Cyclosporin A通过MEK／ERK1／2信号通路调节滋养细胞titin表达

探讨MEK／ERK1／2信号通路在CyclosporinA(CsA)诱导滋养细胞表达titin中的作用。应用RT—PCR、Western blot检测CsA诱导的滋养细胞titin的表达水平,Western blot检测CsA作用于滋养细胞后ERK1／2的活化程度,并观察MEK特异性抑制剂U0126对其mRNA转录的影响。发现CsA以时间和剂量依赖方式诱导titin表达,并刺激滋养细胞ERK1／2的活化,U0126以剂量依赖方式抑制CsA诱导的titin表达。结果表明CsA通过活化MEK／ERK1／2信号通路诱导滋养细胞titin的表达,改变其生物学行为,从而有利于胚胎着床及早期发育。     

Metformin induces differentiation in acute promyelocytic leukemia by activating the MEK/ERK signaling pathway

Recent studies have shown that metformin, a widely used antidiabetic agent, may reduce the risk of cancer development. In this study, we investigated the antitumoral effect of metformin on both acute myeloid leukemia (AML) and acute promyelocytic leukemia (APL) cells. Metformin induced apoptosis with partial differentiation in an APL cell line, NB4, but only displayed a proapoptotic effect on several non-M3 AML cell lines. Further analysis revealed that a strong synergistic effect existed between metformin and all-trans retinoic acid (ATRA) during APL cell maturation and that metformin induced the hyperphosphorylation of extracellular signal-regulated kinase (ERK) in APL cells. U0126, a specific MEK/ERK activation inhibitor, abrogated metformin-induced differentiation. Finally, we found that metformin induced the degradation of the oncoproteins PML-RARα and c-Myc and activated caspase-3. In conclusion, these results suggest that metformin treatment may contribute to the enhancement of ATRA-induced differentiation in APL, which may deepen the understanding of APL maturation and thus provide insight for new therapy strategies.     

Paclitaxel induces prolonged activation of the Ras/MEK/ERK pathway independently of activating the programmed cell death machinery

Paclitaxel is a widely used chemotherapeutic agent and is known to induce programmed cell death (apoptosis) in a variety of cell types, but the precise underlying mechanisms are poorly understood. To elucidate these mechanisms, we challenged human esophageal squamous cancer cell lines with paclitaxel and investigated its effects upon signal transduction pathways. Physiologically relevant concentrations of paclitaxel (1-1,000 nm) induced apoptosis. All three mitogen-activated protein kinase (MAPK) family members, c-Jun N-terminal kinase (JNK), p38 MAPK, and extracellular signal-regulated kinase (ERK) were activated upon paclitaxel treatment. Interestingly, JNK activation and p38 MAPK activation were delayed and peaked at 48 h, whereas ERK activity was sustained over 72 h. In addition, Ras activation and MAPK/ERK kinase (MEK) phosphorylation were observed in concordance with ERK activation. While ERK activation was completely ablated by MEK inhibitors, immunoprecipitation and Western blot analysis revealed that neither MEK-1 nor MEK-2 was involved, but instead another member of the MEK family may potentially participate. Although pretreatment with a general caspase inhibitor, benzyloxycarbonyl-Val-Ala-Asp-fluoromethylketone rescued the cell death, it did not prevent Ras or ERK activation. Furthermore, inhibition of JNK, p38 MAPK, or MEK did not alter PARP cleavage and the cell death induced by paclitaxel. These results in aggregate suggest that the delayed activation of JNK, p38 MAPK, and ERK was not linked to activation of the cell death machinery.     

The D Domain of LRRC4 anchors ERK1/2 in the cytoplasm and competitively inhibits MEK/ERK activation in glioma cells

Background

As a well-characterized key player in various signal transduction networks, extracellular-signal-regulated kinase (ERK1/2) has been widely implicated in the development of many malignancies. We previously found that Leucine-rich repeat containing 4 (LRRC4) was a tumor suppressor and a negative regulator of the ERK/MAPK pathway in glioma tumorigenesis. However, the precise molecular role of LRRC4 in ERK signal transmission is unclear.

