Activation Of Extracellular Signal-regulated Kinase Kinase Research Articles

The role of BDNF transcription in the antidepressant-like effects of 18β-glycyrrhetinic acid in a chronic social defeat stress model

BackgroundXiaoyaosan (XYS), a traditional Chinese medicine formulation, has been used in the treatment of depression. However, no studies have yet identified the active compounds responsible for its antidepressant effects in the brain. Study DesignWe investigated the antidepressants effects of XYS and identified 18β-glycyrrhetinic acid (18β-GA) as the primary compound present in the brain following XYS injection. Furthermore, we explored the molecular mechanisms underlying the antidepressant-like effects of both XYS and 18β-GA. MethodsTo investigate the antidepressant-like effects of XYS and elucidate the associated molecular mechanisms, we employed various methodologies, including cell cultures, the chronic social defeat stress (CSDS) model, behavioral tests, immunoprecipitation, quantitative PCR (qPCR) assays, Western blotting assays, luciferase assays, chromatin immunoprecipitation (ChIP) assays, immunofluorescence staining, and dendritic spine analysis. ResultsWe identified 18β-GA as the primary compound in the brain following XYS injection. In vitro, 18β-GA was found to bind with ERK (extracellular signal-regulated kinase), subsequently activating ERK kinase activity toward both c-Jun and cAMP response element binding protein (CREB). Moreover, 18β-GA activated brain-derived neurotrophic factor (BDNF) transcription by stimulating nuclear factor-erythroid factor 2-related factor 2 (Nrf2), c-Jun, and CREB, while also inhibiting methyl CpG binding protein 2 (MeCP2) both in vitro and in vivo. Chronic intraperitoneal (i.p.) administration of 18β-GA exhibited prophylactic antidepressant-like effects in a CSDS model, primarily by activating BDNF transcription in the medial prefrontal cortex (mPFC). Interestingly, a single i.p. injection of 18β-GA produced rapid and sustained antidepressant-like effects in CSDS-susceptible mice by engaging the BDNF-tropomyosin receptor kinase B (TrkB) signaling pathway in the mPFC. ConclusionThese findings suggest that the activation of BDNF transcription in the mPFC underlies the antidepressant-like effects of 18β-GA, a key component of XYS in the brain.

On the inference of ERK signaling dynamics from protein biosensor measurements.

The extracellular signal-regulated kinase (ERK) signaling pathway plays prominent roles in cell growth, proliferation, and differentiation. ERK signaling is dynamic, involving phosphorylation/dephosphorylation, nucleocytoplasmic shuttling, and interactions with scores of protein substrates in the cytosol and in the nucleus. Live-cell fluorescence microscopy using genetically encoded ERK biosensors offers the potential to infer those dynamics in individual cells. In this study, we have monitored ERK signaling using four commonly used translocation- and Förster resonance energy transfer-based biosensors in a common cell stimulation context. Consistent with previous reports, we found that each biosensor responds with unique kinetics; it is clear that there is not a single dynamic signature characterizing the complexity of ERK phosphorylation, translocation, and kinase activity. In particular, the widely adopted ERK Kinase Translocation Reporter (ERKKTR) gives a readout that reflects ERK activity in both compartments. Mathematical modeling offers an interpretation of the measured ERKKTR kinetics, in relation to cytosolic and nuclear ERK activity, and suggests that biosensor-specific dynamics substantially influence the measured output.

Inhibition of Spred/Sprouty Expression in the Skin of a Contact Dermatitis-Like Model.

We have previously reported that swellings caused by haptens, such as 2,4,6-trinitrochlorobenzene (TNCB), may be associated with the extracellular signal-regulated kinase (ERK)-induced proliferation pathway. However, the involvement of the Spred/Sprouty family as critical negative regulators of the Ras/Raf/ERK signaling pathway at disease sites is not well-established. Thus, in the present study, the effects of hapten-challenge on the expression levels of genes and proteins associated with the Spred/Sprouty family in the ear of mice were investigated. The activation of ERK and epidermal growth factor receptor (EGFR) tyrosine kinase was inhibited by their selective inhibitors, namely, U0126 and PD168393, respectively. Twenty-four hours after the final challenge by the haptens TNCB, 2,4-dinitrofluorobenzene, or oxazolone, ear thickness was augmented by challenge with all haptens and the gene expression levels of Spred1, Spred2, Sprouty1, and Sprouty2 in swelling induced by all haptens were significantly decreased. Furthermore, Spred2, Sprouty1, and Sprouty2 genes were decreased in the epidermis and dermis of the TNCB-challenged ear. In conclusion, it is possible that the mechanism of hapten-challenge-induced skin thickening involves not only the enhancement of cell proliferative functions via the activation of ERK by EGFR tyrosine kinase activation but also the decreases expression of Spred/Sprouty family members.

