Extracellular signal-regulated kinase/90-KDA ribosomal S6 kinase/nuclear factor-kappa B pathway mediates phorbol 12-myristate 13-acetate-induced 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-kappaB (NF-kappaB)-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 IkappaBalpha 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-kappaB 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 IkappaBalpha inhibited PMA-stimulated and RSK1-enhanced megakaryocytic differentiation, indicating that NF-kappaB mediates a signal for megakaryocytic differentiation downstream of RSK1. PMA-stimulated activation of ERK/MAPK, RSK1, and NF-kappaB 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-kappaB pathway mediates PMA-stimulated megakaryocytic differentiation of K562 cells.     

Silencing of membrane-associated sialidase Neu3 diminishes apoptosis resistance and triggers megakaryocytic differentiation of chronic myeloid leukemic cells K562 through the increase of ganglioside GM3

In chronic myeloid leukemia K562 cells, differentiation is also blocked because of low levels of ganglioside GM3, derived by the high expression of sialidase Neu3 active on GM3. In this article, we studied the effects of Neu3 silencing (40-70% and 63-93% decrease in protein content and activity, respectively) in these cells. The effects were as follows: (a) gangliosides GM3, GM1, and sialosylnorhexaosylceramide increased markedly; (b) cell growth and [(3)H]thymidine incorporation diminished relevantly; (c) as mRNA, cyclin D2, and Myc were much less expressed, whereas cyclin D1 was expressed more like its inhibitor p21; (d) as mRNA, pro-apoptotic proteins Bax and Bad increased with concurrent decrease and increase in the anti-apoptotic proteins Bcl-2 and Bcl-XL, respectively; (e) the apoptosis inducers etoposide and staurosporine were active on Neu3 silencing cells but not on mock cells; (f) as mRNA, the megakaryocytic markers CD10, CD44, CD41, and CD61 increased similar to the case of mock cells stimulated with PMA; (g) the signaling cascades mediated by PLC-beta2, PKC, RAF, ERK1/2, RSK90, and JNK were largely activated. The induction of a GM3-rich ganglioside pattern in K562 cells by treatment with brefeldin A elicited a phenotype similar to that of Neu3 silencing cells. In conclusion, upon Neu3 silencing, K562 cells show a decrease in proliferation, propensity to undergo apoptosis, and megakaryocytic differentiation.     

Involvement of nPKC-MAPK pathway in the decrease of nucleophosmin/B23 during megakaryocytic differentiation of human myelogenous leukemia K562 cells

Human myelogenous leukemia K562 cells were induced to undergo megakaryocytic differentiation by long-term treatment with phorbol ester 12-O-tetradecanoyl-phorbol-13-acetate (TPA). The protein level of nucleophosmin/B23 (NPM/B23), a nucleolar protein, was substantially decreased upon TPA treatment. In this study, we found that the proteasome inhibitors blocked the decrease of NPM/B23 protein in response to TPA, suggesting the proteasomes were involved in the downregulation of NPM/B23 upon megakaryocytic differentiation. To investigate the signaling pathway in the downregulation of NPM/B23 during early TPA-induced megakaryocytic differentiation of K562 cells, K562 cells were treated with TPA in the presence of the PKC isozyme-selective inhibitors, GF109203X and Gö 6976, or MEK1 inhibitor, PD98059. The decrease of NPM/B23 protein in the TPA-treated K562 cells was blocked by GF109203X but not by Gö 6976, suggesting the involvement of novel PKCs in the downregulation of NPM/B23 during TPA-induced megakaryocytic differentiation of K562 cells. The application of MEK1 inhibitor PD98059 upon TPA treatment blocked the TPA-induced decrease of NPM/B23 protein and aborted the megakaryocytic differentiation but not to break through the cell growth arrest. Unlike NPM/B23, the degradation of nucleolin in the TPA-treated K562 cells could not be blocked by PD98059 while the TPA-induced megakaryocytic differentiation was abrogated. The decrease of NPM/B23 protein seems to be more correlated with the novel PKC-MAPK-induced megakaryocytic differentiation than another nucleolar protein, nucleolin. Taken together, our results indicated that novel PKC-MAPK pathway was required for the decrease of NPM/B23 during TPA-induced megakaryocytic differentiation.     

Phorbol ester phorbol-12-myristate-13-acetate promotes anchorage-independent growth and survival of melanomas through MEK-independent activation of ERK1/2

The phorbol ester, phorbol-12-myristate-13-acetate (PMA), an activator of PKCs, is known to stimulate the in vitro growth of monolayer cultures of normal human melanocytes whereas it inhibits the growth of most malignant melanoma cell lines. We examined the effect of PMA on proliferation and survival of melanoma cells grown as multicellular aggregates in suspension (spheroids), and aimed to elucidate downstream targets of PKC signaling. In contrast to monolayer cultures, PMA increased cell proliferation as well as protected melanoma cells from suspension-mediated apoptosis (anoikis). Supporting the importance of PKC in anchorage-independent growth, treatment of anoikis-resistant melanoma cell lines with antisense oligonucleotides against PKC-alpha, or the PKC inhibitor G?6976, strongly induced anoikis. PMA induced activation of ERK1/2, but this effect was not prevented by the MEK inhibitors PD98059 or by U0126. Whereas PD98059 treatment alone led to marked activation of the pro-apoptotic Bim and Bad proteins and significantly increased anoikis, these effects were clearly reversed by PMA. In conclusion, our results indicate that the protective effect of PMA on anchorage-independent survival of melanoma cells at least partly is mediated by MEK-independent activation of ERK1/2 and inactivation of downstream pro-apoptotic effector proteins.     

