Signal-regulated Kinase Mitogen-activated Protein Kinase Research Articles

Effect of apigenin on tryptophan metabolic key enzymes expression in lipopolysaccharide-induced microglial cells and its mechanism

[Aims] Flavonoid apigenin (API) has a wide range of biological functions, particularly anti-inflammation. Indoleamine 2,3-dioxygenase (IDO) and 2-Amino-3-carboxymuconate-6-semialdehyde decarboxylase (ACMSD) are important tryptophan metabolic enzymes that play pivotal roles in the production of toxic metabolite quinolinic acid. However, the relationship between inflammation and ACMSD remains unclear. The present study investigated the relationship between inflammation and tryptophan metabolic key enzymes. Similarly, the anti-inflammatory effect of API on important tryptophan metabolic enzymes was examined in lipopolysaccharide (LPS)-treated microglial cells.[Main methods] MG6 cells were exposed to LPS with or without API treatment for 24–48 h. IDO and ACMSD mRNA expression and production of inflammatory mediators were analyzed. Activation of inflammatory signaling pathways, such as mitogen-activated protein kinase (MAPK) and nuclear factor-κB (NF-κB), was also examined to study the mechanism of API in the inflammatory state.[Key findings] LPS suppressed ACMSD expression and enhanced IDO expression. However, API elevated ACMSD mRNA expression and suppressed IDO mRNA expression in LPS-treated MG6 cells. Furthermore, API suppressed interleukin-6 and nitric oxide production, whereas overproduction of inflammatory mediators enhanced IDO expression and assisted tryptophan degradation. API also inhibited activation of extracellular signal-regulated kinase (Erk) and jun N-terminal kinase (JNK) MAPK, and degradation of IκBα.[Significance] These results indicate alteration of ACMSD expression under inflammatory conditions. Moreover, API recovers expression of tryptophan metabolic key enzymes, which may be mediated by inhibition of proinflammatory mediator production via inactivation of Erk, JNK MAPK, and NF-κB pathways in LPS-stimulated microglial cells.

Comparative transcriptomic analysis reveals different host cell responses to Singapore grouper iridovirus and red-spotted grouper nervous necrosis virus

Singapore grouper iridovirus (SGIV) and red-spotted grouper nervous necrosis virus (RGNNV) are important pathogens that cause high mortality and heavy economic losses in grouper aquaculture. Interestingly, SGIV infection in grouper cells induces paraptosis-like cell death, while RGNNV infection induces autophagy and necrosis characterized morphologically by vacuolation of lysosome. Here, a comparative transcriptomic analysis was carried out to identify the different molecular events during SGIV and RGNNV infection in grouper spleen (EAGS) cells. The functional enrichment analysis of DEGs suggested that several signaling pathways were involved in CPE progression and host immune response against SGIV or RGNNV. Most of DEGs featured in the KEGG “lysosome pathway” were up-regulated in RGNNV-infected cells, indicating that RGNNV induced lysosomal vacuolization and autophagy might be due to the disturbance of lysosomal function. More than 100 DEGs in cytoskeleton pathway and mitogen-activated protein kinase (MAPK) signal pathway were identified during SGIV infection, providing additional evidence for the roles of cytoskeleton remodeling in cell rounding during CPE progression and MAPK signaling in SGIV induced cell death. Of note, consistent with changes at the transcriptional levels, the post-translational modifications of MAPK signaling-related proteins were also detected during RGNNV infection, and the inhibitors of extracellular signal-regulated kinase (ERK) and p38 MAPK significantly suppressed viral replication and virus induced vacuoles formation. Moreover, the majority of DEGs in interferon and inflammation signaling were obviously up-regulated during RGNNV infection, but down-regulated during SGIV infection, suggesting that SGIV and RGNNV differently manipulated host immune response in vitro. In addition, purine and pyrimidine metabolism pathways were also differently regulated in SGIV and RGNNV-infection cells. Taken together, our data will provide new insights into understanding the potential mechanisms underlying different host cell responses against fish DNA and RNA virus.

MKP3 Overexpression and ERK1/2 Phosphorylation in Dopaminergic Neurons Regulate DAT Trafficking and Function

