Kisspeptin induces Kiss-1 and GnRH gene expression in mHypoA-55 hypothalamic cell models: Involvement of the ERK and PKA signaling pathways.

mHypoA-55 cells are kisspeptin-expressing neuronal cells originating from the arcuate nucleus of the mouse hypothalamus. These cells are called KNDy neurons because they co-express kisspeptin, neurokinin B, and dynorphin A. In addition, they express gonadotropin-releasing hormone (GnRH). Here, we found that kisspeptin 10 (KP10) increased Kiss-1 (encoding kisspeptin) and GnRH gene expression in kisspeptin receptor (Kiss-1R)-overexpressing mHypoA-55 cells. KP10 greatly increased serum response element (SRE) promoter activity, which is a target of extracellular signal-regulated kinase (ERK) (20.0 ± 2.54-fold). KP10 also increased cAMP-response element (CRE) promoter activity in these cells (2.32 ± 0.36-fold). KP10-increased SRE promoter activity was significantly prevented in the presence of PD098095, a MEK kinase (MEKK) inhibitor, and KP10-induced CRE promoter activity was also inhibited by PD098059. Similarly, H89, a protein kinase A (PKA) inhibitor, significantly inhibited the KP10 induction of SRE and CRE promoters. KP10-induced Kiss-1 and GnRH gene expressions were inhibited in the presence of PD098059. Likewise, H89 significantly inhibited the KP10-induced increase in Kiss-1 and GnRH. Transfection of mHypoA-55 cells with constitutively active MEKK (pFC-MEKK) increased SRE and CRE promoter activities by 9.75 ± 1.77- and 1.36 ± 0.12-fold, respectively. Induction of constitutively active PKA (pFC-PKA) also increased SRE and CRE promoter activities by 2.41 ± 0.42- and 40.71 ± 7.77-fold, respectively. Furthermore, pFC-MEKK and -PKA transfection of mHypoA-55 cells increased both Kiss-1 and GnRH gene expression. Our current observations suggest that KP10 increases both the ERK and PKA pathways and that both pathways mutually interact in mHypoA-55 hypothalamic cells. Activation of both ERK and PKA signaling might be necessary to induce Kiss-1 and GnRH gene expressions.

Salidroside ameliorates pathological cardiac hypertrophy via TLR4-TAK1-dependent signaling.

Salidroside, a prominent active ingredient in traditional Chinese medicines, is garnering increased attention because of its unique pharmacological effects against ischemic heart disease via MAPK signaling, which plays a critical role in regulating the evolution of ventricular hypertrophy. However, the function of Salidroside on myocardial hypertrophy has not yet been elucidated. C57BL/6 mice were subjected to transverse aortic constriction (TAC), and treated with Salidroside (100 mg kg-1  day-1 ) by oral gavage for 3 weeks starting 1 week after surgery. Four weeks after TAC surgery, the mice were subjected to echocardiography and then sacrificed to harvest the hearts for analysis. For in vitro study, neonatal rat cardiomyocytes were used to validate the protective effects of Salidroside in response to Angiotensin II (Ang II, 1 µM) stimulation. Here, we proved that Salidroside dramatically inhibited hypertrophic reactions generated by pressure overload and isoproterenol (ISO) injection. Salidroside prevented the activation of the TAK1-JNK/p38 axis. Salidroside pretreatment of TAK1-inhibited cardiomyocytes shows no additional attenuation of Ang II-induced cardiomyocytes hypertrophy and signaling pathway activation. The overexpression of constitutively active TAK1 removed the protective effects of Salidroside on myocardial hypertrophy. TAC-induced increase of TLR4 protein expression was reduced considerably in the Salidroside treated mice. Transient transfection of small interfering RNA targeting TLR4 (siTLR4) in cardiomyocytes did not further decrease the activation of the TAK1/JNK-p38 axis. In conclusion, Salidroside functioned as a TLR4 inhibitor and displayed anti-hypertrophic action via the TAK1/JNK-p38 pathway.

In-silico genome wide analysis of Mitogen activated protein kinase kinase kinase gene family in C. sinensis.