Methods

The interaction between LRRC4 and ERK1/2 was assessed by co-immunoprecipitation and GST pull-down assays in vivo and in vitro. We also investigated the interaction of LRRC4 and ERK1/2 and the role of the D domain in ERK activation in glioma cells.

Results

Here, we showed that LRRC4 and ERK1/2 interact via the D domain and CD domain, respectively. Following EGF stimuli, the D domain of LRRC4 anchors ERK1/2 in the cytoplasm and abrogates ERK1/2 activation and nuclear translocation. In glioblastoma cells, ectopic LRRC4 expression competitively inhibited the interaction of endogenous mitogen-activated protein kinase (MEK) and ERK1/2. Mutation of the D domain decreased the LRRC4-mediated inhibition of MAPK signaling and its anti-proliferation and anti-invasion roles.

Conclusions

Our results demonstrated that the D domain of LRRC4 anchors ERK1/2 in the cytoplasm and competitively inhibits MEK/ERK activation in glioma cells. These findings identify a new mechanism underlying glioblastoma progression and suggest a novel therapeutic strategy by restoring the activity of LRRC4 to decrease MAPK cascade activation.

     

Leukemia inhibitory factor induces DNA synthesis in Swiss mouse 3T3 cells independently of cyclin D1 expression through a mechanism involving MEK/ERK1/2 activation

Leukemia inhibitory factor (LIF) and oncostatin M (OSM) induce DNA synthesis in Swiss 3T3 cells through common signaling mechanism(s), whereas other related cytokines such as interleukin-6 and ciliary neurotrophic factor do not cause this response. Induction of DNA replication by LIF or prostaglandin F2alpha (PGF2alpha) occurs, in part, through different signaling events. LIF and OSM specifically trigger STAT1 cytoplasmic to nuclear translocation, whereas PGF2alpha fails to do so. However, LIF and PGF2alpha can trigger increases in ERK1/2 activity, which are required for their mitogenic responses because U0126, a MEK1/2 inhibitor, prevents both ERK1/2 activation and induction of DNA synthesis by LIF or PGF2alpha treatment. PGF2alpha induces cyclin D expression and full phosphorylation of retinoblastoma protein. In contrast, LIF fails to promote increases in cyclin D mRNA/protein levels; consequently, LIF induces DNA synthesis without promoting full phosphorylation of retinoblastoma protein (Rb). However, both LIF and PGF2alpha increase cyclin E expression. Furthermore, LIF mitogenic action does not involve protein kinase C (PKC) activation, because a PKC inhibitor does not block this effect. In contrast, PKC activity is required for PGF2alpha mitogenic action. More importantly, the synergistic effect between LIF and PGF2alpha to promote S phase entry is independent of PKC activation. These results show fundamental differences between LIF- and PGF2alpha-dependent mechanism(s) that induce cellular entry into S phase. These findings are critical in understanding how LIF and other related cytokine-regulated events participate in normal cell cycle control and may also provide clues to unravel crucial processes underlying cancerous cell division.     

The Raf/MEK/ERK pathway: new concepts of activation.

The Raf/MEK/ERK signaling was the first MAP kinase cascade to be characterized. It is probably one of the most well known signal transduction pathways among biologists because of its implication in a wide variety of cellular functions as diverse -and occasionally contradictory- as cell proliferation, cell-cycle arrest, terminal differentiation and apoptosis. Discovery and understanding of this pathway have benefited from the combination of both genetic studies in worms and flies and biochemical studies in mammalian cells. However, ten years after, this field is still under debate and new molecular partners in the cascade continue to increase the complexity of its regulation. This review deals with the emergence of new concepts in the activation and regulation of the Raf/MEK/ERK module. In particular, the preponderant role of B-Raf is underlined, and the role of novel regulators such as KSR is discussed.     

