Dephosphorylation-induced EZH2 activation mediated RECK downregulation by ERK1/2 signaling

The reversion-inducing cysteine-rich protein with Kazal motifs (RECK) gene, a widely known cancer inhibitor, could effectively suppress cancer metastasis and angiogenesis. Downregulation or loss of RECK expression frequently occurs during cancer progression. However, the mechanism underlying RECK dysregulation has not been fully elucidated. Herein, we reported for the first time that enhancer of zeste homolog 2 (EZH2), a histone methyltransferase, could epigenetically attenuate RECK expression via catalyzing H3K27 trimethylation (H3K27me3) within the RECK promoter. Furthermore, we also proved, for the first time, the involvement of EZH2 in the inhibition of RECK by extracellular signal-related kinases (ERK)-1/2 signaling. Next, we revealed that the modulation of the enzymic activity of EZH2 resulting from posttranslational phosphorylation at the serine-21 site was responsible for the increased enrichment of H3K27me3 at the RECK promoter region by ERK1/2 signaling. Collectively, the results of our study shed more light on the mechanisms responsible for the dysregulation of RECK by the ERK1/2 pathway.     

Upregulation of Ras/Raf/ERK1/2 signaling and ERK5 in the brain of autistic subjects

Autism is a neurodevelopmental disorder characterized by impairments in social interaction, verbal communication and repetitive behaviors. A number of studies have shown that the Ras/Raf/ERK1/2 (extracellular signal-regulated kinase) signaling pathway plays important roles in the genesis of neural progenitors, learning and memory. Ras/Raf/ERK1/2 and ERK5 have also been shown to have death-promoting apoptotic roles in neural cells. Recent studies have shown a possible association between neural cell death and autism. In addition, two recent studies reported that a deletion of a locus on chromosome 16, which included the mitogen-activated protein kinase 3 (MAPK3) gene that encodes ERK1, is associated with autism. Most recently, our laboratory detected that Ras/Raf/ERK1/2 signaling activities were significantly enhanced in the brain of BTBR mice that model autism, as they exhibit many autism-like behaviors. We thus hypothesized that Ras/Raf/ERK1/2 signaling and ERK5 could be abnormally regulated in the brain of autistic subjects. In this study, we show that the expression of Ras protein was significantly elevated in the frontal cortex of autistic subjects. C-Raf phosphorylation was increased in the frontal cortex, while both C-Raf and A-Raf activities were enhanced in the cerebellum of autistic subjects. We also detected that both the protein expression and activities of ERK1/2 were significantly upregulated in the frontal cortex of autistic subjects, but not in the cerebellum. Furthermore, we showed that ERK5 protein expression is upregulated in both frontal cortex and cerebellum of autistic subjects. These results suggest that the upregulation of Ras/Raf/ERK1/2 signaling and ERK5 activities mainly found in the frontal cortex of autistic subjects may be critically involved in the pathogenesis of autism.     

Baicalein protects against cardiac hypertrophy through blocking MEK‐ERK1/2 signaling

Baicalein, a flavonoid present in the root of Scutellaria baicalensis, is well known for its antibacterial, antiviral, anti‐inflammatory, antithrombotic, and antioxidant effects. Here we show that baicalein also attenuates cardiac hypertrophy. Aortic banding (AB) was performed to induce cardiac hypertrophy secondary to pressure overload in mice. Mouse chow containing 0.05% baicalein (dose: 100 mg/kg/day baicalein) was begun 1 week prior to surgery and continued for 8 weeks after surgery. Our data demonstrated that baicalein prevented cardiac hypertrophy and fibrosis induced by AB, as assessed by echocardiographic and hemodynamic parameters and by pathological and molecular analysis. The inhibitory action of baicalein on cardiac hypertrophy was mediated by effects on mitogen‐activated protein kinase kinase (MEK)‐extracellular signal‐regulated kinases (ERK1/2) signaling and GATA‐4 activation. In vitro studies performed in rat cardiac H9c2 cells confirmed that baicalein attenuated cardiomyocyte hypertrophy induced by angiotensin II, which was associated with inhibiting MEK‐ERK1/2 signaling. In conclusion, our results suggest that baicalein has protective potential for targeting cardiac hypertrophy and fibrosis through suppression of MEK‐ERK1/2 signaling. Baicalein warrants further research as a potential antihypertrophic agent that might be clinically useful to treat cardiac hypertrophy and heart failure. J. Cell. Biochem. 114: 1058–1065, 2013. © 2012 Wiley Periodicals, Inc.     