Mitogen activated protein kinase kinase kinase (MAPKKK) form the upstream component of MAPK cascade. It is well characterized in several plants such as Arabidopsis and rice however the knowledge about MAPKKKs in tea plant is largely unknown. In the present study, MAPKKK genes of tea were obtained through a genome wide search using Arabidopsis thaliana as the reference genome. Among 59 candidate MAPKKK genes in tea, 17 genes were MEKK-like, 31 genes were Raf-like and 11 genes were ZIK- like. Additionally, phylogenetic relationships were established along with structural analysis, which includes gene structure, its location as well as conserved motifs, cis-acting regulatory elements and functional domain signatures that were systematically examined. Also, on the basis of one orthologous gene found between tea and Arabidopsis, functional interaction was carried out in C. sinensis based on an Arabidopsis association model. The expressional profiles indicated major involvement of MAPKKK genes from tea in response to various abiotic stress factors. Taken together, this study provides the targets for additional inclusive identification, functional study, and provides comprehensive knowledge for a better understanding of the MAPKKK cascade regulatory network in C. sinensis.

Cissus subtetragona Planch. Ameliorates Inflammatory Responses in LPS-induced Macrophages, HCl/EtOH-induced Gastritis, and LPS-induced Lung Injury via Attenuation of Src and TAK1.

Several Cissus species have been used and reported to possess medicinal benefits. However, the anti-inflammatory mechanisms of Cissus subtetragona have not been described. In this study, we examined the potential anti-inflammatory effects of C. subtetragona ethanol extract (Cs-EE) in vitro and in vivo, and investigated its molecular mechanism as well as its flavonoid content. Lipopolysaccharide (LPS)-induced macrophage-like RAW264.7 cells and primary macrophages as well as LPS-induced acute lung injury (ALI) and HCl/EtOH-induced acute gastritis mouse models were utilized. Luciferase assays, immunoblotting analyses, overexpression strategies, and cellular thermal shift assay (CETSA) were performed to identify the molecular mechanisms and targets of Cs-EE. Cs-EE concentration-dependently reduced the secretion of NO and PGE2, inhibited the expression of inflammation-related cytokines in LPS-induced RAW264.7 cells, and decreased NF-κB- and AP-1-luciferase activity. Subsequently, we determined that Cs-EE decreased the phosphorylation events of NF-κB and AP-1 pathways. Cs-EE treatment also significantly ameliorated the inflammatory symptoms of HCl/EtOH-induced acute gastritis and LPS-induced ALI mouse models. Overexpression of HA-Src and HA-TAK1 along with CETSA experiments validated that inhibited inflammatory responses are the outcome of attenuation of Src and TAK1 activation. Taken together, these findings suggest that Cs-EE could be utilized as an anti-inflammatory remedy especially targeting against gastritis and acute lung injury by attenuating the activities of Src and TAK1.

TAK1 in the AP-1 pathway is a critical target of Saururus chinensis (Lour.) Baill in its anti-inflammatory action.

ETHNOPHARMACOLOGICAL RELEVANCE: Saururus chinensis (Lour.) Baill (Saururaceae), also known as Asian lizard's tail, is a plant commonly found in East Asia. Its leaves have been used in traditional medicine to treat many diseases such as edema, pneumonia, hypertension, leproma, jaundice, gonorrhea, and rheumatoid arthritis. AIM OF THE STUDY: Based on the efficacies of S. chinensis, the anti-inflammatory effects of this plant and the molecular mechanism were evaluated using the ethanol extract of S. chinensis leaves (Sc-EE). MATERIALS AND METHODS: The production of pro-inflammatory mediators and cytokines in response to Sc-EE was evaluated using Griess and semi-quantitative reverse transcription-polymerase chain reactions. Furthermore, relevant proteins including c-Jun, c-Fos, p38, JNK, ERK, MEK1/2, MKK3/6, MKK4/7, and TAK1 were detected through immunoblotting. RESULTS: Sc-EE diminished production of nitric oxide (NO); decreased expression levels of cyclooxygenase (COX)-2, interleukin (IL)-6, inducible NO synthase (iNOS), and IL-1ß in LPS-stimulated RAW264.7 cells; and attenuated activator protein 1 (AP-1)-mediated luciferase activities. The extract markedly downregulated the phosphorylation of TAK1, upregulated thermal stability of TAK1, and reduced TAK1/AP-1-mediated luciferase activity in LPS-treated RAW264.7 cells and TAK1-overexpressing HEK293T cells. CONCLUSIONS: These results demonstrated that Sc-EE suppresses pro-inflammatory gene expression through blockade of the TAK1/AP-1 pathway in LPS-treated RAW264.7 macrophages, implying that inhibition of TAK1/AP-1 signaling by S. chinensis is a key event in its anti-inflammatory activity.

