ERK-specific Inhibitor U0126 Research Articles

Icaritin alleviates cerebral ischemia‒reperfusion injury by regulating NMDA receptors through ERK signaling

N-methyl-D-aspartate (NMDA) receptors are key signaling molecules that mediate excitotoxicity during cerebral ischemia. GluN2A-containing NMDA receptors, which are mostly located in the intrasynaptic region, mediate normal physiological processes and promote neuronal survival. GluN2B-containing NMDA receptors, which are mostly located in the extrasynaptic region, mediate excitotoxicity injury and promote neuronal death during ischemia. This study investigated the ability of icaritin (ICT) to protect against cerebral ischemia‒reperfusion injury (CI/RI) by regulating GluN2B-containing NMDA receptors through extracellular signaling regulatory kinases/death associated protein kinase 1 (ERK/DAPK1) signaling. A rat CI/RI model was established by transient middle cerebral artery occlusion (tMCAO). Following treatment with ICT and the ERK-specific inhibitor U0126, cerebral infarction, neurological function, and excitotoxicity-related molecule expression were assessed 24 h after reperfusion. ICT treatment significantly decreased cerebral infarct volume, improved neurological function, and regulated NMDA receptor subtype expression and ERK/DAPK1 signaling activation. The ability of ICT to increase GluN2A and postsynaptic density protein 95 (PSD95) mRNA and protein expression, inhibit GluN2B expression, and regulate DAPK1 activation was reversed after administration of the ERK-specific inhibitor U0126. These data indicated that ICT inhibited excitotoxicity injury and exerted a protective effect against CI/RI that was likely mediated by increased ERK signaling pathway activation and regulation of extrasynaptic and intrasynaptic NMDA receptor function, providing a new therapeutic target for ischemic encephalopathy.

Paeoniflorin ameliorates chronic stress-induced depression-like behavior in mice model by affecting ERK1/2 pathway

ABSTRACT Depression is a mental and emotional disorder that has made an opening great burden to the society. Paeoniflorin showed remarkable antidepressant-like effects in multiple animal models with depressive disorders. However, the molecule of paeoniflorin on depression is less studied. This study aims to explore the effect and the molecular mechanism of paeoniflorin on depression in a chronic restraint stress (CRS) mice model. CRS model of C57BL/6 J mice was set up. Sucrose preference test (SPT), tail suspension test (TST), open field test (OFT) and forced swimming test (FST) were used to assess depression symptoms. Immunofluorescence staining, quantitative reverse transcription-polymerase chain reaction (qRT-PCR) and western blotting were implemented to detect the expression changes of the proteins involved in extracellular signal-regulated kinase 1/2 (ERK1/2) signaling pathway. Results showed that paeoniflorin treatment decreased the degree of depression in the CRS mice. Further analysis showed that the expression of ERK1/2 proteins was significantly downregulated, while paeoniflorin could elevate the expression of ERK1/2 proteins in CRS mice. Finally, it showed that inhibiting signaling ERK1/2 pathway could aggravate the depressive behavior when treatment with ERK-specific inhibitor U0126, while the condition could be partially relieved when treated with paeoniflorin. In conclusion, the present study demonstrated that paeoniflorin attenuated chronic stress-induced depression-like behavior in mice by affecting the ERK1/2 pathway. These findings provided the basis for the molecular mechanism of paeoniflorin on the effect of depression, which support paeoniflorin might act as an important drug in the treatment of depression.

Icaritin Alleviates Glutamate-Induced Neuronal Damage by Inactivating GluN2B-Containing NMDARs Through the ERK/DAPK1 Pathway

Excitatory toxicity due to excessive glutamate release is considered the core pathophysiological mechanism of cerebral ischemia. It is primarily mediated by N-methyl-D-aspartate receptors (NMDARs) on neuronal membranes. Our previous studies have found that icaritin (ICT) exhibits neuroprotective effects against cerebral ischemia in rats, but the underlying mechanism is unclear. This study aims to investigate the protective effect of ICT on glutamate-induced neuronal injury and uncover its possible molecular mechanism. An excitatory toxicity injury model was created using rat primary cortical neurons treated with glutamate and glycine. The results showed that ICT has neuroprotective effects on glutamate-treated primary cortical neurons by increasing cell viability while reducing the rate of lactate dehydrogenase (LDH) release and reducing apoptosis. Remarkably, ICT rescued the changes in the ERK/DAPK1 signaling pathway after glutamate treatment by increasing the expression levels of p-ERK, p-DAPK1 and t-DAPK1. In addition, ICT also regulates NMDAR function during glutamate-induced injury by decreasing the expression level of the GluN2B subunit and enhancing the expression level of the GluN2A subunit. As cotreatment with the ERK-specific inhibitor U0126 and ICT abolishes the beneficial effects of ITC on the ERK/DAPK1 pathway, NMDAR subtypes and neuronal cell survival, ERK is recognized as a crucial mediator in the protective mechanism of ICT. In conclusion, our findings demonstrate that ICT has a neuroprotective effect on neuronal damage induced by glutamate, and its mechanism may be related to inactivating GluN2B-containing NMDAR through the ERK/DAPK1 pathway. This study provides a new clue for the prevention and treatment of clinical ischemic cerebrovascular diseases.

