HEMATOPOIESIS

Lyn is required for normal stem cell factor–induced proliferation and chemotaxis of primary hematopoietic cells
Bridget O’Laughlin-Bunner, Nina Radosevic, Marcia L. Taylor, Shivakrupa, Candy DeBerry, Dean D. Metcalfe, Meijuan Zhou,
Clifford Lowell, and Diana Linnekin

Stem cell factor (SCF) binds to c-Kit and is an important mediator of survival, growth, and function of hematopoietic progenitor cells and mast cells. Lyn and other Src family members are activated by SCF and associate with phosphorylated tyrosine residues in the c-Kit juxtamembrane region. However, studies using c-Kit mutants incapable of directly recruiting Src family members suggest this kinase family plays a minimal role in c-Kit stimulus-response coupling mechanisms. The objective of this study was to

specifically target Lyn and subsequently address its role in SCF-mediated responses of primary hematopoietic progenitor cells and mast cells. To this end, a dominant-inhibitory Lyn mutant and Lyndeficient mice were used. Transfection of normal murine mast cells with kinaseinactive Lyn impaired SCF-induced growth. Further, SCF-induced proliferation and chemotaxis of Lyn-deficient mast cells were less than for wild-type mast cells. SCF-induced growth of progenitor cells lacking Lyn was also reduced com-

pared with that of wild-type progenitor cells. Impairment of SCF-mediated responses of Lyn-deficient mast cells and progenitor cells did not result from reductions in surface expression of c-Kit. These studies demonstrate that Lyn is required for normal SCF-mediated responses of primary progenitors and for a differentiated lineage. (Blood. 2001;98:343-350)

© 2001 by The American Society of Hematology

Introduction
Hematopoiesis is regulated by multiple mechanisms, including interaction of cell surface receptors with growth factors and cytokines. These molecules aid in the survival, proliferation, and differentiation of hematopoietic progenitors. One superfamily of receptors that binds hematopoietic growth factors is the receptor tyrosine kinase (RTK) superfamily (reviewed in 1). The cytokine receptor superfamily also plays an important role in this process
(reviewed in 2). c-Kit is an RTK encoded by the c-kit proto-oncogene (reviewed in3-5). The c-Kit ligand is stem cell factor (SCF). The absence of either SCF or c-Kit is lethal, and decreases in expression or function of ligand or receptor results in macrocytic anemia, mast cell deficiency, aberrations in pigmentation, and sterility (reviewed in 3,6,7). Inappropriate expression or constitutive activation of c-Kit has also been associated with human diseases, including mastocytosis, myeloid leukemia, small-cell lung carcinoma, and gastrointestinal stromal cell tumors.8-12
The critical role of c-Kit in normal physiology and the association of activated mutants with human disease highlights the importance of understanding its mechanism of action. Recent work has demonstrated that SCF activates the Ras-Raf-Map kinase cascade, phosphatidylinositol 3 kinase (PI3K), and, in some lineages, the Jak/Stat pathway
(reviewed in 13). SCF induces the association of Src, Lyn, and Fyn with c-Kit and the activation of multiple Src family members.14-20 Reduction in the expression of Lyn using antisense oligonucleotides inhibits
SCF-induced proliferation.15 In addition, treatment of cells with PP1, a
Src family inhibitor, does not alter the in vitro kinase activity of c-Kit but

impairs SCF-induced cell-cycle progression and growth.15,20,21 These data suggest that Lyn has an important function in SCF-induced proliferation in hematopoietic cells. However, several lines of evidence conflict with this hypothesis. First, findings using leukemic cell lines treated with pharmacologic inhibitors and antisense oligonucleotides may not be representative of normal physiology. Second, studies with c-Kit mutants incapable of directly associating with Src family