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• W2040736557 abstract "Two signaling pathways, the extracellular signal-regulated kinase (ERK)/mitogen-activated protein kinase (MAPK)-dependent pathway and the nuclear factor-κB (NF-κB)-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 IκBα kinase, would link two pathways during the differentiation. RSK1 was activated in a time- and dose-dependent manner during the PMA-induced differentiation. Overexpression of wild-type or dominant inhibitory mutant (D205N) of RSK1 enhanced or suppressed PMA-stimulated NF-κB 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 IκBα inhibited PMA-stimulated and RSK1-enhanced megakaryocytic differentiation, indicating that NF-κB mediates a signal for megakaryocytic differentiation downstream of RSK1. PMA-stimulated activation of ERK/MAPK, RSK1, and NF-κB 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-κB pathway mediates PMA-stimulated megakaryocytic differentiation of K562 cells. Two signaling pathways, the extracellular signal-regulated kinase (ERK)/mitogen-activated protein kinase (MAPK)-dependent pathway and the nuclear factor-κB (NF-κB)-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 IκBα kinase, would link two pathways during the differentiation. RSK1 was activated in a time- and dose-dependent manner during the PMA-induced differentiation. Overexpression of wild-type or dominant inhibitory mutant (D205N) of RSK1 enhanced or suppressed PMA-stimulated NF-κB 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 IκBα inhibited PMA-stimulated and RSK1-enhanced megakaryocytic differentiation, indicating that NF-κB mediates a signal for megakaryocytic differentiation downstream of RSK1. PMA-stimulated activation of ERK/MAPK, RSK1, and NF-κB 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-κB 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 1The abbreviations used are:PMAphorbol 12-myristate 13-acetateNF-κBnuclear factor-κBERKextracellular signal-regulated kinaseMAPKmitogen-activated protein kinaseRSK90-kDa ribosomal S6 kinasePBSphosphate-buffered salinePMSFphenylmethylsulfonyl fluorideMOPSmorpholinepropanesulfonic acidGPglycoprotein-induced differentiation of K562 cells (1Alitalo R. Leuk. Res. 1990; 14: 501-514Crossref PubMed Scopus (123) Google Scholar). It has been well documented that K562 cells, human chronic myelogenous leukemic cells carrying Philadelphia chromosome (2Lozzio C.B. Lozzio B.B. Blood. 1975; 45: 321-334Crossref PubMed Google Scholar), can be induced by PMA, a protein kinase C activator, to differentiate into cells with megakaryocytic characteristics (3Colamonici O.R. Trepel J.B. Neckers L.M. Cytometry. 1985; 6: 591-596Crossref PubMed Scopus (12) Google Scholar, 4Sutherland J.A. Turner A.R. Mannoni P. McGann L.E. Turc J.M. J. Biol. Response Modif. 1986; 5: 250-262PubMed Google Scholar, 5Tabilio A. Pelicci P.G. Vinci G. Mannoni P. Civin C.I. Vainchenker W. Testa U. Lipinski M. Rochant H. Breton-Gorius J. Cancer Res. 1983; 43: 4569-4574PubMed Google Scholar, 6Tetteroo P.A. Massaro F. Mulder A. Schreuder van Gelder R. von dem Borne A.E. Leuk. Res. 1984; 8: 197-206Crossref PubMed Scopus (153) Google Scholar). 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-κB (7Kang C.D. Lee B.K. Kim K.W. Kim C.M. Kim S.H. Chung B.S. Biochem. Biophys. Res. Commun. 1996; 221: 95-100Crossref PubMed Scopus (25) Google Scholar), and NF-κB subunit-transfected cells are more sensitive to PMA-induced differentiation than their parental cells, and PMA-induced differentiation was enhanced by the pretreatment with IκBα antisense oligonucleotide (8Kang C.D. Han C.S. Kim K.W. Do I.R. Kim C.M. Kim S.H. Lee E.Y. Chung B.S. Cancer Lett. 1998; 132: 99-106Crossref PubMed Scopus (20) Google Scholar). 