Dopamine is a neurotransmitter that plays a critical role in movement, motivation, and reward. Dopamine neurotransmission is regulated by the dopamine transporter (DAT), which translocates dopamine from the extracellular space to the presynaptic neuron. DAT is a target for psychostimulants such as cocaine, giving DAT a key role in psychostimulant‐use disorders. DAT cell‐surface expression regulated by trafficking to and from the plasma membrane is an important mechanism for controlling DAT function. Two signaling pathways that contribute to DAT trafficking are the protein kinase C (PKC) and mitogen activated protein kinases (MAPKs), including the extracellular signal‐regulated kinase (ERK1/2) and p38 MAPK. ERK1/2 is inactivated by the MAP kinase phosphatase MKP3, which dephosphorylates and inactivates ERK1/2. Previous data from the lab demonstrated that activation of PKC by administration of phorbol 12‐myristate 13‐acetate (PMA) in an in vitro system results in decreased DAT cell‐surface expression, and overexpression of MKP3 attenuates this effect. Furthermore, other studies have shown that ERK1/2 activation, downstream of PKC activation, may lead to phosphorylation of DAT on residue threonine 53 (Thr53), however the significance of this phosphorylation is not completely understood. In order to characterize the contributions of MKP3 and ERK1/2 to DAT phosphorylation and trafficking in vivo, we have developed two viral constructs based on adeno‐associated viral (AAV) vectors that enable cre recombinase (cre)‐dependent expression. One vector cre‐dependently expresses MKP3. The other vector is based on a genetic tool named OptoSOS. This vector enables cre‐dependent, blue‐light induced activation of ERK1/2, and as a result, can spatiotemporally activate intracellular ERK1/2 signaling. Both viral vectors are employed with transgenic rats that express cre in tyrosine hydroxylase‐positive cells (TH‐Cre rats) to drive expression in dopaminergic neurons. From these studies, we find that MKP3 overexpression and resulting ERK1/2 inactivation increases DAT surface expression by blocking the internalization and degradation of the transporter leading to an accumulation of DAT on the cell‐surface. Interestingly, MKP3 overexpression also decreases phosphorylation at Thr53. While we have shown that ERK inactivation results in increased DAT cell‐surface expression, we have employed the OptoSOS system to activate ERK1/2 and assess resulting changes in DAT trafficking. We have confirmed that ERK1/2 is activated by blue‐light in a cre‐dependent manner, and similar to what we observe with MKP3 overexpression, ERK1/2 activation results in changes in DAT cell‐surface expression and phosphorylation. Taken together, our results indicate that MKP3 and ERK1/2 serve critical physiological roles in DAT regulation and can be utilized to investigate the mechanistic relationship between DAT trafficking, phosphorylation, and transporter activity.

Corticosterone potentiates ochratoxin A-induced microglial activation.

Microglial activation in the central nervous system (CNS) has been associated with brain damage and neurodegenerative disorders. Ochratoxin A (OTA) is a mycotoxin that occurs naturally in food and feed and has been associated with neurotoxicity, while corticosteroids are CNS' physiological function modulators. This study examined how OTA affected microglia activation and how corticosteroids influenced microglial neuroinflammation. Murine microglial cells (BV-2) were stimulated by OTA, and the potentiation effects on OTA-induced inflammation were determined by corticosterone pre-treatment. Expressions of pro-inflammatory mediators including tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β), interleukin-6 (IL-6), and inducible nitric oxide synthase (iNOS) were determined. Phosphorylation of mitogen-activated protein kinases (MAPKs) was analyzed by western blotting. OTA significantly increased the mRNA expression of IL-6, TNF-α, IL-1β, and iNOS and also elevated IL-6 and NO levels. Corticosterone pre-treatment enhanced the neuroinflammatory response to OTA in a mineralocorticoid receptor (MR)-dependent mechanism, which is associated with increases in extracellular signal-regulated kinase (ERK) and p38 MAPK activation. In response to OTA, microglial cells produced pro-inflammatory cytokines and NO, while corticosterone increased OTA-induced ERK and p38 MAPK phosphorylation via MR. Findings indicated the direct role of OTA in microglia activation and neuroinflammatory response and suggested that low corticosterone concentrations in the brain exacerbated neurodegeneration.

Regulation of microRNA-126 on the polarization of human macrophages stimulated by Porphyromonas gingivalis lipopolysaccharide

Objective: To study the effect of microRNA-126 (miR-126) on the polarization of human monocyte-derived macrophages stimulated by Porphyromonas gingivalis (Pg) lipopolysaccharide (LPS). Methods: Macrophages derived from human myeloid leukemia mononuclear cells were stimulated by Pg-LPS (5 mg/L) and by Pg-LPS (5 mg/L) after 24 h-transfection of miR-126 mimic or negative control RNA for 48 h, respectively. Real-time quantitative-PCR (qRT-PCR), enzyme-linked immunosorbent assay (ELISA) and Western blotting were conducted to detect the changes in miR-126, pro-inflammatory factor tumor necrosis factor-α (TNF-α), anti-inflammatory factors interleukin-10 (IL-10), inducible nitric oxide synthase (iNOS), arginase-1 (Arg-1) and M1 polarization-related pathways such as nuclear factor kappa-B (NF-κB) and mitogen-activated protein kinase (MAPK) signaling pathways. Results: Compared with non-LPS stimulation group (TNF-α: 1.000±0.020, iNOS: 1.125±0.064, miR-126: 1.004±0.113, IL-10: 1.003±0.053, Arg-1: 1.130±0.061), the mRNA levels of TNF-α (3.105±0.278) and iNOS (4.296±0.003) increased significantly (t=6.53, P=0.003; t=42.63, P<0.001, respectively), while miR-126, IL-10 and Arg-1 expressions (0.451±0.038, 0.545±0.004 and 0.253±0.017) decreased significantly (t=7.95, P=0.001; t=7.36, P=0.002; t=11.94, P<0.001, respectively) after Pg-LPS stimulated by human-derived macrophages for 48 h. The protein expression of iNOS, TNF-α, Arg-1 and IL-10 were consistent at mRNA levels. Meanwhile, the expressions of phospho-NF-κB p65 (p-p65), phospho-extracellular signal-regulated kinase (p-ERK) and phospho-p38 MAPK (p-p38) increased significantly, while the expression of Arg-1 decreased significantly. Compared with the negative controls (scramble RNA) (TNF-α: 1.141±0.197, iNOS: 1.173±0.115, IL-10: 1.032±0.138, Arg-1: 0.933±0.044), the mRNA levels of TNF-α (0.342±0.022) and iNOS (0.588±0.085) expressions significantly decreased (t=5.35, P=0.006; t=5.05, P=0.007), while IL-10 (1.786±0.221) and Arg-1 expressions (2.152±0.229) significantly increased (t=3.71, P=0.021; t=6.21, P=0.003) after Pg-LPS stimulation with miR-126 mimic transfection. The relative protein expressions of iNOS, p-p65, p-ERK and p-p38 significantly decreased (t=13.00, P<0.001; t=6.98, P=0.002; t=10.86, P<0.001; t=8.32, P=0.001), while the protein level of Arg-1 significantly increased (t=12.08, P<0.001). Conclusions: Pg-LPS could promote M1 polarization of macrophages. miR-126 might inhibit the effect of Pg-LPS on the M1 polarization of macrophages through down-regulating NF-κB and MAPK signaling pathways.

