Inhibition Of Extracellular Signal-regulated Kinase Activation Research Articles

ERK Signaling Pathway Is Constitutively Active in NT2D1 Non-Seminoma Cells and Its Inhibition Impairs Basal and HGF-Activated Cell Proliferation.

c-MET/hepatocyte growth factor (HGF) system deregulation is a well-known feature of malignancy in several solid tumors, and for this reason this system and its pathway have been considered as potential targets for therapeutic purposes. In previous manuscripts we reported c-MET/HGF expression and the role in testicular germ cell tumors (TGCTs) derived cell lines. We demonstrated the key role of c-Src and phosphatidylinositol 3-kinase (PI3K)/AKT adaptors in the HGF-dependent malignant behavior of the embryonal carcinoma cell line NT2D1, finding that the inhibition of these onco-adaptor proteins abrogates HGF triggered responses such as proliferation, migration, and invasion. Expanding on these previous studies, herein we investigated the role of mitogen-activated protein kinase (MAPK)/extracellular signal regulated kinase (ERK) pathways in the HGF-dependent and HGF-independent NT2D1 cells biological responses. To inhibit MAPK/ERK pathways we chose a pharmacological approach, by using U0126 inhibitor, and we analyzed cell proliferation, collective migration, and chemotaxis. The administration of U0126 together with HGF reverts the HGF-dependent activation of cell proliferation but, surprisingly, does not exert the same effect on NT2D1 cell migration. In addition, we found that the use of U0126 alone significantly promotes the acquisition of NT2D1 «migrating phenotype», while collective migration of NT2D1 cells was stimulated. Notably, the inhibition of ERK activation in the absence of HGF stimulation resulted in the activation of the AKT-mediated pathway, and this let us speculate that the paradoxical effects obtained by using U0126, which are the increase of collective migration and the acquisition of partial epithelium-mesenchyme transition (pEMT), are the result of compensatory pathways activation. These data highlight how the specific response to pathway inhibitors, should be investigated in depth before setting up therapy.

HMGCS2 Mediation of Ketone Levels Affects Sorafenib Treatment Efficacy in Liver Cancer Cells.

Primary liver cancer is the fifth leading death of cancers in men, and hepatocellular carcinoma (HCC) accounts for approximately 90% of all primary liver cancer cases. Sorafenib is a first-line drug for advanced-stage HCC patients. Sorafenib is a multi-target kinase inhibitor that blocks tumor cell proliferation and angiogenesis. Despite sorafenib treatment extending survival, some patients experience side effects, and sorafenib resistance does occur. 3-Hydroxymethyl glutaryl-CoA synthase 2 (HMGCS2) is the rate-limiting enzyme for ketogenesis, which synthesizes the ketone bodies, β-hydroxybutyrate (β-HB) and acetoacetate (AcAc). β-HB is the most abundant ketone body which is present in a 4:1 ratio compared to AcAc. Recently, ketone body treatment was found to have therapeutic effects against many cancers by causing metabolic alternations and cancer cell apoptosis. Our previous publication showed that HMGCS2 downregulation-mediated ketone body reduction promoted HCC clinicopathological progression through regulating c-Myc/cyclin D1 and caspase-dependent signaling. However, whether HMGCS2-regulated ketone body production alters the sensitivity of human HCC to sorafenib treatment remains unclear. In this study, we showed that HMGCS2 downregulation enhanced the proliferative ability and attenuated the cytotoxic effects of sorafenib by activating expressions of phosphorylated (p)-extracellular signal-regulated kinase (ERK), p-P38, and p-AKT. In contrast, HMGCS2 overexpression decreased cell proliferation and enhanced the cytotoxic effects of sorafenib in HCC cells by inhibiting ERK activation. Furthermore, we showed that knockdown HMGCS2 exhibited the potential migratory ability, as well as decreasing zonula occludens protein (ZO)-1 and increasing c-Myc expression in both sorafenib-treated Huh7 and HepG2 cells. Although HMGCS2 overexpression did not alter the migratory effect, expressions of ZO-1, c-Myc, and N-cadherin decreased in sorafenib-treated HMGCS2-overexpressing HCC cells. Finally, we investigated whether ketone treatment influences sorafenib sensitivity. We showed that β-HB pretreatment decreased cell proliferation and enhanced antiproliferative effect of sorafenib in both Huh7 and HepG2 cells. In conclusion, this study defined the impacts of HMGCS2 expression and ketone body treatment on influencing the sorafenib sensitivity of liver cancer cells.

