Applicable Forensic Biomarker for Drowning Diagnosis: Extracellular Signal-Regulated Kinase 2 (ERK2).

Drowning is a common cause of accidental death worldwide, and it continues to be a serious public health problem. However, diagnosing drowning is a challenging task in forensic investigation because it is difficult to prove actual drowning and other submerged deaths with the autopsy techniques that are currently in use. Here, we show biomarkers that may be helpful for the diagnosis of drowning. We divided the experimental animals into four groups (drowning, postmortem submersion, hypoxia, and control) to evaluate the expression patterns of extracellular signal-regulated kinase 1/2 (ERK1/2). On gene expression analysis, only ERK2 was found to be significantly increased in the drowning groups compared to the other cases. In the immunoblot analysis, phosphorylated ERK2 (p-ERK2) was found to be upregulated in the drowning groups. Immunohistochemical staining also showed that p-ERK in alveolar cells revealed a granular pattern in the drowning groups. However, the expression pattern of ERK2 over time after drowning differed between the freshwater and seawater drowning groups. Taken together, these results indicate that ERK2 may be useful for distinguishing between drowning and postmortem submersion if the postmortem interval (PMI) of drowning is short. Conversely, if the PMI is long from the time that death occurs until the discovery of dead bodies, it is possibly more helpful for identifying between freshwater and seawater drowning.

Histone deacetylase 6 (HDAC6) deacetylates extracellular signal-regulated kinase 1 (ERK1) and thereby stimulates ERK1 activity.

Histone deacetylase 6 (HDAC6), a class IIb HDAC, plays an important role in many biological and pathological processes. Previously, we found that ERK1, a downstream kinase in the mitogen-activated protein kinase signaling pathway, phosphorylates HDAC6, thereby increasing HDAC6-mediated deacetylation of α-tubulin. However, whether HDAC6 reciprocally modulates ERK1 activity is unknown. Here, we report that both ERK1 and -2 are acetylated and that HDAC6 promotes ERK1 activity via deacetylation. Briefly, we found that both ERK1 and -2 physically interact with HDAC6. Endogenous ERK1/2 acetylation levels increased upon treatment with a pan-HDAC inhibitor, an HDAC6-specific inhibitor, or depletion of HDAC6, suggesting that HDAC6 deacetylates ERK1/2. We also noted that the acetyltransferases CREB-binding protein and p300 both can acetylate ERK1/2. Acetylated ERK1 exhibits reduced enzymatic activity toward the transcription factor ELK1, a well-known ERK1 substrate. Furthermore, mass spectrometry analysis indicated Lys-72 as an acetylation site in the ERK1 N terminus, adjacent to Lys-71, which binds to ATP, suggesting that acetylation status of Lys-72 may affect ERK1 ATP binding. Interestingly, an acetylation-mimicking ERK1 mutant (K72Q) exhibited less phosphorylation than the WT enzyme and a deacetylation-mimicking mutant (K72R). Of note, the K72Q mutant displayed decreased enzymatic activity in an in vitro kinase assay and in a cellular luciferase assay compared with the WT and K72R mutant. Taken together, our findings suggest that HDAC6 stimulates ERK1 activity. Along with our previous report that ERK1 promotes HDAC6 activity, we propose that HDAC6 and ERK1 may form a positive feed-forward loop, which might play a role in cancer.

Heme oxygenase 1 regulates apoptosis induced by heat stress in bovine ovarian granulosa cells via the ERK1/2 pathway.