Tetracera loureiri Extract Regulates Lipopolysaccharide-Induced Inflammatory Response Via Nuclear Factor-κB and Mitogen Activated Protein Kinase Signaling Pathways.

Tetracera loureiri (T. loureiri) is a woody climber inhabiting open deciduous or evergreen forests in Southeast Asia. A decoction comprising its stem and other herbs is a traditional Thai remedy for fatigue and jaundice, as well as to promote overall health. Anti-inflammatory effects induced by T. loureiri extract have not been reported. In this study, we investigated the anti-inflammatory effect of an ethanol extract of T. loureiri (ETL) on lipopolysaccharide (LPS)-induced inflammatory response in RAW264.7 macrophages. We found that ETL treatment inhibited the production of nitric oxide (NO) in LPS-stimulated RAW264.7 cells, without affecting cell viability. The effect of ETL on the expression of various pro-inflammatory mediators was analyzed using reverse transcription-polymerase chain reaction (RT-PCR), Western blotting, and enzyme-linked immunosorbent assay (ELISA). We observed that ETL inhibited the expression of inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) at the mRNA and protein levels and decreased the production of prostaglandin E2 (PGE2) by COX-2 in RAW264.7 macrophages. ETL dose-dependently reduced the production of pro-inflammatory cytokines including tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β), and interleukin-6 (IL-6) in LPS-induced RAW264.7 cells, in a dose-dependent manner. Furthermore, ETL suppressed the LPS-induced nuclear translocation of the nuclear factor, NF-κB. Additionally, ETL was found to inhibit the activation of mitogen-activated protein kinases (MAPK), such as extracellular signal-regulated kinase, c-Jun-N-terminal kinase, and p38 MAPK. In conclusion, our findings demonstrate that ETL inhibits the expression of pro-inflammatory mediators and cytokines, thereby downregulating NF-κB and MAPK signaling pathways in LPS-stimulated macrophages, Consequently, ETL is a potential therapeutic agent for the treatment of inflammatory diseases.

Nell-1 attenuates lipopolysaccharide-induced inflammation in human dental pulp cells.

Nel-like molecule type 1 (Nell-1) is a secreted protein that plays an important role in osteoinduction in multiple animal models. A previous study has suggested the anti-inflammatory effect of Nell-1 on bone inflammation inhibition. However, its role in pulpitis has not been investigated. The present study aims to explore the effect of human recombinant Nell-1 (Nell-1) on rat pulp inflammation response, and its effect on lipopolysaccharide-induced inflammation in human dental pulp cells and its related intracellular signaling pathways. 30 Wistar rats with healthy non-carious maxillary first molars were chosen, Nell-1 was absorbed onto a sterile collagen sponge and capped onto exposed pulps. The expression of IL-6 and IL-8 were detected by immunohistochemical staining. Human dental pulp cells (hDPCs) were isolated from healthy extracted premolars and third molars. hDPCs were co-cultured with Escherichia coli lipopolysaccharide (LPS), Nell-1 protein, and mitogen-activated protein kinase (MAPK) inhibitors. The expression of pro-inflammatory cytokines and chemokines, such as IL-6 and IL-8, was examined via quantitative real-time PCR and enzyme-linked immunosorbent assay. The results showed that Nell-1 inhibited the inflammatory response of rat pulp. LPS treatment contributed to the expression of inflammatory factors in hDPCs, whereas Nell-1 obviously suppressed the LPS-induced inflammation. p38 MAPK and extracellular signal-regulated kinase (ERK) MAPK inhibitors attenuated the anti-inflammatory effect of hrNell-1, whereas the c-Jun N-terminal kinases (JNK) MAPK inhibitor exerted minimal effect. Therefore Nell-1 could inhibit LPS-induced inflammation in human dental pulp cells, and this effect may be mediated by p38 and ERK MAPK signaling pathways, but not JNK MAPK signaling pathway.