Regulation of programmed death ligand 1 (PD-L1) expression by TNF-related apoptosis-inducing ligand (TRAIL) in triple-negative breast cancer cells.

Triple-negative breast cancer (TNBC) is an aggressive form of breast cancer that lacks targeted therapies. Previous studies have shown that TNBC cells are highly sensitive to tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL), making it a promising agent for treating TNBC. However, the development of TRAIL resistance limits its further clinical development, and the underlying mechanisms are not fully understood. In this study, we report the role of PD-L1 in TRAIL resistance. Specifically, we found that TRAIL treatment increases PD-L1 expression in TRAIL-sensitive cells and that basal PD-L1 expression is increased in acquired TRAIL-resistant cells. Mechanistically, we found that increased PD-L1 expression was accompanied by increased extracellular signal-regulated kinase (ERK) activation. Using both genetic and pharmacological approaches, we showed that knockdown of ERK by siRNA or inhibition of ERK activation by the mitogen-activated protein kinase kinase inhibitor U0126 decreased PD-L1 expression and increased TRAIL-induced cell death. Furthermore, we found that knockout or knockdown of PD-L1 enhances TRAIL-induced apoptosis, suggesting that PD-L1-mediated TRAIL resistance is independent of its ability to evade immune suppression. Therefore, this study identifies a noncanonical mechanism by which PD-L1 promotes TRAIL resistance, which can be potentially exploited for immune checkpoint therapy.

Inhibition of CXCR4 in Spinal Cord and DRG with AMD3100 Attenuates Colon-Bladder Cross-Organ Sensitization.

BackgroundCross-sensitization of pelvic organs is one theory for why symptoms of gut sickness and interstitial cystitis/bladder pain syndrome overlap. Experimental colitis has been shown to trigger bladder hyperactivity and hyperalgesia in rats. The chemokine receptor CXCR4 plays a key role in bladder function and central sensitization. We aim to study the role of CXCR4 and its inhibitor AMD3100 in colon-bladder cross-organ sensitization.MethodsThe colitis model was established by rectal infusion of trinitrobenzene sulfonic acid. Western blot and immunofluorescence were used to assess the expression and distribution of CXCR4. Intrathecal injection of AMD3100 (a CXCR4 inhibitor) and PD98059 (an ERK inhibitor) were used to inhibit CXCR4 and downstream extracellular signal-regulated kinase (ERK) in the spinal cord and dorsal root ganglion (DRG). Intravesical perfusion of resiniferatoxin was performed to measure the pain behavior counts of rats, and continuous cystometry was performed to evaluate bladder voiding function.ResultsCompared to the control group, CXCR4 was expressed more in bladder mucosa and colon mucosa, L6-S1 dorsal root ganglion (DRG), and the corresponding segment of the spinal dorsal horn (SDH) in rats with colitis. Moreover, intrathecal injection of the AMD3100 suppressed bladder overactivity, bladder hyperalgesia, and mastocytosis symptoms caused by colitis. Furthermore, AMD3100 effectively inhibited ERK activation in the spinal cord induced by experimental colitis. Finally, treatment with PD98059 alleviated bladder overactivity and hyperalgesia caused by colitis.ConclusionIncreased CXCR4 in the DRG and SDH contributes to colon inflammation-induced bladder overactivity and hyperalgesia partly via the phosphorylation of spinal ERK. Treatment targeting the CXCR4/ERK pathway might provide a potential new approach for the comorbidity between the digestive system and the urinary system.

Swine acute diarrhea syndrome coronavirus replication is reduced by inhibition of the extracellular signal-regulated kinase (ERK) signaling pathway

Swine acute diarrhea syndrome coronavirus (SADS-CoV) is a newly discovered enteric coronavirus. We have previously shown that the caspase-dependent FASL-mediated and mitochondrion-mediated apoptotic pathways play a central role in SADS-CoV-induced apoptosis, which facilitates viral replication. However, the roles of intracellular signaling pathways in SADS-CoV-mediated cell apoptosis and the relative advantages that such pathways confer on the host or virus remain largely unknown. In this study, we show that SADS-CoV induces the activation of ERK during infection, irrespective of viral biosynthesis. The knockdown or chemical inhibition of ERK1/2 significantly suppressed viral protein expression and viral progeny production. The inhibition of ERK activation also circumvented SADS-CoV-induced apoptosis. Taken together, these data suggest that ERK activation is important for SADS-CoV replication, and contributes to the virus-mediated changes in host cells. Our findings demonstrate the takeover of a particular host signaling mechanism by SADS-CoV and identify a potential approach to inhibiting viral spread.

