claimed to be associated with caveolar membrane [7, 8], whereas integrin receptors are soluble in 1% Triton X-100 and are considered to be located outside of the caveolar membrane or “raft” [9]
Applying P4, RNA interference, and N-glycosylation processing inhibitor 1-deoxymannojirimycin (DMJ), we found that GM3 and/or CD9/CD81 strongly affect the functional interaction between integrins and FGFR and, affect FGF-independent proliferation in both WI38 and VA13 cells
Summary
Cells—Human embryonal lung diploid fibroblast WI38 and its SV40transformed cell line VA13 were from American Type Culture Collection (ATCC, Manassas, VA). WI38 and VA13 were grown in minimum essential medium (MEM, Invitrogen) supplemented with 10% fetal bovine serum (Hyclone Laboratories, Logan, UT), 1 mM sodium pyruvate, and penicillin/streptomycin at 37 °C, 5% CO2. LN5-producing HDEF cells, kindly donated by Dr William G. Carter (Fred Hutchinson Cancer Research Center, Seattle), were grown in serum-free keratinocyte growth medium (KGM, Cambrex Bioscience, Walkersville, MD) supplemented with SingleQuots® KBM (Cambrex). Reagents—FN from human plasma, poly-L-lysine, DMJ, and swainsonine were from Sigma; siRNA-CD9 and siRNA-CD81 were from Ambion, Austin, TX; D-threo-1-phenyl-2-palmitoylamino-3-pyrrolidino-1propanol (P4) was kindly donated by Dr James A.
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