NF-kB Family Proteins Participate in Multiple Steps of Hematopoiesis through Elimination of Reactive Oxygen Species (ROS).

NF-kB family proteins have been reported to induce the expression of over 150 target genes, thereby crucially regulating immune responses, stress responses, and inflammation. These proteins also play important roles in cell growth and survival in various cell types. However, the precise roles of NF-kB in hematopoiesis and their mechanisms remain undetermined. To examine the roles for NF-kB family proteins in the growth and survival of hematopoietic cells, we expressed dominant negative NF-kB (IkBSR) in a murine IL-3-dependent cell line Ba/F3 using a Lac-inducible system, in which IkBSR was inducibly expressed by the IPTG treatment; this clone was designated Ba/F3/IkBSR. Furthermore, we introduced EPO receptor (R), TPOR, and G-CSFR/gp130 consisting of the extracelluar domain of G-CSFR and cytoplasmic domain of gp130 into this clone. At first, we confirmed that these clones could survive and proliferate under the cultures with IL-3, EPO, TPO, G-CSF, respectively. Although IPTG-induced IkBSR slightly suppressed IL-3- and EPO-dependent growth at low concentrations, it did not affect TPO- or gp130L-dependent growth, suggesting that NF-kB might not be so important for cytokine-dependent growth of hematopoietic cells. In contrast, IkBSR prominently enhanced factor-deprived apoptosis, which was accompanied by the ROS accumulation. To access the roles of ROS in IkBSR-enhanced apoptosis, we overexpressed ROS scavenger enzymes MnSOD and thioredoxin X (TRX) in Ba/F3/IkBSR, respectively. As a result, MnSOD and TRX significantly canceled IkB-SR-enhanced apoptosis, suggesting that ROS would be responsible for this apoptosis. We next analyzed the effects of IkBSR on the growth and survival of normal hematopoietic cells. When IkBSR was introduced into murine Lin⁻Sca-1⁺ hematopoietic stem/progenitor cells with the retrovirus system, it induced apoptosis even in the presence of appropriate cytokines. This apoptosis was also accompanied by the ROS accumulation due to the downregulated expression of anti-oxidants such as glutathione, MnSOD, glutathione peroxidase, and TRX. In addition, the expression of antiapoptotic BCl-2 family members, Bcl-XL, Bcl-2, and A1 was found to be repressed by IkBSR. However, since antioxidants such as MCI (3-methyl-1-phenyl-2-pyrazolin-5-one), N-acetylecysteine and TRX cancelled this apoptosis, ROS were supposed to be more important for IkBSR-induced apoptosis in normal hematopoietic stem/progenitor cells. To further analyze the roles for NF-kB proteins in the development of hematopoietic cells, we expressed IkBSR in an inducible fashion at various stages of hematopoiesis using the OP9 system, in which hematopoietic cells are induced to develop from ES cells. When IkBSR was expressed at the stage of hemangioblasts, IkBSR induced apoptosis and inhibited the development of hematopoietic stem cells, which was also cancelled by MCI. Furthermore, when IkBSR was expressed after the development of hematopoietic stem cells, it also inhibited terminal differentiation towards granulocytes, erythrocytes, and megakaryocytes through ROS-mediated apoptosis; IkBSR inhibited granulopoiesis before the development of myeloblasts, erythropoiesis after the development of proerythroblasts, and megakaryopoiesis during polyploidization of megakaryocytes. These results indicate that NF-kB family proteins play essential roles to prevent apoptosis at multiple steps of hematopoiesis by eliminating ROS.