Involvement of Antioxidant Defenses and NF-κB/ERK Signaling in Anti-Inflammatory Effects of Pterostilbene, a Natural Analogue of Resveratrol

Pterostilbene (PTE), a natural stilbenoid occurring in grapes and berries, is recognized as a dimethylated analogue of resveratrol. This compound shows numerous notable pharmacological activities, including antiaging, anticancer, antidiabetes, antioxidant, and neuroprotection. This study investigates the anti-inflammatory properties of PTE in macrophage cells (RAW 264.7) against the lipoteichoic acid (LTA) stimulation. The expression of inflammatory tumor necrosis factor (TNF-α), interleukin-1β (IL-1 β), and inducible nitric oxide synthase (iNOS) and the content of nitric oxide (NO) were detected in LTA-induced cells. In addition, a Western blot assay was used to detect mitogen-activated protein kinases: extracellular signal-regulated kinase (ERK)1/2, p38 MAPK, and c-Jun N-terminal kinase (JNK). The phosphorylation of IκB and p65 and translocation of nuclear factor kappa B (NF-κB) were assessed by Western blot and immuno-fluorescence staining. The results showed that PTE significantly attenuated NO production and TNF-α, IL-1 β, and iNOS expression in LTA stimulated cells. Among the activation of ERK, JNK, and p38 in cells treated with LTA, PTE at higher concentration had only inhibited ERK activation. However, PTE blocked IκB phosphorylation, phosphorylation and nuclear translocation of p65NF-κB. Fascinatingly, PTE enhanced antioxidant defense molecules as verified by the enhanced heme oxygenase-1 (HO-1) expression, catalase (CAT) antioxidant enzyme, and non-enzymatic antioxidant, and reduced glutathione (GSH) in LTA-induced RAW 264.7 cells. These results suggest that PTE exerts an anti-inflammatory property via attenuating NF-κB/ERK signaling pathways as well as enriching antioxidant defense mechanisms.

Loganin Attenuates the Severity of Acute Kidney Injury Induced by Cisplatin through the Inhibition of ERK Activation in Mice

Cisplatin is the most widely used chemotherapeutic agent. However, it often causes nephrotoxicity, which results in acute kidney injury (AKI). Therefore, we urgently need a drug that can reduce the nephrotoxicity induced by cisplatin. Loganin is a major iridoid glycoside isolated from Corni fructus that has been used as an anti-inflammatory agent in various pathological models. However, the renal protective activity of loganin remains unclear. In this study, to examine the protective effect of loganin on cisplatin-induced AKI, male C57BL/6 mice were orally administered with loganin (1, 10, and 20 mg/kg) 1 h before intraperitoneal injection of cisplatin (10 mg/kg) and sacrificed at three days after the injection. The administration of loganin inhibited the elevation of blood urea nitrogen (BUN) and creatinine (CREA) in serum, which are used as biomarkers of AKI. Moreover, histological kidney injury, proximal tubule damages, and renal cell death, such as apoptosis and ferroptosis, were reduced by loganin treatment. Also, pro-inflammatory cytokines, such as interleukin (IL)-1β, IL-6, and tumor necrosis factor (TNF)-α, reduced by loganin treatment. Furthermore, loganin deactivated the extracellular signal-regulated kinases (ERK) 1 and 2 during AKI. Taken together, our results suggest that loganin may attenuate cisplatin-induced AKI through the inhibition of ERK1/2.

Lanthionine Synthetase C‐like Protein 2: A potential therapeutic target for treating neuropathic pain

Glial activation and over‐production of pro‐inflammatory cytokines and chemokines in the spinal cord are critical mechanisms underlying the genesis of neuropathic pain. While pro‐inflammatory signaling molecules have been extensively investigated, much less is known about anti‐inflammatory signaling pathways and systems. Studies in recent years have demonstrated that lanthionine synthetase C‐like 2 protein (LANCL2) is expressed in cells related to the immune function, like T cells, macrophages, endothelial and epithelial cells, and dendritic cells, suggesting its potential as a target for immunoregulation. In this study, we determined the role of LANCL2 in the genesis of neuropathic pain. Specifically, we show that LANCL2 protein is expressed in spinal dorsal horn and down‐regulated in rats with neuropathic pain. Knockdown of spinal LANCL2 induces mechanical allodynia and thermal hyperalgesia, which is concomitantly accompanied with increases in extracellular signal‐regulated kinase (ERK) activity, tumor necrosis factor alpha (TNF‐α) production, and the activation of astrocytes and microglia in the spinal dorsal horn. Activation of spinal LANCL2 by intrathecal administration of abscisic acid alleviates mechanical and thermal hypersensitivity induced by nerve injury. Pre‐emptive pharmacological activation of LANCL2 prevents the down‐regulation of LANCL2 protein expression, and significantly attenuates the development of mechanical allodynia and thermal hyperalgesia in rats induced by nerve injury. These behavioral changes were accompanied with the suppression of ERK activity and TNF‐α production, and astrocyte and microglia activation in the spinal dorsal horn of rats with nerve injury. Our study provides evidence that LANCL2 may be a potential molecular target for the development of analgesics for neuropathic pain and the management of a wide range of conditions involving neuroinflammation.Support or Funding InformationNS064289; NS107569

