Interferon (IFN)-γ is a proinflammatory cytokine that is linked to erythropoiesis inhibition and may contribute to anemia. However, the mechanism of IFN-γ-inhibited erythropoiesis is unknown. Activin A, a member of the transforming growth factor (TGF)-β superfamily, induces the erythropoiesis of hematopoietic progenitor cells. In this study, a luciferase reporter assay showed that IFN-γ suppressed activin A-induced ζ-globin promoter activation in K562 erythroblast cells in a dose-dependent manner. Activin A reversed the suppressive effect of IFN-γ on the luciferase activity of ζ-globin promoter in a dose-dependent manner. IFN-γ also suppressed the activation of activin A-induced α-globin promoter. IFN-γ reduced the mRNA expression of α-globin, ζ-globin, NF-E2p45, and GATA-1 induced by activin A. The results also showed that IFN-γ induced c-Jun expression when NF-κBp65 and c-Jun bound to two AP-1-binding sites on the c-Jun promoter. The luciferase activity of α-globin and ζ-globin promoters were enhanced by wild-type c-Jun and eliminated by dominant-negative (DN) c-Jun. The suppressive effects of IFN-γ on the mRNA expression of α-globin and ζ-globin were absent in cells expressing DN c-Jun. The ability of NF-E2 to enhance activin A-induced ζ-globin promoter activation decreased when c-Jun was present, and IFN-γ treatment further enhanced the decreasing effect of c-Jun. Chromatin immunoprecipitation revealed that NF-E2p45 bound to the upstream regulatory element (HS-40) of the α-globin gene cluster in response to activin A, whereas c-Jun eliminated this binding. These results suggest that IFN-γ modulates NF-κB/c-Jun to antagonize activin A-mediated NF-E2 transcriptional activity on globin gene expression.
The p38 mitogen-activated protein kinase (p38) is involved in multiple cellular functions such as cell proliferation and differentiation. Previously, we found that activin A mediated hemoglobin synthesis and cell growth inhibition through p38, whereas, basic fibroblast growth factor (bFGF) inactivated p38 to antagonize the activin A effects. In this study, we selected three structurally different histone deacetylase (HDAC) inhibitors, apicidin, MS275, and sodium butyrate that activate p38, to probe the signal pathway from activin A to p38 in chronic myeloid leukemia (CML)-derived K562 cells. HDAC inhibitors and activin A showed additive p38 phosphorylation. The enhanced phosphorylation of p38 was correlated with increased cell differentiation and decreased cell proliferation. The use of p38 inhibitor SB203580 in conjunction with activin A or with the HDAC inhibitors inhibited cell differentiation and restored cell proliferation, indicating that activin A and the HDAC inhibitors exert their effects through p38 activation. However, bFGF did not affect HDAC inhibitors-induced cell differentiation or growth inhibition. Western blots showed that p38 phosphorylation remained at similar levels with or without bFGF in the presence of HDAC inhibitors. Thus, the HDAC inhibitors activate p38 in a manner different from the activin A pathway. Furthermore, mRNA expressions for activin type I, IB, II, and IIB receptors remained constant in the presence of activin A, bFGF, or both activin A and bFGF. These results indicate that bFGF does not directly act on p38 nor on the mRNA expression levels of activin receptors but inhibit activin A activation of p38 upstream of p38 in K562 cells.
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