G-CSF reduces loss of dopaminergic neurons by inhibiting TNF-α and IL-1β in mouse model of Parkinson’s disease

Purpose of the study granulocyte-colony stimulating factor (G-CSF) is a hematopoietic growth factor existing in neutrophils, glial cells and neurons. Increasing researches discovered that G-CSF improved cell survival in neurodegenerative diseases by its anti-inflammatory effect. However, the effect of G-CSF in suppressing inflammation in Parkinson’s disease (PD) remains unclear. Thus, the purpose of this study is to explored the anti-inflammatory effect of G-CSF in mouse model of PD. Materials and methods G-CSF was administrated in the PD model induced by MPTP. Subsequently, the protein of tyrosine hydroxylase (TH), ionized calcium-binding adaptor molecule 1 (Iba-1) and the inflammatory cytokines including tumor necrosis factor-alpha (TNF-α) and interleukin-1 beta (IL-1β) in the midbrain were examined. In addition, the phosphorylated mitogen-activated protein kinases (MAPK) including c-Jun NH2-terminal kinase (JNK), extracellular signal-regulated kinase (ERK) and p38 MAPK in the midbrain were investigated. Results Compared with the MPTP group, the protein of TH in the midbrain was increased, while the Iba-1 and the inflammatory factors were decreased. In addition, the expression of phosphorylated JNK (p-JNK) in the midbrain of the MPTP + G-CSF group was decreased, while the phosphorylated ERK (p-ERK) levels were elevated. Conclusions These findings emphasize that G-CSF inhibited the degradation of DA neurons. The protective effect is associated with the reduction of the inflammatory factors caused by the inhibition of the microglial activation. Moreover, G-CSF may decrease the inflammatory factors through the decrease of P-JNK and the increase of P-ERK.

Garcinol Attenuates Lipoprotein(a)-Induced Oxidative Stress and Inflammatory Cytokine Production in Ventricular Cardiomyocyte through α7-Nicotinic Acetylcholine Receptor-Mediated Inhibition of the p38 MAPK and NF-κB Signaling Pathways.

Garcinol, a nicotinic acetylcholine receptor (nAChR) antagonist, has recently been established as an anti-inflammation agent. However, the molecular mechanism by which garcinol suppresses inflammation in the context of acute myocardial infarction (AMI) remains unclear. Hypothesis: We hypothesized that the administration of physiological doses of garcinol in mice with isoproterenol-induced AMI decreased the effect of lipoprotein(a) (Lp(a))-induced inflammation both in vivo and in vitro via the α7-nAChRs mediated p38 mitogen-activated protein kinase (MAPK)/nuclear factor kappa-light-chain-enhancer of activated B cells (NF-kB) signaling pathway. We analyzed altered reactive oxygen species (ROS) generation, the production of superoxide by mitochondria, cytokine expression patterns, and the role of the p38 MAPK/NF-κB signaling pathway after Lp(a)-stimulated human ventricular cardiomyocyte AC16 cells were treated with increasing doses of garcinol. C-reactive protein (CRP), interleukin (IL)-1β, IL-6, or tumor necrosis factor (TNF)-α production were detected by enzyme-linked immunosorbent assay. The Cell Counting Kit-8 assay was used to evaluate drug cytotoxicity. Western blots and confocal fluorescence microscopy were used to determine altered expression patterns of inflammatory biomarkers. We also examined whether the therapeutic effect of garcinol in AMI was mediated in part by α7-nAChR. Lp(a)-induced inflammatory cardiomyocytes had increased expression of membrane-bound α7-nAChRs in vitro and in vivo. Low-dose garcinol did not affect cardiomyocyte viability but significantly reduced mitochondrial ROS, CRP, IL-1β, IL-6, and TNF-α production in Lp(a)-stimulated cardiomyocytes (p < 0.05). The Lp(a)-induced phosphorylation of p38 MAPKs, CamKII, and NFκB, as well as NFκB-p65 nuclear translocation, was also suppressed (p < 0.05) by garcinol, while the inhibition of p38 MAPK by the inhibitor SB203580 decreased the phosphorylation of extracellular signal-regulated kinase (ERK) and p38 MAPK. Garcinol protected cardiomyocytes by inhibiting apoptosis and inflammation in mice with AMI. Furthermore, garcinol also enhanced the expression of microRNA-205 that suppressed the α7-nAChR-induced p38 MAPK/NF-κB signaling pathway. Garcinol suppresses Lp(a)-induced oxidative stress and inflammatory cytokines by α7-nAChR-mediated inhibition of p38 MAPK/NF-κB signaling in cardiomyocyte AC16 cells and isoproterenol-induced AMI mice.

Withaferin A exerts an anti-obesity effect by increasing energy expenditure through thermogenic gene expression in high-fat diet-fed obese mice

BackgroundThe enhancement of energy expenditure has attracted attention as a therapeutic target for the management of body weight. Withaferin A (WFA), a major constituent of Withania somnifera extract, has been reported to possess anti-obesity properties, however the underlying mechanism remains unknown. PurposeTo investigate whether WFA exerts anti-obesity effects via increased energy expenditure, and if so, to characterize the underlying pathway. MethodsC57BL/6 J mice were fed a high-fat diet (HFD) for 10 weeks, and WFA was orally administered for 7 days. The oxygen consumption rate of mice was measured at 9 weeks using an OxyletPro™ system. Hematoxylin and eosin (H&E), immunohistochemistry, immunoblotting, and real-time PCR methods were used. ResultsTreatment with WFA ameliorated HFD-induced obesity by increasing energy expenditure by improving of mitochondrial activity in brown adipose tissue (BAT) and promotion of subcutaneous white adipose tissue (scWAT) browning via increasing uncoupling protein 1 levels. WFA administration also significantly increased AMP-activated protein kinase (AMPK) phosphorylation in the BAT of obese mice. Additionally, WFA activated mitogen-activated protein kinase (MAPK) signaling, including p38/extracellular signal-regulated kinase MAPK, in both BAT and scWAT. ConclusionWFA enhances energy expenditure and ameliorates obesity via the induction of AMPK and activating p38/extracellular signal-regulated kinase MAPK, which triggers mitochondrial biogenesis and browning-related gene expression.

