Nfix  Promotes Survival of Immature Hematopoietic Cells Via Regulation of C-Mpl

Hematopoietic stem and progenitor cells (HSPC) are necessary for life-long blood production and replenishment of the hematopoietic system during stress. We recently identified the transcription factor nuclear factor I/X ( Nfix) as a critical regulator of hematopoietic stem and progenitor cell (HSPC) survival post-transplantation. Nfix -depleted HSPC are lost from the marrow between 7-10 days post-transplant and residual Nfix -depleted HSPC display elevated levels of apoptosis at these time-points. To further characterize the anti-apoptotic role of Nfix in HSPCs, we overexpressed Nfix in Lineage^- Sca-1⁺ c-Kit⁺ (LSK) cells that were then maintained ex vivo under serum-free conditions with supportive cytokines and monitored the cultures for cell surface phenotype, growth, vector selection, and apoptosis. Ectopic expression of Nfix in primary mouse HSPCs had no significant effect on the expression of lineage markers compared to controls, but did appear to accelerate the loss of immunophenotypic LSK cells, as well as colony forming units (p = 0.023). Nfix -overexpressing HSPCs were extended in their ex vivo culture lifespan from about 20 days to 40 days, and were steadily selected for in culture. HSPCs overexpressing Nfix also displayed hypersensitivity to supportive cytokines and reduced apoptosis when subjected to cytokine deprivation compared to controls (p = 0.032). NFIX has been shown to potentially influence transcription of several known regulators of HSC biology. Here, we show that ectopic Nfix resulted in elevated levels of c-Mpl transcripts and cell surface protein on primary murine HSPCs as well as increased phosphorylation of STAT5, which is known to be activated down-stream of c-MPL. Consequently, Bcl-X_L, an anti-apoptotic factor induced by STAT5, was also significantly upregulated in Nfix -overexpressing cells by two weeks of culture compared to controls (p = 0.0038). Blocking c-MPL signaling by removal of thrombopoietin or addition of a c-MPL neutralizing antibody significantly negated the anti-apoptotic effect of Nfix overexpression on cultured HSPCs (p = 0.045, p = 0.0098, respectively). Further, NFIX was capable of transcriptional activation of a proximal c-Mpl promoter fragment (p = 0.0056). In sum, these data suggest that NFIX-mediated up-regulation of c-Mpl transcription can protect primitive hematopoietic cells from stress ex vivo .