Abstract
We have recently demonstrated that the gene encoding the osteopontin (OPN) protein is activated both by interleukin-3 and granulocyte-macrophage colony-stimulating factor signaling pathways and that, through binding to the cell surface receptor CD44, OPN contributes to the survival activities of interleukin (IL)-3 and GM-CSF (Lin, Y.-H., Huang, C.-J., Chao, J.-R., Chen, S.-T., Lee, S.-F., Yen, J. J.-Y., and Yang-Yen, H.-F. (2000) Mol. Cell. Biol. 20, 2734-2742). In this report, we demonstrate that the CD44-binding domain of OPN involves a region containing amino acid residues from 121 to 140 and that both threonine and serine at positions 137 and 147, respectively, are essential for the survival stimulatory effect of OPN. Substitution of either residue with alanine results into a dominant negative mutant that overrides the survival effect of IL-3. Upon binding to the CD44 receptor, the wild-type OPN but not the inactive mutant induces activation of phosphatidylinositol 3-kinase and Akt. Last, we demonstrate that two waves of Akt activation are detected in IL-3-treated cells and that the survival promoting effect of OPN is mediated predominantly through the phosphatidylinositol 3-kinase/Akt signaling pathway. Together, our results suggest that a positive autoregulatory loop is involved in the survival pathway of IL-3.
Highlights
Both interleukin-3 (IL-3)1 and granulocyte-macrophage colony-stimulating factor (GM-CSF) belong to a family of cytokine growth factors that regulate the proliferation, differentiation, viability, and function of multipotential hematopoietic progenitors as well as of various other hematopoietic cells [1]
We have recently demonstrated that the gene encoding the osteopontin (OPN) protein is activated both by interleukin-3 and granulocyte-macrophage colony-stimulating factor signaling pathways and that, through binding to the cell surface receptor CD44, OPN contributes to the survival activities of interleukin (IL)-3 and GM-CSF
We demonstrate that the survival promoting effect of OPN in IL-3-dependent cells involves activation of the PI3K/Akt signaling pathway
Summary
Cells and Cell Lines—Ba/F3 is a murine IL-3-dependent pro-B-cell line and was maintained in RPMI 1640 supplemented with 10% fetal bovine serum and 1% conditioned medium (CM) from WEHI-3B cells as a source of IL-3. CM Containing OPN—CHOP cells were transiently transfected with various OPN expression vectors by liposome-mediated gene transfer as previously described [26]. We have previously demonstrated that the survival promoting activity of CM from cells transfected with the wild type vector was entirely due to the presence of OPN in the CM [26]. ␣573 cells which lost the ability to synthesize OPN in growth medium containing hGM-CSF were cultured in the presence or absence of CM (30%, v/v) from CHOP cells transiently transfected with wild type or mutant OPN expression vectors. To examine the dominant negative effect of the mutant protein, CM containing wild-type OPN was first mixed with CM (1:1) from CHOP cells transfected with mutant OPN or GFP expression vector prior to being added to the culture
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