Soluble Kit receptor blocks stem cell factor bioactivity in vitro

Stem cell factor (SCF) is a growth factor that promotes the survival, proli feration, and differentiation of hematopoietic cells. SCF and its receptor, Kit, are normally present in both cell surface and soluble forms. Both for ms of Kit can bind SCF. However, the function of soluble Kit is unknown. In order to determine if soluble Kit can modulate SCF activity, we produced a fusion protein, Kit-Fc, comprised of the extracellular domain of murine Ki t and the Fc portion of human IgG, and investigated its ability to bind I-1 25-SCF and to inhibit SCF-stimulated hematopoietic colony growth in vitro. Stable cell lines expressing Kit-Fc were generated and Kit-Fc was purified to greater than 95% purity. Scatchard analysis demonstrated that Kit-Fc bin ds iodinated SCF with high affinity (Kd 570 pM). Kit-Fc also bound to trans membrane SCF displayed on the surface of fibroblasts. The murine mast cell line IC2 was engineered to express murine Kit on the cell surface and was d emonstrated to proliferate in the presence of SCF. Kit-Fc completely blocke d SCF-stimulated proliferation of IC2-Kit cells, but not IL-3-stimulated gr owth of IC2-Kit cells, demonstrating the specificity of Kit-Fc. We investig ated the ability of Kit-Fc to block SCF-stimulated murine hematopoietic col ony growth. Kit-Fc blocked SCF-stimulated erythroid colony growth as effect ively as a neutralizing anti-Kit monoclonal antibody, ACK2, but did not blo ck erythropoietin-stimulated erythroid colony growth. Likewise, Kit-Fc bloc ked SCF-stimulated myeloid colony growth as effectively as ACK2 antibody, b ut did not block IL-3- or GM-CSF-stimulated myeloid colony growth. These re sults indicate that a form of soluble Kit binds SCF with high affinity, and can specifically block the ability of SCF to stimulate hematopoietic colon y growth, suggesting that one function of soluble Kit may be to modulate SC F bioactivity. (C) 2001 Elsevier Science Ltd. All rights reserved.