Heat stress can inhibit follicular development in dairy cows, and thus can affect their reproductive performance. Follicular granulosa cells can synthesize estrogen, that affects the development and differentiation of follicles by apoptosis. Heme oxygenase 1 (HO-1/heat shock protein 32) plays an antiapoptotic and cytoprotective role in various cells during stress-induced apoptosis, but little is known about its definitive function in bovine (ovarian) granulosa cells (bGCs). In our study, the roles and mechanism of HO-1 on the heat stress-induced apoptosis of bGCs were studied. Our results show that the expression of HO-1 was significantly increased under heat stress. Moreover, HO-1 silencing increased apoptosis, whereas its overexpression dampened apoptosis by regulating the expression of Bax/Bcl-2 and the levels of cleaved caspase-3. In addition, HO-1 can also play a cytoprotective role by affecting estrogen levels and decomposing heme to produce biologically active metabolite carbon monoxide (CO). Meanwhile, CO significantly increased the level of HO-1, decreased Bax/Bcl-2 levels, and inhibited the activation of extracellular signal-regulated kinase 1/2 (ERK1/2) signaling pathway. The apoptosis of ovarian GCs can affect the secretion of estrogen and lead to disorder of the ovarian microenvironment, thus affecting the normal function of the ovary. Our results indicate that HO-1 acts as a cytoprotective enzyme and plays a protective role in heat-induced apoptosis of bGCs. In conclusion, HO-1 and its metabolite CO inhibit the apoptosis of bGCs induced by heat stress through the ERK1/2 pathway. The results of this study provide a valuable clue for improving the fertility of heat stressed cows in summer.

The effect of fludrocortisone on the uterine receptivity partially mediated by ERK1/2-mTOR pathway.

Implantation of embryos needs endometrial receptivity. Mineralocorticoids is one of the causes influencing the implantation window. This study targeted to evaluation fludrocortisone different properties on endometrial receptivity. The objective of this study was to assess whether treatment with fludrocortisone could impact the expression of diverse genes and proteins that are involved in uterine receptivity in mice. In this study, 40 female adult BALB/c mice were used. The samples were allocated to four groups of ten. Control group (C) received: vehicle; fludrocortisone group (FCA): received 1.5 mg/kg fludrocortisone; PP242 group (PP242): received 30 mg/kg PP242; fludrocortisone+PP242 group (FCA+PP242): received fludrocortisone and PP242. Mice were killed on window implantation day after mating and confirmed pregnancy. The endometrial epithelium of mouse was collected to assess mRNA expression of leukemia inhibitory factor (LIF), mucin-1 (MUC1), heparin-binding epidermal growth factor (HB-EGF), (Msx.1), miRNA Let-7a, and miRNA 223-3p as well as protein expression of extracellular signal-regulated kinase 1/2 (ERK1/2), mammalian target of rapamycin (mTOR), and eukaryotic translation initiation factor 4E-binding protein 1 (4E-BP1) in the uterine using real-time PCR and western blot, respectively. In comparison with the control group, fludrocortisone administration upregulated the expression of LIF, HB-EGF, Msx.1, miRNA Let-7a, ERK1/2, and mTOR in the epithelial endometrium. The PP242-treated group demonstrated a significant rise in the expression of MUC1, miRNA 223-3p and a remarkable decline in ERK1/2 and p-4E-BP1 levels in comparison with the control group. Combination therapy of (FCA+PP242) resulted in a remarkable rise in LIF, Msx-1, HB-EGF, ERK1/2, and mTOR levels, in comparison with the PP242 group. Furthermore, combination therapy of (FCA+PP242) downregulated the expression of MUC1 in comparison with the PP242-treated group. According to the results, fludrocortisone affected uterine receptivity possibly by means of modulating the expression of genes involved in the uterine receptivity and activation of the ERK1/2-mTOR pathway.

Inhibition of extracellular signal-regulated kinase potentiates the apoptotic and antimetastatic effects of cyclin-dependent kinase inhibitors on metastatic DU145 and PC3 prostate cancer cells.