Overexpression of P16 reversed the MDR1-mediated DDP resistance in the cervical adenocarcinoma by activating the ERK1/2 signaling pathway

BackgroundTo investigate the role of P16 (INK4a)-extracellular signal related kinase 1/2 (ERK1/2) signaling pathway in cisplatin (DDP) resistance induced by multidrug resistance protein 1 (MDR1), also known as P-glycoprotein (P-gp), in cervical adenocarcinoma.MethodsA human DDP-resistant HeLa cell line (HeLa/DDP) was constructed using the combination of incremental and intermittent administration of DDP. Cell Counting Kit-8 (CCK-8) assay was used to measure the IC50 and resistance index (RI) of cells. The morphological changes and population doubling time were observed under an inverted microscope. Plate cloning formation assay was performed to evaluate the cell proliferation and tumorigenic ability. Cell invasion and migration were determined by transwell assays. Besides, the expression of P16, phosphorylated extracellular signal related kinase 1 and 2 (pERK1/2), total ERK1/2 and MDR1 were measured using western blot analysis. The ERK-specific inhibitor U0126 and agonist TPA was used to explore the role of ERK.ResultsThe DDP-resistant cervical adenocarcinoma HeLa/DDP cell line was successfully established, which showed stronger cell growth, invasion, and migration. In the HeLa/DDP cells, pERK1/2 was downregulated, P-gp was upregulated and P16 was downregulated. Overexpression of P16 led to a significant decrease in the proliferation rate, migration ability, and invasion ability of the HeLa/DDP cells. Furthermore, overexpression of P16 increased and the decreased expression of pERK1/2 and P-gp in the HeLa/DDP cells, respectively. Treatment of HeLa/DDP cells transfected with P16 plasmid with ERK-specific inhibitor U0126 significantly decreased the expression of pERK1/2 and increased the expression of P-gp from 6 h to 48 h. Moreover, after 72 h, the expression of pERK1/2 was up-regulated and the expression of P-gp was inhibited.ConclusionOverexpression of P16 could partially reverse the MDR1-mediated DDP resistance in the cervical adenocarcinoma by the enhancement of phosphorylation of ERK signaling pathway, which provided a theoretical basis for the treatment of DDP resistance in cervical adenocarcinoma.

Increased SPK1 expression promotes cell growth by activating the ERK1/2 signaling in non-small-cell lung cancer.

Lung cancer remains the leading cause of cancer-associated mortality in China and the world. Increasing numbers of studies have reported that sphingosine kinase 1 (SPK1) is frequently highly expressed in tumors of various origins, including lung cancer, and its high expression contributes toward tumor progression. However, the clinical significance of SPK1 and its role in the growth and metastasis of non-small-cell lung cancer (NSCLC) remain unclear. In the present study, we found that SPK1 expression was expressed highly in NSCLC tissues and cell lines. Knockdown of SPK1 suppressed cell growth, proliferation, migration, and invasion and increased apoptosis. Moreover, knocking down SPK1 expression inhibited the growth of tumors in nude mice. Mechanistically, silencing the expression of SPK1 inhibited the expression of p-extracellular signal-regulated kinase (ERK). Moreover, the ERK-specific inhibitor U0126 suppressed the expression of the epithelial-mesenchymal transition of lung cancer cells. Together, our findings indicated that SPK1 enhanced tumor growth in lung cancer and induced metastasis by activating the ERK1/2 signaling pathway, indicating its potential application in NSCLC diagnosis and therapy.

Paeoniflorin Ameliorates Chronic Stress-Induced Depression-Like Behaviors and Neuronal Damages in Rats via Activation of the ERK-CREB Pathway.