An absolute role of the PKC-dependent NF-kappaB activation for induction of MMP-9 in hepatocellular carcinoma cells

Matrix metalloproteinases (MMPs) play an important role in inflammation, tumor cell invasion, and metastasis. We found that phorbol-12-myristate-13-acetate (PMA)-stimulated invasion of the hepatocellular carcinoma (HCC) SNU-387 and SNU-398 cells and that PMA induced the secretion of MMP-9 in the cells, but did not induce the secretion of MMP-2. The PMA-induced MMP-9 secretion was abolished by treatment of a pan-protein kinase C (PKC) inhibitor, GF109203X, and an inhibitor of NF-kappaB activation, sulfasalazine, and partly inhibited by treatment of inhibitors of ERK pathway, PD98059 and U0126. In addition, the PMA-stimulated activation of the MMP-9 promoter was completely inhibited by a mutation of the NF-kappaB site within the MMP-9 promoter, but not completely by mutations of two AP-1 sites. Moreover, the MMP-9 induction by HGF and TNF-alpha was also completely inhibited by GF109203X and sulfasalazine, but not by PD98059 and U0126. These data demonstrate that the PKC-dependent NF-kappaB activation is absolute for MMP-9 induction and that the PKC-dependent ERK activation devotes to increase the expression level of MMP-9, in HCC cells.     

A Role for the MEK/MAPK Pathway in PMA-Induced Cell Cycle Arrest: Modulation of Megakaryocytic Differentiation of K562 Cells

In vitromegakaryocytic differentiation of the pluripotent K562 human leukemia cell line is induced by PMA. Treatment of K562 cells with PMA results in growth arrest, polyploidy, morphological changes, and increased cell–cell and cell–substrate adhesion. These PMA-induced changes in K562 cells are preceded by a rapid rise in the activity of MEK (MAP kinase/extracellular regulated kinases) that leads to a sustained activation of ERK2 (extracellular regulated kinase; MAPK). Blockade of MEK1 activation by PD098059, a recently described specific MEK inhibitor [D. T. Dudleyet al.(1995).Proc. Natl. Acad. Sci. USA92, 7686–7689], reverses both the growth arrest and the morphological changes of K562 cells induced by PMA treatment. These changes are not associated with a disruption of PMA-induced down-regulation of BCR-ABL kinase or early integrin signaling events but are associated with a block of the cell-surface expression of the gpIIb/IIIa (CD41) integrin, a cell marker of megakaryocytic differentiation. These results demonstrate that the PMA-induced signaling cascade initiated by protein kinase C activation requires the activity of the MEK/ERK signaling complex to regulate cell cycle arrest, thus regulating the program that leads to the cell-surface expression of markers associated with megakaryocytic differentiation.     

ERK2-Pyruvate Kinase Axis Permits Phorbol 12-Myristate 13-Acetate-induced Megakaryocyte Differentiation in K562 Cells

Metabolic changes that contribute to differentiation are not well understood. Overwhelming evidence shows the critical role of glycolytic enzyme pyruvate kinase (PK) in directing metabolism of proliferating cells. However, its role in metabolism of differentiating cells is unclear. Here we studied the role of PK in phorbol 12-myristate 13-acetate (PMA)-induced megakaryocytic differentiation in human leukemia K562 cells. We observed that PMA treatment decreased cancer-type anabolic metabolism but increased ATP production, along with up-regulated expression of two PK isoforms (PKM2 and PKR) in an ERK2-dependent manner. Interestingly, silencing of PK (PKM2 and PKR) inhibited PMA-induced megakaryocytic differentiation, as revealed by decreased expression of megakaryocytic differentiation marker CD61 and cell cycle behavior. Further, PMA-induced ATP production reduced greatly upon PK silencing, suggesting that PK is required for ATP synthesis. In addition to metabolic effects, PMA treatment also translocated PKM2, but not PKR, into nucleus. ERK1/2 knockdowns independently and together suggested the role of ERK2 in the up-regulation of both the isoforms of PK, proposing a role of ERK2-PK isoform axis in differentiation. Collectively, our findings unravel ERK2 guided PK-dependent metabolic changes during PMA induction, which are important in megakaryocytic differentiation.     

Eicosapentaenoic acid and docosahexaenoic acid modulate MAP kinase (ERK1/ERK2) signaling in human T cells.