Resistance of osteosarcoma cells to the proapoptotic effects of carfilzomib involves activation of mitogen activated protein kinase pathways.

What is the central question of this study? Carfilzomib, a second-generation proteasome inhibitor approved for the treatment of multiple myeloma, shows efficacy against osteosarcoma. However, drug resistance remains a major challenge. What is the role of carfilzomib-induced changes in mitogen-activated protein kinase (MAPK) pathways in the sensitivity of osteosarcoma cells to the proapoptotic effects of the drug? What is the main finding and its importance? The dose-dependent antiapoptotic effects in osteosarcoma are associated with activation of MAPK signalling. Combinational targeting of MAPK signalling pathways can synergistically enhance carfilzomib-induced cell apoptosis, suggesting that MAPK inhibitors in combination with proteasome inhibitors can serve as a novel therapeutic tool for osteosarcoma. Osteosarcoma is the most common primary bone malignancy. Despite efforts to improve outcomes, the overall survival rates for osteosarcoma have remained unchanged over the past three decades. In this study, we assessed the proapoptotic effects of the second-generation proteasome inhibitor carfilzomib on osteosarcoma and investigated the potential mechanisms underlying the synergistic proapoptotic action when combined with mitogen-activated protein kinase (MAPK) inhibitors. We found that carfilzomib alone significantly inhibited cell proliferation and induced apoptosis in a dose-dependent manner, characterized by the induction of cleaved caspase 3 and poly (ADP-ribose) polymerase. More importantly, focusing on the changes of antiapoptotic B-cell lymphoma 2 (Bcl-2) family members and signalling pathways, we found a striking induction of myeloid cell leukaemia 1 (Mcl-1) and the activation of MAPK pathways. Furthermore, we observed that combinational targeting of the MAPK pathways using the specific inhibitors U0126, SP600125 or SB203580 synergistically enhanced carfilzomib-induced cell apoptosis. Notably, we found that the combinational inhibition of extracellular signal-regulated kinase or c-Jun N-terminal kinase MAPK pathways significantly decreased the expression of the three antiapoptotic Bcl-2 family proteins, and in particular this reversed induction of Mcl-1 by carfilzomib. Collectively, our findings show that activation of the MAPK pathways contributes to the mechanisms of drug resistance to carfilzomib. In addition, the synergistic proapoptotic action of MAPK and proteasome inhibitors in osteosarcoma cells suggests that combinational therapy with both drug types may serve as a novel strategy for the clinical management of osteosarcoma.

Role and mechanism of nonreceptor tyrosine kinase Tec in endotoxin/lipopolysaccharide-induced interleukin-8 production in human alveolar epithelial cells A549

