Retinoic acid increases amount of phosphorylated raf; Ectopic expression of cFMS reveals that retinoic acid-induced differentiation is more strongly dependent on ERK2 signaling than induced go arrest is

Summary Retinoic acid is known to cause the myeloid differentiation and G1/0 cell cycle arrest of HL-60 cells in a process that requires mitogen-activated protein/extracellular signal regulated kinase (MEK)-dependent extracellular signal regulated kinase (ERK)2 activation. It has also been shown that ectopic expression of cFMS, a platelet-derived growth factor (PDGF)-family transmembrane tyrosine kinase receptor, enhances retinoic acid-induced differentiation and G1/0 arrest. The mechanism of how the retinoic acid and cFMS signaling pathways intersect is not known. The present data show that the ectopic expression of cFMS results in the differential loss of sensitivity of retinoic acid-induced differentiation or G1/0 arrest to inhibition of ERK2 activation. PD98059 was used to inhibit MEK and consequently ERK2. In wild-type HL-60 cells, PD98059 blocked retinoic acid-induced differentiation; but in cFMS stable transfectants, PD98059 only attenuated the induced differentiation, with the resulting response resembling that of retinoic acid-treated wild-type HL-60. In wild-type HL-60, PD98059 greatly attenuated the retinoic acid-induced G1/0 arrest allied with retinoblastoma (RB) hypophosphorylation; but in cFMS stable transfectants, PD98059 had no inhibitory effect on RB hypophosphorylation and G1/0 arrest. This differential sensitivity to PD98059 and uncoupling of retinoic acid-induced differentiation and G1/0 arrest in cFMS transfectants is associated with changes in mitogen-activated protein kinase signaling molecules. The cFMS transfectants had more activated ERK2 than did the wild-type cells, which surprisingly was not attributable to enhanced mitogen-activated protein-kinase-kinase-kinase (RAF) phosphorylation. Retinoic acid increased the amount of activated ERK2 and phosphorylated RAF in both cell lines. But PD98059 eliminated detectable ERK2 activation, as well as inhibited RAF phosphorylation, in untreated and retinoic acid-treated wild-type HL-60 and cFMS transfectants, consistent with MEK or ERK feedback-regulation of RAF, in all four cases. Since PD98059 blocks the cFMS-conferred enhancement of the retinoic acid-induced differentiation, but not growth arrest, the data indicate that cFMS-enhanced differentiation acts primarily through MEK and ERK2, but cFMS-enhanced G1/0 arrest allied with RB hypophosphorylation depends on another cFMS signal route, which by itself can effect G1/0 arrest without activated ERK2. Ectopic expression of cFMS and differential sensitivity to ERK2 inhibition thus reveal that retinoic acid-induced HL-60 cell differentiation and G1/0 arrest are differentially dependent on ERK2 and can be uncoupled. A significant unanticipated finding was that retinoic acid caused a MEK-dependent increase in the amount of phosphorylated RAF. This increase may help sustain prolonged ERK2 activation.     

EB病毒潜伏膜蛋白1在鼻咽癌细胞中通过ERK介导Ets-1表达

为了探讨EB病毒编码的潜伏膜蛋白1(LMP1)对核转录因子Ets-1表达和活化的影响,并证实细胞外信号调节激酶1/2（ERK1/2）参与了该过程,选用可调控表达LMP1的鼻咽癌细胞系L7,应用蛋白质印迹法检测Ets-1、p-ERK蛋白质表达,免疫共沉淀-蛋白质印迹法检测Ets-1磷酸化状态,使用ERK1/2特异性小分子阻断物PD98059作用后,蛋白质印迹法检测p-ERK、Ets-1表达及磷酸化变化.结果显示：在L7细胞中诱导性LMP1可促进p-ERK、Ets-1蛋白质表达及其苏氨酸残基磷酸化,在一定范围呈时间和剂量效应;通过PD98059对诱导性LMP1作用的干预发现,p-ERK大部分表达被阻断,而Ets-1表达及其苏氨酸磷酸化也被部分阻断,以上结果提示ERK部分介导了LMP1诱导Ets-1表达和活化.     