Sirt2 induces C2C12 myoblasts proliferation by activation of the ERK1/2 pathway

Sirtuins are Class III histone deacetylases （HDACs） that have emerged as important regulators of diverse biological processes, and comprised of seven members （Sirtl to Sirt7） [1]. Sirt2 predominantly resides in the cytoplasm,     

Ras-independent activation of the Raf/MEK/ERK pathway upon calcium-induced differentiation of keratinocytes

MAPKs are crucially involved in the regulation of growth and differentiation of a variety of cells. To elucidate the role of MAPKs in keratinocyte differentiation, activation of ERK, JNK, and p38 in response to stimulation with extracellular calcium was analyzed. We provide evidence that calcium-induced differentiation of keratinocytes is associated with rapid and transient activation of the Raf/MEK/ERK pathway. Stimulation of keratinocytes with extracellular calcium resulted in activation of Raf isozymes and their downstream effector ERK within 10-15 min, but did not increase JNK or p38 activity. Calcium-induced ERK activation differed in kinetics from mitogenic ERK activation by epidermal growth factor and could be modulated by alterations of intracellular calcium levels. Interestingly, calcium stimulation led to down-regulation of Ras activity at the same time that ERK activation was initiated. Expression of a dominant-negative mutant of Ras also did not significantly impair calcium-induced ERK activation, indicating that calcium-mediated ERK activation does not require active Ras. Despite the transient nature of ERK activation, calcium-induced expression of the cyclin-dependent kinase inhibitor p21/Cip1 and the differentiation marker involucrin was sensitive to MEK inhibition, which suggests a role for the Raf/MEK/ERK pathway in early stages of keratinocyte differentiation.     

FGF-2 and TPA induce matrix metalloproteinase-9 secretion in MCF-7 cells through PKC activation of the Ras/ERK pathway

Matrix metalloproteinases (MMPs) play an important role in cancer metastasis. Here, we investigated the effect of fibroblast growth factor-2 (FGF-2) and 12-O-tetradecanoylphorbol-13-acetate (TPA) on the secretion of type IV collagenases (MMP-2, MMP-9) in breast cancer MCF-7 cells. As shown by gelatin zymography, both FGF-2 and TPA stimulated the secretion of MMP-9 in MCF-7 cells while they did not change the level of MMP-2 secretion. Signaling cascade studies indicated that both FGF-2 and TPA induced Ras activation, c-Raf phosphorylation, mitogen-activated protein kinase/ERK kinase (MEK(1/2)) phosphorylation, and extracellular signal-regulated kinase (ERK(1/2)) phosphorylation. The FGF-2- and TPA-induced MMP-9 secretion was significantly inhibited by transient transfection of MCF-7 cells with dominant negative Ras (Ras-N17) and by treatment with MEK(1/2) inhibitor PD98059. A pan-protein kinase C (PKC) inhibitor, GF109203X, was found to totally abolish the FGF-2- and TPA-induced MMP-9 secretion and ERK(1/2) phosphorylation. Use of isoform-specific PKC inhibitors such as Rotllerin and G?6976 suggested, moreover, that the PKC-delta isoform is a likely component of FGF-2 and TPA trophic signaling. These results demonstrated that FGF-2 and TPA induce MMP-9 secretion in MCF-7 cells mainly through PKC-dependent activation of the Ras/ERK(1/2) signaling pathway.     

Endothelin-1 induces expression of matrix-associated genes in lung fibroblasts through MEK/ERK

The endothelins are a family of endothelium-derived peptides that possess a variety of biological activities, including potent vasoconstriction. Endothelin-1 (ET-1) is up-regulated during tissue repair and pulmonary fibrosis. Here, we use genome-wide expression array analysis to show that the addition of ET-1 (100 nm, 4 h) to normal lung fibroblasts directly induces expression of matrix and matrix-associated genes, including the profibrotic protein CCN2 (connective tissue growth factor, or CTGF). ET-1 induces the MEK/ERK MAP kinase pathway in fibroblasts. Blockade of the MEK/ERK kinase pathway with U0126 abrogates the ability of ET-1 to induce expression of matrix and matrix-associated mRNAs and the CCN2 protein. The CCN2 promoter possesses an ET-1 response element, which maps to the previously identified basal control element-1 (BCE-1) site. Our results suggest that ET-1 induces a program of matrix synthesis in lung fibroblasts and that ET-1 may play a key role in connective tissue deposition during wound repair and in pulmonary fibrosis.     