Neuronal damage is related to the onset and treatment of depressive disorders. Antidepressant-like effects have been elicited by paeoniflorin on animal models. The aim of this study is to demonstrate whether the neuroprotective effect of paeoniflorin on rats suffered from chronic unpredictable mild stress (CUMS) was regulated by the ERK-CREB signaling pathway. Results showed that paeoniflorin not only ameliorated depressive-like behavior with low locomotor activity and prolonged immobility duration in our forced swimming test but also reduced sucrose consumption. Paeoniflorin treatment decreased the degree of neuronal damage in the hippocampus of the model rats. Conversely, it markedly increased the mRNA levels of ERK1, ERK2, and CREB and the levels of ERK, p-ERK, CREB, and p-CREB protein expression in the hippocampus. Blockade of the ERK-CREB axis with the ERK-specific inhibitor U0126 repressed the neuroprotective and antidepressant-like effects of paeoniflorin on rats in the setting of chronic-mild-stress and abolished the recoveries of p-ERK mediated by paeoniflorin treatment. Thus, paeoniflorin possibly exerted a neuroprotective effect modulated by the ERK-CREB signaling pathway on CUMS-induced hippocampal damage in rats.

Inhibition of ERK signaling potentiates the anti-tumor activity of FL118 on hepatocellular carcinoma cells

Background: A novel camptothecin analogue, FL118, has been shown to exhibit superior anticancer activity over irinotecan on colon and lung cancer cells. It was reported that FL118 mainly performs its broadspectrum anticancer activity through inducing cancer cell apoptosis. Accumulated evidence indicated that ERK pathway was constitutively activated in liver cancer, thereby promoting tumorigenesis, development, metastasis and recurrence. In this study, we aimed to investigate the anticancer potency of FL118 on liver cancer cell lines and the effect of FL118 on extracellular regulated protein kinase (ERK) pathway. Methods: Viability of PLC and Huh7 cells was detected by MTT assay. Western blot assay was performed to assess phosphorylated and total ERK protein. And cell apoptosis was measured by Hochest-PI staining. Results: Our results revealed that FL118 can inhibit PLC and Huh7 cell viability and markedly induce their apoptosis. Western blot results indicated that ERK pathway was activated in huh7 and PLC cells after administration of FL118. In addition, the pro-apoptotic and antiviability ability of FL118 can be potentiated by ERK-specific inhibitor U0126. Conclusions: Our study confirmed good anticancer activity of FL118 on liver cancer, and targeting ERK pathway can significantly improve its anticancer potency.

Effects of High Glucose on Cell Viability and Differentiation in Primary Cultured Schwann Cells: Potential Role of ERK Signaling Pathway.

Diabetic peripheral neuropathy (DPN) is one of the most common complications of diabetes mellitus and hyperglycemia is considered to be the major factor in the development and progression of DPN. Because of the contribution of Schwann cells (SCs) to the pathology of DPN, we investigated the effects of high glucose on cell proliferation, apoptosis and differentiation in primary cultured SCs. Cell Counting Kit-8 (CCK-8) assay and Hoechst staining showed that high glucose inhibited SCs proliferation and increased apoptosis ratio in time and concentration dependent manner. Western blot and real-time quantitative PCR analysis revealed that the major myelin proteins and genes expressions including P0, MAG and Krox-20, were downregulated time dependently in SCs exposed to high glucose from 48 to 96h. To further elucidate the underlying pathogenic mechanisms, we also explored the role of ERK signaling pathway in high glucose induced SC injury, which has been proved to drive demyelination of peripheral nerves. The western blot analysis showed that compared with control group phosphorylation level of ERK was increased by 14.3% in SCs exposed to high glucose for 72h (P<0.01). Using immunocytochemistry analysis, we observed that the ERK specific inhibitor U0126 blocked the ERK activation induced by high glucose and reversed the inhibitory effect of high glucose on P0 expression. Taken together, these results suggest that high glucose can cause damage in primary cultured SCs and may exert the inhibitory effect on SC differentiation and myelination through ERK signaling activation.

Estrogen receptor alpha promotes smoking-carcinogen-induced lung carcinogenesis via cytochrome P450 1B1.