Oxidative stress in the skin: Impact and related protection.

Skin, our first interface to the external environment, is subjected to oxidative stress caused by a variety of factors such as solar ultraviolet, infrared and visible light, environmental pollution, including ozone and particulate matters, and psychological stress. Excessive reactive species, including reactive oxygen species and reactive nitrogen species, exacerbate skin pigmentation and aging, which further lead to skin tone unevenness, pigmentary disorder, skin roughness and wrinkles. Besides these, skin microbiota are also a very important factor ensuring the proper functions of skin. While environmental factors such as UV and pollutants impact skin microbiota compositions, skin dysbiosis results in various skin conditions. In this review, we summarize the generation of oxidative stress from exogenous and endogenous sources. We further introduce current knowledge on the possible roles of oxidative stress in skin pigmentation and aging, specifically with emphasis on oxidative stress and skin pigmentation. Meanwhile, we summarize the science and rationale of using three well-known antioxidants, namely vitamin C, resveratrol and ferulic acid, in the treatment of hyperpigmentation. Finally, we discuss the strategy for preventing oxidative stress-induced skin pigmentation and aging.

Ligand-Specific Signaling Profiles and Resensitization Mechanisms of the Neuromedin U2 Receptor.

The structurally related, but distinct neuropeptides, neuromedin U (NmU) and neuromedin S (NmS) are ligands of two G protein-coupled NmU receptors (NMU1 and NMU2). Hypothalamic NMU2 regulates feeding behavior and energy expenditure and has therapeutic potential as an anti-obesity target, making an understanding of its signaling and regulation of particular interest. NMU2 binds both NmU and NmS with high affinity, resulting in receptor-ligand co-internalization. We have investigated whether receptor trafficking events post-internalization are biased by the ligand bound and can therefore influence signaling function. Using recombinant cell lines expressing human NMU2, we demonstrate that acute Ca2+ signaling responses to NmU or NmS are indistinguishable and that restoration of responsiveness (resensitization) requires receptor internalization and endosomal acidification. The rate of NMU2 resensitization is faster following NmU compared with NmS exposure, but is similar if endothelin-converting enzyme-1 activity is inhibited or knocked down. Although acute activation of extracellular signal-regulated kinase (ERK) is also similar, activation by NMU2 is longer lasting if NmS is the ligand. Furthermore, when cells are briefly challenged before removal of free, but not receptor-bound ligand, activation of ERK and p38 mitogen-activated protein kinase by NmS is more sustained. However, only NmU responses are potentiated and extended by endothelin-converting enzyme-1 inhibition. These data indicate that differential intracellular ligand processing produces different signaling and receptor resensitization profiles and add to the findings of other studies demonstrating that intracellular ligand processing can shape receptor behavior and signal transduction.

Electroacupuncture relieves depression-like symptoms in rats exposed to chronic unpredictable mild stress by activating ERK signaling pathway

Electroacupuncture (EA) has been shown to alleviate the symptoms associated with major depressive disorder; however, the underlying mechanisms remain unclear. While the mainstay treatment for depression are pharmacological agents that modulate serotonergic and/or noradrenergic activity of the brain, recent data suggest that, neurotrophins may play a larger role in the pathogenesis of depression and may offer better therapeutic potential in alleviating symptoms associated with depression. One downstream target of neurotrophins is the extracellular signal-regulated kinase (ERK)/Mitogen-activated protein kinase (MAPK) cascade, a major mediator of cellular stress often associated with clinical depression. In this study, we assessed whether the efficacy of EA is due to regulation of these novel pathways using an animal model of depression induced by chronic unpredictable mild stress (CUMS). We found that EA stimulation at specific locations, Baihui (GV20), and Yintang (GV29) ameliorated the behavioral responses of CUMS, which included reduced locomotion, decreased sucrose intake and weight loss. Furthermore, EA increased the activation of ERK and ribosomal s6 kinase (RSK) levels under stress. Both the behavioral and biochemical responses to EA were attenuated with administration of ERK inhibitor, suggesting that EA improves depression-like symptoms in stressed rats, in part, by activation of ERK signaling.

