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

Hirohiko Shibayama, Naoyuki Anzai, Stephen E. Braun, Seiji Fukuda, Charlie Mantel, Hal E. Broxmeyer

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32 Scopus citations

Abstract

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; α4β1 integrins) and VLA-5 (α5β1 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-β1 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.

Original languageEnglish (US)
Pages (from-to)1540-1548
Number of pages9
JournalBlood
Volume93
Issue number5
StatePublished - Mar 1 1999

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ASJC Scopus subject areas

  • Biochemistry
  • Immunology
  • Hematology
  • Cell Biology

Cite this

Shibayama, H., Anzai, N., Braun, S. E., Fukuda, S., Mantel, C., & Broxmeyer, H. E. (1999). H-Ras is involved in the inside-out signaling pathway of interleukin-3- induced integrin activation. Blood, 93(5), 1540-1548.