Cerebral protection of epigallocatechin gallate (EGCG) via preservation of mitochondrial function and ERK inhibition in a rat resuscitation model.

BackgroundVarious and opposite roles of epigallocatechin gallate (EGCG) have been reported in different studies. We aimed to investigate how EGCG affects the cerebral injury in a cardiac arrest/cardiopulmonary resuscitation (CA/CPR) model of rat.MethodsThe rats which were subjected to CA/CPR randomly received low dose of EGCG (3 mg/kg, Low-EGCG group, n=16), high dose of EGCG (9 mg/kg, High-EGCG group, n=16) and equal volume of 0.9% saline solution (NS group, n=16) at the first minute after return of spontaneous circulation (ROSC). The rats underwent anesthesia and intubation were defined as Sham group (n=16). Twenty-four hours after ROSC, neural defect score (NDS), ROS fluorescence intensity, degree of mitochondrial permeability transition pore (mPTP) opening, ATP contents and mitochondrial ATP synthase expression were evaluated in the four groups. The expression of extracellular signal-regulated kinase (ERK) activity and cleaved-caspase 3 were also detected by Western blot.ResultsCA/CPR induced severe ischemia-reperfusion injury (IRI), resulted in mitochondrial dysfunction and upregulated phosphorylation of ERK. EGCG dose-dependently alleviated the IRI after CA/CPR, inhibited ERK activity and restored mitochondrial function and, as indicated by improved NDS, reduced ROS level, decreased mPTP opening, elevated ATP content, increased ATPase expression and downregulated cleaved-caspase 3 level.ConclusionEGCG alleviated global cerebral IRI by restoring mitochondrial dysfunction and ERK modulation in a rat CA/CPR model, which might make it a potential candidate agent against IRI after CA/CPR in the future. Further study is needed to determine whether higher dosage of EGCG might aggravate cerebral IRI post-CA/CPR.

Ajoene, a Major Organosulfide Found in Crushed Garlic, Induces NAD(P)H:quinone Oxidoreductase Expression Through Nuclear Factor E2-related Factor-2 Activation in Human Breast Epithelial Cells

BackgroundNAD(P)H:quinone oxidoreductase-1 (NQO1) is a widely-distributed flavin adenine dinucleotide-dependent flavoprotein that promotes obligatory 2-electron reductions of quinones, quinoneimines, nitroaromatics, and azo dyes. This reduces quinone levels and thereby minimizes generation of excess reactive oxygen species (ROS) formed by redox cycling, and concurrent depletion of intracellular thiol pools. Ajoene is derived from crushed garlic. It is formed by a reaction involving two allicin molecules, and is composed of allyl sulfide and vinyl disulfide. Ajoene is present in two isomers, E- and Z-form.MethodsExpression of antioxidant enzymes and nuclear factor E2-related factor-2 (Nrf2) was measured by Western blot analysis. NQO1 promoter activity was assessed by the luciferase reporter gene assay. ROS accumulation was monitored by using the fluorescence-generating probe 2′,7′-dichlorofluorescein diacetate. The intracellular glutathione levels were measured by using a commercially available kit.ResultsZ-ajoene significantly up-regulated the expression of representative antioxidant enzyme NQO1 in non-tumorigenic breast epithelial MCF-10A cells at non-toxic concentrations. Z-ajoene enhanced up-regulation and nuclear translocation of Nrf2, which plays a pivotal role in the induction of many genes encoding antioxidant enzymes and other cytoprotective proteins. Z-ajoene treatment also increased the activity of nqo1-promoter harboring antioxidant response element consensus sequences in MCF-10A cells. Silencing of Nrf2 by small interfering RNA abrogated ajoene-induced expression of NQO1. Z-ajoene activated extracellular signal-regulated kinase (ERK). Inhibition of ERK activation by U0126 abrogated ability of Z-ajoene to activate Nrf2 and to induce NQO1 expression. Intracellular ROS accumulation was observed after treatment with Z-ajoene, whereas the E-isoform was not effective. The inhibition of ROS by treatment with N-acetylcysteine, a radical scavenger, abrogated Z-ajoene-induced expression of NQO1 as well as activation of ERK and Nrf2, suggesting that Z-ajoene augments the Nrf2-dependent antioxidant defense via ROS generation and ERK activation.ConclusionsZ-ajoene induces NQO1 expression in MCF-10A cells through ROS-mediated activation of Nrf2.

