The Role of CD69 in TNFalpha-Inhibited Erythroid Differentiation

The erythroid differentiation of hematopoietic progenitor cells is induced by erythropoietin (EPO). The proinflammatory cytokine tumor necrosis factor alpha (TNFα) plays a physiological role in cell proliferation, apoptosis, inflammation and tumorigenesis. Anemia is considered as a common symptom induced by inflammation, cancer and hematopoietic diseases. Inflammation, cancer and hematopoietic disease related anemia were shown to be induced by TNFα. However, the molecular mechanism of TNFα-inhibited erythroid differentiation in hematopoietic progenitor cells has not been elucidated. EPO was used in the treatment of inflammation, cancer and hematopoietic disease related anemia. However, the serious side effects and resistance of EPO were recently described in distinct studies. Indeed, several studies showed the increased mortality came from accelerated progression of cancer. Unexpectedly, the use of EPO in patients might increase the risk of cancer-associated death. Thus, the molecular mechanisms of TNFα-inhibited erythroid differentiation need to be further elucidated in order to identify potential target molecules for inflammation and cancer as well as hematopoietic disease related anemia. Our preliminary results revealed that TNFα suppressed EPO-induced erythorid differentiation and α-globin promoter in hematopoietic progenitor cells TF-1. These two cytokines can affect the regulation of erythroid differentiation in TF-1 cells may be used as a tool to understand the molecular mechanisms of TNFα-inhibited erythroid differentiation. We have used the DNA microarray analysis to screen for genes involved in TNFα-inhibited erythroid differentiation. We found one gene, CD69, was down-regulated by EPO; the expression level and promoter activity of CD69 were up-regulated by the combination of TNFα with EPO. CD69 is a cell surface receptor rapidly induced after leukocyte activation and plays a key modulator of inflammatory responses. Our preliminary results revealed that the binding of NF-κB p65 to the CD69 promoter induced by TNFα in TF-1 cells was demonstrated by the chromatin immunoprecipitation (ChIP) assay. Furthermore, EPO-induced α-globin promoter activity was reduced in CD69 over-expressing cells. CD69 over-expression induced tyrosine phosphorylated signals in TF-1 cells. The tyrosine phosphorylated signals were further enhanced by CD69 cross-linking with CD69 antibody. Taken together, these results imply that CD69 may be played a novel role in TNFα-inhibited erythroid differentiation. In this proposal, we will study the role of CD69 in TNFα-inhibited erythroid differentiation. The objectives of this program project are: (1) to identify the role of CD69 in TNFα-inhibited erythroid differentiation (2) to study the molecular mechanism of CD69-inhibited erythroid differentiation (3) to delineate how TNFα and EPO regulate CD69 gene expression