Plasminogen/plasmin regulates c-fos and egr-1 expression via the MEK/ERK pathway

In this study, we showed that plasminogen (Plg) and plasmin (Pla) bind to lysine-binding sites on cell surface and trigger a signaling pathway that activates the mitogen-activated protein kinase (MAPK) MEK and ERK1/2, which in turn leads to the expression of the primary response genes c-fos and early growth response gene egr-1. Our data show that the Plg/Pla-stimulated steady-state mRNA levels of both genes reached a maximum by 30 min and then returned to basal levels by 1h. The gene induction was sensitive to both pharmacological and genetic inhibition of MEK. Leupeptin, a serine protease inhibitor, suppressed Pla but not Plg-induced c-fos and egr-1 expression, emphasizing the role played by the serine protease activity associated with Pla. Pre-incubation with cholera toxin completely blocked the Plg/Pla-induced gene expression, suggesting that another signaling pathway, which recruits G protein-coupled receptors, may also be involved. Furthermore, Plg/Pla also stimulated AP-1 and EGR-1 DNA-binding activities, which were abrogated by pharmacological inhibition of MEK. Altogether, these results suggest that Plg/Pla stimulates c-fos and egr-1 expression via activation of the MEK/ERK pathway.     

The activation of MEK/ERK signaling pathway by bone morphogenetic protein 4 to increase hepatocellular carcinoma cell proliferation and migration

Hepatocellular carcinoma (HCC) is one of the most common visceral malignancies worldwide, with a very high incidence and poor prognosis. Bone morphogenesis protein 4 (BMP4), which belongs to the TGF-β superfamily of proteins, is a multifunctional cytokine, which exerts its biologic effects through SMAD- and non-SMAD-dependent pathways, and is also known to be involved in human carcinogenesis. However, the effects of the BMP4 signaling in liver carcinogenesis are not yet clearly defined. Here, we first show that BMP4 and its receptor, BMPR1A, are overexpressed in a majority of primary HCCs and that it promotes the growth and migration of HCC cell lines in vitro. We also establish that BMP4 can induce HCC cyclin-dependent kinase (CDK)1 and cyclin B1 upregulation to accelerate cell-cycle progression. Our study indicates that the induction of HCC cell proliferation is independent of the SMAD signaling pathway, as Smad4 knockdown of HCC cell lines still leads to the upregulation of CDK1 and cyclin B1 expression after BMP4 treatment. Using mitogen-activated protein/extracellular signal-regulated kinase (MEK) selective inhibitors, the induction of CDK1, cyclin B1 mRNA and protein were shown to be dependent on the activation of MEK/extracellular signal-regulated kinase (ERK) signaling. In vivo xenograft studies confirmed that the BMPR1A-knockdown cells were significantly less tumorigenic than the control groups. Our findings show that the upregulation of BMP4 and BMPR1A in HCC promotes the proliferation and metastasis of HCC cells and that CDK1 and cyclin B1 are important SMAD-independent molecular targets in BMP4 signaling pathways, during the HCC tumorigenesis. It is proposed that BMP4 signaling pathways may have potential as new therapeutic targets in HCC treatment.     

Suppression of MEK/ERK signaling pathway enhances cisplatin-induced NF-kappaB activation by protein phosphatase 4-mediated NF-kappaB p65 Thr dephosphorylation