The MEK/ERK pathway acts upstream of NF kappa B1 (p50) homodimer activity and Bcl-2 expression in a murine B-cell lymphoma cell line. MEK inhibition restores radiation-induced apoptosis

In a previously published report (Kurland, J. F., Kodym, R., Story, M. D., Spurgers, K. B., McDonnell, T. J., and Meyn, R. E. (2001) J. Biol. Chem. 276, 45380-45386), we described the NF kappa B status for two murine B-cell lymphoma cell lines, LY-as (apoptosis-sensitive) and LY-ar (apoptosis-refractory) and provided evidence that NF kappa B1 (p50) homodimers contribute to the expression of Bcl-2 in the LY-ar line. In the present study, we investigated the upstream signals leading to p50 homodimer activation and Bcl-2 expression. We found that in LY-ar cells, ERK1 and ERK2 were constitutively phosphorylated, whereas LY-as cells had no detectable ERK1 or ERK2 phosphorylation. Treatment of LY-ar cells with the MEK inhibitors PD 98059, U0126, and PD 184352 led to a loss of phosphorylated ERK1 and ERK2, a reversal of nuclear p50 homodimer DNA binding, and a decrease in Bcl-2 protein expression. Similarly, activation of the MEK/ERK pathway in LY-as cells by phorbol ester led to Bcl-2 expression that could be blocked by PD 98059. Furthermore, treatment of LY-ar cells with tumor necrosis factor-alpha, an I kappa B kinase activator, did not alter the suppressive effect of PD 98059 on p50 homodimer activity, suggesting an I kappa B kinase-independent pathway for p50 homodimer activation. Lastly, all three MEK inhibitors sensitized LY-ar cells to radiation-induced apoptosis. We conclude that the MEK/ERK pathway acts upstream of p50 homodimer activity and Bcl-2 expression in this B-cell lymphoma cell system and suggest that the use of MEK inhibitors could be useful clinically in combination with ionizing radiation to treat lymphoid malignancies.     

Regulation of epidermal growth factor-induced connexin 43 gap junction communication by big mitogen-activated protein kinase1/ERK5 but not ERK1/2 kinase activation

The gap junction protein, Cx43, plays a pivotal role in coupling cells electrically and metabolically, and the putative phosphorylation sites that modulate its function are reflected as changes in gap junction communication. Growth factor stimulation has been correlated with a decrease in gap junction communication and a parallel activation of ERK1/2; the inhibition of epidermal growth factor (EGF)-induced Cx43 gap junction uncoupling was observed by using the MEK1/2 inhibitor, PD98059. Because 1) BMK1/ERK5, another MAPK family member also activated by growth factors, possesses a phosphorylation motif similar to ERK1/2, and 2) it has been reported that PD98059 can inhibit not only MEK1/2-ERK1/2 but also MEK5-BMK1 activation, we investigated whether BMK1 can regulate EGF-induced Cx43 gap junction uncoupling and phosphorylation, comparing this to the role of ERK1/2 on Cx43 function and phosphorylation induced by EGF. Selective activation or inactivation of ERK1/2 by using a constitutively active form or a dominant negative form of MEK1 did not regulate Cx43 gap junction coupling. In contrast, we found that BMK1, selectively activated by constitutively active MEK5alpha, induced gap junction uncoupling, and the inhibition of BMK1 activation by transfection of dominant negative BMK1 prevented EGF-induced gap junction uncoupling. Activated BMK1 selectively phosphorylates Cx43 on Ser-255 in vitro and in vivo, but not on S279/S282, which are reported as the consensus phosphorylation sites for MAPK. Furthermore, by co-immunoprecipitation, we found that BMK1 directly associates with Cx43 in vivo. These data indicate that BMK1 is more important than ERK1/2 in EGF-mediated Cx43 gap junction uncoupling by association and Cx43 Ser- 255 phosphorylation.     