Objective: To investigate the role and mechanism of nonreceptor tyrosine kinase Tec in the production of pro-inflammatory cytokine interleukin-8 (IL-8) induced by endotoxin/lipopolysaccharide (LPS) in human alveolar epithelial cells A549. Methods: Human alveolar epithelial cells A549 were routinely cultured and passaged in Roswell Park Memorial Institute-1640 medium containing 10% fetal bovine serum. The second or third passage of cells were collected for subsequent experiments. (1) Cells were collected and divided into 6 groups with 4 wells in each group according to the random number table. Cells in blank control group were routinely cultured for 2 h. Cells in simple LPS group were routinely cultured for 1 h and then stimulated by 1 μg/mL LPS for 1 h. Cells in simple LFM-A13 group were cultured with conventional culture medium adding 75 μmol/L LFM-A13 for 1 h and then cultured with replaced conventional culture medium for 1 h. Cells in 25 μmol/L LFM-A13+ LPS group, 75 μmol/L LFM-A13+ LPS group, and 100 μmol/L LFM-A13+ LPS group were cultured with conventional culture medium adding 25, 75, and 100 μmol/L LFM-A13 respectively for 1 h and then all stimulated by 1 μg/mL LPS added into the replaced conventional culture medium for 1 h. The protein expression of Tec in cells of each group was detected by Western blotting, and the content of IL-8 in cell culture supernatant of each group was determined by enzyme-linked immunosorbent assay. (2) Cells were collected and divided into 5 groups with 4 wells in each group according to the random number table. Cells in blank control group were routinely cultured for 2 h. Cells in small interfering RNA (siRNA) control+ LPS group were transfected with empty lentivirus for 10 h and then stimulated by 1 μg/mL LPS added into the conventional culture medium for 2 h. Cells in Tec mus-298 RNA interference (RNAi)+ LPS group, Tec mus-299 RNAi+ LPS group, and Tec mus-300 RNAi+ LPS group were transfected with lentivirus loaded with Tec mus-298 RNAi, Tec mus-299 RNAi, and Tec mus-300 RNAi respectively for 10 h and then stimulated by 1 μg/mL LPS added into the conventional culture medium for 2 h. The protein expression of Tec in cells of each group was detected by Western blotting to screen Tec-siRNA with the best silencing effect on Tec gene. (3) Cells were collected and divided into 4 groups with 4 wells in each group according to the random number table. Cells in blank control group were routinely cultured for 2 h. Cells in virus control group were transfected with empty lentivirus for 10 h and then routinely cultured for 2 h. Cells in simple LPS group were stimulated by 1 μg/mL LPS added into the conventional culture medium for 2 h. Cells in Tec-siRNA+ LPS group were transfected with lentivirus loaded with Tec-siRNA with the best silencing effect on Tec gene for 10 h and then stimulated by 1 μg/mL LPS added into the conventional culture medium for 2 h. The protein expressions of p38 mitogen-activated protein kinase (MAPK) and extracellular signal-regulated kinase (ERK) MAPK of cells in each group were detected by Western blotting. Data were processed with one-way analysis of variance and the least significant difference-t test. Results: (1) Compared with that of blank control group, the protein expression of Tec of cells in simple LPS group was obviously increased (t=9.72, P 0.05]. Compared with that of simple LPS group, the content of IL-8 in culture supernatant of cells in 25 μmol/L LFM-A13+ LPS group was not obviously changed [(204±38) pg/mL, t=0.54, P>0.05], but the content of IL-8 in culture supernatant of cells in 75 μmol/L LFM-A13+ LPS group and 100 μmol/L LFM-A13+ LPS group was obviously decreased [(144±44), (137±51) pg/mL, t=3.63, 2.55, P 0.05). The protein expression of Tec was the lowest in cells of Tec mus-298 RNAi+ LPS group, so Tec mus-298 RNAi was used in subsequent experiment. (3) Compared with 1.16±0.16 and 0.78±0.11 of blank control group, the protein expressions of p38 MAPK and ERK MAPK of cells in virus control group were not obviously changed (1.66±0.13, 0.89±0.11, t=11.09, 3.60, P>0.05), but the protein expressions of p38 MAPK and ERK MAPK of cells in simple LPS group were obviously increased (2.83±0.29, 1.86±0.37, t=9.70, 7.23, P<0.05). Compared with those of simple LPS group, the protein expression of p38 MAPK and protein expression of ERK MAPK of cells in Tec-siRNA+ LPS group were obviously decreased (0.69±0.16, 1.03±0.24, t=13.78, 4.12, P<0.05 or P<0.01). Conclusions: Tec may mediate the production and release of pro-inflammatory cytokine IL-8 from human alveolar epithelial cells A549 induced by LPS via the p38 MAPK and ERK MAPK signal pathways.

The ERK MAPK Pathway Is Essential for Skeletal Development and Homeostasis.

Mitogen-activated protein kinases (MAPKs) are a family of protein kinases that function as key signal transducers of a wide spectrum of extracellular stimuli, including growth factors and pro-inflammatory cytokines. Dysregulation of the extracellular signal-regulated kinase (ERK) MAPK pathway is associated with human skeletal abnormalities including Noonan syndrome, neurofibromatosis type 1, and cardiofaciocutaneous syndrome. Here, we demonstrate that ERK activation in osteoprogenitors is required for bone formation during skeletal development and homeostasis. Deletion of Mek1 and Mek2, kinases upstream of ERK MAPK, in osteoprogenitors (Mek1OsxMek2−/−), resulted in severe osteopenia and cleidocranial dysplasia (CCD), similar to that seen in humans and mice with impaired RUNX2 function. Additionally, tamoxifen-induced deletion of Mek1 and Mek2 in osteoprogenitors in adult mice (Mek1Osx-ERTMek2−/−) significantly reduced bone mass. Mechanistically, this corresponded to decreased activation of osteoblast master regulators, including RUNX2, ATF4, and β-catenin. Finally, we identified potential regulators of osteoblast differentiation in the ERK MAPK pathway using unbiased phospho-mass spectrometry. These observations demonstrate essential roles of ERK activation in osteogenesis and bone formation.

Induction of functional erythropoietin and erythropoietin receptor gene expression by gamma-aminobutyric acid and piperine in kidney epithelial cells

AimsThe aim of this study was to evaluate gamma-aminobutyric acid (GABA)- and piperine-induced erythropoietin (EPO) and EPO-receptor expression. Materials and methodsThe effect of GABA and piperine on cell viability was examined using kidney epithelial cells. Expression levels of EPO and EPO-R mRNA and protein were evaluated in response to GABA and piperine treatments. GABA- and piperine-mediated activation of the mitogen-activated protein kinase (MAPK) signaling pathway was investigated. Additionally, EPO function was evaluated using conditioned media containing EPO. The GABA receptor type involved in this process was identified. Key findingsMessenger RNA and protein expression levels of EPO and EPO-R significantly increased in response to treatment with GABA, piperine, or the combination of both, compared with control. GABA plus piperine synergistically enhanced EPO and EPO-R expression through p38 and c-Jun N-terminal kinase (JNK) MAPK signaling pathways, but not through the extracellular signal-regulated kinase (ERK) MAPK pathway. SB203580 and SP600125 (p38 and JNK pathway inhibitors, respectively) attenuated GABA plus piperine-induced EPO and EPO-R expression. Treatment of macrophages with EPO-containing conditioned media induced mRNA expression of interleukin (IL)-10 and nuclear factor (NF)-κB due to the interaction between EPO and EPO-R. Interestingly, GABA-induced EPO and EPO-R expression was mediated through GABAA, not GABAB, receptor activation. SignificanceThese findings demonstrate that GABA plus piperine-mediated p38 and JNK MAPK activation increases EPO and EPO-R expression, resulting in up-regulation of IL-10 and NF-κB.