Metformin Restrains Pancreatic Duodenal Homeobox-1 (PDX-1) Function by Inhibiting ERK Signaling in Pancreatic Ductal Adenocarcinoma.

Pancreatic ductal adenocarcinoma (PDAC) is one of the most potent and perilous diseases known, with a median survival rate of 3-5 months due to the combination of only advanced stage diagnosis and ineffective therapeutic options. Metformin (1,1-Dimethylbiguanide hydrochloride), the leading drug used for type 2 diabetes mellitus, emerges as a potential therapy for PDAC and other human cancers. Metformin exerts its anticancer action via a variety of adenosine monophosphate (AMP)-activated protein kinase (AMPK)- dependent and/or AMPK-independent mechanisms. We present data here showing that metformin downregulated pancreatic transcription factor pancreatic duodenal homeobox-1 (PDX-1), suggesting a potential novel mechanism by which metformin exerts its anticancer action. Metformin inhibited PDX-1 expression at both protein and mRNA levels and PDX-1 transactivity as well in PDAC cells. Extracellular signal-regulated kinase (ERK) was identified as a PDX-1-interacting protein by antibody array screening in GFP-PDX-1 stable HEK293 cells. Co-transfection of ERK1 with PDX-1 resulted in an enhanced PDX-1 expression in HEK293 cells in a dose-dependent manner. Immunoprecipitation/Western blotting analysis confirmed the ERK-PDX-1 interaction in PANC-1 cells stimulated by epidermal growth factor (EGF). EGF induced an enhanced PDX-1 expression in PANC-1 cells and this stimulation was inhibited by MEK inhibitor PD0325901. Metformin inhibited EGF-stimulated PDX-1 expression with an accompanied inhibition of ERK kinase activation in PANC- 1 cells. Taken together, our studies show that PDX-1 is a potential novel target for metformin in PDAC cells and that metformin may exert its anticancer action in PDAC by down-regulating PDX-1 via a mechanism involving inhibition of ERK signaling.

Abstract 2600: Targeting cancer stem cell through blocking the Erk pathway with Kazinol-E from Broussonetia kazinoki

Abstract Growing evidence suggests that a small population of cells, called cancer stem cells (CSCs) contribute to the heterogeneity, recurrence and resistance to chemotherapy in breast cancer. In this regard, CSC is considered to be a good target to cure breast cancers. The extracellular signal-regulated kinase (ERK) signaling pathway is one of the major determinants in the control of diverse cellular process including the maintenance of CSCs. Kazinol-E is an antioxidant flavan purified from the root bark of Broussonetia kazinoki (Moraceae, paper mulberry). In this study, we found the anti CSC effect of Kazinol-E through targeting ERK pathway. It decreased the population of a breast cancer cell (MCF-7) CSCs which are CD44+/CD24- or aldehyde dehydrogenase positive, at the concentration that does not influence the growth of general MCF-7 cells cultured with serum. It targeted and inhibited ERK kinase activities in vivo in both CSC and in regular cultured cells suggesting the CSCs depend more than the rest of cancer cells do on Erk signaling for the survival. In vitro kinase assay using purified Erk-1 and p90RSK2, as the substrate, and the molecular docking study suggest that the Kazinol-E suppress the Erk activity by direct binding to the ATP binding pocket of Erk-1. Interestingly, a Kazinol-C which is very similar with Kazinol-E in structure did not show the Erk-1 inhibition nor anti CSC activity. These results collectively suggest that Kazinol-E may be a specific anti CSC agent targeting Erk pathway. Citation Format: Yu-Chae Jung, Seula Han, Li Hua, Hui-Yuan Zhao, Cheol-Jung Lee, Yong-Yeon Cho, Raok Jeon, Jae-Ha Ryu, Woo-Young Kim. Targeting cancer stem cell through blocking the Erk pathway with Kazinol-E from Broussonetia kazinoki. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2600. doi:10.1158/1538-7445.AM2015-2600

Phosphoproteomic Analysis of KSHV-Infected Cells Reveals Roles of ORF45-Activated RSK during Lytic Replication.