Xinyang Tablet inhibits MLK3-mediated pyroptosis to attenuate inflammation and cardiac dysfunction in pressure overload.

ETHNOPHARMACOLOGICAL RELEVANCE: Xinyang tablet (XYT) has been traditionally used in the treatment of cardiovascular diseases (CVDs). Our previous study indicated that XYT exhibited protective effects in heart failure (HF). AIM OF THE STUDY: The aim of the present study was to determine the protective effects of XYT in pressure overload induced HF and to elucidate its underlying mechanisms of action. MATERIALS AND METHODS: We analyzed XYT content using high-performance liquid chromatography (HPLC.). Mice were subjected to transverse aortic constriction (TAC) to generate pressure overload-induced cardiac remodeling and were then orally administered XYT or URMC-099 for 1 week after the operation. HL1 mouse cardiomyoblasts were induced by lipopolysaccharides (LPS) to trigger pyroptosis and were then treated with XYT or URMC-099. We used echocardiography (ECG), hematoxylin and eosin (H&E) staining, Masson's trichrome staining and a terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling (TUNEL) assay to evaluate the effects of XYT. Messenger ribonucleic acid (mRNA) levels of collagen metabolism biomarkers and inflammation-related factors were detected. We determined protein levels of inflammation- and pyroptosis-related signaling pathway members via Western blot (WB). Caspase-1 activity was measured in cell lysate using a Caspase-1 Activity Assay Kit. Subsequently, to define the candidate ingredients in XYT that regulate mixed-lineage kinase-3 (MLK3), we used molecular docking (MD) to predict and evaluate binding affinity with MLK3. Finally, we screened 24 active potential compounds that regulate MLK3 via MD. RESULTS: ECG, H&E staining, Masson's trichrome staining and TUNEL assay results showed that XYT remarkably improved heart function, amelorated myocardial fibrosis and inhibited apoptosis in vivo. Moreover, it reduced expression of proteins or mRNAs related to collagen metabolism, including collagen type 1 (COL1), fibronectin (FN), alpha smooth-muscle actin (α-SMA), and matrix metalloproteinases-2 and -9 (MMP-2, MMP-9). XYT also inhibited inflammation and the induction of pyroptosis at an early stage, as well as attenuated inflammation and pyroptosis levels in vitro. CONCLUSION: Our data indicated that XYT exerted protective effects against pressure overload induced myocardial fibrosis (MF), which might be associated with the induction of pyroptosis-mediated MLK3 signaling.

Licochalcone A inhibits proliferation and promotes apoptosis of colon cancer cell by targeting programmed cell death-ligand 1 via the NF-κB and Ras/Raf/MEK pathways.

ETHNOPHARMACOLOGICAL RELEVANCE: Glycyrrhiza glabra L., a traditional medicinal, has a history of thousands of years. It is widely used in clinic and has been listed in Chinese Pharmacopoeia. Licochalcone A is a phenolic chalcone compound and a characteristic chalcone of Glycyrrhiza glabra L. It has many pharmacological activities, such as anti-cancer, anti-inflammatory, anti-viral and anti-angiogenic activities. AIM OF THE STUDY: In this study, we explored the anti-tumor activity and potential mechanism of licochalcone A in vitro and in vivo. MATERIALS AND METHODS: In vitro, the mechanism of licochalcone A at inhibiting PD-L1 expression was investigated by molecular docking, western blotting, RT-PCR, flow cytometry, immunofluorescence and immunoprecipitation assays. The co-culture model of T cells and tumor cells was used to detect the activity of cytotoxic T lymphocytes. Colony formation, EdU labelling and apoptosis assays were used to detect changes in cellular proliferation and apoptosis. In vivo, anti-tumor activity of licochalcone A was assessed in a xenograft model of HCT116 cells. RESULTS: In the present study, we found that licochalcone A suppressed the expression of programmed cell death ligand-1 (PD-L1), which plays a key role in regulating the immune response. In addition, licochalcone A inhibited the expressions of p65 and Ras. Immunoprecipitation experiment showed that licochalcone A suppressed the expression of PD-L1 by blocking the interaction between p65 and Ras. In the co-culture model of T cells and tumor cells, licochalcone A pretreatment enhanced the activity of cytotoxic T lymphocytes and restored the ability to kill tumor cells. In addition, we showed that licochalcone A inhibited cell proliferation and promoted cell apoptosis by targeting PD-L1. In vivo xenograft assay confirmed that licochalcone A inhibited the growth of tumor xenografts. CONCLUSION: In general, these results reveal the previously unknown properties of licochalcone A and provide new insights into the anticancer mechanism of this compound.

