HoxB4 Effects on Hematopoietic Stem Cells Are Dependent on STAT3 Activity; A Potential Convergence of Self-Renewing Signals.

We previously showed that STAT3 activation can promote hematopoietic stem cell (HSC) self-renewal in a physiologically regulated manner as observed for induction ofHoxB4 in hematopoietic cells. In the current study, we investigated whether functional interplay of the two molecules can be found for similar phenotypes in HSCs. First, we examined whether STAT3 activation and HoxB4 induction can cause additive effects through each independent down-stream targets. Retroviral vector encoding constitutively activated STAT3 (STAT3-C-GFP) and HoxB4 (HoxB4-YFP) were infected into 5-FUbone marrow cells (BMCs) and each single or double-infected cells were transplanted into lethally irradiated recipients. However, BMCs transduced with both STAT3-C and HoxB4exhibited comparable extent of enhancing effects on repopulation (42.8% ) over control group (MIG/MIY; 16.2%) as those single transduced with STAT3-C (41.4%) or HoxB4alone (46%) as determined by % donor cell engraftment in the recipient 12 weeks after transplantation. Similarly, ex-vivo culture of double-infected BMCs for 16 days caused comparable level expansion of Lin-Sca-1+ (LS) cells over control group as those infected by STAT3-C or HoxB4 alone (51-folds vs. 44-folds or 62-folds for STAT3-C/HoxB4 vs. STAT3-C or HoxB4, respectively). These results indicated redundant effects of STAT3-Cand HoxB4 up-regulation rather than two independent down-stream effects. Interestingly, LS cells transduced with STAT3-C did not exhibit any induction of HoxB4, whereas LS cells infected with HoxB4 exhibited up-regulation of STAT3 transcripts. Furthermore, HoxB4-mediated enhancing effects on repopulation was ameliorated when BMCs were co-infected with HoxB4 and dominant-negative STAT3 (dnSTAT3) as evidenced by decreased % repopulation of donor cells (45.8%, 22.8%, 6.1 % for HoxB4, HoxB4/dnSTAT3, and MIG/MIY). Expansion of un-differentiated hematopoietic cells during 16days ex-vivo culture was similarly inhibited by co-expression of HoxB4 and dnSTAT3(75 and 15 folds over control group for HoxB4 and HoxB4/dnSTAT3), indicating thatSTAT3 activity plays a down-stream signal for HoxB4-mediated enhancing effects on HSCs. Microarray based expression analysis of STAT3-C transduced LS cells compared to MIG-transduced LS cells revealed 58 genes with significant expression changes (1.8fold, p<0.05), including Sca-1, podocalyxin-like1, glypican 4 and sprouty homolog 1. Interestingly, significant numbers of the genes induced by STAT3-C were also induced in the HoxB4-transduced LS cells, further confirming the down-stream redundant activation of target genes. Taken together, these results demonstrate a molecular interplay of STAT3and HoxB4 to regulate HSC self-renewal in a manner that STAT3 plays a down-stream ofHoxB4 effects. Moreover, taking that HoxB4 induction had been frequently observed in the cells stimulated by other up-stream self-renewing signals, our studies provide a further convergence of molecular signals regulating HSC self-renewal.