Kaposi’s Sarcoma-Associated Herpesvirus (KSHV) is an oncogenic virus which has adapted unique mechanisms to modulate the cellular microenvironment of its human host. The pathogenesis of KSHV is intimately linked to its manipulation of cellular signaling pathways, including the extracellular signal-regulated kinase (ERK) mitogen-activated protein kinase (MAPK) pathway. We have previously shown that KSHV ORF45 contributes to the sustained activation of both ERK and p90 ribosomal S6 kinase (RSK, a major functional mediator of ERK/MAPK signaling) during KSHV lytic replication. ORF45-activated RSK is required for optimal KSHV lytic gene expression and progeny virion production, though the underlying mechanisms downstream of this activation are still unclear. We hypothesized that the activation of RSK by ORF45 causes differential phosphorylation of cellular and viral substrates, affecting biological processes essential for efficient KSHV lytic replication. Accordingly, we observed widespread and significant differences in protein phosphorylation upon induction of lytic replication. Mass-spectrometry-based phosphoproteomic screening identified putative substrates of ORF45-activated RSK in KSHV-infected cells. Bioinformatic analyses revealed that nuclear proteins, including several transcriptional regulators, were overrepresented among these candidates. We validated the ORF45/RSK-dependent phosphorylation of several putative substrates by employing KSHV BAC mutagenesis, kinase inhibitor treatments, and/or CRISPR-mediated knockout of RSK in KSHV-infected cells. Furthermore, we assessed the consequences of knocking out these substrates on ORF45/RSK-dependent regulation of gene expression and KSHV progeny virion production. Finally, we show data to support that ORF45 regulates the translational efficiency of a subset of viral/cellular genes with complex secondary structure in their 5’ UTR. Altogether, these data shed light on the mechanisms by which KSHV ORF45 manipulates components of the host cell machinery via modulation of RSK activity. Thus, this study has important implications for the pathobiology of KSHV and other diseases in which RSK activity is dysregulated.

Deoxynivalenol induces ectodomain shedding of TNF receptor 1 and thereby inhibits the TNF-α-induced NF-κB signaling pathway

Trichothecene mycotoxins are known to inhibit eukaryotic translation and to trigger the ribotoxic stress response, which regulates gene expression via the activation of the mitogen-activated protein (MAP) kinase superfamily. In this study, we found that deoxynivalenol induced the ectodomain shedding of tumor necrosis factor (TNF) receptor 1 (TNFRSF1A) and thereby inhibited the TNF-α-induced signaling pathway. In human lung carcinoma A549 cells, deoxynivalenol and 3-acetyldeoxynivalenol inhibited the expression of intercellular adhesion molecule-1 (ICAM-1) induced by TNF-α more strongly than that induced by interleukin 1α (IL-1α), whereas T-2 toxin and verrucarin A exerted nonselective inhibitory effects. Deoxynivalenol and 3-acetyldeoxynivalenol also inhibited the nuclear factor κB (NF-κB) signaling pathway induced by TNF-α, but not that induced by IL-1α. Consistent with these findings, deoxynivalenol and 3-acetyldeoxynivalenol induced the ectodomain shedding of TNF receptor 1 by TNF-α-converting enzyme (TACE), also known as a disintegrin and metalloproteinase 17 (ADAM17). In addition to the TACE inhibitor TAPI-2, the MAP kinase or extracellular signal-regulated kinase (ERK) kinase (MEK) inhibitor U0126 and the p38 MAP kinase inhibitor SB203580, but not the c-Jun N-terminal kinase (JNK) inhibitor SP600125, suppressed the ectodomain shedding of TNF receptor 1 induced by deoxynivalenol and reversed its selective inhibition of TNF-α-induced ICAM-1 expression. Our results demonstrate that deoxynivalenol induces the TACE-dependent ectodomain shedding of TNF receptor 1 via the activation of ERK and p38 MAP kinase, and thereby inhibits the TNF-α-induced NF-κB signaling pathway.

Glutathione-S-transferase M1 regulation of diesel exhaust particle-induced pro-inflammatory mediator expression in normal human bronchial epithelial cells