Purvalanol and roscovitine are specific cyclin-dependent kinase (CDK) inhibitors, which have antiproliferative and apoptotic effects on various types of cancer. Although, the apoptotic accomplishment of purvalanol and roscovitine was elucidated at the molecular level, the underlying exact of drug-induced apoptosis through mitogen-activated protein kinase (MAPK) signaling still speculative. In addition, the role of CDK inhibitors in the downregulation of extracellular signal-regulated kinase 1/2 (ERK1/2)-mediated epithelial-mesenchymal transition (EMT) remains unclear. Here, we investigated the potential effect of each CDK inhibitors on cell proliferation, migration, and generation of reactive oxygen species due to the inhibition of MAPKs in metastatic DU145 and PC3 prostate cancer cells. We reported that purvalanol and roscovitine induced mitochondria membrane potential loss-dependent apoptotic cell death, which was also characterized by activation of several caspases, cleavage of poly (ADP-ribose) polymerase-1 in DU145 and PC3 cells. Cotreatment of either purvalanol or roscovitine with ERK1/2 inhibitor, U0126, synergistically suppressed cell proliferation, and induced apoptotic action. Also, ERK1/2 inhibition potentiated the effect of each CDK inhibitor on the downregulation of EMT processes via increasing the epithelial marker and decreasing mesenchymal markers through reduction of Wnt signaling regulators in DU145 cells. This study provides biological evidence about purvalanol and roscovitine have apoptotic and antimetastatic effects via MAPK signaling on prostate cancer cell by activation of GSK3ß signaling and inhibition of phosphoinositide-3-kinase/AKT (PI3K/AKT) pathways involved in the EMT process.

Acute physical exercise increases leptin-induced hypothalamic extracellular signal-regulated kinase1/2 phosphorylation and thermogenesis of obese mice.

The obesity is a result of energy imbalance and the increase in thermogenesis seems an interesting alternative for the treatment of this disease. The mechanism of energy expenditure through thermogenesis is tightly articulated in the hypothalamus by leptin. The hypothalamic extracellular signal-regulated kinase-1/2 (ERK1/2) is a key mediator of the thermoregulatory effect of leptin and mediates the sympathetic signal to the brown adipose tissue (BAT). In this context, physical exercise is one of the main interventions for the treatment of obesity. Thus, this study aimed to verify the effects of acute physical exercise on leptin-induced hypothalamic ERK1/2 phosphorylation and thermogenesis in obese mice. Here we showed that acute physical exercise reduced the fasting glucose of obese mice and increased leptin-induced hypothalamic p-ERK1/2 and uncoupling protein 1 (UCP1) content in BAT ( P < 0.05). These molecular changes are accompanied by an increased oxygen uptake (VO 2 ) and heat production in obese exercised mice ( P < 0.05). The increased energy expenditure in the obese exercised animals occurred independently of changes in spontaneous activity. Thus, this is the first study demonstrating that acute physical exercise can increase leptin-induced hypothalamic ERK1/2 phosphorylation and energy expenditure of obese mice.

Tg737 acts as a key driver of invasion and migration in liver cancer stem cells and correlates with poor prognosis in patients with hepatocellular carcinoma.

We previously demonstrated that the Tg737 gene plays a critical role in the carcinogenesis of hepatocellular carcinoma (HCC). However, few systematic investigations have focused on the biological function of Tg737 in the invasion and migration of liver cancer stem cells (LCSCs) and on its clinical significance. In this study, Tg737 overexpression was achieved via gene transfection in MHCC97-H side population (SP) cells, which are considered a model for LCSCs in scientific studies. Tg737 overexpression significantly inhibited the invasion and migration of SP cells in an extracellular signal-regulated kinase1/2 (ERK1/2)/matrix metalloproteinase-2 (MMP-2)-dependent manner. Furthermore, Tg737 expression was frequently decreased in HCC tissues relative to that in adjacent noncancerous liver tissues. This decreased expression was significantly associated with tumor differentiation, the American Joint Committee on Cancer (AJCC) stage, metastasis, tumor size, vascular invasion, alpha-fetoprotein (AFP) levels, and tumor number. Moreover, multivariate Cox regression analyses demonstrated that Tg737 expression was an independent factor for predicting the overall survival of HCC patients. Notably, Kaplan-Meier analysis further showed that overall survival was significantly worse among patients with low Tg737 expression. Collectively, our findings demonstrated that Tg737 is a poor prognostic marker in patients with HCC, which may be due to its ability to promote LCSCs invasion and migration. These results provide a basis for investigating of Tg737 as a novel prognostic biomarker and therapeutic target.

