Signal transduction from membrane-bound macrophage colony-stimulating factor

Abstract

Macrophage colony-stimulating factor (M-CSF) exists in different forms, which bind the same membrane receptor (M-CSFR), due to alternative splicing and post-translational modifications. Membrane bound M-CSF (mM-CSF) was highly expressed in some malignancies including J6-1, J6-2 (leukemia), SMMC7721, BEL7402 (hepatoma) cell lines and specimens from patients with leukemia, Hodgkin's Disease (HD), hepatoma, etc. (Zheng GG et al. Hematologica 1999, 84:951). Furthermore, we demonstrated that mM-CSF and its receptor played adhesion molecule-like roles in J6-1 cells and the activation of mM-CSF by recombinant soluble receptor (M-CSFsR) resulted in the decrease of cytoplasmic pH, mobilization of cytosolic calcium, tyrosine-phosphorylation on cytoplasmic protiens, etc. (Zheng GG et al. Leukemia Res 2000, in press). In the present study, we used cytochalasin B (CB) to study whether the integrity of cytoskeleton is necessary for mM-CSF signaling. The results showed that one hour's treatment of J6-1 cells by CB before exposed to M-CSFsR blocked the tyrosine-phosphorylation on cytoplasmic proteins with MW of 45 and 55-90kD, while decreased the sensitivity of cytoplasmic pH changes, but showed no obvious influence on cytosolic calcium mobilization. The initial activation process of most membrane proteins by their ligands involves dimerization or cluster of the membrane protein and cross-linking of the membrane protein by specific antibody can mimic the effects of their natural ligands, while dimerization or cluster is not indispensable for the signaling of some membrane proteins. We used a monoclonal antibody (McAb) B5, which was developed in our lab belonging to IgM subtype and recognizes N-terminal part of mM-CSF, to cross-link mM-CSF on J6-1 cell surface before underwent phospho-tyrosine Western blot analysis. While F(ab')2 fragment of B5 caused no obvious tyrosine phosphorylation when compared with BSA control, 10 minutes' treatment by B5 dose-dependently caused tyrosine phosphorylation on cytoplasmic proteins with MW of approximately 45 and 50-90kD, which mimicked the effects of M-CSFsR. The above results suggested that the initiation of mM-CSF signaling need dimerization/cluster of them on cell surface and cytoskeleton take part in some, but not all, of the downstream signal events.