Role of Atypical p50:p50:Bcl-3 NFκlB Complex in Differentiation of Erythroid Cells.

We recently reported that mouse and human primary erythroid progenitor cells and erythroid cell lines synthesize and respond to Tumor Necrosis Factor-alpha (TNF-α). The nuclear transcriptional control complex, NFκlB is central in signaling downstream from TNF-α; so we began to study the function of NFκlB in erythroid cells. We made three very interesting initial findings: 1) first we found that NFκlB binding to DNA increased very slowly in HCD57 erythroid cells treated with erythropoietin (EPO, the hormone required for red blood cell development). An inhibitory effect of adding a neutralizing antibody to TNF-α on EPO-stimulated NFκlB DNA suggested this increase in NFκlB was due to TNF-α rather than direct EPO signaling. 2) We also found that NFκlB binding to DNA increased 10-fold or greater during erythroid differentiation. We found greatly increased NFκlB DNA binding in HCD57 cells that differentiated due to over-expression of JunB, F-MEL induced by DMSO, or human UT7-EPO or murine HCD57 cells induced to differentiate with hemin. 3) Surprisingly, we found that the NFκlB DNA binding complex in mouse primary erythroid cells and the erythroid cells lines tested was almost exclusively composed of the atypical p50/p50/Bcl3 NFκlB rather than the canonical p65/p50 or the non-canonical p65/p52 NFκlB. When we begin to study the biological significance of this atypical NFκlB in EPO-mediated erythroid differentiation in vivo using genetic tools, we found marked deficiencies in the development of erythroid cells in either the nfkb1−/− mice (p50−/−) or the bcl3 −/− mice. The nfkb1−/− mice were mildly anemic. The number of red blood cells in the circulation of these mice was statistically lower than in control mice. The number of CFU-e was also reduced in nfkb1−/− mice. Using the Ter-119 and CD71 staining method, we noted that proerythroblasts and immature erythroid cells increased and mature erythroblasts decreased in either non-anemic bone marrow or anemic spleens of nfkb1−/− mice. Forward scatter of Ter-119+ cells also showed an increased size of the average immature erythroid cell in the bone a marrow of nfkb1 −/− mice, suggesting a block in differentiation and continued cell cycling of the immature erythroblasts. Similar erythroid defects were observed in the spleens of anemic bcl-3−/− mice. nfkb1−/− mice and bcl-3−/− mice are also apparently unable to produce new reticulocytes as effectively as wild type mice after induction of anemia. Our working hypothesis is without expression of either p50 or Bcl-3 NFκlB proteins, immature erythroid cells continue to proliferate and ineffectively differentiate. In summary, the atypical p50/p50/Bcl-3 NFκlB complex appears necessary for maximal differentiation of immature erythroid cells.