COMMD7 functions as molecular target in pancreatic ductal adenocarcinoma.

Our previous studies provided evidence that COMMD7 was associated with tumor progression in human solid cancer. Herein, we aimed to investigate its expression pattern, clinical significance, and biological function in pancreatic ductal adenocarcinoma (PDAC). We found that high COMMD7 expression was specifically detected in PDAC tissues and PDAC cell lines. In addition, COMMD7 overexpression positively correlated with histological differentiation and tumor node metastasis (TNM) stage. Patients with high COMMD7 expression had significantly poorer overall survival, and high COMMD7 expression was an independent predictor of poor prognosis. To further explore the regulatory mechanism of COMMD7, we used stable short hairpin RNA (shRNA)-mediated knockdown and divided the work into in vitro and in vivo experiments. In vitro, the anti-proliferation effects of COMMD7 inhibition were observed under long-time stress conditions, which correlated with cyclin D1 and Bcl-2 downregulation and Bax upregulation. We found that under short-time stress conditions, decreased COMMD7 expression also inhibited PDAC cell invasion in vitro which decreased the secretion of matrix metalloproteinase 2 (MMP-2). Moreover, extracellular signal-regulated kinase1/2 (ERK1/2) was identified as a direct target of COMMD7. The inhibition of ERK1/2 activity under short- or long-time stress conditions using specific inhibitors in COMMD7 inhibition cells all exhibited a strong tumorigenic role. In vivo, COMMD7 was sufficient to impair tumor growth. Our results suggest that COMMD7 plays an important role in the late progression of PDAC and is a potential novel target. © 2016 Wiley Periodicals, Inc.

Multivariate signaling regulation by SHP2 differentially controls proliferation and therapeutic response in glioma cells.

Information from multiple signaling axes is integrated in the determination of cellular phenotypes. Here, we demonstrate this aspect of cellular decision making in glioblastoma multiforme (GBM) cells by investigating the multivariate signaling regulatory functions of the protein tyrosine phosphatase SHP2 (also known as PTPN11). Specifically, we demonstrate that the ability of SHP2 to simultaneously drive ERK1/2 and antagonize STAT3 pathway activities produces qualitatively different effects on the phenotypes of proliferation and resistance to EGFR and c-MET co-inhibition. Whereas the ERK1/2 and STAT3 pathways independently promote proliferation and resistance to EGFR and c-MET co-inhibition, SHP2-driven ERK1/2 activity is dominant in driving cellular proliferation and SHP2-mediated antagonism of STAT3 phosphorylation prevails in the promotion of GBM cell death in response to EGFR and c-MET co-inhibition. Interestingly, the extent of these SHP2 signaling regulatory functions is diminished in glioblastoma cells that express sufficiently high levels of the EGFR variant III (EGFRvIII) mutant, which is commonly expressed in GBM. In cells and tumors that express EGFRvIII, SHP2 also antagonizes the phosphorylation of EGFRvIII and c-MET and drives expression of HIF-1α and HIF-2α, adding complexity to the evolving understanding of the regulatory functions of SHP2 in GBM.

Hesperidin, A Popular Antioxidant Inhibits Melanogenesis via Erk1/2 Mediated MITF Degradation.

Regulation of melanogenesis has been the focus of treatment for hyperpigmentary skin disorders. Although hesperidin is one of the most well-known, naturally occurring flavonoids with antioxidant and anti-inflammatory effect, its anti-melanogenic effect is not known. The present study aims to determine the anti-melanogenic effect of hespiridin as well as its underlying molecular mechanisms. Melanin contents were measured in normal human melanocytes and B16F10 melanoma cells. Protein and mRNA levels of tyrosinase, microphthalmia-associated transcription factor (MITF), tyrosinase related protein-1 (TRP-1) and TRP-2 were determined. Melanogenesis-regulating signals were examined. In results, hesperidin strongly inhibited melanin synthesis and tyrosinase activity. Hesperidin decreased tyrosinase, TRP-1, and TRP-2 protein expression but increased phospho-extracellular signal-regulated kinase 1/2 (p-Erk1/2) expression. Specific inhibitor of Erk1/2 or proteasome inhibitor reversed the inhibition of melanogenesis induced by hesperidin. Taken together, hesperidin, a popular antioxidant, stimulated Erk1/2 phosphorylation which subsequently degraded MITF which resulted in suppression of melanogenic enzymes and melanin synthesis.

