Activation Of Extracellular Regulated Protein Kinase Research Articles

DUSP2 Deletion Inhibits Macrophage Migration by Inhibiting ERK Activation in Zebrafish

Dual-specificity phosphatase 2 (DUSP2) regulates the activation of members of the mitogen-activated protein kinase (MAPK) family, which is involved in a variety of cellular processes including cell proliferation, differentiation, apoptosis, and migration. DUSP2 also regulates the expression of inflammatory mediators in macrophages; however, it remains unknown whether DUSP2 participates in macrophage migration. Here, using the tail fin injury model in zebrafish larvae, we found that the deletion of DUSP2 inhibited the expression of pro-inflammatory cytokines and macrophage chemokines. Moreover, live imaging results showed that the migration of macrophages to the injury site was inhibited after DUSP2 deletion. This inhibitory effect was mediated through the reduced activation of extracellular regulated protein kinases (ERK) in DUSP2 knockout zebrafish.

Aloperine inhibits RANKL-induced osteoclast differentiation via suppressing the MAPK signaling pathways

To figure out the molecular mechanism of aloperine (ALO) on receptor activator of nuclear factor (NF)-kappa-B (κb) ligand (RANKL)-caused osteoclast differentiation. The histopathological analysis and tartrate-resistant acid phosphatase (TRAP) staining assays were applied to check the extent of bone loss of the femur. Then, the protein expressions of nitric oxide synthase 2, glutathione reductase, nuclear erythroid 2-related factor 2, heme oxygenase-1, nicotinamide adenine dinucleotide phosphate (NADPH) oxidase 1, and catalase were checked in RAW264.7, a macrophage cell line, by enzyme-linked-immunosorbent serologic assay and Western blot analysis. Further, the TRAP staining and quantitative polymerase chain reaction assay were applied to characterize the level of RAW264.7 osteoclast differentiation. Besides, Western blot assay was used to check the protein expressions of extracellular regulated protein kinases (ERK), c-Jun N-terminal kinase (JNK), and P38 in RAW264.7. Finally, ERK activation blocker U0126 was used to inhibit mitogen-activated protein kinase (MAPK) signaling pathway to determine the effect of MAPK signaling pathway on RAW264.7 osteoclast differentiation. In this study, the results demonstrated that ALO could inhibit lipopolysaccharide-induced bone loss in vivo in a dose-dependent manner, with significant inhibitory effects at high doses. Further research indicated that ALO could inhibit RANKL-induced oxidative stress and osteoclast differentiation of RAW264.7. Then, ALO could inactivate MAPK signaling pathway. Finally, the results showed that inhibition of MAPK signaling pathway could increase ALO inhibition of RANKL-caused osteoclast differentiation. ALO inhibits RANKL-caused osteoclast differentiation by suppressing the MAPK signaling pathways.

Sanggenon C Suppresses Tumorigenesis of Gastric Cancer by Blocking ERK-Drp1-Mediated Mitochondrial Fission.

Sanggenon C is a flavonoid extracted from the root bark of white mulberry, which is a traditional Chinese medicine with anti-inflammatory, antioxidative, and antitumor pharmacological effects. In this study, sanggenon C was found to inhibit human gastric cancer (GC) cell proliferation and colony formation, induce GC cell cycle arrest in the G0-G1 phase, and promote GC cell apoptosis. Moreover, sanggenon C was found to decrease the level of mitochondrial membrane potential in GC cells and inhibit mitochondrial fission. Mechanistically, RNA sequencing, bioinformatics analysis, and a series of functional analyses confirmed that sanggenon C inhibited mitochondrial fission to induce apoptosis by blocking the extracellular regulated protein kinases (ERK) signaling pathway, and constitutive activation of ERK significantly abrogated these effects. Finally, sanggenon C was found to suppress the growth of tumor xenografts in nude mice without obvious side effects to the vital organs of animals. This study reveals that sanggenon C could be a novel therapeutic strategy for GC treatment.