Smoking carcinogen N-nitrosamines such as 4-methylnitrosamino-l-3-pyridyl-butanone (NNK) require metabolic activation to exert their genotoxicity. The first activation step is mainly catalyzed by cytochrome P450 (CYP) family. Estrogen receptor α (ERα) plays a role in lung pathology. The association between them is unknown. In this study, we explored the relationship and function of CYP1B1 and ERα in NNK-induced lung tumorigenesis. CYP1B1 and ERα expression was analyzed in human lung cancer tissues and NNK-induced lung tumor of A/J mice. Cell lines NCI-H23 and NCI-H460 were employed to further study the responsible mechanisms using various cellular and molecular approaches. Our in vivo experiments demonstrated that CYP1B1 and ERα were over-expressed at the early stage of NNK-induced lung tumorigenesis. Microarray analysis found that ERα was involved in the extracellular-signal-regulated kinase (ERK)/MAPK pathway. NNK activated RAS/ERK/AP1 as it remarkably increased the levels of p-ERK, c-Fos, and c-Jun but inhibited multiple negative regulators of Ras/ERK/AP1, Pdcd4, Spry1, Spry2, and Btg2 through up-regulating miR-21. Both CYP1B1 siRNA and ERK-specific inhibitor U0126 suppressed NNK-mediated ERα up-regulation, suggesting that ERα was downstream of CYP1B1 and ERK. ERK inactivation led to the accumulation of CYP1B1, indicating that CYP1B1 was upstream of ERK activation. Inhibition of ERK or ERα decreased NNK-induced cell proliferation. Blockage of CYP1B1 or ERα induced apoptosis of lung cancer cells. Collectively, NNK-mediated ERα induction is via CYP1B1 and ERK and contributes to the lung carcinogenesis. The inhibition of CYP1B1, ERK, or ERα may arrest the lung cancer cell growth, implicating a pivotal strategy for the treatment of lung cancer. Smoking carcinogen NNK requires metabolic activation to exert their genotoxicity. CYP1B1 is the enzyme to catalyze NNK. NNK activates CYP1B1 and ERK to induce ERα. Inhibition of CYP1B1, ERK, or ERα arrests the lung cancer cell growth.

Role of endothelin B receptor in oligodendroglioma proliferation and survival: In vitro and in vivo evidence

In this study, the role of the endothelinB receptor (ETBR) in oligodendroglioma cell proliferation and survival was investigated invitro and invivo. The overexpression and knockdown of ETBR was conducted in Hs683 human oligodendroglioma cells, and cell proliferation and activation (phosphorylation) of extracellular signal-regulated kinase (ERK) were measured invitro. An orthotopic xenograft oligodendroglioma mouse model was established. Mouse survival times and immunohistochemical Ki67 staining in the xenografts were examined. Invitro experiments demonstrated that the overexpression of ETBR significantly enhanced the proliferation of oligodendroglioma cells and the activation of ERK compared with the controls, which was eliminated by the selective ETBR inhibitor BQ788 and ERK-specific inhibitor U0126, but not selective endothelinA receptor inhibitor BQ123. By contrast, the knockdown of endogenous ETBR markedly decreased oligodendroglioma cell proliferation and the activation of ERK compared with the controls. Overexpression of ETBR significantly increased immunohistochemical Ki67 staining in the Hs683 cell orthotopic xenograft and decreased animal survival. By contrast, knockdown of ETBR significantly decreased Ki67 staining and increased mouse survival times. Intratumoral injection of BQ788, but not BQ123, significantly decreased Ki67 staining and prolonged mouse survival times. In conclusion, ETBR was demonstrated to mediate the proliferation of oligodendroglioma cells according to an ERK-dependent mechanism. Using an orthotopic xenograft oligodendroglioma mouse model, it was demonstrated invivo that ETBR promotes oligodendroglioma proliferation and that the selective ETBR antagonist effectively inhibits the proliferation of oligodendroglioma cells and prolongs survival times. This study provides a novel insight into the role of ETBR in oligodendroglioma proliferation and survival, and provides the first invivo evidence that ETBR-specific antagonists are a potential therapeutic alternative for oligodendrogliomas.

Cardioprotective effect of Danshensu against myocardial ischemia/reperfusion injury and inhibits apoptosis of H9c2 cardiomyocytes via Akt and ERK1/2 phosphorylation

Danshensu, as the effective component of Salvia miltiorrhiza (Danshen), has been widely used in clinic for treatment of cardiovascular diseases in China. In the present study, we aimed to confirm the cardioprotective effect of Danshensu from myocardial ischemia/reperfusion (MI/R) injury in vivo, and investigate the potential molecular mechanisms against simulated ischemia/reperfusion (SI/R) induced cardiomyocytes apoptosis in vitro. The rat model of MI/R injury was induced by a transient vessel occlusion for 30min and reperfusion for 3h, Danshensu significantly reduced myocardium infarct size and the production of creatine kinase-MB (CK-MB), cardiac troponin (cTnI) in serum. In vitro experiment, H9c2 cardiomyocytes were incubated with vehicle or ischemic buffer during hypoxia, and then it was reoxygenated with or without Danshensu. Danshensu markedly improved cell viability and decreased lactate dehydrogenase (LDH) release. We confirmed anti-apoptotic effect of Danshensu both by flow-cytometric analysis and terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay, and this effect was associated with the increase of Bcl-2/Bax ratio and the decrease of active caspase-3 expression. Western blot analysis also showed that Danshensu increased phosphorylation of Akt and extracellular signal-related kinase 1/2 (ERK1/2) in H9c2 cells, and the protective effects of Danshensu were partially inhibited by phosphatidylinositol 3′-kinase (PI3K) specific inhibitor wortmannin or ERK specific inhibitor U0126. Our results suggested that Danshensu could provide significant cardioprotection against MI/R injury, and the potential mechanisms might to suppression of cardiomyocytes apoptosis through activating the PI3K/Akt and ERK1/2 signaling pathways.