Serotonin acts as a novel regulator of interleukin-6 secretion in osteocytes through the activation of the 5-HT(2B) receptor and the ERK1/2 signalling pathway.

Interleukin-6 (IL-6) is a potent stimulator of osteoclastic bone resorption. Osteocyte secretion of IL-6 plays an important role in bone metabolism. Serotonin (5-HT) has recently been reported to regulate bone metabolism. The aim of this study was to evaluate the effect of serotonin on osteocyte expression of IL-6. The requirement for the 5-HT receptor(s) and the role of the extracellular signal-regulated kinase 1/2 (ERK1/2) in serotonin-induced IL-6 synthesis were examined. In this study, real-time PCR and ELISA were used to analyse IL-6 gene and protein expression in serotonin-stimulated MLO-Y4 cells. ERK1/2 pathway activation was determined by Western blot. We found that serotonin significantly activated the ERK1/2 pathway and induced IL-6 mRNA expression and protein synthesis in cultured MLO-Y4 cells. However, these effects were abolished by pre-treatment of MLO-Y4 cells with a 5-HT2B receptor antagonist, RS127445 or the ERK1/2 inhibitor, PD98059. Our results indicate that serotonin stimulates osteocyte secretion of IL-6 and that this effect is associated with activation of 5-HT2B receptor and the ERK1/2 pathway. These findings provide support for a role of serotonin in bone metabolism by indicating serotonin regulates bone remodelling by mediating an inflammatory cytokine.

CAMSAP3 depletion induces lung cancer cell senescence-associated phenotypes through extracellular signal-regulated kinase inactivation.

BACKGROUND: Cellular senescence is an aging-related process found in cancer cells that contributes to irreversible growth arrest and tumor aggressiveness. Recently, calmodulin-regulated spectrin-associated protein 3 (CAMSAP3), a minus-end microtubule-stabilizing protein, has received increasing attention in cancer cell biology. However, the biological role of CAMSAP3 on senescence in human lung cancer remains incompletely understood. METHODS: The function of CAMSAP3 on the regulation of cellular senescence-associated phenotypes in human non-small cell lung cancer H460 cells were determined in CAMSAP3 deletion (H460/C3ko) cells. The effects of CAMSAP3 on cell proliferation were investigated using MTT and colony formation assays. The cell cycle activity was evaluated by flow cytometry and the senescence-associated phenotypes were observed by SA-ß-Gal staining. Quantitative RT-PCR and westen blot were used to evaluate the expression of cell cycle and senescence markers. Moreover, the interaction of CAMSAP3-ERK1/2 and possible partner protein was quantified using immunoprecipitation/mass spectrometry and immunofluorescence. Lastly, an xenograft model were performed. RESULTS: CAMSAP3 knockout promotes lung cancer cell senescence-associated phenotypes and induces G1 cell cycle arrest. Mechanistic investigation revealed that phosphorylated ERK (p-ERK) was markedly downregulated in CAMSAP3-deleted cells, suppressing cyclin D1 expression levels, and full-length CAMSAP3 abrogated these phenotypes. Proteomic analysis demonstrated that vimentin, an intermediate filament protein, is required as a scaffold for CAMSAP3-modulating ERK signaling. Furthermore, an in vivo tumor xenograft experiment showed that tumor initiation is potentially delayed in CAMSAP3 knockout tumors with the downregulation of p-ERK and cyclin D1, resulting in a senescence-like phenotype. CONCLUSION: This study is the first to report an intriguing role of CAMSAP3 in lung carcinoma cell senescence-associated phenotypes via the modulation of p-ERK/cyclin D1 signaling.