Sphingosine 1-phosphate receptor 1 governs endothelial barrier function and angiogenesis by upregulating endoglin signaling

BackgroundThe sphingosine 1-phosphate (S1P)/S1P receptor (S1pr) 1 signaling plays an essential role in regulating vascular integrity and angiogenesis. We have previously shown that cell-surface expression of endoglin (Eng) is sustained by S1P/S1pr1 signaling in endothelial cells (ECs). However, whether S1pr1 mediates Eng signaling, or vice versa, remains unknown.MethodsS1pr1 inhibitors were used to study whether pharmacological inhibition induces basal vascular leakage in vivo. An acute respiratory distress syndrome (ARDS) model was used to study whether S1pr1 inhibition evoked greater inflammation in lungs. A S1pr1 inhibitor, a bone morphogenetic protein 9 (BMP9) blocking antibody, or lentivirus-mediated expression of soluble extracellular domain of Eng (sEng) were used to test whether blocking both S1P/S1pr1 and BMP9/Eng signaling axes would impose any interaction in retinal angiogenesis. To clarify whether S1P and BMP9 function in a linear pathway, a study of trans-endothelial electrical resistance (TEER) measurement was carried out using a mouse islet EC line MS1; time course studies were executed to exam downstream effectors of S1P and BMP9 signaling pathways in ECs; two stable MS1 cell lines were generated, one with overexpression of human S1PR1 and the other with knockdown of Eng, to validate S1pr1 and Eng were the key players for the crosstalk. Inhibitor of extracellular regulated protein kinases (ERK) was used to check whether this signaling was involved in S1P-induced cell-surface localization of Eng.ResultsThe present study elucidated that S1pr1 and Eng are both pivotal for angiogenesis in the postnatal mouse retina, and that the activation of S1pr1 or Eng increases vascular barrier function. Activation of S1pr1 enhanced the phosphorylation of Smad family members 1, 5, and 8 (pSmad1/5/8), while the inhibition of S1pr1 reduced the levels of pSma1/5/8 induced by BMP9 treatment. Activation or loss of Eng did not affect S1pr1 signaling. Moreover, activation of ERK was involved in promoting EC-surface expression of Eng by S1pr1.ConclusionsOur data demonstrates for the first time that there exists a linear pathway of S1pr1-Eng signaling axis in ECs, which governs vascular homeostasis. Functional BMP9/Eng signaling requires S1P/S1pr1 activation, and S1pr1 signaling acts as a vascular protection mechanism upstream of Eng.

GluN2A/ERK/CREB Signaling Pathway Involved in Electroacupuncture Regulating Hypothalamic-Pituitary-Adrenal Axis Hyperactivity.

The hyperactivity of the hypothalamic-pituitary-adrenal (HPA) axis caused by stress will inevitably disrupt the homeostasis of the neuroendocrine system and damage physiological functions. It has been demonstrated that electroacupuncture (EA) can modulate HPA axis hyperactivity during the perioperative period. As the initiating factor of the HPA axis, hypothalamic corticotrophin-releasing hormone (CRH) is the critical molecule affected by EA. However, the mechanism by which EA reduces CRH synthesis and secretion remains unclear. Activated N-methyl-D-aspartate receptor (NMDAR) has been linked to over-secretion of hypothalamic CRH induced by stress. To determine whether NMDAR is involved in EA regulating the over-expression of CRH, a surgical model of partial hepatectomy (HT) was established in our experiment. The effect of EA on hypothalamic NMDAR expression in HT mice was examined. Then, we investigated whether the extracellular regulated protein kinases (ERK)/cyclic adenosine monophosphate response element-binding protein (CREB) signaling pathway mediated by NMDAR was involved in EA regulating HPA axis hyperactivity. It was found that surgery enhanced the expression of hypothalamic CRH and caused HPA axis hyperactivity. Intriguingly, EA effectively suppressed the expression of CRH and decreased the activation of GluN2A (NMDAR subunit), ERK, and CREB in HT mice. GluN2A, ERK, and CREB antagonists had similar effects on normalizing the expression of CRH and HPA axis function compared with EA. Our findings suggested that surgery enhanced the activation of the hypothalamic GluN2A/ERK/CREB signaling pathway, thus promoting the synthesis and secretion of CRH. EA suppressed the phosphorylation of GluN2A, ERK, and CREB in mice that had undergone surgery, indicating that the GluN2A/ERK/CREB signaling pathway was involved in EA alleviating HPA axis hyperactivity.