Oxidative stress-induced apoptosis is mediated by ERK1/2 phosphorylation

Oxidative stress is known to induce apoptosis in a wide variety of cell types, apparently by modulating intracellular signaling pathways. High concentrations of H2O2 have been found to induce apoptosis in L929 mouse fibroblast cells. To elucidate the mechanisms of H2O2-mediated apoptosis, ERK1/2, p38-MAPK, and JNK1/2 phosphorylation was examined, and ERK1/2 and JNK1/2 were found to be activated by H2O2. Inhibition of ERK1/2 activation by treatment of L929 cells with PD98059 or dominant-negative ERK2 transfection blocked H2O2-induced apoptosis, while inhibition of JNK1/2 by dominant-negative JNK1 or JNK2 or MKK4 or MKK7 transfection did not affect H2O2-mediated apoptosis. H2O2-mediated ERK1/2 activation was not only Ras-Raf dependent, but also both tyrosine kinase (PDGFbeta receptor and Src) and PKCdelta dependent. H2O2-mediated PKCdelta-dependent and tyrosine kinase-dependent ERK1/2 activations were independent from each other. Based on the above results, we suggest for the first time that oxidative damage-induced apoptosis is mediated by ERK1/2 phosphorylation which is not only Ras-Raf dependent, but also both tyrosine kinase and PKCdelta dependent.     

Involvement of ERK1/2 signaling pathway in DJ-1-induced neuroprotection against oxidative stress

Parkinson’s disease (PD) is a progressive neurodegenerative disorder. Although the precise mechanism remains unclear, mounting evidence suggests that oxidative stress plays an important role in the pathogenesis of PD. DJ-1 gene is associated with oxidative stress and mutations in DJ-1 are involved in an autosomal recessive, early onset familial form of PD. The ERK1/2 signaling pathway contributes to neuroprotection during oxidative stress. However, the correlation between DJ-1 and the ERK1/2 signaling pathway remains unknown. To test for an association of DJ-1 with the ERK1/2 signaling pathway, we transfected wild-type and L166P mutated DJ-1 into COS-7 and MN9D cells. The results showed that over-expression of WT-DJ-1 dramatically enhanced the phosphorylation of ERK1/2 and its upstream kinase MEK1/2. Meanwhile, WT-DJ-1, but not L166P-DJ-1 inhibited the expression of protein phosphatase 2A (PP2A), an inhibitor of the ERK1/2 signaling pathway. Moreover, over-expression of WT-DJ-1 increased cell viability and decreased cell sensitivity to H₂O₂-induced neurotoxicity. Inhibition of the ERK1/2 signaling pathway with a MEK1/2 inhibitor reversed these changes. We conclude that DJ-1 does affect the ERK1/2 signaling pathway and change the susceptibility of cells to oxidative stress.     

Curcumin induces apoptosis in JAK2‐mutated cells by the inhibition of JAK2/STAT and mTORC1 pathways

Myeloproliferative neoplasms are chronic myeloid cancers divided in Philadelphia positive and negative. The JAK2 V617F is the most common mutation in Philadelphia negative patients and results in a constitutive activation of the JAK/STAT pathway, conferring a proliferative advantage and apoptosis inhibition. Recent studies identified a functional crosstalk between the JAK/STAT and mTOR pathways. The identification of an effective therapy is often difficult, so the availability of new therapeutic approaches might be attractive. Previous studies showed that curcumin, the active principle of the Curcuma longa, can suppress JAK2/STAT pathways in different type of cancer and injuries. In this study, we investigated the anti‐proliferative and pro‐apoptotic effects of curcumin in JAK2 V617F‐mutated cells. HEL cell line and cells from patients JAK2 V617F mutated have been incubated with increasing concentrations of curcumin for different time. Apoptosis and proliferation were evaluated. Subsequently, JAK2/STAT and AKT/mTOR pathways were investigated at both RNA and protein levels. We found that curcumin induces apoptosis and inhibition of proliferation in HEL cells. Furthermore, we showed that curcumin inhibits JAK2/STAT and mTORC1 pathways in JAK2 V617F‐mutated cells. This inhibition suggests that curcumin could represent an alternative strategy to be explored for the treatment of patients with myeloproliferative neoplasms.     

Glutamate accelerates RPE cell proliferation through ERK1/2 activation via distinct receptor-specific mechanisms

The proliferation and migration of Retinal Pigment Epithelium cells resulting from an epithelial-mesenchymal transition plays a key role in proliferative vitreoretinopathy, which leads to retinal detachment and the loss of vision. In neurons, glutamate has been shown to activate the Ras/Raf/MEK/ERK cascade, which participates in the regulation of proliferation, differentiation, and survival processes. Although glutamate-stimulation and the activation of ERK1/2 by different stimuli have been shown to promote RPE cell proliferation, the signaling pathway(s) linking these effects has not been established. We analyzed the molecular mechanisms leading to glutamate-induced proliferation by determining ERK1/2 and CREB phoshporylation in chick RPE cells in primary culture and the human-derived RPE cell line ARPE-19. This study shows for the first time, that glutamate promotes RPE cell proliferation by activating two distinct signaling pathways linked to selective glutamate receptor subtypes. Results demonstrate that glutamate stimulates RPE cell proliferation as well as ERK and CREB phosphorylation. These effects were mimicked by the mGluR agonist ACPD and by NMDA, and were prevented by the respective receptor inhibitors MCPG and MK-801, indicating a cause-effect relationship between these processes. Whereas mGluR promoted proliferation by activating the MEK/ERK/CREB cascade, NMDA stimulated proliferation through the MEK-independent activation of Ca(2+)/calmodulin-dependent kinases. The blockage of both signaling pathways to proliferation by KN-62 suggests the involvement of CaMKs in the control of glutamate-induced proliferation at a common step, downstream of CREB, possibly the regulation of cell cycle progression. Based on these findings, the participation of glutamate in the development of PVR can be considered.     