Inhibition of Inflammatory Responses by Centella asiatica via Suppression of IRAK1-TAK1 in Mouse Macrophages.

Centella asiatica (L.) Urb. (C. asiatica) has been widely treated for inflammation-related diseases in China for thousands of years. While C. asiatica showed relevant effects as traditional medicine, the mechanism of C. asiatica suppressing inflammation has not been thoroughly investigated. Therefore, this study was conducted to reveal the anti-inflammatory mechanism of methanol fraction from C. asiatica (MCA) at the molecular level in murine macrophages. Levels of inflammation-related mediators were observed with treatment of MCA. MCA significantly suppressed nitric oxide production and iNOS expression in RAW 264.7 macrophages. Prostaglandin E2 production was alleviated by MCA via the downregulation of cyclooxygenase-2. MCA treatment also reduced pro-inflammatory tumor necrosis factor-[Formula: see text] and interleukin (IL)-6 levels. LPS/D-GalN-induced acute hepatitis in mouse was alleviated by MCA treatment. In addition, MCA decreased the phosphorylation of inhibitory [Formula: see text]B[Formula: see text] (I[Formula: see text]B[Formula: see text]) at Ser32/36 and thereby blocked I[Formula: see text]B[Formula: see text] degradation. TXY motif phosphorylation in the activation loops of mitogen-activated protein kinases (MAPKs) was also suppressed by MCA treatment. Further investigation revealed that MCA inhibited transforming growth factor-[Formula: see text]-activated kinase 1 (TAK1) phosphorylation and IL-1 receptor-associated kinase (IRAK1) degradation, the upstream kinases activating nuclear factor [Formula: see text]B and MAPKs. Taken together, MCA exhibited anti-inflammatory properties via the downregulation of IRAK1-TAK1 signaling pathways.

Curcumin ameliorates peritoneal fibrosis via inhibition of transforming growth factor-activated kinase 1 (TAK1) pathway in a rat model of peritoneal dialysis.

BACKGROUND: Peritoneal fibrosis (PF) remains a serious complication of long-term peritoneal dialysis (PD). The goal of this study was to investigate the anti-fibrotic effects of curcumin on the PF response to PD and its' mechanism. METHODS: Male Sprague-Dawley rats were infused with 20 mL of 4.25% glucose-based standard PD fluid for 8 consecutive weeks to establish PF model and then divided into five groups: Control, received sham operation and 0.9% physiological saline; PD, received 4.25% standard PD fluid; Curcumin, PD rats injected intraperitoeally with curcumin for 8 weeks at doses of 10, 20 or 40 mg/kg. Masson's staining was performed to evaluate the extent of PF. Peritoneal Equilibration Test (PET) was conducted to assess ultrafiltration volume (UFV) and mass transfer of glucose (MTG), quantitative RT-PCR, and immunohistochemistry or western blotting were performed to measure the expression levels of inflammation and fibrosis-associated factors. We also detected the TGF-ß1 in peritoneal fluid by ELISA. RESULTS: Compared with the control group, the PD rats showed decreased UFV (2.54 ± 0.48 to 9.87 ± 0.78 mL, p < 0.05] and increased MTG (18.99 ± 0.86 to 10.85 ± 0.65 mmol/kg, p < 0.05) as well as obvious fibroproliferative response, with markedly increased peritoneal thickness (178.33 ± 4.42 to 25.26 ± 0.32um, p < 0.05) and higher expression of a-SMA, collagen I and TGF-ß1. Treatment with curcumin significantly increased UFV, reduced MTG and peritoneal thickness of PD rats. The elevated TGF-ß1 in peritoneal fluid of PD rats was significantly decreased by curcumin. It attenuated the increase in protein and mRNA of TGF-ß1, α-SMA and collagen I in peritoneum of PD rats. The mRNA expressions of TAK1, JNK and p38, as well as the protein expressions of p-TAK1, p-JNK and p-p38 in peritoneum of PD rats were reduced by curcumin. CONCLUSIONS: Present results demonstrate that curcumin showed a protective effect on PD-related PF and suggest an implication of TAK1, p38 and JNK pathway in mediating the benefical effects of curcumin.

