Extracellular Signal-regulated Kinase/90-kDa Ribosomal S6 Kinase/Nuclear Factor-κB Pathway Mediates Phorbol 12-Myristate 13-Acetate-induced Megakaryocytic Differentiation of K562 Cells

Abstract

Two signaling pathways, the extracellular signal-regulated kinase (ERK)/mitogen-activated protein kinase (MAPK)-dependent pathway and the nuclear factor-B (NF-B)-dependent pathway, have been known to mediate megakaryocytic differentiation of K562 cells induced by phorbol 12-myristate 13-acetate (PMA). In this study, we examined whether 90-kDa ribosomal S6 kinase (RSK), known as a substrate of ERK/MAPK and a signal-inducible IB␣ kinase, would link two pathways during the differentiation. RSK1 was activated in a time-and dosedependent manner during the PMA-induced differentiation. Overexpression of wild-type or dominant inhibitory mutant (D205N) of RSK1 enhanced or suppressed PMAstimulated NF-B activation and megakaryocytic differentiation as shown by morphology, nonspecific esterase activity, and expression of the CD41 megakaryocytic marker, respectively. In addition, overexpression of the dominant inhibitory mutant (S32A/S36A) of IB␣ inhibited PMA-stimulated and RSK1-enhanced megakaryocytic differentiation, indicating that NF-B mediates a signal for megakaryocytic differentiation downstream of RSK1. PMA-stimulated activation of ERK/MAPK, RSK1, and NF-B and the PMA-induced megakaryocytic differentiation were prevented by pretreatment with PD98059, a specific inhibitor of the mitogen-activated ERK kinase (MEK). Therefore, these results demonstrate that the sequential ERK/RSK1/NF-B pathway mediates PMA-stimulated megakaryocytic differentiation of K562 cells.Hematopoiesis in vertebrates, which is a complicated multistep process, is a paradigm for the development of diverse specialized cell types from multipotent progenitors. How specific signaling pathways control hematopoiesis is a major focus in the area of hematopoiesis. A widely used model system for studying early steps in megakaryocytic differentiation of hematopoietic stem cells is PMA 1 -induced differentiation of K562 cells (1). It has been well documented that K562 cells, human chronic myelogenous leukemic cells carrying Philadelphia chromosome (2), can be induced by PMA, a protein kinase C activator, to differentiate into cells with megakaryocytic characteristics (3-6). These include changes in cell morphology and adhesive properties, cell growth arrest, and expression of markers associated with the megakaryocytes. Despite recent advances in studying the molecular events associated with PMA-induced megakaryocytic differentiation of K562 cells, the exact signaling mechanism is not fully understood. We have previously shown that PMA-induced differentiation of K562 cells is inhibited by the treatment with pyrrolidine dithiocarbamate, an inhibitor of NF-B (7), and NF-B subunit-transfected cells are more sensitive to PMA-induced differentiation than their parental cells, and PMA-induced differentiation was enhanced by the pretreatment with IB␣ antisense oligonucleotide (8). In contrast, it has been reported recently that overexpression of the constitutively active mutants of MEK induce megakaryocytic differentiation in K562 cells, and blockade of MEK activatio...

Signaling Events Involved in Macrophage Chemokine Expression in Response to Monosodium Urate Crystals

“…and STAT1␣ Tyr 701 in interferon-␥-stimulated M (34). Based on these data and in agreement with other independent studies (35)(36)(37)(38), subtoxic and specific concentrations of inhibitors against the MEK1/2-ERK1/2 pathway were selected to perform the experiments presented below. As depicted in Fig.…”

A survey of the signaling pathways involved in megakaryocytic differentiation of the human K562 leukemia cell line by molecular and c-DNA array analysis

“…K562 cells can undergo further differentiation in both megakaryocytic and erythroid lineages depending on the stimulus. Phorbol esters such as PMA-stimulated megakaryocytic differentiation (Shelly et al, 1998;Kim et al, 2001;Dorsey et al, 2002;Pettiford and Herbst, 2003), whereas hemin, hydroxyurea, Ara-C or as reported more recently imatinib mesylate (gleevec) induced erythroid differentiation of this cell line (Belhacene et al, 1998;Park et al, 2001;Jacquel et al, 2003;Kawano et al, 2004a, b). Megakaryocytic differentiation of K562 cells induced by PMA mimics, in part, the physiologic process that takes place in the bone marrow in response to a variety of stimuli (Long et al, 1990).…”

90-kDa Ribosomal S6 Kinase Is a Direct Target for the Nuclear Fibroblast Growth Factor Receptor 1 (FGFR1)

“…The present findings expand the role of nuclear FGFR1 to include activation of RSK1, which in turn activates CREB. Nuclear RSK phosphorylates and activates a number of additional transcriptional factors including ER81 (53), c-Fos (27, 28), C/EBP␤ (29), estrogen receptor (31), and IB␣ (30,54). Transcriptional factors that, similar to CREB, are regulated by nuclear FGFR1 include AP1 and NFB.…”