Epac1‐induced cellular proliferation in prostate cancer cells is mediated by B‐Raf/ERK and mTOR signaling cascades

cAMP‐dependent, PKA‐independent effects on cell proliferation are mediated by cAMP binding to EPAC and activation of Rap signaling. In this report, we employed the analogue 8‐CPT‐2‐O‐Me‐cAMP to study binding to EPAC and subsequent activation of B‐Raf/ERK and mTOR signaling in human cancer cells. This compound significantly stimulated DNA synthesis, protein synthesis, and cellular proliferation of human 1‐LN prostate cancer cells. By study of phosphorylation‐dependent activation, we demonstrate that EPAC‐mediated cellular effects require activation of the B‐Raf/ERK and mTOR signaling cascades. RNAi directed against EPAC gene expression as well as inhibitors of ERK, PI 3‐kinase, and mTOR were employed to further demonstrate the role of these pathways in regulating prostate cancer cell proliferation. These studies were then extended to several other human prostate cancer cell lines and melanoma cells with comparable results. We conclude that B‐Raf/ERK and mTOR signaling play an essential role in cAMP‐dependent, but PKA‐independent, proliferation of cancer cells. J. Cell. Biochem. 108: 998–1011, 2009. © 2009 Wiley‐Liss, Inc.     

Role of ERK1/2 signaling during EGF-induced inhibition of palatal fusion

During mammalian palatal fusion, the medial edge epithelial (MEE) cells must stop DNA synthesis prior to the initial contact of opposing palatal shelves and thereafter selectively disappear from the midline. Exogenous EGF has been shown to inhibit the cessation of DNA synthesis and induce cleft palate; however, the precise intracellular mechanism has not been determined. We hypothesized that EGF signaling acting via ERK1/2 would maintain MEE DNA synthesis and cell proliferation and consequently inhibit the process of palatal fusion. Palatal shelves from E13 mouse embryos were maintained in organ cultures and stimulated with EGF. EGF-treated palates failed to fuse with intact MEE and had significant ERK1/2 phosphorylation. Both EGF-induced ERK1/2 phosphorylation and BrdU-incorporation were localized in the nucleus of MEE cells. Subsequent inhibition assays using U0126, a specific inhibitor of ERK1/2 phosphorylation, were conducted. U0126 inhibited EGF-induced ERK1/2 phosphorylation in a dose-dependent manner and consequently MEE cells stopped proliferation. The threshold of ERK1/2 inactivation to stop MEE DNA synthesis coincides with the level required to rescue the EGF-induced cleft palate phenotype. These results indicate that EGF-induced inhibition of palatal fusion is dependent on nuclear ERK1/2 activation and that this mechanism must be tightly regulated during normal palatal fusion.     

Association of upregulated Ras/Raf/ERK1/2 signaling with autism

Autism is a neurodevelopmental disorder characterized by impairments in social interaction, verbal communication and repetitive behaviors. BTBR mouse is currently used as a model for understanding mechanisms that may be responsible for the pathogenesis of autism. Growing evidence suggests that Ras/Raf/ERK1/2 signaling plays death-promoting apoptotic roles in neural cells. Recent studies showed a possible association between neural cell death and autism. In addition, two studies reported that a deletion of a locus on chromosome 16, which includes the MAPK3 gene that encodes ERK1, is associated with autism. We thus hypothesized that Ras/Raf/ERK1/2 signaling could be abnormally regulated in the brain of BTBR mice that models autism. In this study, we show that expression of Ras protein was significantly elevated in frontal cortex and cerebellum of BTBR mice as compared with B6 mice. The phosphorylations of A-Raf, B-Raf and C-Raf were all significantly increased in frontal cortex of BTBR mice. However, only C-Raf phosphorylation was increased in the cerebellum of BTBR mice. In addition, we further detected that the activities of both MEK1/2 and ERK1/2, which are the downstream kinases of Ras/Raf signaling, were significantly enhanced in the frontal cortex. We also detected that ERK1/2 is significantly over-expressed in frontal cortex of autistic subjects. Our results indicate that Ras/Raf/ERK1/2 signaling is upregulated in the frontal cortex of BTBR mice that model autism. These findings, together with the enhanced ERK1/2 expression in autistic frontal cortex, imply that Ras/Raf/ERK1/2 signaling activities could be increased in autistic brain and involved in the pathogenesis of autism.