1732-P: ERK1 and ERK2 Are Required for Lipopolysaccharide-Induced Production of Cytokines and Chemokines by Macrophages

Low-grade chronic inflammation may be a factor in the development of insulin resistance. Studies suggest that chronic inflammation may be due in part to changes in composition and function of gut microflora, which provide an intestinal barrier preventing bacterial lipopolysaccharide (LPS) release. Animal and human studies demonstrate that obesity increases gut permeability leading to elevated plasma LPS levels resulting in inflammation and metabolic dysfunction. We hypothesized that insulin resistance could be produced by inflammatory factors secreted by macrophages when exposed to gut-released LPS. We examined the extracellular signal-regulated kinase (ERK) signaling pathway, which may be responsible for the macrophage inflammatory response. We examined whether inhibition of ERK activity blocked LPS-mediated responses in bone marrow derived macrophages (BMDM). To determine which ERK isoform is involved in the regulation of inflammatory factor production, we used siRNA to knockdown ERK1, ERK2, or both. BMDM were treated with LPS (100 ng/ml, 6hr). LPS induced TNF-α, RANTES, MCP-1, and IFN-β production of 106371±18250 pg/ml, 11827 ± 1168 pg/ml, 851 ± 73 pg/ml, and 454 ± 46 pg/ml, respectively. Knockdown of ERK1 decreased the release of TNF-α and RANTES to 26534 ± 1471 pg/ml and 5938 ± 806 pg/ml, respectively. Knockdown of ERK2 decreased the release of TNF-α and RANTES to 11600±3610 pg/ml and 7289 ± 265 pg/ml, respectively. Knockdown of either ERK1 or ERK2 did not decrease the release of MCP-1 and IFN-β. However, double knockdown of ERK1 and ERK2 had the greatest inhibition of TNF-α, RANTES, MCP-1, and IFN-β release (not detected, 1540 ± 240 pg/ml, 549 ± 32 pg/ml, and 31 ± 1 pg/ml, respectively). In summary, knockdown of both ERK isoforms is necessary to completely abrogate the LPS effect in macrophages. We propose that ERK positively regulates LPS-mediated inflammatory responses and inhibition of ERK signaling may protect against development of insulin resistance. Disclosure P.F. Chang: None. S.M. Reyna: None. Funding National Institutes of Health; University of Texas Rio Grande Valley

Haem oxygenase-1 induction prevents glucocorticoid-induced osteoblast apoptosis through activation of extracellular signal–regulated kinase1/2 signalling pathway

BackgroundHigh-dose glucocorticoid (GC) therapy always causes osteoporosis partly by inducing osteoblast apoptosis. However, the underlying mechanisms of GC-induced apoptosis remain elusive. Haem oxygenase-1 (HO-1) is a cytoprotective protein that rescues cells from H2O2 or high glucose–induced apoptosis. In bone metabolism, HO-1 also participates in osteoclast and osteoblast differentiation. ObjectiveThe present study aimed to investigate the protective role of HO-1 against GC-induced osteoblast apoptosis and to elucidate the underlying mechanism. MethodsMouse osteoblastic MC3T3-E1 cells were treated with dexamethasone (Dex) for 24 h in the presence or absence of cobalt (III) protoporphyrin IX chloride (CoPP, an inducer of HO-1). In some experiments, U0126 was added to the culture 1 h before CoPP treatment. The induction of apoptosis was determined by flow cytometry. Cell viability was evaluated using a cell counting kit-8 (CCK-8) assay. The expression levels of Bax and bcl-2 were measured by real-time polymerase chain reaction and Western blot. HO-1, extracellular signal–regulated kinase (ERK)-1/2 and pERK1/2 protein levels were measured by Western blot analysis. ResultsDex promoted apoptosis and inhibited cell viability in MC3T3-E1 cells. In addition, Dex significantly increased Bax expression and reduced Bcl-2 expression. The expression of HO-1 was also reduced after Dex treatment. HO-1 induction by CoPP significantly attenuated Dex-induced apoptosis as evidenced by Annexin V/PI staining. The mRNA expression level of antiapoptotic gene Bcl-2 was also increased after CoPP treatment. Moreover, CoPP treatment increased the phosphorylation of ERK1/2. U0126, an inhibitor of ERK activation, significantly abrogated the protective effects of CoPP. ConclusionOur results demonstrate that HO-1 induction by CoPP can attenuate Dex-induced apoptosis of mouse osteoblastic MC3T3-E1 cells. The antiapoptotic effect of HO-1 induction may be correlated with the activation of ERK1/2 signalling pathway.The translational potential of this article: HO-1 induction by CoPP can prevent GC-induced osteoblast apoptosis. Our findings will highlight the therapeutic potential of HO-1 induction in GC-induced osteoporosis.