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 activation by PD98059 reverses both the growth arrest and the morphological changes of K562 cells induced by PMA treatment (9Whalen A.M. Galasinski S.C. Shapiro P.S. Nahreini T.S. Ahn N.G. Mol. Cell. Biol. 1997; 17: 1947-1958Crossref PubMed Scopus (203) Google Scholar, 10Herrera R. Hubbell S. Decker S. Petruzzelli L. Exp. Cell Res. 1998; 238: 407-414Crossref PubMed Scopus (89) Google Scholar, 11Racke F.K. Lewandowska K. Goueli S. Goldfarb A.N. J. Biol. Chem. 1997; 272: 23366-23370Abstract Full Text Full Text PDF PubMed Scopus (163) Google Scholar). This suggested that a sustained activation of the ERK/MAPK pathway is necessary to induce a differentiation program along the megakaryocyte lineage in K562 cells.In our previous study (8Kang C.D. Han C.S. Kim K.W. Do I.R. Kim C.M. Kim S.H. Lee E.Y. Chung B.S. Cancer Lett. 1998; 132: 99-106Crossref PubMed Scopus (20) Google Scholar), although there were no differences in the basal or PMA-stimulated activities of ERK/MAPK between the parental and NF-κB subunit-transfected K562 cells, PMA-induced differentiation was almost completely inhibited in both parental and NF-κB subunit-transfected cells after preventing PMA-stimulated activation of MAP kinase by the pretreatment of PD98059. Therefore, it is likely that the MAP kinase pathway is required for the PMA-induced megakaryocytic differentiation of K562 cells, suggesting that activation of MAP kinase works through NF-κB activation, or activation of both NF-κB and MAP kinase pathways are involved.Two recent reports have shown that the 90-kDa ribosomal S6 kinase (RSK1) is an essential kinase required for phosphorylation and subsequent degradation of IκBα in response to mitogens, including PMA (12Ghoda L. Lin X. Greene W.C. J. Biol. Chem. 1997; 272: 21281-21288Crossref PubMed Scopus (181) Google Scholar, 13Schouten G.J. Vertegaal A.C. Whiteside S.T. Israel A. Toebes M. Dorsman J.C. van der Eb A.J. Zantema A. EMBO J. 1997; 16: 3133-3144Crossref PubMed Scopus (205) Google Scholar). Therefore, RSK1 is a good candidate to connect the ERK/MAPK pathway and the NF-κB pathway in PMA-induced megakaryocytic differentiation of K562 cells. In this study, the role of RSK1 in PMA-induced megakaryocytic differentiation of K562 cells was investigated.MATERIALS AND METHODSCell CultureK562 cells were grown in suspension in RPMI 1640 medium (Life Technologies, Inc.) supplemented with 10% (v/v) heat-inactivated fetal bovine serum (Life Technologies, Inc.), 100 units/ml penicillin, and 100 μg/ml streptomycin (Sigma). For the experiment, exponentially growing K562 cells were collected (1,200 × g, 10 min) and resuspended in fresh culture medium.Ribosomal S6 Kinase AssayThe S6 kinase assays were performed using S6 kinase Assay Kit from Upstate biotechnology, Inc., according to manufacturer's protocol. Cells (1 × 106/ml) were harvested, washed twice in ice-cold PBS, and then scraped into 150 μl of lysis buffer (20 mm Tris, pH 7.4, 150 mm NaCl, 5 mm EDTA, 1 mmEGTA, 1% Triton X-100, 2.5 mm sodium pyrophosphate, 1 mm β-glycerophosphate, 1 mmNa3Vo4, 1 μg/ml leupeptin, and 1 mm PMSF). After incubation for 30 min at 4 °C, lysates were centrifuged at 20,000 × g for 20 min at 4 °C. Cell lysates (200 μg of protein) were mixed with 2 μg of anti-p90RsK1 antibody (rabbit polyclonal IgG) and then immunoprecipitated with gentle rocking overnight at 4 °C. The immunocomplexes were collected by protein A-Sepharose beads for 3 h at 4 °C.For in vitro kinase assay, the immunoprecipitates/protein A-Sepharose beads were washed once with ice-cold cell lysis buffer and three times with an assay dilution buffer (ADB: 20 mm MOPS, pH 7.2, 25 mm β-glycerol phosphate, 5 mmEGTA, 1 mm sodium orthovanadate, and 1 mmdithiothreitol). The immunoprecipitates/protein A-Sepharose beads were then resuspended in ADB containing substrate mixture (50 μm substrate peptide), inhibitor mixture (4 μm protein kinase C inhibitor peptide, 0.4 μm protein kinase A inhibitor peptide, and 4 μm Compound 24571 in ADB), and [γ-32P]ATP mixture. The reaction