We previously reported that suppression of the MEK/ERK pathway increases drug resistance of SiHa cells. In this study, we further characterized the underlying mechanism of this phenomenon. Pretreatment of SiHa cells with MEK/ERK inhibitor enhanced cisplatin-induced NF-kappaB activation. However, results of immunoblotting analysis showed that neither cisplatin nor MEK/ERK inhibitors induced marked IkappaBalpha degradation, suggesting that suppression of the MEK/ERK signaling pathway may enhance cisplatin-induced NF-kappaB activation via mechanisms other than the conventional pathway. Previous findings that protein phosphatase 4 (PP4), a nuclear serine/threonine phosphatase, directly interacts with and activates NF-kappaB led us to examine the phosphorylation status of NF-kappaB p65. Coincident with activation of NF-kappaB, cisplatin induced Ser phosphorylation but decreased Thr phosphorylation of NF-kappaB p65. Suppression of the MEK/ERK pathway further enhanced cisplatin-induced Thr dephosphorylation but did not affect cisplatin-induced Ser phosphorylation of NF-kappaB p65. Further, in parallel with Thr dephosphorylation, the protein level of nuclear PP4 was increased in cisplatin-treated cells and was further increased by suppression of the MEK/ERK pathway. SiHa cells were then transfected by a sense or an antisense PP4 gene. PP4-overexpressing cells showed a decrease in Thr phosphorylation of NF-kappaB p65 to nearly undetectable levels, and both basal and cisplatin-induced NF-kappaB activities were higher than those in parental cells. By contrast, cisplatin, either alone or with MEK/ERK inhibitors, induced little NF-kappaB activation in antisense PP4-transfected cells. Coprecipitated complex kinase assay revealed a fragment of NF-kappaB p65 (amino acids 279-444) to contain potential phosphorylation sites that directly interact with PP4. Further studies by site-directed mutagenesis suggested that Thr(435) was the major phosphorylation site.     

c-Raf/MEK/ERK pathway controls protein kinase C-mediated p70S6K activation in adult cardiac muscle cells

p70S6 kinase (S6K1) plays a pivotal role in hypertrophic cardiac growth via ribosomal biogenesis. In pressure-overloaded myocardium, we show S6K1 activation accompanied by activation of protein kinase C (PKC), c-Raf, and mitogen-activated protein kinases (MAPKs). To explore the importance of the c-Raf/MAPK kinase (MEK)/MAPK pathway, we stimulated adult feline cardiomyocytes with 12-O-tetradecanoylphorbol-13-acetate (TPA), insulin, or forskolin to activate PKC, phosphatidylinositol-3-OH kinase, or protein kinase A (PKA), respectively. These treatments resulted in S6K1 activation with Thr-389 phosphorylation as well as mammalian target of rapamycin (mTOR) and S6 protein phosphorylation. Thr-421/Ser-424 phosphorylation of S6K1 was observed predominantly in TPA-treated cells. Dominant negative c-Raf expression or a MEK1/2 inhibitor (U0126) treatment showed a profound blocking effect only on the TPA-stimulated phosphorylation of S6K1 and mTOR. Whereas p38 MAPK inhibitors exhibited only partial effect, MAPK-phosphatase-3 expression significantly blocked the TPA-stimulated S6K1 and mTOR phosphorylation. Inhibition of mTOR with rapamycin blocked the Thr-389 but not the Thr-421/Ser-424 phosphorylation of S6K1. Therefore, during PKC activation, the c-Raf/MEK/extracellular signal-regulated kinase-1/2 (ERK1/2) pathway mediates both the Thr-421/Ser-424 and the Thr-389 phosphorylation in an mTOR-independent and -dependent manner, respectively. Together, our in vivo and in vitro studies indicate that the PKC/c-Raf/MEK/ERK pathway plays a major role in the S6K1 activation in hypertrophic cardiac growth.     

Growth arrest signaling of the Raf/MEK/ERK pathway in cancer

The Raf/MEK/extraceUular signal-regulated kinase （ERK） pathway has a pivotal role in facilitating cell proliferation, and its deregulated activation is a central signature of many epithelial cancers. However paradoxically, sustained activity of Raf/MEK/ERK can also result in growth arrest in many different cell types. This anti-proliferative Raf/MEK/ERK signaling also has physiological significance, as exemplified by its potential as a tumor suppressive mechanism. Therefore, significant questions include in which cell types and by what mechanisms this pathway can mediate such an opposing context of signaling. Particularly, our understating of the role of ERK1 and ERK2, the focal points of pathway signaling, in growth arrest signaling is still limited. This review discusses these aspects of Raf/MEK/ ERK-mediated growth arrest signaling.