Activator of G protein signaling 3 modulates prostate tumor development and progression

Prostate cancer (PCa) is a leading cause of cancer death among men, with greater prevalence of the disease among the African American population in the USA. Activator of G-protein signaling 3 (AGS3/G-protein signaling modulator 1) was shown to be overexpressed in prostate adenocarcinoma relative to the prostate gland. In this study, we investigated the correlation between AGS3 overexpression and PCa malignancy. Immunoblotting analysis and real-time quantitative-PCR showed increase in AGS3 expression in the metastatic cell lines LNCaP (~3-fold), MDA PCa 2b (~2-fold), DU 145 (~2-fold) and TRAMP-C1 (~20-fold) but not in PC3 (~1-fold), relative to control RWPE-1. Overexpression of AGS3 in PC3, LNCaP and MDA PCa 2b enhanced tumor growth. AGS3 contains seven tetratricopeptide repeats (TPR) and four G-protein regulatory (GPR) motifs. Overexpression of the TPR or the GPR motifs in PC3 cells had no effect in tumor growth. Depletion of AGS3 in the TRAMP-C1 cells (TRAMP-C1-AGS3-/-) decreased cell proliferation and delayed wound healing and tumor growth in both C57BL/6 (~3-fold) and nude mice xenografts, relative to control TRAMP-C1 cells. TRAMP-C1-AGS3-/- tumors also exhibited a marked increase (~5-fold) in both extracellular signal-regulated kinase (ERK) 1/2 and P38 mitogen-activated protein kinase (MAPK) activation, which correlated with a significant increase (~3-fold) in androgen receptor (AR) expression, relative to TRAMP-C1 xenografts. Interestingly, overexpression of AGS3 in TRAMP-C1-AGS3-/- cells inhibited ERK activation and AR overexpression as compared with control TRAMP-C1 cells. Taken together, the data indicate that the effect of AGS3 in prostate cancer development and progression is probably mediated via a MAPK/AR-dependent pathway.

Density-dependent ERK MAPK expression regulates MMP-9 and influences growth.

Previous work has shown that expression of the extracellular signal-regulated kinase (ERK) is decreased by high density in normal fibroblast cells, and this was correlated with increased expression of mitogen-activated protein kinase phosphatases. Because of these differences in ERK regulation upon contact inhibition, it is likely that other cellular responses may be influenced by the attainment of a contact-inhibited state. Expression of matrix metalloproteinase-9 and cadherin cleavage were both found to be decreased upon reaching high culture density. Inhibition of ERK activity with the MEK inhibitor PD98059 resulted in increased expression of cadherins, while constitutive activation of ERK through the use of expression of an ERK construct with a D319N sevenmaker mutation resulted in decreased expression of cadherins and enhanced colony formation of HT-1080 fibrosarcoma cells. Taken together, these results corroborate a role for the regulation of ERK upon the attainment of a contact-inhibited state with increased expression of cadherins.

Analysis of Retinoic Acid Receptor Signaling in Colorectal Cancer.

Retinoic acid receptor (RAR) signaling plays an important role in embryonic development and homeostasis of many tissues. At the cellular level, activation of RAR signaling often induces cell cycle arrest, differentiation, and apoptosis in many types of cells. Consequently, loss of normal RAR function in the presence of physiological levels of retinoic acid (RA) is often observed in cancers, and pharmacological reactivation of RAR signaling has been considered a promising strategy for cancer therapy and prevention. One of important mechanisms that regulate RAR activity in cancer cells is cross-talk with growth factor signaling, where activation of extracellular signal-regulated kinase (ERK) plays a major role in suppressing RAR transcriptional activity downstream of growth factor receptors. Conversely, strong activation of RAR can induce suppression of ERK activity by inducing expression of a phosphatase specific for ERK to exert tumor-suppressive activity in colorectal cancer. Here, we describe the basic methods to analyze interactions between RAR and ERK signaling in colorectal cancer cells.