Extracellular Hsp70 modulates the inflammatory response of cigarette smoke extract in NCI-H292 cells.

What is the central question of this study? Does extracellular heat shock protein 70 (eHsp70) alter cigarette smoke extract (CSE)-induced inflammatory responses in NCI-H292 bronchial epithelial cells? What is the main finding and its importance? eHsp70 modulates inflammatory responses and TLR2, TLR4 and Hsp70 gene expression, and protects NCI-H292 cells against CSE-induced cytotoxicity. eHsp70 might be implicated in development of inflammatory diseases affected by cigarette smoke, such as COPD. One of the major risk factors for development of chronic obstructive pulmonary disease (COPD) is cigarette smoke. Extracellular Hsp70 (eHsp70) is increased in sera of COPD patients, and can act as damage-associated molecular pattern (DAMP). In this study, we explored inflammatory parameters (cytokine concentrations, Toll-like receptor (TLR) 2 and 4 and Hsp70 expression, mitogen-activated protein kinases (MAPKs) and nuclear factor κB (NF-κB) activation, and cytotoxicity) after exposure of bronchial-epithelial NCI-H292 cells to cigarette smoke extract (CSE) alone (2.5 and 15%) and in combinations with recombinant human (rh) Hsp70 (0.3, 1 and 3μgml-1 ). We applied specific MAPKs, NF-κB and Hsp70 inhibitors to elucidate rhHsp70 inflammation-associated responses. CSE alone and combinations of 15% CSE with rhHsp70 stimulated IL-1α, IL-6 and IL-8 release. However, rhHsp70 applied with 2.5% CSE decreased secretion of cytokines indicating antagonistic effects. Individual and combined treatments with 2.5% CSE suppressed TLR2 expression. CSE at 15% induced TLR2 and TLR4 gene expression, whereas rhHsp70 abolished that effect. rhHsp70 and 15% CSE alone reduced, while their combination increased, intracellular Hsp70 mRNA level. CSE alone and in combination with rhHsp70 activated extracellular signal-regulated kinase and p38 MAPKs, while inhibition of MAPKs, NF-κB and Hsp70 attenuated IL-6 and IL-8 secretion. CSE at 15% reduced cell viability and induced apoptosis, as shown by MTS and caspases-3/7 assays. CSE at 2.5% alone stimulated lactate dehydrogenase release, but cellular membrane integrity remained intact in co-treatments with rhHsp70. rhHsp70 might modulate the inflammatory response of CSE and could also protect NCI-H292 cells against CSE cytotoxicity. Those effects are implemented via MAPK and NF-κB signalling pathways.

Increase in soluble protein oligomers triggers the innate immune system promoting inflammation and vascular dysfunction in the pathogenesis of sepsis

Sepsis is a profoundly morbid and life-threatening condition, and an increasingly alarming burden on modern healthcare economies. Patients with septic shock exhibit persistent hypotension despite adequate volume resuscitation requiring pharmacological vasoconstrictors, but the molecular mechanisms of this phenomenon remain unclear. The accumulation of misfolded proteins is linked to numerous diseases, and it has been observed that soluble oligomeric protein intermediates are the primary cytotoxic species in these conditions. Oligomeric protein assemblies have been shown to bind and activate a variety of pattern recognition receptors (PRRs) including formyl peptide receptor (FPR). While inhibition of endoplasmic reticulum (ER) stress and stabilization of protein homeostasis have been promising lines of inquiry regarding sepsis therapy, little attention has been given to the potential effects that the accumulation of misfolded proteins may have in driving sepsis pathogenesis. Here we propose that in sepsis, there is an accumulation of toxic misfolded proteins in the form of soluble protein oligomers (SPOs) that contribute to the inflammation and vascular dysfunction observed in sepsis via the activation of one or more PRRs including FPR. Our laboratory has shown increased levels of SPOs in the heart and intrarenal arteries of septic mice. We have also observed that exposure of resistance arteries and vascular smooth muscle cells to SPOs is associated with increased mitogen-activated protein kinase (MAPK) signaling including phosphorylated extracellular signal-regulated kinase (p-ERK) and p-P38 MAPK pathways, and that this response is abolished with the knockout of FPR. This hypothesis has promising clinical implications as it proposes a novel mechanism that can be exploited as a therapeutic target in sepsis.

Cafestol Inhibits Cyclic-Strain-Induced Interleukin-8, Intercellular Adhesion Molecule-1, and Monocyte Chemoattractant Protein-1 Production in Vascular Endothelial Cells.

