Studies using primary cells from mice with germline or tissue-specific deletion of SOCS3 indicate that SOCS3 is a negative regulator of IL-6, LIF, Leptin and G-CSF-induced cellular responses. Mice with selective deletion of SOCS3 gene in blood and endothelial cells develop a fatal inflammatory disease characterized by tissue infiltration with neutrophils and macrophages, and display hyper-responsiveness to G-CSF in vitro and in vivo (

). Previous structure-function studies in cell lines have suggested that SOCS3 negatively regulates cytokine signalling by targeting the activated receptor for ubiquination and proteasomal degradation. An alternative model proposes that SOCS3 selectively inactivates phosphorylated STAT3, or targets phosphorylated STAT3 for ubiquination and proteasomal degradation. To test in primary cells which model best fits physiology, we investigated signal transduction and cellular responses of SOCS3-deficient and wild-type lin- kit+ progenitor cells after stimulation with G-CSF, IL-6 and other haemopoietic cytokines. Activation of the STAT3, STAT5 and MAP kinase pathways was monitored by immunoblotting with phospho-specific antibodies, and quantified by flow cytometry after stimulating cells for between 5 min and 4 hours. Proliferation and differentiation were quantified in liquid and agar cultures stimulated with SCF, IL-3, GM-CSF, G-CSF, IL-6 and IL-11 alone, or in combination with SCF for 4 or 7 days. No differences in signalling, proliferation or differentiation between SOCS3-deficient and wild-type cells were observed after stimulation with SCF, IL-3, GM-CSF or combinations of these. However, marked differences were observed for G-CSF and IL-6, with increased cellular output and a skewing of differentiation away from purely neutrophil to a mixed neutrophil and macrophage output. Both cytokines induced prolonged phosphorylation of STAT3 in the absence of SOCS3 (consistent with previous observations in macrophages and neutrophils), but no perturbations in MAP Kinase activation. STAT5 phosphorylation was not detectable after stimulation with G-CSF or IL-6 in either genotype, but was robust and equal between genotypes after stimulation with IL-3. Stimulation with either G-CSF or IL-6 for 4 hours resulted in consistent differences between genotypes in transcriptional profiles, and increased transcription of CEBPβ was identified as a candidate regulator of the observed altered differentiation. Most intriguingly, in the absence of SOCS3, progenitor cells failed to show any synergy (or even additive effect) in proliferation when cultured in SCF plus IL-6, and the skewing of differentiation towards macrophages was magnified. As for individual cytokines, no differences in signalling pathway activation other than STAT3 were observed when progenitors were incubated with combinations of SCF and IL-6 or G-CSF. We conclude that SOCS3 primarily regulates STAT3 activation downstream of receptor activation by G-CSF or IL-6, rather than inhibiting all signal transduction from the activated receptors. These observations argue that further structure-function studies are required to define the relative importance of ubiquination and kinase inhibition as physiological mechanisms for SOCS3’s suppressor activities. They also provide new insight to the molecular regulation of synergy between SCF and IL-6.

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