Astaxanthin alleviates neuropathic pain by inhibiting the MAPKs and NF-κB pathways.

Neuropathic pain is a complex condition that usually lasts a lifetime and has a major negative impact on life after injury. Improving pain management is an important and unmet need. Astaxanthin (AST) is a natural marine medicine with effective antioxidant and anti-inflammatory properties and neuroprotective effects. However, few mechanisms can explain the role of AST in the treatment of neuropathic pain. In the present study, we examined its potential to eliminate spinal nerve ligation (SNL) damage by inhibiting the phosphorylation of extracellular signal-regulated kinase (ERK)1/2, phosphorylation of p38 mitogen-activated protein kinase (p38 MAPK), nuclear factor-κB (NF-κB) p65 and the inflammatory response. The results of behavior tests indicated the promising role of AST in analgesic effect in SNL mice. AST decreased the neuronal and non-neuronal activation, the levels of the inflammatory signaling mediators (p-ERK1/2 p-p38 MAPK and NF-κB p65) and inflammatory cytokine expression (interleukin [IL]-1, IL-17, IL-6, and tumor necrosis factor-α [TNF-α]. These results suggest that AST is a promising candidate to reduce nociceptive hypersensitization after SNL.

TIPE3 promotes non-small cell lung cancer progression via the protein kinase B/extracellular signal-regulated kinase 1/2-glycogen synthase kinase 3ß-ß-catenin/Snail axis.

BACKGROUND: Tumor necrosis factor-α-induced protein 8-like 3 (TNFAIP8L3, also called TIPE3) has been shown to activate PI3K-AKT and MEK-ERK pathways. However, the roles of TIPE3 in progression of lung cancer are largely unknown. METHODS: Immunohistochemistry and western blotting were carried out to analyze the expression of TIPE3 in lung cancer clinical tissues and cells. TIPE3-overexpressing and knock-down NSCLC cell lines were established by transfer of TIPE3 coding sequence and shRNA, respectively. In vitro functional assays were performed to assess the effects of TIPE3 on proliferation and metastasis of NSCLC cells. Tumor xenograft mouse model was used to examine the roles of TIPE3 in growth of NSCLC cells in vivo. Western blotting, immunofluorescence, and immunohistochemistry were conducted to evaluate the association of TIPE3 and molecules related to AKT/ERK1/2-GSK3ß-ß-catenin/Snail pathway. PI3K, MEK, or GSK3ß kinase and proteasome inhibition assays as well as ß-Trcp and STUB1 siRNA assays were employed to determine the contribution of AKT/ERK1/2-GSK3ß signaling and ubiquitin-proteasome pathway to the regulatory effects of TIPE3 on expression of ß-catenin, Snail1, and Slug. RESULTS: We demonstrated that TIPE3 was elevated in lung cancer tissues and cells. The expression level of TIPE3 was positively correlated with malignant clinicopathological characteristics of lung cancer patients, such as tumor size, pathologic stage, and lymph node metastasis. Knockdown of TIPE3 suppressed the proliferation and growth of NSCLC cells as well as their migration and invasion ability, whereas TIPE3 overexpression facilitated these biological processes. Mechanistic data showed that TIPE3 promoted AKT and ERK1/2 signaling, inactivated GSK3ß activity, and enhanced the expression and transcriptional activity of ß-catenin, Snail1, and Slug in NSCLC cells. Kinase or proteasome inhibition and ß-Trcp or STUB1 knockdown assays further revealed that TIPE3 upregulated ß-catenin, Snail1, and Slug via the AKT/ERK1/2-GSK3ß pathway, in an ubiquitin-proteasome-dependent manner. More importantly, clinical data demonstrated that the expression level of TIPE3 was positively associated with the activation of AKT/ERK1/2-GSK3ß-ß-catenin/Snail pathway in lung cancer. CONCLUSIONS: Our findings indicate that upregulation of TIPE3 promotes the progression of human NSCLC considerably by activating ß-catenin, Snail1, and Slug transcriptional signaling via the AKT/ERK1/2-GSK3ß axis. Therefore, TIPE3 may represent a potential therapeutic target for NSCLC.