The ScFRK2 mitogen-activated protein kinase kinase kinase (MAP3K) is involved in early embryo sac development in Solanum chacoense.

Fertilization-related kinase (FRK) is a group of the mitogen-activated protein kinase kinase kinase (MAP3K or MEKK) that has proliferated in Solanaceae species. Studies on the wild potato Solanum chacoense have shown that three ScFRKs are directly involved in female gametophyte development. Decreasing the expression of ScFRK1 and ScFRK3 by RNA interference lead to embryonic sac development arrest at the functional megaspore (FM) stage. As for ScFRK2, the first FRK studied, antisense and co-suppression lines showed no abnormality, while overexpression lines lead to a drastic decrease in seed numbers, presumably caused by a conversion of the ovule into a carpel-like structure. Here we show that in ScFRK2 overexpression lines, carpel-like structures from the ovule cannot explain the drastic decrease in seeds considering the low percentage of these carpel-like structures but occurs in early ovule development as observed in Scfrk1 and Scfrk3 knockdown mutants were most ovules are arrested at the FM stage. The highly similar phenotype from knockdown mutants (Scfrk1 and Scfrk3) and ScFRK2 overexpression lines suggests that these MAP kinases could operate antagonistically through a balance between ScFRK1 and 3 on one side and ScFRK2 on the other. This study strongly suggests the importance of the FRK family expression levels during early stages of ovule development in Solanum chacoense embryo sac.

Primary study on the hypoglycemic mechanism of 5rolGLP-HV in STZ-induced type 2 diabetes mellitus mice.

5rolGLP-HV is a promising dual-function peptide for the treatment of diabetes and thrombosis simultaneously. For investigating the therapeutic mechanism of 5rolGLP-HV for type 2 diabetes mellitus (T2DM), STZ-induced diabetic mice were established and treated with 5rolGLP-HV. The results showed that daily water and food intake, blood glucose, serum and pancreatic insulin levels significantly decreased after 5rolGLP-HV treatment with various oral concentrations, and 16 mg/kg was the optimal dose for controlling diabetes. 5rolGLP-HV treatment decreased the MDA levels and the T-SOD activity in serum and pancreatic of diabetic mice (but not up to significant difference), and significantly increased the expression of signal pathways related genes of rolGLP-1, also the density of insulin expression and the numbers of apoptosis cells in islets of diabetic mice were significantly decreased in comparison to the negative diabetic mice. These effects above may be clarified the hypoglycemic mechanisms of 5rolGLP-HV, and 5rolGLP-HV may be as a potential drug for diabetes in future.

Resibufogenin suppresses transforming growth factor-ß-activated kinase 1-mediated nuclear factor-κB activity through protein kinase C-dependent inhibition of glycogen synthase kinase 3.

Resibufogenin (RB), one of the major active compounds of the traditional Chinese medicine Chansu, has received considerable attention for its potency in cancer therapy. However, the anticancer effects and the underlying mechanisms of RB on pancreatic cancer remain elusive. Here, we found that RB inhibited the viability and induces caspase-dependent apoptosis in human pancreatic cancer cells Panc-1 and Aspc. Resibufogenin-induced apoptosis was through inhibition of constitutive nuclear factor-κB (NF-κB) activity and its target genes' expression, which was caused by downregulation of transforming growth factor-ß-activated kinase 1 (TAK1) levels and suppression of IκB kinase activity in Panc-1 and Aspc cells. This induction of TAK1-mediated NF-κB inactivation by RB was associated with increased glycogen synthase kinase-3 (GSK-3) phosphorylation and subsequent suppression of its activity. Moreover, RB-induced GSK-3 phosphorylation/inactivation acted through activation of protein kinase C but not Akt. Finally, RB suppressed human pancreatic tumor xenograft growth in athymic nude mice. Thus, our findings reveal a novel mechanism by which RB suppresses TAK1-mediated NF-κB activity through protein kinase C-dependent inhibition of GSK-3. Our findings provide a rationale for the potential application of RB in pancreatic cancer therapy.

