H-Ras is involved in the inside-out signaling pathway of interleukin-3-induced integrin activation

The proto-oncogene product, p21(ras), has been implicated in the cellular m echanism of adhesion, although its precise role has been controversial. Num erous cytokines and growth-factors activate Ras, which is an important comp onent of their growth-promoting signaling pathways. On the other hand, the role of Ras in cytokine-induced adhesion has not been elucidated. We theref ore investigated the function of H-Ras in the inside-out signaling pathway of interleukin-3 (IL-3)-induced integrin activation in the murine Baf3 cell line after transfection of cells with either constitutively active, domina nt-negative, or wild-type H-Ras cDNAs. Adhesion of Baf3 cells to fibronecti n was induced by IL-3 in a dose-dependent manner via very late antigen-4 (V LA-4; alpha 4 beta 1 integrins) and VLA-5 (alpha 5 beta 1 integrins) activa tion. On the other hand, IL-4 did not induce the adhesion of Baf3 cells to fibronectin, although IL-4 did stimulate the cell proliferation of Baf3 cel ls. Constitutively active H-Ras-transfected Baf3 cells adhered to fibronect in without IL-3 stimulation through VLA-4 and VLA-5, whereas dominant-negat ive H-Ras-transfected Baf3 cells showed significantly less adhesion induced by IL-3 compared with wild-type and constitutively active H-Ras-transfecte d Baf3 cells. Anti-beta 1 integrin antibody (clone; 9EG7), which is known t o change integrin conformation and activate integrins, induced the adhesion of dominant-negative H-Ras-transfected Baf3 cells as much as the other typ es of H-Ras-transfected Baf3 cells. 8-Br-cAMP, Dibutyryl-cAMP, Ras-Raf-1 pa thway inhibitors, and PD98059, a MAPK kinase inhibitor, suppressed prolifer ation and phosphorylation of MAPK detected by Western blotting with anti-ph ospho-MAPK antibody, but not adhesion of any type of H-Ras-transfected Baf3 cells, whereas U-73122, a phospholipase C (PLC) inhibitor, suppressed adhe sion of these cells completely. These data indicate that H-Ras and PLC, but not Raf-1, MAPK kinase, or the MAPK pathway, are involved in the inside-ou t signaling pathway of IL-3-induced VLA-4 and VLA-5 activation in Baf3 cell s. (C) 1999 by The American Society of Hematology.