BackgroundDiesel exhaust particles (DEP) contribute substantially to ambient particulate matter (PM) air pollution in urban areas. Inhalation of PM has been associated with increased incidence of lung disease in susceptible populations. We have demonstrated that the glutathione S-transferase M1 (GSTM1) null genotype could aggravate DEP-induced airway inflammation in human subjects. Given the critical role airway epithelial cells play in the pathogenesis of airway inflammation, we established the GSTM1 deficiency condition in primary bronchial epithelial cells from human volunteers with GSTM1 sufficient genotype (GSTM1+) using GSTM1 shRNA to determine whether GSTM1 deficiency could exaggerate DEP-induced expression of interleukin-8 (IL-8) and IL-1β proteins. Furthermore, the mechanisms underlying GSTM1 regulation of DEP-induced IL-8 and IL-1β expression were also investigated.MethodsIL-8 and IL-1β protein levels were measured using enzyme-linked immunosorbent assay. GSTM1 deficiency in primary human bronchial epithelial cells was achieved using lentiviral GSTM1 shRNA particles and verified using real-time polymerase chain reaction and immunoblotting. Intracellular reactive oxygen species (ROS) production was evaluated using flow cytometry. Phosphorylation of protein kinases was detected using immunoblotting.ResultsExposure of primary human bronchial epithelial cells (GSTM1+) to 25-100 μg/ml DEP for 24 h significantly increased IL-8 and IL-1β protein expression. Knockdown of GSTM1 in these cells further elevated DEP-induced IL-8 and IL-1β expression, implying that GSTM1 deficiency aggravated DEP-induced pro-inflammatory response. DEP stimulation induced the phosphorylation of extracellular signal-regulated kinase (ERK) and Akt, the downstream kinase of phosphoinositide 3-kinase (PI3K), in GSTM1+ bronchial epithelial cells. Pharmacological inhibition of ERK kinase and PI3K activity blocked DEP-induced IL-8 and IL-1β expression. DEP-induced ERK and Akt phosphorylation could be increased by GSTM1 knockdown. In addition, pretreatment of HBEC with the antioxidant N-acetyl cysteine significantly inhibited DEP-induced ERK and Akt phosphorylation, and subsequent IL-8 and IL-1β expression.ConclusionGSTM1 regulates DEP-induced IL-8 and IL-1β expression in primary human bronchial epithelial cells by modulation of ROS, ERK and Akt signaling.

Nicotinamide Phosphoribosyltransferase Is Essential for Interleukin-1β-mediated Dedifferentiation of Articular Chondrocytes via SIRT1 and Extracellular Signal-regulated Kinase (ERK) Complex Signaling

Although much is known about interleukin (IL)-1β and its role as a key mediator of cartilage destruction in osteoarthritis, only limited information is available on IL-1β signaling in chondrocyte dedifferentiation. Here, we have characterized the molecular mechanisms leading to the dedifferentiation of primary cultured articular chondrocytes by IL-1β treatment. IL-1β or lipopolysaccharide, but not phorbol 12-myristate 13-acetate, retinoic acid, or epidermal growth factor, induced nicotinamide phosphoribosyltransferase (NAMPT) expression, showing the association of inflammatory cytokines with NAMPT regulation. SIRT1, in turn, was activated NAMPT-dependently, without any alteration in the expression level. Activation or inhibition of SIRT1 oppositevely regulates IL-1β-mediated chondrocyte dedifferentiation, suggesting this protein as a key regulator of chondrocytes phenotype. SIRT1 activation promotes induction of ERK and p38 kinase activities, but not JNK, in response to IL-1β. Subsequently, ERK and p38 kinase activated by SIRT1 also induce SIRT1 activation, forming a positive feedback loop to sustain downstream signaling of these kinases. Moreover, we found that the SIRT1-ERK complex, but not SIRT1-p38, is engaged in IL-1β-induced chondrocyte dedifferentiation via a Sox-9-mediated mechanism. JNK is activated by IL-1β and modulates dedifferentiation of chondrocytes, but this pathway is independent on NAMPT-SIRT1 signaling. Based on these findings, we propose that IL-1β induces dedifferentiation of articular chondrocytes by up-regulation of SIRT1 activity enhanced by both NAMPT and ERK signaling.

A Feedback Loop between Androgen Receptor and ERK Signaling in Estrogen Receptor-Negative Breast Cancer

Estrogen receptor (ER)-negative breast cancer is heterogeneous, and the biology of this disease has remained poorly understood. Molecular apocrine is a subtype of ER-negative breast cancer that is characterized by the overexpression of steroid-response genes such as AR and a high rate of ErbB2 amplification. In this study, we have identified a positive feedback loop between the AR and extracellular signal-regulated kinase (ERK) signaling pathways in molecular apocrine breast cancer. In this process, AR regulates ERK phosphorylation and kinase activity. In addition, AR inhibition results in the down-regulation of ERK target proteins phospho-RSK1, phospho-Elk-1, and c-Fos using an in vivo molecular apocrine model. Furthermore, we show that AR-mediated induction of ERK requires ErbB2, and AR activity, in turn, regulates ErbB2 expression as an AR target gene. These findings suggest that ErbB2 is an upstream connector between the AR and ERK signaling pathways. Another feature of this feedback loop is an ERK-mediated regulation of AR. In this respect, the inhibition of ERK phosphorylation reduces AR expression and CREB1-mediated transcriptional regulation of AR acts as a downstream connector between the AR and ERK signaling pathways in molecular apocrine cells. Finally, we demonstrate that AR-positive staining is associated with the overexpression of ERK signaling targets phospho-Elk-1 and c-Fos in ER-negative breast tumors, which further supports a cross-regulation between the AR and ERK signaling pathways in molecular apocrine subtype. This study demonstrates an AR-ERK feedback loop in ER-negative breast cancer with significant biologic and therapeutic implications in this disease.