mixture was incubated for 10 min at 30 °C, and then 20 μl of the reaction mixture was spotted onto p81 phosphocellulose squares (1.5 × 1.5 cm, Upstate biotechnology, Inc.). The papers were washed five times for 5 min each with 0.75% phosphoric acid and once for 3 min with acetone. Dried papers were wrapped, and radioactivity was detected with a Molecular Imager (GS-525, Bio-Rad).Plasmids and DNA TransfectionpcDNA3-Rsk1, pcDNA3-Rsk1- D205N, and pCMV-IκBα-32A36A (kindly donated by Dr. Zantema, Leiden University, Netherlands) have been described elsewhere (13Schouten G.J. Vertegaal A.C. Whiteside S.T. Israel A. Toebes M. Dorsman J.C. van der Eb A.J. Zantema A. EMBO J. 1997; 16: 3133-3144Crossref PubMed Scopus (205) Google Scholar). pcDNA3-Rsk1-D205N has a mutated ATP binding site of the amino-terminal kinase domain of RSK1, and pCMV-IκBα-S32AS36A contains mutated alanines at the serine phosphorylation sites. pCMV-p50 and -p65 are expression plasmids in which the p50 and p65 subunits of NF-κB were cloned inHindIII/XbaI of pRc/CMV (Invitrogen), respectively (8Kang C.D. Han C.S. Kim K.W. Do I.R. Kim C.M. Kim S.H. Lee E.Y. Chung B.S. Cancer Lett. 1998; 132: 99-106Crossref PubMed Scopus (20) Google Scholar). Electroporation was carried out using an Electroporator II (Invitrogen) at 1,500 V/cm, 50 microfarads condition. After electroporation, stable transformants were selected with G418 (475 μg/ml) and identified by Western blot analysis using polyclonal antibody against RSK1 (Upstate biotechnology, Inc.) and IκBα (Santa Cruz Biotechnology, Inc., Santa Cruz, CA).Preparation of Nuclear Extracts and Electrophoretic Mobility Shift AssaysNuclear extracts were prepared as described previously (8Kang C.D. Han C.S. Kim K.W. Do I.R. Kim C.M. Kim S.H. Lee E.Y. Chung B.S. Cancer Lett. 1998; 132: 99-106Crossref PubMed Scopus (20) Google Scholar). 2 × 106 cells were harvested and washed with cold phosphate-buffered saline and resuspended in 50 μl of lysis buffer (10 mm HEPES, pH 7.9, 1.5 mmMgCl2, 10 mm KCl, 0.5 mmdithiothreitol, and 0.5 mm PMSF). The cells were allowed to be swollen on ice for 10 min, after which the cells were resuspended in 30 μl of lysis buffer containing 0.05% Nonidet P-40. Then the tube was vigorously mixed on a vortex machine three times for 10 s, and the homogenate was centrifuged at 250 × g for 10 min to pellet the nuclei. The nuclear pellet was resuspended in 40 μl of ice-cold nuclear extraction buffer (5 mm HEPES, pH 7.9, 26% glycerol (v/v), 1.5 mm MgCl2, 0.2 mm EDTA, 0.5 mm dithiothreitol, and 0.5 mm PMSF), incubated on ice for 30 min with intermittent mixing, and centrifuged at 24,000 × g for 20 min at 4 °C. The nuclear extract was either used immediately or stored at −70 °C for later use.Electrophoretic mobility shift assay was performed by incubating 10 μg of nuclear extract for 20 min at room temperature with 17.5 fmol of 32P-end labeled 22-mer double-stranded NF-κB oligonucleotide (5′-AGT TGA GGG GAT TTT CCC AGG C-3′) from the κ light chain enhancer (15Lenardo M.J. Baltimore D. Cell. 1989; 58: 227-229Abstract Full Text PDF PubMed Scopus (1253) Google Scholar) according to the manufacturer's manual (Promega, Co.). For gel mobility supershift assay, 1 μg of anti-p50 antibody was incubated with whole cell extracts for 20 min at 21 °C prior to NF-κB binding reaction.Nonspecific Esterase StainingThe cytocentrifuged slides were stained with a nonspecific esterase staining kit (Muto Pure Chemicals, Ltd., Tokyo, Japan) according to manufacturer's protocol. Nonspecific esterase-stained slides were examined microscopically for intracellular dark red granules.Kinase Assays for MAP Kinase/ERK ActivityMAP kinase activity was measured with a p44/42 MAP Kinase Assay Kit (New England Biolabs, Inc.) according to manufacturer's protocol. Cells were lysed in buffer containing 20 mm Tris, pH 7.5, 150 mm NaCl, 1 mm EDTA, 1 mm EGTA, 1% Triton X-100, 2.5 mm sodium pyrophosphate, 