Gold nanoparticles synergize with bacterial lipopolysaccharide to enhance class A scavenger receptor dependent particle uptake in neutrophils and augment neutrophil extracellular traps formation

Gold nanoparticles (AuNPs) are extensively utilized in biomedical fields. However, their potential interaction with host cells has not been comprehensively elucidated. In this study, we demonstrated a size-dependent effect of AuNPs to synergize with bacterial lipopolysaccharide (LPS) in promoting neutrophil extracellular traps (NETs) release in human peripheral neutrophils. Mechanistically, LPS was more efficient to contact with 10 nm AuNPs and promote their uptake in neutrophils compared to 40 and 100 nm AuNPs, leading to a synergistic upregulation of class A scavenger receptor (SRA) which mediated AuNPs uptake and triggered activation of extracellular regulated protein kinase (ERK) and p38. Blocking SRA or inhibiting ERK and p38 activation remarkably abrogated the effect of AuNPs and LPS to induce NETs formation. Further experiments demonstrated that AuNPs and LPS augmented the production of cytosolic reactive oxygen species (ROS) in p38 and ERK dependent manner, through upregulating and activating NADPH oxidase 2 (NOX2). Accordingly, scavenging of ROS or inhibiting the NOX2 dampened NETs release induced by combined AuNPs and LPS treatment. AuNPs and LPS also synergized to upregulate reactive oxygen species modulator 1 (ROMO1) via activating ERK, thereby increasing mitochondrial ROS generation and promoting the release of NETs. In summary, we provide new evidences about the synergy of AuNPs and LPS to augment cellular responses in neutrophils, which implicates the need to consider the amplifying effect by pathogenic stimuli when utilizing nanomaterials in infectious or inflammatory conditions.

Norcantharidin induces G2/M arrest and apoptosis via activation of ERK and JNK, but not p38 signaling in human renal cell carcinoma ACHN cells.

Renal cell carcinoma (RCC) is generally acknowledged as the most resistant primary malignancy unresponsive to conventional radiotherapy and chemotherapy treatments. Norcantharidin (NCTD), a therapeutic compound derived from medicinal plants, has been shown to trigger apoptosis, as well as antimetastatic and antioxidant activities in several tumor cells. However, NCTD's mechanism of antitumor activity in the RCC cell line remains unclear. In this study, we report that NCTD led to a time- and dose-dependent inhibition of cell proliferation. It had also markedly induced apoptosis and G2/M phase cell cycle arrest in a dose-dependent manner by decreasing the expressions of pro-caspase-3, pro-caspase-9, cyclin B1, and pCDC25C while increasing active caspase-3, cleaved-PARP, P21, and pCDC2 levels. Interestingly, NCTD treatment provoked the phosphorylation of extracellular-regulated protein kinase (ERK) and c-Jun-N-terminal kinase (JNK), but not of p38 MAPK. Moreover, SCH772984 and SP600125, ERK and JNK inhibitors, respectively, could partially abolish NCTD-induced apoptosis and G2/M phase cell cycle arrest. Collectively, these findings suggest that NCTD might activate JNK and ERK signaling pathways, consequently inducing apoptosis and G2/M arrest through the modulation of related proteins. This study provided evidence that NCTD is a promising therapeutic drug for the treatment of RCC.

SYPL1 Inhibits Apoptosis in Pancreatic Ductal Adenocarcinoma via Suppression of ROS-Induced ERK Activation.