Regulatory role of heme oxygenase-1 in silica-induced lung injury

BackgroundSilicosis, a progressive inflammatory lung disease attributed mainly to occupational exposure to silica dust, shows loss of lung function even after cessation of exposure. In addition to conventional evaluation methods such as chest X-ray, computed tomography, and spirometry, we identified heme oxygenase (HO)-1, an inducible antioxidant, as a potential biomarker to identify at-risk patients. We found that HO-1 was critical in attenuating the disease progression of silicosis; however, the key signaling pathway has not yet been elucidated. Here, we report the critical pathway after silica exposure, focusing on the role of silica-derived reactive oxygen species (ROS) signaling and its attenuation, which is mediated by HO-1 induction, in vivo and in vitro.MethodsNormal bronchial epithelial cells and a macrophage cell line, as well as a murine silicosis model generated by intratracheal administration of 2.5 mg of crystalline silica, were used in this study. The pathways activated in response to silica exposure, including the mitogen-activated protein kinase (MAPK) signaling pathway, were examined and compared with or without super-induction of HO-1.ResultsThe murine silicosis model was first assessed for the evaluation of activated pathways after silica exposure, focusing on ROS-MAPK activation. In the murine model, increased expression of HO-1 in the lungs was observed after silica-instillation. Moreover, silica-medicated activation of extracellular signal-regulated kinase (ERK) in the lungs was attenuated in response to silica-induced HO-1 upregulation. Activation of other MAPKs, such as p38 and c-Jun N-terminal kinase pathways, after silica exposure was not significantly different irrespective of HO-1 induction. Further in vitro studies showed that 1) silica-induced HO-1 was significantly attenuated by inhibiting ERK activation, and 2) carbon monoxide and bilirubin as final byproducts of HO-1 could inhibit ERK activation. Taken together, silica-induced HO-1 upregulation was mediated by ERK activation, and HO-1 further regulates ERK activation via its final byproducts, carbon monoxide and bilirubin.ConclusionsThis is the first study to demonstrate the regulatory role of HO-1 in silicosis. This finding could contribute to the development of a treatment strategy of monitoring HO-1 levels as a marker of therapeutic intervention.

Up-regulation of DUSP5 and DUSP6 by gonadotropin-releasing hormone in cultured hypothalamic neurons, GT1-7 cells.

Gonadotropin-releasing hormone (GnRH) is secreted from hypothalamic neurons (GnRH neurons) and stimulates anterior pituitary gonadotrophs to synthesize and secrete gonadotropins. In addition to gonadotrophs, GnRH neurons also express GnRH receptors, and the autocrine action of GnRH is reportedly involved in the regulation of functions of GnRH neurons. There is accumulating evidence that extracellular signal-regulated kinase (ERK), one of mitogen-activated protein kinases (MAPKs), is activated by GnRH and involved in various effects of GnRH in GnRH neurons. In the present study, we performed microarray analysis to examine the types of genes whose expression was regulated by GnRH in immortalized mouse GnRH neurons (GT1-7 cells). We found that 257 genes among 55,681 genes examined were up-regulated after 30-min treatment of GT1-7 cells with GnRH. These up-regulated genes included four dual-specificity MAPK phosphatases (DUSPs), DUSP1, DUSP2, DUSP5, and DUSP6. Reverse transcription-polymerase chain reaction analysis confirmed that the mRNA levels of DUSP5 and DUSP6 were robustly increased within 30 min. U0126, an inhibitor of ERK activation, completely inhibited the increases in the mRNA levels of DUSP5 and DUSP6. Immunoblotting analysis revealed that ERK activation peaked at 5 min and declined steeply at 60 min, whereas DUSP5 and DUSP6 proteins were increased from 60 min. It was notable that down-regulation of DUSP6 augmented GnRH-induced ERK activation approximately 1.7-fold at 60 min. These results suggested that the up-regulation of DUSP6 regulates the duration of ERK activation at least in part.

The C-terminus of the amelogenin peptide influences the proliferation of human bone marrow mesenchymal stem cells.

