Interferon-γ suppresses activin A/NF-E2 induction of erythroid gene expression through the NF-κB/c-Jun pathway.

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)-ß 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.

Basic fibroblast growth factor inhibits p38-mediated cell differentiation and growth inhibition by activin A but not by histone deacetylase inhibitors in CML cells.

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.

Activin A induces erythroid gene expressions and inhibits mitogenic cytokine-mediated K562 colony formation by activating p38 MAPK.

Activin A, a member of the transforming growth factor (TGF)-beta superfamily, is involved in the regulation of erythroid differentiation. Previous studies have shown that activin A inhibited the colony-forming activity of mouse Friend erythroleukemia cells, however, the mechanism remains unknown. First, we show herein that activin A induced the expression and activated the promoters of alpha-globin and zeta-globin in K562 cells, confirming that activin A induces erythroid differentiation in K562 cells. The p38 mitogen activated protein kinase (MAPK) inhibitor, SB203580, inhibited and the extracellular signal regulated kinase (ERK) inhibitor, PD98059, enhanced the expression and promoter activities of alpha-globin and zeta-globin by activin A, indicating that p38 MAPK and ERK are crucial for activin A-induced erythroid genes expression. Second, SB203580 inhibited the inhibitory effect of activin A on the colony-forming activity of K562 cells using the methylcellulose colony assay, indicating that activin A inhibits K562 colony formation by activating p38 MAPK. In addition, mitogenic cytokines SCF, IL-3, and GM-CSF induced colony formation of K562 cells that could be inhibited by PD98059 or enhanced by SB203580, respectively, indicating that these mitogenic cytokines induce K562 colony formation by activating ERK and inactivating p38 MAPK. Furthermore, activin A reduced the induction effect of these mitogenic cytokines on K562 colony formation in a dose-dependent manner. The inhibition of p38 MAPK reverted the inhibitory effect of activin A on mitogenic cytokine-mediated K562 colony formation. We conclude that activin A can regulate the same pathway via p38 MAPK to coordinate cell proliferation and differentiation of K562 cells.