1 mmβ-glycerophosphate, 1 mm Na3VO4, 1 μg/ml leupeptin, and 1 mm PMSF for 15 min at 4 °C. 200 μl of cell lysate (∼200 μg of protein) were mixed with monoclonal phospho-MAPK antibody (1:50 dilution) and incubated with gentle rocking for overnight at 4 °C. Immunoprecipitates were collected by protein A-Sepharose beads (10–20 μl) for 2 h at 4 °C. Beads were washed twice with cold lysis buffer and twice with 500 μl of kinase buffer (20 mm Tris, pH 7.5, 5 mm β-glycerophosphate, 2 mm dithiothreitol, 0.1 mm Na3VO4, and 10 mm MgCl2). Kinase assay was performed by incubating the suspended pellet with kinase buffer containing 100 μm ATP and GST-Elk1 fusion protein for 30 min at 30 °C. The samples were analyzed by 12% SDS-polyacrylamide gel electrophoresis. Phospho(Ser383)-Elk1 was detected with specific antibody using Western blot analysis.Flow Cytometric AnalysisCells were harvested after treatment with PMA for 72 h and washed in PBS with 1% bovine serum albumin (PBS-B). 2–5 × 105 cells were incubated at 30 °C in 100 μl of PBS-B containing 10 μl anti-CD41-PE or anti-CD61-FITC (Becton-Dickinson, Mountain View, CA). Cells were washed in PBS-B, resuspended in PBS with 1% glycerol, and analyzed with a fluorescence-activated cell sorter using CellQuest 8.6 (Becton-Dickinson, Mountain View, CA).RESULTSActivation of RSK1 after Treatment of PMAAs we mentioned previously, the ribosomal S6 kinase (RSK1) is a good candidate connecting the ERK/MAPK pathway and the NF-κB pathway, both of which are known to be involved in PMA-induced megakaryocytic differentiation of K562 cells. Therefore, we examined whether RSK1 could be activated by PMA in K562 cells.To measure the PMA-stimulated RSK1 activity, an immune complex kinase assay was done. When K562 cells were treated with various concentrations of PMA for 1 h, RSK1 was activated in a dose-dependent manner up to 10 nm of PMA (Fig.1 A). After treatment with 10 nm PMA for the indicated time (Fig. 1 B), a sustained activation of RSK1 was shown over at least 12 h with ∼10-fold activation at 1 h peak time. This result is consistent with sustained activation of the ERK/MAPK during PMA-induced megakaryocytic differentiation of K562 cells (11Racke F.K. Lewandowska K. Goueli S. Goldfarb A.N. J. Biol. Chem. 1997; 272: 23366-23370Abstract Full Text Full Text PDF PubMed Scopus (163) Google Scholar).Isolation of Wild-type or Dominant Inhibitory Mutant of RSK1-transfected K562 CellsTo clarify the discrepancy between our previous study showing that activation of NF-κB mediates the PMA-induced differentiation of K562 cells (8Kang C.D. Han C.S. Kim K.W. Do I.R. Kim C.M. Kim S.H. Lee E.Y. Chung B.S. Cancer Lett. 1998; 132: 99-106Crossref PubMed Scopus (20) Google Scholar) and other studies showing that a sustained activation of the ERK/MAPK pathway is required for induction of differentiation along the megakaryocyte lineage in K562 cells (9Whalen A.M. Galasinski S.C. Shapiro P.S. Nahreini T.S. Ahn N.G. Mol. Cell. Biol. 1997; 17: 1947-1958Crossref PubMed Scopus (203) Google Scholar, 10Herrera R. Hubbell S. Decker S. Petruzzelli L. Exp. Cell Res. 1998; 238: 407-414Crossref PubMed Scopus (89) Google Scholar, 11Racke F.K. Lewandowska K. Goueli S. Goldfarb A.N. J. Biol. Chem. 1997; 272: 23366-23370Abstract Full Text Full Text PDF PubMed Scopus (163) Google Scholar), K562 cells were transfected with wild-type RSK1 or a dominant inhibitory mutant (D205N) of RSK1 and then stable transformants were isolated. After stimulation of PMA, K562-RSK1 or K562-RSK1 (D205N) cells have higher or lower RSK1 activity compared with parental K562 cells, respectively (Fig.2 A). During the transfection of wild-type RSK1, we observed some adherent cells with a morphological resemblance to the PMA-stimulated population (Fig.2 B), suggesting that the RSK1-dependent pathway might be involved in megakaryocytic differentiation of K562 cells. Practically, these cells, which may