Synaptophysin-like 1 (SYPL1) is a neuroendocrine-related protein. The role of SYPL1 in pancreatic ductal adenocarcinoma (PDAC) and the underlying molecular mechanism remain unclarified. Here, after analyzing five datasets (GSE15471, GSE16515, GSE28735, TCGA, and PACA-AU) and 78 PDAC patients from Sun Yat-sen University Cancer Center, we demonstrated that SYPL1 was upregulated in PDAC and that a high level of SYPL1 indicated poor prognosis. Bioinformatics analysis implied that SYPL1 was related to cell proliferation and cell death. To validate these findings, gain-of-function and loss-of-function experiments were carried out, and we found that SYPL1 promoted cell proliferation in vitro and in vivo and that it protected cells from apoptosis. Mechanistic studies revealed that sustained extracellular-regulated protein kinase (ERK) activation was responsible for the cell death resulting from knockdown of SYPL1. In addition, bioinformatics analysis showed that the expression of SYPL1 positively correlated with antioxidant activity. Reactive oxygen species (ROS) were upregulated in cells with SYPL1 knockdown and vice versa. Upregulated ROS led to ERK activation and cell death. These results suggest that SYPL1 plays a vital role in PDAC and promotes cancer cell survival by suppressing ROS-induced ERK activation.

DAXX mediates high phosphate-induced endothelial cell apoptosis in vitro through activating ERK signaling.

Backgroud and PurposeHyperphosphatemia, which is a high inorganic phosphate (Pi) level in the serum, promotes endothelial cells dysfunction and is associated with cardiovascular diseases in patients with chronic kidney diseases (CKD). However, the underlying mechanism of high Pi-induced endothelia cell apoptosis remains unclear.MethodsHuman umbilical vein endothelial cells (HUVECs) were treated with normal Pi (1.0 mM) and high Pi (3.0 mM), and then cell apoptosis, abnormal gene expression and potential signaling pathway involvement in simulated hyperphosphatemia were examined using flow cytometry, quantitative PCR (qPCR) and western blot analysis. A two-step 5/6 nephrectomy was carried out to induce CKD and biochemical measurements were taken.ResultsThe rat model of CKD revealed that hyperphosphatemia is correlated with an increased death-domain associated protein (DAXX) expression in endothelial cells. In vitro, high Pi increased the mRNA and protein expression level of DAXX in HUVECs, effects that were reversed by additional phosphonoformic acid treatment. Functionally, high Pi resulted in a significantly increased apoptosis in HUVECs, whereas DAXX knockdown markedly repressed high Pi-induced cell apoptosis, indicating that DAXX mediated high Pi-induced endothelial cell apoptosis. High Pi treatment and DAXX overexpression induced the activation of extracellular regulated protein kinases (ERKs), while DAXX knockdown inhibited high Pi-induced ERKs activation. Finally, we demonstrated that DAXX overexpression induced HUVECs apoptosis in the presence of normal Pi, whereas additional treatment with U0126 (a specific ERK inhibitor) reversed that effect.ConclusionUpregulated DAXX promoted high Pi-induced HUVECs apoptosis by activating ERK signaling and indicated that the DAXX/ERK signaling axis may be served as a potential target for CKD therapy.

Dual-specificity phosphatase 15 (DUSP15) in the nucleus accumbens is a novel negative regulator of morphine-associated contextual memory.

Drug relapse among addicts often occurs due to the learned association between drug-paired cues and the rewarding effects of these drugs, such as morphine. Contextual memory associated with morphine has a central role in maintenance and relapse. We showed that morphine-conditioned place preference (CPP) activates extracellular-regulated protein kinase (ERK) in the nucleus accumbens (NAc). The main enzymes that mediate ERK dephosphorylation are members of the dual-specificity phosphatase (DUSP) superfamily. It is unclear which members regulate the morphine CPP-induced activation of ERK. After screening, DUSP15 was found to be decreased during both morphine CPP expression and the reinstatement period. Intra-NAc infusions of AAV-DUSP15 (overexpression) not only prevented the expression of morphine-induced CPP but also facilitated extinction, inhibited reinstatement, and abolished ERK activation. However, after repeated morphine exposure and withdrawal in mice, there was no change in the expression of p-ERK and DUSP15, and the overexpression of DUSP15 in the NAc did not improve the impaired spatial memory or anxiety-like behaviour induced by morphine. Together, these findings indicate that DUSP15 not only prevents the expression of drug-paired contextual memory but also promotes the extinction of existing addiction memories, thus providing a novel therapeutic target for the treatment of drug addiction.