Calycosin Suppresses Epithelial Derived Initiative Key Factors and Maintains Epithelial Barrier in Allergic Inflammation via TLR4 Mediated NF-κB Pathway.

BACKGROUND/AIMS: Calycosin is a bioactive component of Astragali Radix, a Chinese herb for treating allergy. We have previously demonstrated that calycosin effectively inhibited allergic inflammation efficiently. The aim of this study was to explore the mechanism of calycosin on epithelial cells in allergic inflammation. METHODS: An initial stage of atopic dermatitis (AD) model in which mice were just sensitized with FITC, was established in vivo and immortalized human keratinocytes (HaCaT cells) were utilized in vitro. Initiative key cytokines, TSLP and IL-33, were measured by ELISA, qPCR, immunofluorescence and Western blot. The junctions in epithelial cells were observed by electron microscopy and tight junctions (TJs) (Occludin and ZO-1) were assessed by Western blot and immunofluorescence. TLR4, MyD88, TAK1, TIRAP and NF-κB were measured by qPCR or Western blot. RESULTS: The results showed that TSLP and IL-33 were inhibited significantly by calycosin in the initial stage of AD model. Simultaneously, calycosin attenuated the separated gap among the epithelial cells and increased the expression of TJs. TSLP/IL-33 and TJs were similarly affected in LPS-stimulated HaCaT cells in vitro. Meanwhile, calycosin not only inhibited the expressions of TLR4, MyD88, TAK1 and TIRAP, but also reduced NF-κB activation in vitro and in vivo. An NF-κB inhibitor enhanced the expressions of TJs and reduced that of TSLP/IL-33 in LPS-stimulated HaCaT cells. CONCLUSION: These results indicated that calycosin reduced the secretion of TSLP/IL-33 and attenuated the disruption of epithelial TJs by inhibiting TLR4 mediated NF-κB signaling pathway. These findings help to understand the beneficial effects of calycosin on AD, and to develop effective preventive or therapeutic strategies to combat this disease and other epithelial barrier deletion-mediated allergic diseases.

TAK1 determines susceptibility to endoplasmic reticulum stress and leptin resistance in the hypothalamus.

Sustained endoplasmic reticulum (ER) stress disrupts normal cellular homeostasis and leads to the development of many types of human diseases, including metabolic disorders. TAK1 (also known as MAP3K7) is a member of the mitogen-activated protein kinase kinase kinase (MAP3K) family and is activated by a diverse set of inflammatory stimuli. Here, we demonstrate that TAK1 regulates ER stress and metabolic signaling through modulation of lipid biogenesis. We found that deletion of Tak1 increased ER volume and facilitated ER-stress tolerance in cultured cells, which was mediated by upregulation of sterol-regulatory-element-binding protein (SREBP)-dependent lipogenesis. In the in vivo setting, central nervous system (CNS)-specific Tak1 deletion upregulated SREBP-target lipogenic genes and blocked ER stress in the hypothalamus. Furthermore, CNS-specific Tak1 deletion prevented ER-stress-induced hypothalamic leptin resistance and hyperphagic obesity under a high-fat diet (HFD). Thus, TAK1 is a crucial regulator of ER stress in vivo, which could be a target for alleviation of ER stress and its associated disease conditions.

A novel NF-κB/YY1/microRNA-10a regulatory circuit in fibroblast-like synoviocytes regulates inflammation in rheumatoid arthritis.