be high expressors of RSK1, could not be expanded due to a severely retarded growth rate. On the other hand, all isolated RSK1 transfectants showed no or very weak evidence of differentiation, such as morphological change, nonspecific esterase activity, or expression of surface markers (see Figs 5 and7 B). Empty vectors did not affect the PMA-induced megakaryocytic differentiation of K562 cells (data not shown).Figure 2Isolation of wild-type or dominant inhibitory mutant (D205N) of RSK1-transfected K562 cells. A, comparison of RSK1 activities among parental and wild-type or dominant negative inhibitory mutant (D205N) of RSK1-transfected K562 cells. Of the isolated clones, high expressors of each insert were selected, and their RSK1 activities were compared with that of parental K562 cells after treatment of PMA for 1 h. B, adhesion and spreading of K562 cells following transfection with wild-type RSK1. Cells in the culture dish were visualized by inverted microscopy.View Large Image Figure ViewerDownload (PPT)Figure 5Effect of RSK1 and NF-κB activity on the PMA-induced megakaryocytic differentiation of K562 cells. The parental, RSK1-transfected, RSK1 (D205N)-transfected, IκBα (S32A/S36A)-transfected, or RSK1/IκBα (S32A/S36A)-transfected K562 cells were cultured in the absence (left panels) or presence (right panels) of 10 nm PMA. After 3 days cells in the culture dish were photographed for morphological assay (A), and cytospin preparations were stained to examine nonspecific esterase activity (B). C, adhesion and spreading of K562 cells following transfection with both subunits of NF-κB. After 72 h of transfection, cells in culture dish were visualized by inverted microscopy.View Large Image Figure ViewerDownload (PPT)Figure 7Flow cytometric analysis of the effect of overexpression of RSK1, dominant inhibitory mutants of RSK1 or IκBα, and PD98059 on CD41 expression. To analyze the effect of overexpression of RSK1, dominant inhibitory mutants of RSK1 or IκBα on CD41 expression, the parental (A), RSK1-transfected (B), RSK1 (D205N)-transfected (C), IκBα (S32A/S36A)-transfected (D), or RSK1/IκBα (S32A/S36A)-transfected (E) cells were cultured in the absence (thin solid lines) or presence (thick solid lines) of 10 nm PMA for 3 days. For analysis of the effect of PD98059 on CD41 expression (F), the parental (thin solid line) and RSK1-transfected (thick solid line) cells were treated as described in the legend of Fig. 6. Dashed lines show isotype controls.View Large Image Figure ViewerDownload (PPT)Effect of Wild-type or Dominant Inhibitory Mutant of RSK1 on PMA-induced Activation of NF-κB in K562 CellsSince we have suggested that PMA-induced megakaryocytic differentiation of K562 cells is dependent on the NF-κB pathway (8Kang C.D. Han C.S. Kim K.W. Do I.R. Kim C.M. Kim S.H. Lee E.Y. Chung B.S. Cancer Lett. 1998; 132: 99-106Crossref PubMed Scopus (20) Google Scholar), a NF-κB mobility shift assay was done to examine whether NF-κB activity of K562 cells would be modulated after transfection with wild-type RSK1 or the dominant inhibitory mutant (D205N) of RSK1. Overexpression of RSK1 or RSK1 (D205N) enhanced or suppressed PMA-stimulated NF-κB activity, respectively (Fig. 3, upper panel). Consistently, degradation of IκBα was enhanced or suppressed by overexpression of RSK1 or RSK1 (D205N) (Fig. 3,lower panel). These results are consistent with the RSK1 activities of K562-RSK1 and K562-RSK1 (D205N) cells (Fig.2 A), indicating that activity of NF-κB could be modulated downstream of RSK1 in K562 cells.Figure 3Effect of wild-type or dominant inhibitory mutant of RSK1 on PMA-induced activation of NF-κB in K562 cells. Upper panel, activities of NF-κB after treatment with PMA in the parental and wild-type or dominant negative inhibitory mutant (D205N) of RSK1-transfected K562 cells. Nuclear extracts were prepared after treatment of PMA for 1 h, and 10 μg of nuclear extracts