Moderate coffee consumption is inversely associated with cardiovascular disease mortality; however, mechanisms underlying this causal effect remain unclear. Cafestol, a diterpene found in coffee, has various properties, including an anti-inflammatory property. This study investigated the effect of cafestol on cyclic-strain-induced inflammatory molecule secretion in vascular endothelial cells. Cells were cultured under static or cyclic strain conditions, and the secretion of inflammatory molecules was determined using enzyme-linked immunosorbent assay. The effects of cafestol on mitogen-activated protein kinases (MAPK), heme oxygenase-1 (HO-1), and sirtuin 1 (Sirt1) signaling pathways were examined using Western blotting and specific inhibitors. Cafestol attenuated cyclic-strain-stimulated intercellular adhesion molecule-1 (ICAM-1), monocyte chemoattractant protein- (MCP-) 1, and interleukin- (IL-) 8 secretion. Cafestol inhibited the cyclic-strain-induced phosphorylation of extracellular signal-regulated kinase and p38 MAPK. By contrast, cafestol upregulated cyclic-strain-induced HO-1 and Sirt1 expression. The addition of zinc protoporphyrin IX, sirtinol, or Sirt1 silencing (transfected with Sirt1 siRNA) significantly attenuated cafestol-mediated modulatory effects on cyclic-strain-stimulated ICAM-1, MCP-1, and IL-8 secretion. This is the first study to report that cafestol inhibited cyclic-strain-induced inflammatory molecule secretion, possibly through the activation of HO-1 and Sirt1 in endothelial cells. The results provide valuable insights into molecular pathways that may contribute to the effects of cafestol.

Spatial and temporal signal processing and decision making by MAPK pathways.

Mitogen-activated protein kinase (MAPK) pathways are conserved from yeast to man and regulate a variety of cellular processes, including proliferation and differentiation. Recent developments show how MAPK pathways perform exquisite spatial and temporal signal processing and underscores the importance of studying the dynamics of signaling pathways to understand their physiological response. The importance of dynamic mechanisms that process input signals into graded downstream responses has been demonstrated in the pheromone-induced and osmotic stress-induced MAPK pathways in yeast and in the mammalian extracellular signal-regulated kinase MAPK pathway. Particularly, recent studies in the yeast pheromone response have shown how positive feedback generates switches, negative feedback enables gradient detection, and coherent feedforward regulation underlies cellular memory. More generally, a new wave of quantitative single-cell studies has begun to elucidate how signaling dynamics determine cell physiology and represents a paradigm shift from descriptive to predictive biology.

Neuroprotective effects of erythropoietin against hypoxic injury via modulation of the mitogen-activated protein kinase pathway and apoptosis.

PurposeHypoxic-ischemic encephalopathy is a significant cause of neonatal morbidity and mortality. Erythropoietin (EPO) is emerging as a therapeutic candidate for neuroprotection. Therefore, this study was designed to determine the neuroprotective role of recombinant human EPO (rHuEPO) and the possible mechanisms by which mitogen-activated protein kinase (MAPK) signaling pathway including extracellular signal-regulated kinase (ERK1/2), JNK, and p38 MAPK is modulated in cultured cortical neuronal cells and astrocytes.MethodsPrimary neuronal cells and astrocytes were prepared from cortices of ICR mouse embryos and divided into the normoxic, hypoxia (H), and hypoxia-pretreated with EPO (H+EPO) groups. The phosphorylation of MAPK pathway was quantified using western blot, and the apoptosis was assessed by caspase-3 measurement and terminal deoxynucleotidyl transferase dUTP nick end labeling assay.ResultsAll MAPK pathway signals were activated by hypoxia in the neuronal cells and astrocytes (P<0.05). In the neuronal cells, phosphorylation of ERK-1/-2 and apoptosis were significantly decreased in the H+EPO group at 15 hours after hypoxia (P<0.05). In the astrocytes, phosphorylation of ERK-1/-2, p38 MAPK, and apoptosis was reduced in the H+EPO group at 15 hours after hypoxia (P<0.05).ConclusionPretreatment with rHuEPO exerts neuroprotective effects against hypoxic injury reducing apoptosis by caspase-dependent mechanisms. Pathologic, persistent ERK activation after hypoxic injury may be attenuateed by pretreatment with EPO supporting that EPO may regulate apoptosis by affecting ERK pathways.

Promotion of metastasis of thyroid cancer cells via NRP-2-mediated induction.

Tumor-node-metastasis is one of the leading causes of morbidity and mortality in thyroid cancer patients. Upregulation of vascular endothelial growth factor-C (VEGF-C) increases the migratory ability of thyroid cancer cells to lymph nodes. Expression of neuropilin-2 (NRP-2), the co-receptor of VEGF-C, has been reported to be correlated with lymph node metastasis in human thyroid cancer. The present study investigated the role of VEGF-C/NRP-2 signaling in the regulation of metastasis of two different types of human thyroid cancer cells. The results indicated that the VEGF-C/NRP-2 axis significantly promoted the metastatic activities of papillary thyroid carcinoma cells through the activation of the mitogen-activated protein kinase (MAPK) kinase (MEK)/extracellular signal-regulated kinase and p38 MAPK signaling cascades. However, neither MEK or p38 MAPK inhibitors produced significant inhibition of the migratory activity and invasiveness regulated by the VEGF-C/NRP-2 axis in follicular thyroid carcinoma cells. Finally, VEGF-C/NRP-2-mediated invasion and migration of thyroid cancer cells required the expression of NRP-2. The present results demonstrate that the promotion of metastasis by VEGF-C is mainly due to the upregulation of NRP-2 in thyroid cancer cells, and this metastatic activity regulated by the VEGF-C/NRP-2 axis provides further insight into the process of tumor metastasis.