ERK Inhibition by SU1498 Alters Expression of Bcl‐2 Family Proteins in Breast Cancer Cells

The extracellular signal‐regulated kinase (ERK) pathway is vital to the cell survival. We have demonstrated the ability of SU1498, an inhibitor of ERK kinase activity, to sensitize breast cancer cells with constitutively active ERK to apoptosis. To identify the mechanisms involve, the expression of the antiapoptotic Bcl‐2 and Bcl‐xL and the proapoptotic Bad, Bax, Bik and Bak were examined under apoptotic conditions. One breast cell line (MCF‐10A) and three breast cancer cell lines (MCF 7, MDA‐MB‐231 and MDA‐MB‐468) were used to examine the effects of SU1498 and serum starvation on the expression of the Bcl‐2 family proteins. The MCF‐10A and MCF7 cells exhibit normal ERK regulation while the MDA‐MB‐231 and MDA‐MB‐468 cells lines have a constitutively activated ERK. Across all the cell lines, the combined treatments resulted in either decreased expression, or no changes in the expression, of the antiapoptotic Bcl2 proteins. The expression of the proapoptotic proteins Bad or Bak were dramatically upregulated only in those cells with constitutive ERK activation, while they tended to demonstrate reduced expression in the cells lines with normal ERK regulation. These cell lines with upregulate Bad and/or Bak expression were also those most sensitive to SU1498 induced cell death. The ability of SU1498 to sensitize breast cancer cells to apoptosis by inhibition of ERK may have therapeutic uses.Supported by NSERC

Dedifferentiation of Human Epidermal Keratinocytes Induced by UV In Vitro

Dedifferentiation is an important biological phenomenon and is understood as a process in which cells develop in a reverse order from a more differentiated to a less differentiated state. In the present study, we observed that after UV treatment, the surviving keratinocytes underwent the reversion from differentiated state to dedifferentiated state, evidenced by the changes from three levels, including phenotype, morphology and function. First, the mature keratinocytes acquired a dedifferentiated phenotype indicated by reexpression of transit-amplifying (TA) cell markers, including CK14 and β1 integrin. Second, the cells experienced morphological changes during the process of dedifferentiation. Cells treated with UV were small and had a high nuclear to cytoplasmic ratio, whereas cells without treatment had well-developed cellular organelles and abundant tonofilaments. Third, after UV treatment, the cells regained strong proliferation capacity. These dedifferentiation-derived stem cells formed colonies with defined edges developed and presented multiple-layer growing profile under three-dimensional culture condition. The skin equivalent (SE) produced with UV-treated keratinocytes also showed a well-organized structure with four stratifications. We also report the signaling pathway involved in the dedifferentiation process. The extracellular signal-regulated kinase (ERK) pathway regulates the phenotype reversion of keratinocytes into progenitor cells. Inhibition of ERK kinase activities with a specific inhibitor (PD98059) substantially blocked phosphorylation of ERK1/2 and human keratinocyte dedifferentiation. These data collectively provide a proof-of-concept that UV treatment of HEKs is capable of inducing a phenotype reversion from an adult differentiated state to an immature-like dedifferentiated state via ERK Mitogen-Activated Protein Kinases (MAPK)-dependent pathway. It may offer the direct evidence for the existence of dedifferentiation and the underlying mechanisms involved in the process, which may bring a new insight for the regenerative medicine.