Inhaled gold nanoparticles cause cerebral edema and upregulate endothelial aquaporin 1 expression, involving caveolin 1 dependent repression of extracellular regulated protein kinase activity

BackgroundGold nanoparticles (Au-NPs) have extensive applications in electronics and biomedicine, resulting in increased exposure and prompting safety concerns for human health. After absorption, nanoparticles enter circulation and effect endothelial cells. We previously showed that exposure to Au-NPs (40–50 nm) collapsed endothelial tight junctions and increased their paracellular permeability. Inhaled nanoparticles have gained significant attention due to their biodistribution in the brain; however, little is known regarding their role in cerebral edema. The present study investigated the expression of aquaporin 1 (AQP1) in the cerebral endothelial cell line, bEnd.3, stimulated by Au-NPs.ResultsWe found that treatment with Au-NPs induced AQP1 expression and increased endothelial permeability to water. Au-NP exposure rapidly boosted the phosphorylation levels of focal adhesion kinase (FAK) and AKT, increased the accumulation of caveolin 1 (Cav1), and reduced the activity of extracellular regulated protein kinases (ERK). The inhibition of AKT (GDC-0068) or FAK (PF-573228) not only rescued ERK activity but also prevented AQP1 induction, whereas Au-NP-mediated Cav1 accumulation remained unaltered. Neither these signaling molecules nor AQP1 expression responded to Au-NPs while Cav1 was silenced. Inhibition of ERK activity (U0126) remarkably enhanced Cav1 and AQP1 expression in bEnd.3 cells. These data demonstrate that Au-NP-mediated AQP1 induction is Cav1 dependent, but requires the repression on ERK activity. Mice receiving intranasally administered Au-NPs displayed cerebral edema, significantly augmented AQP1 protein levels; furthermore, mild focal lesions were observed in the cerebral parenchyma.ConclusionsThese data suggest that the subacute exposure of nanoparticles might induce cerebral edema, involving the Cav1 dependent accumulation on endothelial AQP1.

Eclalbasaponin I causes mitophagy to repress oxidative stress-induced apoptosis via activation of p38 and ERK in SH-SY5Y cells

Oxidative stress accompanying excessive accumulation of reactive oxygen species (ROS) and mitochondrial dysfunction leads to the occurrence of neurodegenerative diseases. Our previous study showed that Eclalbasaponin I (EcI), a triterpene saponin isolated from Aralia elata (Miq.) Seem. (A. elata), repressed oxidative stress in human neuroblastoma SH-SY5Y cells. However, the detailed mechanism remains unclear. In this study, pretreatment with EcI in SH-SY5Y cells significantly activated the p38-mitogenactivated protein kinase (p38), the extracellular regulated protein kinase (ERK), whereas it did not affect the c-jun NH2 terminal kinases (JNK). In accordance with the initial findings, EcI-induced neuroprotective effect was attenuated by SB203580 (SB, a p38 inhibitor) or FR180204 (FR, an ERK inhibitor), being further confirmed by specific small interfering RNA (siRNA). Inhibition of either p38 or ERK up-regulated the apoptosis induction in EcI- and H2O2-cotreated cells. Furthermore, p38 or ERK suppression enhanced intracellular and mitochondrial ROS generation, decreased the activities of endogenous antioxidant defences as well as the mitochondrial membrane potential (MMP), resulting in dysfunction of mitochondria. In addition, EcI-induced autophagy and mitophagy were obviously down-regulated when p38 or ERK activation was blocked. Cumulatively, these findings supported that EcI-caused mitophagy contributed to the neuroprotective effect through p38 or ERK activation. Mitophagy induction might be an effective therapeutic intervention in neurodegenerative diseases.

Involvement of calcium in 50-Hz magnetic field-induced activation of sphingosine kinase 1 signaling pathway.