Histone deacetylase 1 and 2 differentially regulate apoptosis by opposing effects on extracellular signal-regulated kinase 1/2

Histone deacetylases (HDACs) are epigenetic regulators that are important for the control of various pathophysiological events. We found that HDAC inhibitors completely abolished transforming growth factor-β1 (TGF-β1)-induced apoptosis in AML-12 and primary mouse hepatocytes. Expression of a dominant-negative mutant of HDAC1 or downregulation of HDAC1 by RNAi both suppressed TGF-β1-induced apoptosis. In addition, overexpression of HDAC1 enhanced TGF-β1-induced apoptosis, and the rescue of HDAC1 expression in HDAC1 RNAi cells restored the apoptotic response of cells to TGF-β1. These data indicate that HDAC1 functions as a proapoptotic factor in TGF-β1-induced apoptosis. In contrast, downregulation of HDAC2 by RNAi increased spontaneous apoptosis and markedly enhanced TGF-β1-induced apoptosis, suggesting that HDAC2 has a reciprocal role in controlling cell survival. Furthermore, inhibition of extracellular signal-regulated kinase 1/2 (ERK1/2) by MEK1 inhibitor PD98059 or expression of a kinase-dead mutant of MEK1 restored the apoptotic response to TGF-β1 in HDAC1 RNAi cells. Strikingly, HDAC2 RNAi caused an inhibition of ERK1/2, and the spontaneous apoptosis can be abolished by reactivation of ERK1/2. Taken together, our data demonstrate that HDAC1 and 2 reciprocally affect cell viability by differential regulation of ERK1/2; these observations provide insight into the roles and potential mechanisms of HDAC1 and 2 in apoptosis.     

Activation of the ERK pathway and atypical protein kinase C isoforms in exercise- and aminoimidazole-4-carboxamide-1-beta-D-riboside (AICAR)-stimulated glucose transport

Exercise increases glucose transport in muscle by activating 5'-AMP-activated protein kinase (AMPK), but subsequent events are unclear. Presently, we examined the possibility that AMPK increases glucose transport through atypical protein kinase Cs (aPKCs) by activating proline-rich tyrosine kinase-2 (PYK2), ERK pathway components, and phospholipase D (PLD). In mice, treadmill exercise rapidly activated ERK and aPKCs in mouse vastus lateralis muscles. In rat extensor digitorum longus (EDL) muscles, (a) AMPK activator, 5-aminoimidazole-4-carboxamide-1-beta-d-riboside (AICAR), activated PYK2, ERK and aPKCs; (b) effects of AICAR on ERK and aPKCs were blocked by tyrosine kinase inhibitor, genistein, and MEK1 inhibitor, PD98059; and (c) effects of AICAR on aPKCs and 2-deoxyglucose (2-DOG) uptake were inhibited by genistein, PD98059, and PLD-inhibitor, 1-butanol. Similarly, in L6 myotubes, (a) AICAR activated PYK2, ERK, PLD, and aPKCs; (b) effects of AICAR on ERK were inhibited by genistein, PD98059, and expression of dominant-negative PYK2; (c) effects of AICAR on PLD were inhibited by MEK1 inhibitor UO126; (d) effects of AICAR on aPKCs were inhibited by genistein, PD98059, 1-butanol, and expression of dominant-negative forms of PYK2, GRB2, SOS, RAS, RAF, and ERK; and (e) effects of AICAR on 2DOG uptake/GLUT4 translocation were inhibited by genistein, PD98059, UO126, 1-butanol, cell-permeable myristoylated PKC-zeta pseudosubstrate, and expression of kinase-inactive RAF, ERK, and PKC-zeta. AMPK activator dinitrophenol had effects on ERK, aPKCs, and 2-DOG uptake similar to those of AICAR. Our findings suggest that effects of exercise on glucose transport that are dependent on AMPK are mediated via PYK2, the ERK pathway, PLD, and aPKCs.     