The main etiopathogenesis of rheumatoid arthritis (RA) is overexpressed inflammatory cytokines and tissue injury mediated by persistent NF-κB activation. MicroRNAs widely participate in the regulation of target gene expression and play important roles in various diseases. Here, we explored the mechanisms of microRNAs in RA. We found that microRNA (miR)-10a was downregulated in the fibroblast-like synoviocytes (FLSs) of RA patients compared with osteoarthritis (OA) controls, and this downregulation could be triggered by TNF-α and IL-1ß in an NF-κB-dependent manner through promoting the expression of the YingYang 1 (YY1) transcription factor. Downregulated miR-10a could accelerate IκB degradation and NF-κB activation by targeting IRAK4, TAK1 and BTRC. This miR-10a-mediated NF-κB activation then significantly promoted the production of various inflammatory cytokines, including TNF-α, IL-1ß, IL-6, IL-8, and MCP-1, and matrix metalloproteinase (MMP)-1 and MMP-13. In addition, transfection of a miR-10a inhibitor accelerated the proliferation and migration of FLSs. Collectively, our data demonstrates the existence of a novel NF-κB/YY1/miR-10a/NF-κB regulatory circuit that promotes the excessive secretion of NF-κB-mediated inflammatory cytokines and the proliferation and migration of RA FLSs. Thus, miR-10a acts as a switch to control this regulatory circuit and may serve as a diagnostic and therapeutic target for RA treatment.

Genome-wide analysis of MAPKKKs shows expansion and evolution of a new MEKK class involved in solanaceous species sexual reproduction.

BACKGROUND: Members of the plant MAP Kinases superfamily have been mostly studied in Arabidopsis thaliana and little is known in most other species. In Solanum chacoense, a wild species close to the common potato, it had been reported that members of a specific group in the MEKK subfamily, namely ScFRK1 and ScFRK2, are involved in male and female reproductive development. Apart from these two kinases, almost nothing is known about the roles of this peculiar family. METHODS: MEKKs were identified using BLAST and hidden Markov model (HMM) to build profiles using the 21 MEKKs from A. thaliana. Following protein sequence alignments, the neighbor-joining method was used to reconstruct phylogenetic trees of the MEKK subfamily. Kinase subdomains sequence logos were generated with WebLogo in order to pinpoint FRK distinct motifs. Codon alignments of the FRKs kinase subdomains and maximum-likelihood phylogenetic trees were used in the codon substitution models of the codeml program in the PAML package to detect selective pressure between FRK groups. RESULTS: With the recent progress in Next-Generation Sequencing technologies, the genomes and transcriptomes of numerous plant species have been recently sequenced, giving access to a vast amount of data. With the aim of finding all members of the MEKK subfamily members in plants, we screened the genomes of 15 species from different clades of the plant kingdom. Interestingly, the whole MEKK subfamily has significantly expanded throughout evolution, especially in solanaceous species. This holds true for members of the FRK class, which have also strongly expanded and diverged. CONCLUSIONS: Expansion and rapid evolution of the FRK class members in solanaceous species support the hypothesis that they have acquired new roles, mainly in male and female reproductive development.

Picroside II Inhibits the MEK-ERK1/2-COX2 Signal Pathway to Prevent Cerebral Ischemic Injury in Rats.

The objective of this study is to explore the neuroprotective effect and mechanism of picroside II on ERK1/2-COX2 signal transduction pathway after cerebral ischemic injury in rats. Focal cerebral ischemic models were established by inserting monofilament threads into the middle cerebral artery in 200 Wistar rats. Twenty four rats were randomly selected into control group, while the other rats were randomly divided into six groups: model group, picroside group, lipopolysaccharide (LPS) with picroside group, U0126 with picroside group, LPS group, and U0126 group with each group containing three subgroups with ischemia at 6, 12, and 24 h. Neurobehavioral function in the rats was evaluated by modified neurological severity score points (mNSS) test; structure of neurons was observed using hematoxylin-eosin (HE) staining; apoptotic cells were counted using terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay; expressions of phosphorylated mitogen/extracellular signal-regulated kinase kinas1/2 (pMEK1/2), phosphorylated extracellular signal-regulated protein kinase1/2 (pERK1/2), and cyclooxygenase (COX2) in the cortex were determined using immunohistochemistry (IHC) and Western blot (WB); and real-time PCR was used to determine the level of COX2 mRNA. The neurological behavioral malfunction appeared in all rats with middle cerebral artery occlusion (MCAO). In the model group, neuron damage was extensive, while the neurobehavioral function score, apoptotic cell index, expression of pMEK1/2, pERK1/2, and COX2 and the level of COX2 mRNA increased significantly when compared to the control group. The peak COX2 mRNA level was in ischemia 12 h, prior to the peak in COX2 protein expression. In the picroside and U0126 groups, the neurological behavioral function was improved, and the number of apoptotic cells and the expression of pMEK1/2, pERK1/2, and COX2 decreased significantly when compared to the model group. In the LPS with picroside group, at ischemia 6 h neuron damage was extensive, and pMEK1/2, pERK1/2, and COX2 expression were much higher than in the model group. But at ischemia 12 and 24 h, the expression of pMEK1/2, pERK1/2, and COX2 decreased slightly, and the neurobehavioral function also improved slightly. In LPS group, neuron damage was extensive, pMEK1/2, pERK1/2, and COX2 expression was still at a high level, and COX2 mRNA peak arrived at ischemic 12 h. Picroside II downregulates COX2 expression after MCAO by inhibiting MEK-ERK1/2 in rats to protect neurons from apoptosis and inflammation.