Previously, we found that exposure to a 50-Hz magnetic field (MF) could induce human amniotic epithelial (FL) cell proliferation and sphingosine kinase 1 (SK1) activation, but the mechanism was not clearly understood. In the present study, the possible signaling pathways which were involved in SK1 activation induced by 50-Hz MF exposure were investigated. Results showed that MF exposure increased intracellular Ca2+ which was dependent on the L-type calcium channel, and induced Ca2+ -dependent phosphorylation of extracellular regulated protein kinase (ERK), SK1, and protein kinase C α (PKCα). Also, treatment with U0126, an inhibitor of ERK, could block MF-induced SK1 phosphorylation, but had no effect on PKCα phosphorylation. Also, the inhibitor of PKCα, Gö6976, had no effect on MF-induced SK1 activation in FL cells. In addition, the activation of ERK and PKCα could be abolished by SKI II, the inhibitor of SK1. In conclusion, the intracellular Ca2+ mediated the 50-Hz MF-induced SK1 activation which enhanced PKCα phosphorylation, and there might be a feedback mechanism between SK1 and ERK activation in responding to MF exposure in FL cells. Bioelectromagnetics. 9999:XX-XX, 2019. © 2019 Bioelectromagnetics Society.

Telmisartan Attenuates Uric Acid-Induced Epithelial-Mesenchymal Transition in Renal Tubular Cells.

We examined whether and how uric acid induces epithelial to mesenchymal transition (EMT) in renal tubular cells, along with the mechanism by which telmisartan acts on uric acid-induced renal injury. Rat renal proximal tubular epithelial cells (NRK-52E) were exposed to various concentrations of uric acid in the presence or absence of telmisartan. Treatment with uric acid increased the expression of α-SMA, decreased the expression of E-cadherin, and promoted EMT in NRK-52E cells. Uric acid treatment also led to increased endothelin-1 (ET-1) production, activation of extracellular-regulated protein kinase 1/2 (ERK1/2), and the upregulation of nicotinamide adenine dinucleotide phosphate oxidase 4 (NOX4). Use of ET-1 receptor inhibitor (BQ123 or BQ788) could inhibit uric acid-induced EMT in NRK-52E cells. Pretreatment with the ERK inhibitor (U0126 or PD98059) suppressed the release of ET-1 and EMT induced by uric acid. Additionally, pretreatment with a traditional antioxidant (diphenylene iodonium or apocynin) inhibited the activation of ERK1/2, release of ET-1, and uric acid-induced EMT in NRK-52E cells. These findings suggested that uric acid-induced EMT in renal tubular epithelial cells occurs through NADPH oxidase-mediated ERK1/2 activation and the subsequent release of ET-1. Furthermore, telmisartan (102 nmol/L to 104 nmol/L) inhibited the expression of NOX4, intracellular reactive oxygen species (ROS), activation of ERK1/2, and the release of ET-1 in a dose-dependent manner, thereby preventing uric acid-induced EMT in NRK-52E. In conclusion, telmisartan could ameliorate uric acid-induced EMT in NRK-52E cells likely through inhibition of the NADPH oxidase/ERK1/2/ET-1 pathway.

Mechanical stresses induce paracrine β-2 microglobulin from cardiomyocytes to activate cardiac fibroblasts through epidermal growth factor receptor.

By employing a proteomic analysis on supernatant of mechanically stretched cardiomyocytes, we found that stretch induced a significantly high level of β-2 microglobulin (β2M), a non-glycosylated protein, which is related to inflammatory diseases but rarely known in cardiovascular diseases. The present data showed that serum β2M level was increased in patients with hypertension and further increased in patients with chronic heart failure (HF) as compared with control group, and the high level of serum β2M level correlated to cardiac dysfunction in these patients. In pressure overload mice model by transverse aortic constriction (TAC), β2M levels in serum and heart tissue increased progressively in a time-dependent manner. Exogenous β2M showed pro-fibrotic effects in cultured cardiac fibroblasts but few effects in cardiomyocytes. Adeno-associated virus 9 (AAV9)-mediated knockdown of β2M significantly reduced cardiac β2M level and inhibited myocardial fibrosis and cardiac dysfunction but not cardiac hypertrophy at 4 weeks after TAC. In vitro, mechanical stretch induced the rapid secretion of β2M mainly from cardiomyocytes by activation of extracellular-regulated protein kinase (ERK). Conditional medium (CM) from mechanically stretched cardiomyocytes activated cultured cardiac fibroblasts, and the effect was partly abolished by CM from β2M-knockdown cardiomyocytes. In vivo, knockdown of β2M inhibited the increase in phosphorylation of epidermal growth factor receptor (EGFR) induced by TAC. In cultured cardiac fibroblasts, inhibition of EGFR significantly attenuated the β2M-induced the activation of EGFR and pro-fibrotic responses. The present study suggests that β2M is a paracrine pro-fibrotic mediator and associated with cardiac dysfunction in response to pressure overload.