High intensity ERK signal mediates hepatocyte growth factor-induced proliferation inhibition of the human hepatocellular carcinoma cell line HepG2

Hepatocyte growth factor (HGF) induces growth stimulation of a variety of cell types, but it also induces growth inhibition of several types of tumor cell lines. The molecular mechanism of the HGF-induced growth inhibition of tumor cells remains obscure. We have investigated the intracellular signaling pathway involved in the antiproliferative effect of HGF on the human hepatocellular carcinoma cell line HepG2. HGF induced strong activation of ERK in HepG2 cells. Although the serum-dependent proliferation of HepG2 cells was inhibited by the MEK inhibitor PD98059 in a dose-dependent manner, 10 microM PD98059 reduced the HGF-induced strong activation of ERK to a weak activation; and as a result, the proliferation inhibited by HGF was completely restored. Above or below this specific concentration, the restoration was incomplete. Expression of constitutively activated Ha-Ras, which induces strong activation of ERK, led to the proliferation inhibition of HepG2 cells, as was observed in HGF-treated HepG2 cells. This inhibition was suppressed by the MEK inhibitor. Furthermore, HGF treatment and expression of constitutively activated Ha-Ras changed the hyperphosphorylated form of the retinoblastoma tumor suppressor gene product pRb to the hypophosphorylated form. This change was inhibited by the same concentration of MEK inhibitor needed to suppress the proliferation inhibition. These results suggest that ERK activity is required for both the stimulation and inhibition of proliferation of HepG2 cells; that the level of ERK activity determines the opposing proliferation responses; and that HGF-induced proliferation inhibition is caused by cell cycle arrest, which results from pRb being maintained in its active hypophosphorylated form via a high-intensity ERK signal in HepG2 cells.     

A differential role of extracellular signal-regulated kinase in stimulated PC12 pheochromocytoma cell movement

Rat pheochromocytoma PC12 cells have been widely used as a cell system for study of growth factor-stimulated cell functions. We report here that nerve growth factor (NGF) stimulated both chemotaxis (directional migration) and chemokinesis (random migration) of PC12 cells. Treatment with a MEK1/2-specific inhibitor (PD98059) or expression of a dominant negative variant of Ras differentially inhibited NGF-stimulated chemotaxis but not chemokinesis of PC12 cells. Priming of PC12 cells with NGF resulted in reduced extracellular signal-regulated kinase (ERK) activation and loss of chemotactic, but not chemokinetic, response. In addition, NGF stimulation of ERK is known to involve an early transient phase of activation followed by a late sustained phase of activation; in contrast, epidermal growth factor (EGF) elicits only early transient ERK activation. We observed that like NGF, EGF also stimulated both chemotaxis and chemokinesis, and treatment with PD98059 abolished the EGF-stimulated chemotaxis. Therefore, the early transient phase of ERK activation functioned in signaling chemotaxis; the late sustained phase of ERK activation did not seem to have an essential role. In addition, our results suggested that chemotactic signaling required a threshold level of ERK activation; at below threshold level of ERK activation, chemotaxis would not occur.     

Blockade of the extracellular signal-regulated kinase pathway induces marked G1 cell cycle arrest and apoptosis in tumor cells in which the pathway is constitutively activated: up-regulation of p27(Kip1)

