Differential Roles of SOCS Family Members in EpoR Signal Transduction

To elucidate the roles of suppressor of cytokine signaling (SOCS) family members in erythropoietin (EPO) signaling, we explored SOCS gene regulation, mRNA stability, and protein function in two EPO-responsive hematopoietic cell lines. Using two independent approaches, one involving inhibition of specific signaling molecules and the other employing cell lines that express particular EpoR mutants and thereby activate only subsets of signaling cascades, we demonstrate that induction of SOCS1, SOCS2, SOCS3, and cytokine-inducible SH2-containing protein (CIS) in response to EPO stimulation appears to depend on Stat5 but not on mitogen-activated protein kinase (MAPK) or phosphatidylinositol 3-kinase (PI3K). SOCS4 expression, in contrast, does not appear to be EPO inducible. Furthermore, we show differential stabilities of SOCS transcripts, with SOCS2 the longest-lived and SOCS1 and CIS the least stable, and provide evidence in support of EPO-independent expression of SOCS3 and SOCS4. In order to understand the effects of SOCS on EPO-mediated effects, we generated multiple stable cell lines that inducibly express particular SOCS proteins. Overexpression of SOCS1, SOCS3, or CIS negatively regulates EPO-mediated cell proliferation, Stat5 phosphorylation, and activation of a Stat-dependent luciferase reporter. In contrast, SOCS2 is less effective, and SOCS4 is ineffective at counteracting EPO-mediated events. Thus, we have demonstrated differential regulation and function of various SOCS family members in EPO-dependent hematopoietic cells.