Low-intensity pulsed ultrasound in combination with SonoVue induces cytotoxicity of human renal glomerular endothelial cells via repression of the ERK1/2 signaling pathway

Objectives: Low-intensity pulsed ultrasound (LIPUS) and SonoVue have been used widely for diagnosis and therapeutic treatment. The effects of LIPUS and SonoVue on the microvascular system and underlying molecular mechanisms have not been established.Methods: Cultured human renal glomerular endothelial cells (HRGECs) were treated with 5-min ultrasonic irradiation, 20% SonoVue or the combination of both treatments. Cell proliferation, viablity, and apoptosis were measured by MTT assay, Trypan blue exclusion assay and flow cytometry, respectively. Activation of extracellular regulated protein kinases (ERK) were examined by Western blot.Results: We found that LIPUS and SonoVue alone do not induce cytotoxicity of HRGECs; however, the combination of the two treatments reduces cell proliferation and increases cell death. In addition, the combination of LIPUS and SonoVue suppressed the activation of ERK 1/2 in HRGRCs. With pretreatment of the inhibitor of ERK1/2 signaling, PD98059, LIPUS, and SonoVue does not induce additional cell death and inhibition of proliferation.Conclusions: LIPUS combined with SonoVue induces cytotoxicity of HRGECs via repression of the ERK1/2 signaling pathway.

Cystamine slows but not inverses the progression of monocrotaline-induced pulmonary arterial hypertension in rats.

Tissue transglutaminase (TG2) plays an important role in pulmonary arterial hypertension (PAH). Previous research indicate that TG2 and protein serotonylation catalyzed by TG2 are upregulated in PAH. Serotonin transporter inhibitor fluoxetine ameliorates PAH via inhibition of protein serotonylation. It is still unknown whether PAH is inhibited through direct inhibition of TG2. Therefore, the present study aimed to investigate the effects of TG2 inhibitor cystamine on monocrotaline-induced PAH in rats. Rats were treated with monocrotaline (60 mg·kg-1, i.p.) in combination with or without cystamine (20, 40 mg·kg-1·day-1, p.o.). The results showed that compared with monocrotaline alone, combination of monocrotaline with cystamine (40 mg·kg-1·day-1, p.o.) relieved right ventricle hypertrophy, inhibited pulmonary arteriolar remodeling, and downregulated protein expression of TG2, phosphorylated protein kinase B (Akt), and extracellular regulated protein kinase (ERK) at day 21. However, except for TG2 expression, these changes were not significantly inhibited by cystamine at day 35. In addition, cystamine dose-dependently enhanced the survival rate of rats injected with monocrotaline at day 35. The findings suggest that cystamine slows but not reverses monocrotaline-induced PAH in rats, which was largely associated with the inhibition of TG2 protein expression and Akt and ERK activation.

HMGB1-mediated autophagy attenuates gemcitabine-induced apoptosis in bladder cancer cells involving JNK and ERK activation