Delphinidin Attenuates Neoplastic Transformation in JB6 Cl41 Mouse Epidermal Cells by Blocking Raf/Mitogen-Activated Protein Kinase Kinase/Extracellular Signal-Regulated Kinase Signaling

Recent studies suggest that anthocyanidins play a pivotal role in the chemopreventive effects of fruits and vegetables. However, the underlying molecular mechanisms and cellular targets remain unknown. Neoplastic transformation of cells and inflammation are considered to be major events contributing to carcinogenesis. Here, we report that delphinidin, a major dietary anthocyanidin, inhibits tumor promoter-induced transformation and cyclooxygenase-2 (COX-2) expression in JB6 promotion-sensitive mouse skin epidermal (JB6 P+) cells by directly targeting Raf and mitogen-activated protein kinase kinase (MEK). Delphinidin inhibited 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced neoplastic transformation and COX-2 expression at both the protein and transcriptional levels. The activation of activator protein-1 and nuclear factor-kappaB induced by TPA was dose dependently inhibited by delphinidin treatment. Delphinidin strongly suppressed Raf1 and MEK1 kinase activities and subsequently attenuated TPA-induced phosphorylation of MEK, extracellular signal-regulated kinase (ERK), p90RSK, and MSK. Although delphinidin suppressed ERK and c-Jun NH(2)-terminal kinase activities, it was more effective at inhibiting Raf1 or MEK1 activities. Pull-down and competition assays revealed that delphinidin binds with Raf1 or MEK1 noncompetitively with ATP. Delphinidin also dose dependently suppressed JB6 P+ cell transformation induced by epidermal growth factor and H-Ras, both of which are involved in the activation of Raf/MEK/ERK signaling. Together, these findings suggested that the targeted inhibition of Raf1 and MEK activities and COX-2 expression by delphinidin contribute to the chemopreventive potential of fruits and vegetables.

Hepatitis B Virus-induced hFGL2 Transcription Is Dependent on c-Ets-2 and MAPK Signal Pathway

Fibrinogen-like protein 2 (FGL2)/fibroleukin plays a pivotal role in the pathogenesis of experimental and human fulminant and chronic viral hepatitis. To define the transcription factor(s) and upstream signal transduction pathways involved in the transcription of human FGL2 (hFGL2) in response to hepatitis B (HB) virus, hepatitis B core (HBc), hepatitis B virus S protein (HBs), or hepatitis B virus X protein (HBx) protein, expression plasmids were cotransfected with an hFGL2 promoter luciferase reporter construct into Chinese hamster ovary and HepG2 cells, respectively. HBc and HBx proteins, but not HBs protein, enhanced hFGL2 transcription in both cell lines. A strong regulatory region from -712 to -568 (relative to the transcriptional starting site) was shown to be responsible for hFGL2 gene transcription in response to both HBc and HBx proteins. c-Ets-2 was shown to be translocated to the nucleus in association with hFGL2 expression in response to both HBc and HBx proteins. Short hairpin RNA (shRNA) interference of c-Ets-2 expression inhibited hFGL2 gene transcription by 64.8 and 60.0% in response to HBc and HBx, respectively. c-Ets-2 protein was highly expressed in peripheral blood mononuclear cells from patients with severe chronic hepatitis B (CHB) in contrast to patients with mild CHB. Increased phosphorylation of ERK and JNK was detected in peripheral blood mononuclear cells from patients with severe CHB. ERK inhibitor PD098059 or ERK shRNA abolished the nuclear c-Ets-2 DNA binding activity and hFGL2 induction in response to HBc, whereas JNK inhibitor SP600125 or JNK shRNA abolished the nuclear c-Ets-2 DNA binding activity and hFGL2 induction in response to HBx. In conclusion, HBc and HBx proteins enhance transcription of hFGL2 through c-Ets-2 dependent on MAPK signal pathways.

Requirements of calcium fluxes and ERK kinase activation for glucose- and interleukin-1β-induced β-cell apoptosis

Increasing evidence indicates that beta-cell apoptosis and impaired secretory function were partly mediated by interleukin (IL)-1beta and/or high-glucose-induced beta-cell production of IL-1beta. However, the specific signal transduction pathways and molecular events involved in beta-cell dysfunction remain largely unresolved. In this study, we investigated whether Ca(2+) and extracellular signal-regulated kinase (ERK) activation plays a role for IL-1beta action in rat islets. Exposure of rat islets for 4 days to 33.3 mM glucose and 140 ng/ml IL-1beta- induced beta-cell apoptosis and impaired glucose-stimulated insulin secretion. By Western blotting with phosphospecific antibodies, glucose and IL-1beta were shown to activate ERK. Ca(2+) channel blocker nimodipine or ERK inhibitor PD98059 prevented glucose- and IL-1beta-induced ERK activation, beta-cell apoptosis, and impaired function. Furthermore, treatment with Ca(2+) ionophore ionomycin, or exposure to thapsigargin, an inhibitor of sarco(endo)plasmic reticulum Ca(2+) ATPase, all caused an amplification of IL-1beta-induced ERK activation in rat islet. On the other hand, a chelator of intracellular free Ca(2+) [bis-(o-aminophenoxy)-N,N,N',N'-tetraacetic acid-acetoxymethyl] (BAPTA/AM) and an inhibitor of calmodulin (W7) diminished IL-1beta-induced phosphorylation of ERK. Finally, islet release of IL-1beta in response to high glucose could be abrogated by nimodipine, mibefradil, or PD98059. Together, these data suggest that glucose- and IL-1beta-induced beta-cell secretory dysfunction and apoptosis are Ca(2+) influx and ERK dependent in rat islets.

