Molecular studies on G-CSF receptor signaling in granulocytes and regulation of Fcgamma receptor function in macrophages: (Roles for a novel protein LRG and inositol phosphatase SHIP-2 respectively)

Neutrophils and macrophages are two major cell types involved in innate immune response. They both have specific surface receptors, which, in the presence of corresponding stimulants, will transduce unique signals that are critical for their survival, differentiation, and functions.; In the first part, we characterized the physiological roles of a novel protein, LRG, in G-CSFR signaling pathways in granulocytes. We showed that the transcription factors PU.1 and C/EBPepsilon, that regulate the expression of multiple myeloid-specific genes also bind to the LRG promoter, and LRG localizes to the same cytoplasmic compartment as myeloperoxidase. Stable transfection of LRG into 32Dc13 cells resulted in accelerated G-CSF-mediated neutrophil differentiation and induction of CD11b and CD13 expression, which was found to correlate with increased level of phospho-Stat3 but not with PU.1 or p27 kip1 levels. In contrast, constitutive expression of LRG in 32Dwt18 cells expressing a chimeric Epo/G-CSF receptor consisting of the EpoR extracellular domain fused to the G-CSFR transmembrane and cytoplasmic domains failed to do so in response to Epo stimulation. Collectively, these findings suggest a role for LRG in modulating neutrophil differentiation and activation via non-redundant G-CSFR signals.; In the second part, we studied the regulation of FcgammaR-mediated phagocytosis of IgG-coated particles in macrophages, which involve inositol phosphatases PTEN and SHIP-1. We have analyzed the role of SHIP-2, an inositol phosphatase with high level homology to SHIP-1, in phagocytosis using independent cell models that allow for manipulation of SHIP-2 function without influencing SHIP-1. We presented evidence that SHIP-2 translocates to the site of phagocytosis and downregulates FcgammaR-mediated phagocytosis. Our data indicated that SHIP-2 must contain both the N-terminal SH2 domain and the C-terminal proline-rich domain to mediate its inhibitory effect. The effect of SHIP-2 is independent of SHIP-1, as overexpression of dominant-negative SHIP-2 in SHIP-1 deficient primary macrophages resulted in enhanced phagocytic efficiency. Likewise, specific knockdown of SHIP-2 expression using siRNA resulted in enhanced phagocytosis. Finally, analysis of the molecular mechanism of SHIP-2 downregulation of phagocytosis revealed that SHIP-2 downregulates upstream activation of Rae. These data suggest that SHIP-2 is a novel negative regulator of FcgammaR-mediated phagocytosis independent of SHIP-1.; Taken together, we concluded that specific receptors have unique downstream signaling events which are under tight control.