This study was conducted on human Jurkat T cell lines to elucidate the role of EPA and DHA, n-3 PUFA, in the modulation of two mitogen-activated protein (MAP) kinases, that is, extracellular signal-regulated kinases 1 and 2 (ERK1 and ERK2). The n-3 PUFA alone failed to induce phosphorylation of ERK1/ERK2. We stimulated the MAP kinase pathway with anti-CD3 antibodies and phorbol 12-myristate 13-acetate (PMA), which act upstream of the MAP kinase (MAPK)/ERK kinase (MEK) as U0126, an MEK inhibitor, abolished the actions of these two agents on MAP kinase activation. EPA and DHA diminished the PMA- and anti-CD3-induced phosphorylation of ERK1/ERK2 in Jurkat T cells. In the present study, PMA acts mainly via protein kinase C (PKC) whereas anti-CD3 antibodies act via PKC-dependent and -independent mechanisms. Furthermore, DHA and EPA inhibited PMA-stimulated PKC enzyme activity. EPA and DHA also significantly curtailed PMA- and ionomycin-stimulated T cell blastogenesis. Together these results suggest that EPA and DHA modulate ERK1/ERK2 activation upstream of MEK via PKC-dependent and -independent pathways and that these actions may be implicated in n-3 PUFA-induced immunosuppression.     

Activation of P2Y2 receptor induces c-FOS protein through a pathway involving mitogen-activated protein kinases and phosphoinositide 3-kinases in HeLa cells

The effects of P2Y2 purinoceptor activation on c-Fos expression and the signaling pathways evoked by extracellular ATP/UTP in HeLa cells were investigated. We found that P2Y2 activation induced c-Fos protein and phosphorylated the extracellular signal-regulated kinases 1 and 2 (ERK1/2). The P2Y2-stimulated c-Fos induction was partly blocked (a) by U73122, a phospholipase C inhibitor, (b) by G?6976, a conventional PKC inhibitor, (c) by PD098059, a mitogen-activated protein kinase kinase inhibitor, and, moreover, (d) by the inhibitors of phosphoinositide 3-kinases (PI3K), LY294002 and wortmannin. When G?6976 and PD098059, or G?6976 and wortmannin, were combined there was a totally inhibition of P2Y2-induced c-Fos increase. Either U73122 or G?6976 did not inhibit ERK1/2 phosphorylation induced by ATP/UTP, while it was inhibited by LY294002 (or wortmannin) and by staurosporine. Additionally, wortmannin inhibited the cytosol-to-membrane translocation of PKC- epsilon induced by ATP/UTP. These data indicated that agonist-induced PI3K and downstream PKC- epsilon activation mediated the effect of ATP/UTP on ERK1/2 activation. To test the biological consequences of ERK1/2 activation, the effect of P2Y2 on cell functions were examined. P2Y2 stimulation increased cell proliferation and this effect was attenuated by PD098059 in a dose-dependent manner, thereby indicating that the ERK pathway mediates mitogenic signaling by P2Y2. In conclusion, the activation of conventional PKCs through P2Y2 receptor acts in concert with ERK and PI3K/PKC- epsilon pathways to induce c-Fos protein and HeLa cell proliferation.     

Role of PKC and MAPK in cytosolic PLA2 phosphorylation and arachadonic acid release in primary murine astrocytes

Although Group IV cytosolic phospholipase A2 (cPLA2) in astrocytes has been implicated in a number of neurodegenerative diseases, mechanisms leading to its activation and release of arachidonic acid (AA) have not been clearly elucidated. In primary murine astrocytes, phorbol myristate acetate (PMA) and ATP stimulated phosphorylation of ERK1/2 and cPLA2 as well as evoked AA release. However, complete inhibition of phospho-ERK by U0126, an inhibitor of mitogen-activated protein kinase kinase (MEK), did not completely inhibit PMA-stimulated cPLA2 and AA release. Epidermal growth factor (EGF) also stimulated phosphorylation of ERK1/2 and cPLA2[largely through a protein kinase C (PKC)-independent pathway], but EGF did not evoke AA release. These results suggest that phosphorylation of cPLA2 due to phospho-ERK is not sufficient to evoke AA release. However, complete inhibition of ATP-induced cPLA2 phosphorylation and AA release was observed when astrocytes were treated with GF109203x, a general PKC inhibitor, together with U0126, indicating the important role for both PKC and ERK in mediating the ATP-induced AA response. There is evidence that PMA and ATP stimulated AA release through different PKC isoforms in astrocytes. In agreement with the sensitivity of PMA-induced responses to PKC down-regulation, prolonged treatment with PMA resulted in down-regulation of PKCalpha and epsilon in these cells. Furthermore, PMA but not ATP stimulated rapid translocation of PKCalpha from cytosol to membranes. Together, our results provided evidence for an important role of PKC in mediating cPLA2 phosphorylation and AA release in astrocytes through both ERK1/2-dependent and ERK1/2-independent pathways.