ABSTRACT
Signal transduction via the interferon-gamma (IFN-gamma) receptor requires the tyrosine phosphorylation of signal transducers and activators of transcription (Stats). Whereas tyrosine phosphorylation of Stat1 occurs in all cells, activation of Stat5 by IFN-gamma is cell type-restricted. Here we investigated the mechanism of Stat5 activation by the IFN-gamma receptor. In transfection assays both Stat5 isoforms, Stat5a and Stat5b, were phosphorylated on tyrosine in response to IFN-gamma. Stat5 activation required the presence of tyrosine 420 (Tyr-420) in the murine IFNGR1 receptor chain, which also serves as the Stat1 binding site. Moreover, a peptide including Tyr-440, the Stat1 binding site of the human IFNGR1 chain, conferred the ability upon a synthetic receptor to activate Stat5. Suppressor of cytokine signaling 3 (SOCS3) inhibited the activation of Stat5 by the IFN-gamma receptor, and the Tyr-440-containing peptide stretch was sufficient for repression. SOCS3 expression had little effect on the activity of Jak kinases not associated with cytokine receptors. In IFN-gamma-treated, Stat1-deficient fibroblasts Stat5 was inefficient in inducing transcription of a Stat-dependent reporter gene, suggesting it does not per se make a major contribution to the expression of IFN-gamma-responsive genes.
Subject(s)
DNA-Binding Proteins/metabolism , Milk Proteins , Protein Isoforms/metabolism , Proteins/metabolism , Receptors, Interferon/metabolism , Repressor Proteins , Trans-Activators/metabolism , Transcription Factors , Animals , Binding Sites , Cell Line , DNA-Binding Proteins/antagonists & inhibitors , Humans , Interferon-gamma/metabolism , Protein Isoforms/antagonists & inhibitors , Receptors, Interferon/chemistry , STAT1 Transcription Factor , STAT5 Transcription Factor , Suppressor of Cytokine Signaling 3 Protein , Suppressor of Cytokine Signaling Proteins , Trans-Activators/antagonists & inhibitors , Transcription, Genetic , Tumor Suppressor ProteinsABSTRACT
Stat5 is activated by multiple receptors of hematopoietic cytokines. To study its role during hematopoiesis, we have generated primary chicken myeloblasts expressing different dominant-negative (dn) alleles of Stat5. This caused a striking inability to generate mature cells, due to massive apoptosis during differentiation. Bcl-2 was able to rescue differentiating cells expressing dnStat5 from apoptosis, suggesting that during cytokine-dependent differentiation the main function of the protein is to ensure cell survival. Our findings with dnStat5-expressing chicken myeloblasts were confirmed with primary hematopoietic cells from Stat5a/Stat5b-deficient mice. Bone marrow cells from these animals displayed a strong increase in apoptotic cell death during GM-CSF-dependent functional maturation in vitro. The antiapoptotic protein Bcl-x was induced by GM-CSF and IL-3 in a Stat5-dependent fashion. Ectopic expression of Bcl-x rescued Stat5-deficient bone marrow cells from apoptosis, indicating that Stat5 promotes the survival of myeloid progenitor cells through its ability to induce transcription of the bcl-x gene. Finally, the recruitment of myeloid cells to inflammatory sites was found strongly impeded in Stat5-deficient mice. Taken together, our findings suggest that Stat5 may promote cytokine-dependent survival and proliferation of differentiating myeloid progenitor cells in stress or pathological situations, such as inflammation.
Subject(s)
Apoptosis/physiology , DNA-Binding Proteins/physiology , Hematopoiesis/physiology , Milk Proteins , Trans-Activators/physiology , Alleles , Animals , Apoptosis/genetics , Bone Marrow Cells/physiology , Cell Differentiation/genetics , Cell Differentiation/physiology , Cell Division/genetics , Cell Division/physiology , Cell Line, Transformed , Cell Survival , Cells, Cultured , Chickens , DNA-Binding Proteins/biosynthesis , DNA-Binding Proteins/deficiency , Hematopoiesis/genetics , Inflammation/pathology , Mice , Mice, Inbred C57BL , Mice, Knockout , Proto-Oncogene Proteins c-bcl-2/genetics , Proto-Oncogene Proteins c-bcl-2/physiology , STAT5 Transcription Factor , Trans-Activators/biosynthesis , Trans-Activators/deficiency , bcl-X ProteinABSTRACT
We have investigated the activation of the STAT5 protein during the differentiation of myelomonocytic cells. In the human U937 promonocytic cell line, STAT5 activation occurred in response to several inducers of monocytic differentiation (phorbol ester, 1alpha,25-dihydroxyvitamin D3, and retinoic acid). In the promyelocytic HL60 cell line, STAT5 was activated in the course of either phorbol ester-induced monocytic differentiation or DMSO-induced granulocytic differentiation. To test for involvement of STAT5 in the differentiation of primary nonimmortalized cells, chicken myeloid progenitor cells transfected with the ts21-E26 avian retrovirus were studied. At 37 degrees C the temperature-sensitive oncoprotein of the ts21-E26 virus (p135(gag-myb-ets)) installs a differentiation block that is released by a shift to the nonpermissive temperature (42 degrees C). Both proliferation and differentiation of ts21-E26-transfected myeloblasts require the continuous presence of chicken myelomonocytic growth factor (cMGF). Addition of cMGF to growth factor-starved cells rapidly caused STAT5 tyrosine phosphorylation, DNA binding, and transcriptional activity at both permissive and nonpermissive temperatures. Moreover, the temperature shift-induced onset of myelomonocytic differentiation strictly correlated with the appearance of activated STAT5 in ts21-E26-infected myeloblasts, but not in cells infected with wtE26 retrovirus. These data position STAT5 in a nuclear signaling cascade induced by the cMGF receptor and suggest a contribution of STAT5 to the process of myelomonocytic differentiation or to the functional changes that accompany the maturation of myeloid progenitor cells to a terminally differentiated stage.
