TNF-α/Fas-RIP-1-Induced Cell Death Signaling Separates Hematopoietic Stem Cells/Progenitors Into Two Distinct Populations

Abstract 393 Tumor necrosis factor-α (TNF-α) and Fas ligand (FasL) have been found to induce a negative regulatory effects on hematopoiesis and have been implicated in the pathogenesis of human bone marrow failure (BMF) syndromes. However, the molecular mechanism by which these factors inhibit hematopoiesis is still not completely known. We previously reported that Tak1-knockout mice (Tak1−/−) develop BMF due to the mass apoptosis of hematopoietic cells, including hematopoietic stem cells and progenitors (HSC/Ps). Taking advantage of this mouse model, we studied the effects of TNF-α and Fas-induced death signaling on HSC/Ps by examining their contributions to the development of BMF syndromes in Tak1−/− mice. To do so, TNF-α and Fas-induced signaling were genetically inactivated in Tak1−/− HSC/Ps in order to examine to what degree both the apoptosis of HSC/Ps and BMF in vivo can be prevented. We found that complete inactivation of TNF-α signaling by the deletion of both Tnfr1 and Tnfr2 (TNF receptors 1 and 2) is able to protect up to 30–40% of Tak1−/− HSC/Ps from apoptosis. In vitro studies suggested that Fas signaling also contributes to less than 10% of Tak−/− HSC/P death. However, since Fas works on the same population of cells as TNF-α, and because TNF-α signaling is dominant in vivo, inactivation of Fas signaling failed to inhibit the apoptosis of HSC/Ps and BM damage in Tak1−/− mice. In addition, inhibition of RIP-1 (Receptor-Interacting Protein-1) activity by the specific inhibitor Nec-1 (Necrostatin-1) but not inhibition of FADD/caspase-8 signaling was able to protect the same percentage of the Tak−/− HSC/Ps from death as complete inactivation of TNF-α signaling did, but was unable to further improve the survival of Tak1−/−Tnfr1−/−r2−/− HSC/Ps (Tak1, Tnfr1 and r2 compound mutant). This suggests that TNF-α, acting through RIP-1, induces death in 30 to 40% of HSC/Ps. To investigate the causes of apoptosis in the remainder of cells, we looked for factors which either protect Tak1−/−Tnfr1−/−r2−/− HSC/Ps from death or further induce such death. We found that the expression of major pro-survival genes is significantly down-regulated in Tak1−/− HSC/Ps. The survival of the Tak1−/−Tnfr1−/−r2−/− HSC/Ps can be further improved by transducing the over-expression of dominant negative (DN)-caspase-9, as well as by Bcl-xl. Our studies suggest that there is heterogeneity in BM HSC/Ps. Only a portion of HSC/Ps is responsive to TNFα/Fas-RIP-1-induced cell death, whereas the death of the remaining HSC/Ps is induced by an intrinsic apoptotic mechanism. Tak1 is involved in mediating hematopoietic cytokine- and pro-inflammatory factor-induced survival signaling, protecting against both the TNF-α/Fas-RIP-1-dependent and independent death of HSC/Ps. Disclosures: No relevant conflicts of interest to declare.