Interleukin-6 silences y gene expression via the jak/stat signal transduction pathway

We have previously shown that bipotential K562 cells treated with interleukin-6 (IL6) exhibit a concentration dependant decrease in y globin mRNA levels with a concurrent increase in the platelet specific marker glycoprotein lib (J. Biol. Chem. 272:230, 1997). This suggests that IL-6 may be involved in erythroid versus megakaryocytic lineage commitment. We subsequently tested the effects of two known megakaryocyte inducers, thrombopoietin and 1L-11 in K562 cells. Neither agent had an effect on y mRNA levels, suggesting a unique role for IL-6 in y gene silencing. IL-6 is known to phosphorylate the transcription factors Jak2, Stall and StatS to activate gene expression. To gain further insight into the mechanism for y gene silencing by IL-6 we examined the Jak/Stat signal transduction pathway. K562 cells were grown in serum-free media for 24 hours, then treated for 5 minutes with genistein (10nM), a general tyrosine kinase inhibitor, followed by the addition of 1L6 (100ng/ml). y mRNA levels were calculated as a ratio of GAPD mRNA and compared to untreated cells. K562 cells treated with either genistein alone or followed by IL-6, showed a 4.3-fold increase in y mRNA levels, demonstrating the ability of genistein to abrogate the inhibitory effect of IL-6 on y gene expression. These results suggest that Stat proteins may bind in the y promoter to alter y gene expression. Gel mobility shift assay (GMS A) experiments were completed to identify potential Stall and/or Stat3 binding sites. A sequence located in the y gene from+1 to +25 (AyStatS) contained a potential Stat3 site that was tested further. We observed decreased binding for a major DNA-protein complex with IL-6 (100ng/ml) treated K562 nuclear extract at 2 and 4 hours, however pre-treatment with AG-490 (25|iM), a Jak2 specific inhibitor, for 16 hours prevented the decreased binding observed with IL6 treatment alone. Supershift experiments with monoclonal IgG antibodies to Stall and Stal3 revealed lhal Ihe DNA-prolein complex established with the AyStat3 probe contained both Stall and Stat3 proteins. These experiments suggest that Stat3 binding in the y gene promoter may mediate the inhibitory effect of IL-6 on y gene expression. In other systems, an inhibitory splice variant, Stat3β has been identified. Experiments will be completed to test whether the Stat3β variant mediates y gene silencing by IL-6.