Activin A is a member of the transforming growth factor (TGF)-beta superfamily that regulates cell proliferation and differentiation. Using the p38 inhibitor SB203580, our previous studies demonstrated that p38 was involved in activin A-mediated hemoglobin (Hb) synthesis in K562 cells. SB203580 is an inhibitor of p38alpha and p38beta isoforms. In this study, we show that p38alpha and p38beta mRNA were expressed in K562 cells and that activin A activated the kinase activities of these isoforms. To investigate the roles of p38alpha and p38beta isoforms in activin A-mediated erythroid differentiation, we generated stable clones that over-expressed the dominant negative p38 isoforms p38alpha(AF) and p38beta(AF) in K562 cells. The expressions of either p38alpha(AF) or p38beta(AF) reduced activin A-induced p38 activation, Hb synthesis, and zeta-globin promoter activity. Similarly, down-regulation of either p38alpha or p38beta by isoform-specific siRNAs also reduced activin A-induced zeta-globin promoter activity. Co-expressions of p38alpha(AF) and p38beta(AF), together, greatly inhibited the transcription activity of the zeta-globin promoter. Conversely, expression of mitogen-activated protein kinase kinase (MKK) 6b(E), a constitutive activator of p38, significantly activated zeta-globin promoter. Co-expressions of either p38alpha or p38beta with MKK6b had a similar activation of zeta-globin promoter. Activin A induction of erythroid differentiation was inhibited by follistatin. Activin A-induced phosphorylation of MKK6 and p38 was also inhibited by follistatin. Moreover, over-expression of MKK6b(E) reverted follistatin inhibition of activin A-induced zeta-globin promoter activity. These results demonstrate that activin A induces erythroid differentiation of K562 cells through activation of MKK6-p38alpha/p38beta pathway and follistatin inhibits those effects.
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.
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