Amelogenins are a family of enamel matrix proteins that are important for formation of enamel. Amelogenins may induce division of mesenchymal stem cells (MSCs), among others. Recently, the C-terminus of the amelogenin peptide (AMG-CP) has been shown to enhance the proliferation of cementoblast lineage cells. The role of the amelogenin peptide on the proliferation of human MSCs and related alterations in the intracellular signaling pathway were studied. MSCs were exposed to AMG-CP in vitro. The MTS and 5-bromo-2'-deoxyuridine (BrdU) assays were used to determine proliferation. Expression of the amelogenin receptor, lysosomal-associated membrane protein 1 (LAMP1), was examined in MSCs with western blotting. Binding of AMG-CP to LAMP1 was inhibited with anti-LAMP1 antibody. Components of the mitogen-activated protein kinase (MAPK)-extracellular signal-regulated kinase (ERK) pathway were studied with western blotting and enzyme-linked immunosorbent assay, and U0126, an MAPK inhibitor, was used to inhibit ERK activity. MSC proliferation was significantly increased in the presence of AMG-CP and significantly inhibited by anti-LAMP1 antibody or U0126. Increased phosphorylated ERK1/2 was observed in the presence of AMG-CP, and decreased phosphorylated ERK1/2 was seen in the presence of anti-LAMP1 antibody or U0126. A C-terminal amelogenin variant increased the proliferation of MSCs via an interaction with LAMP1 and the MAPK-ERK signaling pathway, indicating the possibility of using MSCs for tissue regeneration in the craniofacial region.

AS1041, a Novel Synthesized Derivative of Marine Natural Compound Aspergiolide A, Arrests Cell Cycle, Induces Apoptosis, and Inhibits ERK Activation in K562 Cells.

AS1041 is a novel synthesized anthraquinone lactone derivative of marine natural compound aspergiolide A (ASP-A) with new structure skeleton and marked cytotoxicity in cancer cells. To study its cytotoxicity in detail, we evaluated its activity on human K562 chronic myelogenous leukemia cells and investigated the related molecule mechanisms. AS1041 significantly inhibited the proliferation and colony formation of K562 cells. Moreover, AS1041 arrested cell cycle progression at G2/M phase in a concentration-dependent manner, and also caused concentration- and time-dependent induction of apoptosis. In addition, the molecular mechanisms investigation showed that AS1041 did not localize in the cellular nucleus and did not affect topoisomerases I or II. However, AS1041 could inactivate extracellular signal-regulated kinase (ERK) and contribute to AS1041-induced apoptosis. We concluded that AS1041 was cytotoxic to K562 leukemia cells and the cytotoxicity related to the cell cycle arrest, apoptosis induction, and ERK inhibition. These results implied that AS1041 was a novel derivative of ASP-A with significant cytotoxicity to chronic myelogenous leukemia cells and may have therapeutic potential for the treatment of cancer and leukemia.

Osthole, a Coumadin Analog from Cnidium monnieri (L.) Cusson, Ameliorates Nucleus Pulposus-Induced Radicular Inflammatory Pain by Inhibiting the Activation of Extracellular Signal-Regulated Kinase in Rats

Aim: This study was aimed at assessing the role of extracellular signal regulated kinase (ERK) in mechanical allodynia resulting from lumbar disc herniation (LDH) and exploring the osthole's anti-nociceptive effect on ERK activation. Methods: Radicular pain was generated by applying nucleus pulposus (NP) to the L₅ dorsal root ganglion (DRG). Allodynia was measured using Von Frey filaments to calculate the mechanical pain threshold. Phosphorylated ERK and total ERK protein in the lumbar spinal dorsal horn was detected by using the Western blot technique. Cyclooxygenase 2 (COX-2) mRNA was assessed by real-time reverse-transcription polymerase chain reaction. Results: The application of NP to L₅ DRG induced mechanical hypersensitivity which lasted for at least 28 days, and a significant increase of ERK phosphorylation in the ipsilateral spinal dorsal horn from postoperative day (POD) 1 to POD 21. ERK inhibitor attenuated NP-induced hyperalgesia compared to the dimethyl sulfoxide-(vehicle control) administered group (p < 0.05). Epidural treatment with osthole could ameliorate NP-evoked hyperalgesia by suppressing the activation of ERK rather than decreasing the expression of ERK protein. Osthole could also inhibit the increased expression of COX-2 mRNA in spinal dorsal horn, which was a known downstream effect of ERK signaling pathway. Conclusions: Our results suggest that ERK activation in the spinal dorsal horn plays a vital role in NP-evoked hyperalgesia. Osthole exerts analgesic effect on radicular inflammatory pain in LDH rat model, by down-regulating the mRNA expression of the target gene of COX-2 via inhibiting ERK activation in the spinal dorsal horn.