Epigallocatechin-3-Gallate and PEDF 335 Peptide, 67LR Activators, Attenuate Vasogenic Edema, and Astroglial Degeneration Following Status Epilepticus.

Non-integrin 67-kDa laminin receptor (67LR) is involved in cell adherence to the basement membrane, and it regulates the interactions between laminin and other receptors. The dysfunction of 67LR leads to serum extravasation via blood-brain barrier (BBB) disruption. Polyphenol (-)-epigallocatechin-3-O-gallate (EGCG) and pigment epithelium-derived factor (PEDF) bind to 67LR and inhibit neovascularization. Therefore, in the present study, we investigated the effects of EGCG and NU335, a PEDF-derive peptide, on BBB integrity and their possible underlying mechanisms against vasogenic edema formation induced by status epilepticus (SE, a prolonged seizure activity). Following SE, both EGCG and NU335 attenuated serum extravasation and astroglial degeneration in the rat piriform cortex (PC). Both EGCG and NU335 reversely regulated phosphatidylinositol 3 kinase (PI3K)/AKT-eNOS (endothelial nitric oxide synthase) mediated BBB permeability and aquaporin 4 (AQP4) expression in endothelial cells and astrocytes through the p38 mitogen-activated protein kinase (p38 MAPK) and extracellular signal-regulated kinase 1/2 (ERK1/2) signaling pathways, respectively. Furthermore, EGCG and NU335 decreased p47Phox (a nicotinamide adenine dinucleotide phosphate oxidase subunit) expression in astrocytes under physiological and post-SE conditions. Therefore, we suggest that EGCG and PEDF derivatives may activate 67LR and its downstream effectors, and they may be considerable anti-vasogenic edema agents.

Disrupting the vicious cycle created by NOX activation in sickle erythrocytes exposed to hypoxia/reoxygenation prevents adhesion and vasoocclusion.

In sickle cell disease (SCD), recurrent painful vasoocclusive crisis are likely caused by repeated episodes of hypoxia and reoxygenation. The sickle erythrocyte (SSRBC) adhesion plays an active role in vasoocclusion. However, the effect of prolonged reoxygenation after hypoxic stress on the molecular mechanisms in SSRBCs involved in onset of episodic vasoocclusion remain unclear. Exposure of human SSRBCs to hypoxia followed by 2 h reoxygenation, increased reactive oxygen species (ROS) production. Using specific pharmacological inhibitors, we show that excess ROS production in both reticulocytes and mature SSRBCs is regulated by NADPH oxidases (NOXs), the mitogen-activated protein kinase (ERK1/2), and G-protein coupled-receptor kinase 2 (GRK2). Consequently, SSRBC ROS create an intracellular positive feedback loop with ERK1/2 and GRK2 to mediate SSRBC adhesion to endothelium in vitro, and vasoocclusion in a mouse model of vasoocclusion in vivo. Importantly, reducing ROS levels in SSRBCs with redox-active manganese (Mn) porphyrins, commonly known as mimics of superoxide dismutase (SOD), disrupted the cycle created by ROS by affecting NOX and GRK2 activities and ERK1/2 phosphorylation, thus abrogating RBC-endothelial interactions. Inhibition adhesion assays show that LW (ICAM-4, CD242) blood group glycoprotein and CD44 are the RBC adhesion molecules mediating endothelial binding. Conversely, hypoxia/reoxygenation of normal RBCs failed to activate this feedback loop, and adhesion. These findings provide novel insights into the pathophysiological significance of the deleterious cycle created by NOX-dependent ROS, GRK2 and ERK1/2 within SSRBCs activated by hypoxia/reoxygenation, and involved in SSRBC adhesion and vasoocclusion. Thus, this loop in SSRBCs, which can be disrupted by Mn porphyrins, likely drives the profound SCD vasculopathy, and may point to new therapeutic targets to prevent chronic vasoocclusive events.