Nocodazole increases the ERK activity to enhance MKP-1 expression which inhibits p38 activation induced by TNF-α

The mitogen-activated protein kinase p38 plays key roles in cell progression, differentiation, inflammation, and apoptosis. p38 is activated by a variety of extracellular stimuli such as UV and proinflammatory cytokine tumor necrosis factor alpha (TNF-α). It has been demonstrated that destruction of microtubules with different reagents led to impaired p38 activation in response to various extracellular stimuli. However, several other groups have reported that microtubule-interfering agents stimulate the activation of MAPK superfamily members including p38 in certain cell context. The discrepancy suggests that destruction of microtubules stimulates the activation of MAPK superfamily members and thereby induces certain feedback inhibitor(s) of p38 signaling. In this article, we report that nocodazole, a widely used microtubule-interfering agent, antagonized UV- or TNF-α-induced p38 activation, even though this drug by itself weakly activated p38. The RNA synthesis inhibitor actinomycin D, but not p38-specific inhibitor SB203580, reversed the inhibitory effect of nocodazole on TNF-α-induced p38 activation. Nocodazole also weakly activated JNK, but significantly activated ERK. The inhibition by nocodazole of TNF-α-induced p38 activation was abolished by ERK-specific inhibitor U0126. Further exploration revealed that nocodazole significantly enhanced MKP-1 expression via the ERK activity. Thus, nocodazole increases the ERK activity to enhance MKP-1 expression which inhibits p38 activation induced by TNF-α.

Lumican induces human corneal epithelial cell migration and integrin expression via ERK 1/2 signaling

Lumican is a major proteoglycans of the human cornea. Lumican knock-out mice have been shown to lose corneal transparency and to display delayed wound healing. The purpose of this study was to define the role of lumican in corneal epithelial cell migration. Over-expression of lumican in human corneal epithelial (HCE-T) cells increased both cell migration and proliferation, and increased levels of integrins α2 and β1. ERK 1/2 was also activated in lumican over-expressed cells. When we treated HCE-T cells with the ERK-specific inhibitor U0126, cell migration and the expression of integrin β1 were completely blocked. These data provide evidence that lumican stimulates cell migration in the corneal epithelium by activating ERK 1/2, and point to a novel signaling pathway implicated in corneal epithelial cell migration.

Basic Fibroblast Growth Factor-induced Neuronal Differentiation of Mouse Bone Marrow Stromal Cells Requires FGFR-1, MAPK/ERK, and Transcription Factor AP-1

It has been reported recently that bone marrow stromal cells (BMSCs) are able to differentiate into various neural cells both in vivo and in vitro (Egusa, H., Schweizer, F. E., Wang, C. C., Matsuka, Y., and Nishimura, I. (2005) J. Biol. Chem. 280, 23691-23697). However, the underlying mechanisms remain largely unknown. In this report, we have demonstrated that basic fibroblast growth factor (bFGF) alone effectively induces mouse BMSC neuronal differentiation. These differentiated neuronal cells exhibit characteristic electrophysiological properties and elevated levels of the neuronal differentiation marker, growth-associated protein-43 (GAP-43). To explore possible signaling pathways, we first analyzed the expression of various FGF receptors in mouse BMSCs. FGF receptor-1, -2, and -3 were detected, but only FGFR-1 was shown to be activated by bFGF. Small interfering RNA knock down of FGFR-1 in BMSCs significantly inhibited neuronal differentiation. Moreover, we have shown that the mitogen-activated protein kinase (ERK1/2) is persistently activated and blockage of ERK activity with the ERK-specific inhibitor U0126 prevents neuronal differentiation. It appears that activation of ERK cascade and neuronal differentiation of BMSCs induced by bFGF are independent of Ras activity but require functions of phospholipase C-gamma pathway. Lastly, we examined the role of the immediate-early transcription factors AP-1 and NF-kappaB and have found that phospholipase C-gamma-dependent c-Jun and ERK-dependent c-fos, but not the NF-kappaB, are strongly activated by bFGF, which in turn regulates the neuronal differentiation of BMSCs.