were used for mobility shift assay. For supershift assay (last lane), anti-p50 antibody was incubated with nuclear extracts prior to the NF-κB binding reaction. Lower panel, Western blot analysis of the cytosolic IκBα level after treatment of PMA.View Large Image Figure ViewerDownload (PPT)The Effect of RSK1 and NF-κB Activities on the PMA-induced Megakaryocytic Differentiation of K562 CellsIf RSK1 mediates PMA-induced megakaryocytic differentiation of K562 cells via NF-κB activation, inhibition of NF-κB should lead to blocking of RSK1-mediated NF-κB activation and differentiation of K562 cells induced by PMA. To address this hypothesis, we isolated a double transfectant, which expresses both a dominant inhibitory mutant of IκBα and a wild-type RSK1 as much as each single transfectant (Fig.4 A). K562 and K562-IκBα (S32A/S36A) cells had comparable activities of RSK1, and K562-RSK1 and K562-RSK1/IκBα (S32A/S36A) cells had comparable activities of RSK1 (Fig. 4 B). As shown in Fig. 4 C, overexpression of IκBα (S32A/S36A) inhibited both PMA-stimulated NF-κB activation and RSK1-mediated NF-κB activation.Figure 4Effect of dominant inhibitory mutant (S32A/S36A) of IκBα on PMA-stimulated and RSK1-mediated activation of NF-κB. A, isolation of IκBα (S32A/S36A) or RSK1/IκBα (S32A/S36A)-transfected K562 cells. A double transfectant, which expressed both a dominant inhibitory mutant of IκBα (upper panel) and RSK1 (lower panel) as much as each single transfectant, was isolated. B,comparison of RSK1 activities. The parental K562 cells and transfectants were treated with PMA for 1 h, and the S6 kinase assay was done as described under “Materials and Methods.”C, effect of dominant inhibitory mutant (S32A/S36A) of IκBα on PMA-stimulated and RSK1-mediated activation of NF-κB. NF-κB DNA binding activity was assayed as described in the legend of Fig. 3.View Large Image Figure ViewerDownload (PPT)Then, we have examined the effect of RSK1 and NF-κB activities on PMA-induced megakaryocytic differentiation of K562 cells. PMA-induced differentiation of K562 cells was enhanced or suppressed by overexpression of RSK1 or RSK1 (D205N), respectively, as shown by morphology (Fig. 5 A) and nonspecific esterase assay (Fig. 5 B), indicating that PMA-induced differentiation of K562 cells could be affected by RSK1 activity. Overexpression of IκBα (S32A/S36A) prevented morphological change and activation of nonspecific esterase induced by PMA and also inhibited RSK1-mediated differentiation of K562 cells (Fig. 5, A and B). In addition, when K562 cells were transfected transiently with both subunits of NF-κB, some cells underwent morphological changes and were adherent to the culture dish (Fig. 5 C). These results demonstrate that the RSK/NF-κB pathway mediates PMA-induced megakaryocytic differentiation of K562 cells.Effect of ERK Activity on RSK1 Activation and RSK1-mediated NF-κB Activation of K562 CellsIf the sequential ERK/RSK/NF-κB pathway plays a role in PMA-induced megakaryocytic differentiation of K562 cells, pretreatment with PD98059, a specific inhibitor of MEK that prevents the activation of MEK and subsequently the activation of ERK/MAPK, would lead to prevention of the activation of the ERK/RSK/NF-κB pathway and subsequent megakaryocytic differentiation of K562 cells induced by PMA. When PMA-stimulated activation of ERK was prevented by the pretreatment with PD98059 in K562 and K562-RSK1 cells (Fig. 6 A), PMA-stimulated activation of RSK1 and NF-κB was also prevented (Fig. 6, Band C, respectively), indicating that activation of RSK1 and NF-κB depends on ERK/MAPK activation. In addition, PMA-induced megakaryocytic differentiation was prevented by the pretreatment with PD98059 in both K562 and K562-RSK1 cells (Fig. 6 D). From these results, it could be concluded that PMA-stimulated activation of RSK1 and NF-κB is ERK-dependent, and sequential activation of ERK, RSK1, and NF-κB mediates