Constitutive activation of the ERK pathway is associated with the neoplastic phenotype of a relatively large number of human tumor cells. Blockade of the ERK pathway by treatment with PD98059, a specific inhibitor of mitogen-activated protein (MAP) kinase/ERK kinase (MEK), completely suppressed the growth of tumor cells in which the pathway is constitutively activated (RPMI-SE and HT1080 cells). Consistent with its prominent antiproliferative effect, PD98059 induced a remarkable G(1) cell cycle arrest, followed by a modest apoptotic response, in these tumor cells. Selective up-regulation of p27(Kip1) was observed after PD98059 treatment of RPMI-SE and HT1080 cells. Overexpression in RPMI-SE cells of either a kinase-negative form of MEK1 or wild-type MAP kinase phosphatase-3 also induced up-regulation of p27(Kip1). The up-regulation of p27(Kip1) correlated with increased association of p27(Kip1) with cyclin E-cyclin-dependent kinase (CDK) 2 complexes, a concomitant inhibition of cyclin E-CDK2 kinase activity, and a consequent decrease in the phosphorylation state of retinoblastoma protein, which would culminate in the marked G(1) cell cycle arrest observed in these tumor cells. These results suggest that the complete growth suppression that follows specific blockade of the ERK pathway in tumor cells in which the pathway is constitutively activated is mediated by up-regulation of p27(Kip1).     

The extracellular signal-regulated kinase pathway is required for activation-induced cell death of T cells

T cells can undergo activation-induced cell death (AICD) upon stimulation of the T cell receptor-CD3 complex. We found that the extracellular signal-regulated kinase (ERK) pathway is activated during AICD. Transient transfection of a dominant interfering mutant of mitogen-activated/extracellular signal-regulated receptor protein kinase kinase (MEK1) demonstrated that down-regulation of the ERK pathway inhibited FasL expression during AICD, whereas activation of the ERK pathway with a constitutively active MEK1 resulted in increased expression of FasL. We also found that pretreatment with the specific MEK1 inhibitor PD98059 prevented the induction of FasL expression during AICD and inhibited AICD. However, PD98059 had no effect on other apoptotic stimuli. We found only very weak ERK activity during Fas-mediated apoptosis (induced by Fas cross-linking). Furthermore, preincubation with the MEK1 inhibitor did not inhibit Fas-mediated apoptosis. Finally, we also demonstrated that pretreatment with the MEK1 inhibitor could delay and decrease the expression of the orphan nuclear steroid receptor Nur77, which has been shown to be essential for AICD. In conclusion, this study demonstrates that the ERK pathway is required for AICD of T cells and appears to regulate the induction of Nur77 and FasL expression during AICD.     

Glial cell‐derived neurotrophic factor increases migration of human chondrosarcoma cells via ERK and NF‐κB pathways

Invasion of tumor cells is the primary cause of therapeutic failure in the treatment of malignant chondrosarcomas. Glial cell‐derived neurotrophic factor (GDNF) plays a crucial role in migration and metastasis of human cancer cells. Integrins are the major adhesive molecules in mammalian cells. Here we found that GDNF directed the migration and increased cell surface expression of αv and β3 integrin in human chondrosarcoma cells. Pretreated of JJ012 cells with MAPK kinase (MEK) inhibitors PD98059 or U0126 inhibited the GDNF‐mediated migration and integrin expression. Stimulation of cells with GDNF increased the phosphorylation of MEK and extracellular signal‐regulating kinase (ERK). In addition, NF‐κB inhibitor (PDTC) or IκB protease inhibitor (TPCK) also inhibited GDNF‐mediated cells migration and integrin up‐regulation. Stimulation of cells with GDNF induced IκB kinase (IKKα/β) phosphorylation, IκB phosphorylation, p65 Ser⁵³⁶ phosphorylation, and κB‐luciferase activity. Furthermore, the GDNF‐mediated increasing of κB‐luciferase activity was inhibited by PD98059, U0126, PDTC and TPCK or MEK, ERK, IKKα, and IKKβ mutants. Taken together, these results suggest that the GDNF acts through MEK/ERK, which in turn activates IKKα/β and NF‐κB, resulting in the activations of αvβ3 integrin and contributing the migration of human chondrosarcoma cells. J. Cell. Physiol. 220: 499–507, 2009. © 2009 Wiley‐Liss, Inc.     