Astragaloside IV Attenuates Glutamate-Induced Neurotoxicity in PC12 Cells through Raf-MEK-ERK Pathway.

Astragaloside IV (AGS-IV) is a main active ingredient of Astragalus membranaceus Bunge, a medicinal herb prescribed as an immunostimulant, hepatoprotective, antiperspirant, a diuretic or a tonic as documented in Chinese Materia Medica. In the present study, we employed a high-throughput comparative proteomic approach based on 2D-nano-LC-MS/MS to investigate the possible mechanism of action involved in the neuroprotective effect of AGS-IV against glutamate-induced neurotoxicity in PC12 cells. Differential proteins were identified, among which 13 proteins survived the stringent filter criteria and were further included for functional discussion. Two proteins (vimentin and Gap43) were randomly selected, and their expression levels were further confirmed by western blots analysis. The results matched well with those of proteomics. Furthermore, network analysis of protein-protein interactions (PPI) and pathways enrichment with AGS-IV associated proteins were carried out to illustrate its underlying molecular mechanism. Proteins associated with signal transduction, immune system, signaling molecules and interaction, and energy metabolism play important roles in neuroprotective effect of AGS-IV and Raf-MEK-ERK pathway was involved in the neuroprotective effect of AGS-IV against glutamate-induced neurotoxicity in PC12 cells. This study demonstrates that comparative proteomics based on shotgun approach is a valuable tool for molecular mechanism studies, since it allows the simultaneously evaluate the global proteins alterations.

Cloning and analysis of the soybean MEKK gene.

In this paper, homologous cloning methods were used to clone the soybean GmMEKK gene, which possesses a high degree of similarity to Arabidopsis thaliana AtMEKK1. AtMEKK1 is formed by 595 amino acids, and its secondary structure is formed by 38 irregular curls, 24 α helix, 14 ß, with S-TKc domain, transmembrane domain and does not have membrane spanning domain and signal peptide. GmMEKK-GFP subcellular localization fusion and prokaryotic expression vectors were generated and it was revealed that GmMEKK encodes a highly conserved 66.8-kDa nuclear protein that is expressed in soybean roots, stem floral pieces, and leaves. A real-time quantitative PCR analysis of GmMEKK under different abiotic stresses revealed that the expression level of GmMEKK increased under drought and low phosphorus and nitrogen conditions. Taken together, these data suggest that GmMEKK may play an important role in the soybean abiotic stress response.

The Hippo effector YAP promotes resistance to RAF- and MEK-targeted cancer therapies.

Resistance to RAF- and MEK-targeted therapy is a major clinical challenge. RAF and MEK inhibitors are initially but only transiently effective in some but not all patients with BRAF gene mutation and are largely ineffective in those with RAS gene mutation because of resistance. Through a genetic screen in BRAF-mutant tumor cells, we show that the Hippo pathway effector YAP (encoded by YAP1) acts as a parallel survival input to promote resistance to RAF and MEK inhibitor therapy. Combined YAP and RAF or MEK inhibition was synthetically lethal not only in several BRAF-mutant tumor types but also in RAS-mutant tumors. Increased YAP in tumors harboring BRAF V600E was a biomarker of worse initial response to RAF and MEK inhibition in patients, establishing the clinical relevance of our findings. Our data identify YAP as a new mechanism of resistance to RAF- and MEK-targeted therapy. The findings unveil the synthetic lethality of combined suppression of YAP and RAF or MEK as a promising strategy to enhance treatment response and patient survival.