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 mechanism of adhesion, although its precise role has been controversial. Numerous cytokines and growth-factors activate Ras, which is an important component of their growth-promoting signaling pathways. On the other hand, the role of Ras in cytokine-induced adhesion has not been elucidated. We therefore 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, dominant-negative, or wild-type H-Ras cDNAs. Adhesion of Baf3 cells to fibronectin was induced by IL-3 in a dose-dependent manner via very late antigen-4 (VLA-4; alpha4beta1 integrins) and VLA-5 (alpha5beta1 integrins) activation. 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 cells. Constitutively active H-Ras-transfected Baf3 cells adhered to fibronectin without IL-3 stimulation through VLA-4 and VLA-5, whereas dominant-negative H-Ras-transfected Baf3 cells showed significantly less adhesion induced by IL-3 compared with wild-type and constitutively active H-Ras-transfected Baf3 cells. Anti-beta1 integrin antibody (clone; 9EG7), which is known to change integrin conformation and activate integrins, induced the adhesion of dominant-negative H-Ras-transfected Baf3 cells as much as the other types of H-Ras-transfected Baf3 cells. 8-Br-cAMP, Dibutyryl-cAMP, Ras-Raf-1 pathway inhibitors, and PD98059, a MAPK kinase inhibitor, suppressed proliferation and phosphorylation of MAPK detected by Western blotting with anti-phospho-MAPK antibody, but not adhesion of any type of H-Ras-transfected Baf3 cells, whereas U-73122, a phospholipase C (PLC) inhibitor, suppressed adhesion 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-out signaling pathway of IL-3-induced VLA-4 and VLA-5 activation in Baf3 cells.
Departments of Microbiology/Immunology, Medicine, and the Walther Oncology Center, Indiana University School of Medicine, Indianapolis, IN, USA.
H
H
Shibayama
N
N
Anzai
S E
SE
Braun
S
S
Fukuda
C
C
Mantel
H E
HE
Broxmeyer
D000818:Animals; D002448:Cell Adhesion / Q000502:physiology; D002460:Cell Line; D004305:Dose-Response Relationship, Drug; D011905:Genes, ras; D006412:Hematopoietic Stem Cells / Q000166:cytology / Q000502:physiology*; D039041:Integrin alpha4beta1; D016023:Integrins / Q000502:physiology*; D007377:Interleukin-3 / Q000494:pharmacology*; D051379:Mice; D015689:Oncogene Protein p21(ras) / Q000502:physiology*; D017469:Receptors, Fibronectin / Q000502:physiology*; D016201:Receptors, Lymphocyte Homing / Q000502:physiology*; D015398:Signal Transduction / Q000502:physiology*
D039041:Integrin alpha4beta1; D016023:Integrins; D007377:Interleukin-3; D017469:Receptors, Fibronectin; D016201:Receptors, Lymphocyte Homing; D015689:Oncogene Protein p21(ras)