PMA-induced megakaryocytic differentiation of K562 cells.Figure 6Effect of PD98059 on RSK1 and NF-κB activities and PMA-induced megakaryocytic differentiation in RSK1-transfected K562cells. After pretreatment of 40 μm PD98059 for 2 h, the parental and RSK1-transfected cells were treated with PMA for 1 h, and ERK (A), RSK1 (B), and NF-κB (C) activities were compared. For morphological and nonspecific esterase activity assays (D), the parental and RSK1-transfected cells were treated with 10 nm PMA after pretreatment of 40 μm PD98059 for 2 h. After 3 days cells in the culture dish were photographed for morphological assay (upper panels), and cytospin preparations were stained to examine nonspecific esterase activity (lower panels). For untreated and PMA-treated controls, refer to the legend of Fig. 5.View Large Image Figure ViewerDownload (PPT)Flow Cytometric Analysis of the Effect of Overexpression of RSK1, Dominant Inhibitory Mutants of RSK1 and IκBα and PD98059 on CD41 ExpressionTo evaluate the role of the ERK/RSK/NF-κB pathway in PMA-induced megakaryocytic differentiation of K562 more specifically, flow cytometric analysis on expression of CD41, a megakaryocytic surface marker, was performed (Fig. 7). Consistent with the morphology and nonspecific esterase assay, overexpression of RSK1 enhanced the expression of CD41 induced by PMA (Fig. 7 B), while overexpression of RSK1 (D205N) suppressed the expression of CD41 (Fig. 7 C). Overexpression of IκBα (S32A/S36A) inhibited the expression of CD41 induced by PMA (Fig.7 D) and enhanced by overexpression of RSK1 (Fig.7 E). In addition, PMA-induced CD41 expression was prevented by the pretreatment with PD98059 in both parental and RSK1-transfected K562 cells (Fig. 7 F). Similar results were observed in CD61 expression (data not shown). These results confirmed that PMA-induced megakaryocytic differentiation is dependent on the ERK/RSK/NF-κB pathway.DISCUSSIONThe signaling mechanism that mediates the PMA-induced megakaryocytic differentiation of K562 cells is still controversial. We have proposed that PMA-induced megakaryocytic differentiation of K562 cells is dependent on the NF-κB pathway (8Kang C.D. Han C.S. Kim K.W. Do I.R. Kim C.M. Kim S.H. Lee E.Y. Chung B.S. Cancer Lett. 1998; 132: 99-106Crossref PubMed Scopus (20) Google Scholar). In contrast, others (9Whalen A.M. Galasinski S.C. Shapiro P.S. Nahreini T.S. Ahn N.G. Mol. Cell. Biol. 1997; 17: 1947-1958Crossref PubMed Scopus (203) Google Scholar, 10Herrera R. Hubbell S. Decker S. Petruzzelli L. Exp. Cell Res. 1998; 238: 407-414Crossref PubMed Scopus (89) Google Scholar, 11Racke F.K. Lewandowska K. Goueli S. Goldfarb A.N. J. Biol. Chem. 1997; 272: 23366-23370Abstract Full Text Full Text PDF PubMed Scopus (163) Google Scholar) have suggested recently that a sustained activation of the ERK/MAPK pathway is required to induce a differentiation program along the megakaryocyte lineage in K562 cells. In this study, we have demonstrated that RSK1 plays a role bridging ERK and NF-κB pathways during the PMA-induced megakaryocytic differentiation of K562 cells.Since RSK is activated by ERK/MAPK (16Blenis J. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 5889-5892Crossref PubMed Scopus (1152) Google Scholar), RSK1 activity was assayed during PMA-induced differentiation of K562 cells. After treatment with PMA, a sustained activation of RSK1 was observed over at least 12 h with a 1-h peak time after stimulation. It has been shown that a sustained activation of the ERK/MAPK pathway is required for megakaryocytic differentiation of K562 cells (11Racke F.K. Lewandowska K. Goueli S. Goldfarb A.N. J. Biol. Chem. 1997; 272: 23366-23370Abstract Full Text Full Text PDF PubMed Scopus (163) Google Scholar). Since RSK is a target molecule of ERK/MAPK (17Sturgill T.W. Ray L.B. Erikson E. Maller J.L. Nature. 1988; 334: 715" @default.
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