High-mobility group box 1 (HMGB1) has been found to mediate autophagy during chemotherapy in several cancers. However, whether HMGB1plays a role in autophagy and chemoresistance in bladder cancer is elusive. In this report, HMGB1 expression was found to be increased in 30 primary bladder cancer tissue specimens compared to their matched adjacent non-tumor tissues. While gemcitabine induced apoptotic cell death, it also induced HMGB1 expression and autophagy in bladder cancer T24 and BIU-87 cells. Suppressing HMGB1 expression with siRNA strongly potentiated gemcitabine-induced apoptosis. HMGB1 siRNA or autophagy inhibitors suppressed gemcitabine-induced autophagy. Further, gemcitabine activated c-Jun N-terminal kinase (JNK) and extracellular regulated protein kinase (ERK) and Bcl-2 phosphorylation, and blocking ERK and JNK inhibited autophagy and increased apoptosis in gemcitabine-treated cells. Interestingly, suppressing HMGB1 expression attenuated gemcitabine-induced ERK and JNK activation and Bcl-2 phosphorylation. Thus, our results suggest that while gemcitabine kills bladder cancer cells through apoptosis, a cytoprotective autophagy is also induced involving HMGB1-mediated JNK and ERK to counteract the cytotoxicity of gemcitabine, and intervention targeting this pathway may improve the anticancer efficacy of gemcitabine against bladder cancer.

Caffeic acid attenuated acetaminophen-induced hepatotoxicity by inhibiting ERK1/2-mediated early growth response-1 transcriptional activation

Caffeic acid (CA) is a natural compound abundant in fruits, coffee and plants. This study aims to investigate the involved mechanism of the therapeutic detoxification of CA against acetaminophen (APAP)-induced hepatotoxicity. CA (10, 30 mg/kg) was orally given to mice at 1 h after mice were pre-administrated with APAP (300 mg/kg). The therapeutic detoxification of CA against APAP-induced hepatotoxicity was observed by detecting serum aminotransferases, liver malondialdehyde (MDA) amount and liver histological evaluation in vivo. CA reduced APAP-induced increase in the mRNA expression of early growth response 1 (Egr1) in hepatocytes, and inhibited APAP-induced Egr1 transcriptional activation in vitro and in vivo. CA reduced the increased expression of growth arrest and DNA-damage-inducible protein (Gadd45)α induced by APAP in hepatocytes. Moreover, Egr1 siRNA reduced Gadd45α expression and reversed APAP-induced cytotoxicity in hepatocytes. Further results showed that CA blocked APAP-induced activation of extracellular-regulated protein kinase (ERK1/2) signaling cascade in vivo and in vitro. In addition, the application of ERK1/2 inhibitors (PD98059 and U0126) abrogated the nuclear translocation of Egr1 induced by APAP in hepatocytes. In conclusion, this study demonstrated the therapeutic detoxification of CA against APAP-induced liver injury, and the inhibition of CA on ERK1/2-mediated Egr1 transcriptional activation was involved in this process.

Induction of dissociated cytokine profiles by ISRAA with selective critical involvement of ERK1/2 in its signaling functions.

The immune system-released activating agent (ISRAA) is an immune mediator activated as a result of a nerve stimulus initiated by immune challenge. We have previously demonstrated that ISRAA and tumor necrosis factor (TNF) receptor 1 (TNFR1) share an interspecies-conserved motif (72% homology) that induces the apoptosis and proliferation of human peripheral blood mononuclear cells (hPBMCs) in a dose-dependent manner. In the present study, cytokine profiles were examined in response to the stimulation of hPBMCs with ISRAA. Furthermore, the signaling pathways induced by ISRAA were mapped. The results revealed high measurable levels of TNF-α, interleukin (IL)-6, IL-8, IL-10 and interferon (IFN)-γ, but not IL-4, IL-17 (IL-17A) or transforming growth factor (TGF)-β. The analysis of signaling pathways revealed the activation of extracellular-regulated protein kinase (ERK)1/2 as a downstream signal in the mitogen‑activated protein kinase (MAPK) pathway during TNF‑α and IL-6 production and apoptosis, but not during proliferation following stimulation with ISRAA by triggering the Fas-associated protein with death domain (FADD). STAT3 was found to be unphosphorylated in the ISRAA‑stimulated hPBMCs, and STAT3 was ubiquitously expressed in unstimulated cells, suggesting that ISRAA has a protein inhibitor of activated STAT (PIAS)-like activity, by functioning as a negative regulator of the effects of STAT3 on the Janus kinase (JAK)/STAT pathway. The determination of the nature of cytokine responses together with the signaling pathways of cellular activity induced by ISRAA paves the way for the investigation of a potential target of ISRAA and for the development of novel therapeutic approaches for the treatment of immune-regulated disorders.