Involvement of PKCα and ERK1/2 signaling pathways in EGCG's protection against stress-induced neural injuries in Wistar rats.

Stress-induced neural injuries are closely linked to the pathogenesis of various neuropsychiatric disorders and psychosomatic diseases. We and others have previously demonstrated certain protective effects of epigallocatechin-3-gallate (EGCG) in stress-induced cerebral impairments, but the underlying protective mechanisms still remain poorly elucidated. Here we provide evidence to support the possible involvement of PKCα and extracellular signal-regulated kinase 1/2 (ERK1/2) signaling pathways in EGCG-mediated protection against restraint stress-induced neural injuries in rats. In both open-field and step-through behavioral tests, the restraint stress-induced neuronal impairments were significantly ameliorated by administration of EGCG or green tea polyphenols (GTPs), which was associated with a partial restoration of normal plasma glucocorticoid, dopamine and serotonin levels. Furthermore, the stress-induced decrease of PKCα and ERK1/2 expression and phosphorylation was significantly attenuated by EGCG and to a less extent by GTP administration. Additionally, EGCG supplementation restored the production of adenosine triphosphate (ATP) and the expression of a key regulator of cellular energy metabolism, the peroxisome proliferators-activated receptor-γ coactivator-1α (PGC-1α), in stressed animals. In conclusion, PKCα and ERK1/2 signaling pathways as well as PGC-1α-mediated ATP production might be involved in EGCG-mediated protection against stress-induced neural injuries.

Computational design, chemical synthesis, and biological evaluation of a novel ERK inhibitor (BL-EI001) with apoptosis-inducing mechanisms in breast cancer.

Extracellular signal-regulated kinase1/2 (ERK1/2) plays a crucial role in the resistance of apoptosis in carcinogenesis; however, its targeted small-molecule inhibitors still remain to be discovered. Thus, in this study, we computationally and experimentally screened a series of small-molecule inhibitors targeting ERK toward different types of human breast cancer cells. Subsequently, we synthesized some candidate ERK inhibitors, identified a novel ERK inhibitor (BL-EI001) with anti-proliferative activities, and analyzed the BL-EI001/ERK complex. Moreover, we found that BL-EI001 induced breast cancer cell apoptosis via mitochondrial pathway but independent on Ras/Raf/MEK pathway. In addition, we carried out proteomics analyses for exploring some possible BL-EI001-induced apoptotic pathways, and further found that BL-EI001-induced apoptosis affected ERK phosphorylation in breast cancer. Further, we found that BL-EI001 bear anti-tumor activities without remarkable toxicities, and also induced mitochondrial apoptosis by targeting ERK in vivo. Taken together, these results demonstrate that in silico design and experimental discovery of a synthesized small-molecule ERK inhibitor (BL-EI001)as a potential novel apoptosis-inducing drug in the treatment of breast cancer.

ERK activation is required for hydrostatic pressure-induced tensile changes in engineered articular cartilage.

The objective of this study was to identify ERK 1/2 involvement in the changes in compressive and tensile mechanical properties associated with hydrostatic pressure treatment of self-assembled cartilage constructs. In study 1, ERK 1/2 phosphorylation was detected by immunoblot, following application of hydrostatic pressure (1 h of static 10 MPa) applied at days 10-14 of self-assembly culture. In study 2, ERK 1/2 activation was blocked during hydrostatic pressure application on days 10-14. With pharmacological inhibition of the ERK pathway by the MEK1/ERK inhibitor U0126 during hydrostatic pressure application on days 10-14, the increase in Young's modulus induced by hydrostatic pressure was blocked. Furthermore, this reduction in Young's modulus with U0126 treatment during hydrostatic pressure application corresponded to a decrease in total collagen expression. However, U0126 did not inhibit the increase in aggregate modulus or GAG induced by hydrostatic pressure. These findings demonstrate a link between hydrostatic pressure application, ERK signalling and changes in the biomechanical properties of a tissue-engineered construct.