Activin A induction of erythroid differentiation through MKK6‐p38α/p38β pathway is inhibited by follistatin

Abstract

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.

Activin A induction of erythroid differentiation sensitizes K562 chronic myeloid leukemia cells to a subtoxic concentration of imatinib

“…Cells were cultured in RPMI 1640 medium supplemented with 10% fetal bovine serum, 2 mM L-glutamine, 100 U/ml penicillin, and 100 g/ml streptomycin in a 5% CO 2 incubator at 37°C. K562/p38␣(AF)1 and K562/p38␤(AF)1 cells stably expressing dominant-negative (AF) forms of p38␣ and p38␤, respectively, in K562 cells are described elsewhere (27). The medium used to maintain K562/p38␣(AF)1 and K562/p38␤(AF)1 cells was the same as that used to maintain parental K562 cells, except for inclusion of 200 g/ml G418 in the medium.…”

“…Our previous studies showed that activin A induced erythroid differentiation through p38␣ and p38␤ (27). K562/p38␣(AF)1 and K562/p38␤(AF)1 clones stably expressing dominant-negative p38␣ and p38␤, respectively, which we previously established and characterized (27), were used for our next study.…”

“…We and others previously reported that activin A induced the erythroid differentiation of CML cells (10,25). In addition, our previous study showed that activin A induces erythroid differentiation of CML cells through activation of the MAPK kinase 6 (MKK6)-p38 MAPK pathway (27).…”

MPT0B169 and MPT0B002, New Tubulin Inhibitors, Induce Growth Inhibition, G2/M Cell Cycle Arrest, and Apoptosis in Human Colorectal Cancer Cells

“…Total cell extracts were prepared, and Western blotting was performed as previously described [12]. Equal amounts of protein were resolved using sodium dodecyl sulfate polyacrylamide gel electrophoresis.…”

MPT0B169, a New Tubulin Inhibitor, Inhibits Cell Growth and Induces G2/M Arrest in Nonresistant and Paclitaxel-Resistant Cancer Cells

“…Total cell extracts were prepared as described [16]. Protein lysate (30 μg) was resolved using SDS-PAGE, and the protein bands were transferred to a PVDF membrane (Millipore, Bedford, Mass., USA).…”