Extracellular signal-regulated protein kinases mediate H(+),K(+)-ATPase alpha-subunit gene expression.

Extracellular signal-regulated protein kinases (ERKs) are important in many cellular functions. We and others have previously reported that prolonged exposure of gastric parietal cells to epidermal growth factor (EGF) enhanced gastric acid secretion stimulated by secretagogues via ERKs. In this study, we examined whether ERKs regulated H(+),K(+)-ATPase alpha-subunit gene expression using a gastric cancer cell line, AGS. EGF induced ERK activity time- and dose-dependently with a maximal effect observed at 10 min and 10 nM, respectively. The MEK inhibitors, U0126 and PD-98059, dose-dependently inhibited the ERK activity stimulated by EGF. To test H(+),K(+)-ATPase alpha-subunit gene expression, we transfected AGS cells with a plasmid containing a canine H(+),K(+)-ATPase alpha-subunit gene promoter fused to a luciferase reporter gene. EGF induced luciferase activity in transfected cells; this effect was inhibited by the MEK inhibitors, suggesting that EGF-induced gene expression involved the ERK pathway. When AGS cells were transfected with the reporter plasmids in conjunction with an expression vector encoding constitutively active MEK1, luciferase activity was strongly enhanced; this effect was attenuated by the MEK inhibitors or by an additional cotransfection of dominant negative MEK1. Taken together, our results led us to conclude that the ERK pathway may mediate H(+),K(+)-ATPase alpha-subunit gene expression, contributing to gastric acid secretion in parietal cells.     

Basal Extracellular Signal-Regulated Kinase Activity Modulates Cell-Cell and Cell-Matrix Interactions

Suppression of the basal extracellular signal-regulated kinase (ERK) activity in PC12 cells markedly altered their phenotype. Wild-type cells grew in a dissociated pattern adherent to the substrate. The stable expression of an ERK inhibitory mutant resulted in the formation of calcium-dependent aggregates which were less adherent to the substrate. Concomitantly, the cells reorganized their actin cytoskeleton and increased their expression of several adherens junction proteins, particularly cadherin. Metabolic labeling demonstrated an increased synthesis of cadherin and β-catenin in these cells. Nontransfected PC12 cells and a ras-transformed MDCK cell line also formed aggregates and increased their expression of adherens junction proteins following treatment with the selective MEK inhibitor PD98059. A peptide containing the HAV cadherin recognition sequence attenuated the aggregation. These studies suggest that in PC12 and epithelial cells, ERKs are pivotally positioned to enhance substrate interactions when active or to release homotypic interactions when suppressed.     

MEK/ERK信号通路对人结膜上皮细胞增殖的影响及其机制研究

目的:探讨MEK/ERK信号通路对人结膜上皮细胞增殖的影响及其可能的机制。方法:采用不同浓度(0、12.5、25、50、100μmol/L)的MEK抑制剂PD98059处理人结膜上皮细胞(HConEpiC),通过CCK-8法检测不同浓度PD98059作用不同时间(0、12、24、48 h)对人结膜上皮细胞增殖的影响,Western blot检测不同浓度PD98059对人结膜上皮细胞ERK1/2、P-ERK1/2表达的影响。结果:相比对照组(0μmol/L),不同浓度(12.5、25、50、100μmol/L)PD98059处理后的人结膜上皮细胞增殖率明显下降,呈剂量-效应关系,且随处理时间增加(12、24、48 h)其抑制作用也显著增强,差异均有统计学意义(P0.05)。不同浓度PD98059处理人结膜上皮细胞24 h后,其ERK及p-ERK1/2表达随处理浓度增加而降低,与对照组(0μmol/L)相比差异有统计学意义(P0.05),且二者表达量与细胞增值抑制率均呈显著负相关(r=-0.995、r=-0.968,P0.05)。结论:PD98059可抑制人结膜上皮细胞增殖,这可能与其下调ERK表达和减少其活化有关。