Even Ephemeral Endotoxin Exposure Establishes Endotoxin Tolerance

Macrophages previously exposed to bacterial lipopolysaccharide (LPS) develop a "tolerant" response with decreased extracellular signal-regulated kinase (ERK) activation in response to LPS rechallenge. Prior work using 21-hour LPS pretreatment showed that 100 ng/mL of LPS-inhibited tumor necrosis factor (TNF) release, whereas very low dose LPS (1 ng/mL) augmented TNF release. Endotoxin tolerance was also associated with alterations in activation of ERK and p38 kinase when cells were restimulated with LPS. We hypothesized that the interval after pretreatment, before LPS rechallenge, modulates macrophage response to LPS. RAW 264.7 macrophage-like cells were pretreated for 4 hours in 0 ng/mL (none), 1 ng/mL, 10 ng/mL, or 100 ng/mL of Escherichia coli 0111:B4 LPS. After 4 hour pretreatment, medium was discarded. Cells were rechallenged immediately or 21 hours later with 0 ng/mL, 1 ng/mL, 10 ng/mL, or 100 ng/mL LPS. Supernatant TNF secretion at 3 hour was measured using enzyme-linked immunosorbent assay. Active phospho-ERK was examined by Western blot using specific monoclonal antibodies 30 minutes after LPS rechallenge. Statistical analysis by chi and student's t test. When macrophages were pretreated for 4 hour and incubated overnight (21-hour interval) 1 ng/mL of LPS augmented and 100 ng/mL inhibited TNF release with LPS rechallenge. In contrast, with immediate rechallenge, we saw additive effects with 100 ng/mL LPS and no difference with 1 ng/mL LPS versus no pretreatment. Western blot revealed that even with immediate rechallenge "tolerant" macrophages were unable to activate ERK. A short LPS exposure is sufficient to induce alterations in ERK activation in macrophages, but longer intervals are required to express altered cytokine release. In conjunction with other recent findings, these results suggest that both pretreatment dose and interval modulate macrophage responsiveness to LPS rechallenge.

In Vivo and In Vitro Characterization of a Novel Neuroprotective Strategy for Stroke: Ischemic Postconditioning

As clinical trials of pharmacological neuroprotective strategies in stroke have been disappointing, attention has turned to the brain's own endogenous strategies for neuroprotection. Recently, a hypothesis has been offered that modified reperfusion subsequent to a prolonged ischemic episode may also confer ischemic neuroprotection, a phenomenon termed 'postconditioning'. Here we characterize both in vivo and in vitro models of postconditioning in the brain and offer data suggesting a biological mechanism for protection. Postconditioning treatment reduced infarct volume by up to 50% in vivo and by approximately 30% in vitro. A duration of 10 mins of postconditioning ischemia after 10 mins of reperfusion produced the most effective postconditioning condition both in vivo and in vitro. The degree of neuroprotection after postconditioning was equivalent to that observed in models of ischemic preconditioning. However, subjecting the brain to both preconditioning as well as postconditioning did not cause greater protection than each treatment alone. The prosurvival protein kinases extracellular signal-regulated kinase (ERK), p38 mitogen-activated protein kinase (MAPK), and Akt show prolonged phosphorylation in the cortex of postconditioned rats. Neuroprotection after postconditioning was inhibited only in the presence of LY294002, which blocks Akt activation, but not U0126 or SB203580, which block ERK and P38 MAP kinase activity. In contrast, preconditioning-induced protection was blocked by LY294002, U0126, and SB203580. Our data suggest that postconditioning may represent a novel neuroprotective approach for focal ischemia/reperfusion, and one that is mediated, at least in part, by the activation of the protein kinase Akt.