Gremlin-1 promotes IL-1ß-stimulated chondrocyte inflammation and extracellular matrix degradation via activation of the MAPK signaling pathway.

The role and mechanism of Gremlin-1 in osteoarthritis (OA) were expected to be probed in this study. Firstly, an in vitro OA model was constructed by stimulating human chondrocyte cell line CHON-001 with IL-1ß. Next, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and quantitative reverse transcriptase polymerase chain reaction (qRT-PCR) were utilized for assessing the effect of IL-1ß with different concentrations (5, 10, and 20 ng/mL) on the activity and Gremlin-1 messenger RNA of CHON-001 cells, respectively. Besides, the influence of knocking down/over-expressing Gremlin-1 on the inflammatory factors (IL-6, TNF-α, IL-18 and PGE2), oxidative stress-related substances (malondialdehyde [MDA]; superoxide dismutase [SOD]; lactate dehydrogenase [LDH]), extracellular matrix (ECM) degradation-related proteins, and mitogen-activated protein kinase (MAPK) pathway proteins in IL-1ß-stimulated CHON-001 cells were tested by enzyme-linked immunosorbent assay, related kits, qRT-PCR, and western blot, respectively. IL-1ß inhibited CHON-001 cell proliferation and upregulated Gremlin-1 expression in a concentration-dependent manner. Overexpression of Gremlin-1 increased the IL-6, TNF-α, IL-18, PGE2, and MDA levels, enhanced the LDH activity, and decreased the SOD activity in IL-1ß-induced CHON-001 cells; while the effect of Gremlin-1 knockdown on the above factors was in contrast with that of the overexpression. Furthermore, overexpression of Gremlin-1 upregulated protein expression of matrix metalloproteinase (MMP)-3, MMP-13, and ADAMTS4 while downregulated protein expression of collagen III, aggrecan, and SOX-9 in IL-1ß-stimulated CHON-001 cells. Besides, overexpression of Gremlin-1 increased the p-p38/p38 value while decreased the p-JNK/JNK value in L-1ß-stimulated CHON-001 cells; however, knockdown of Gremlin-1 reversed the above results. Gremlin-1 may promote IL-1ß-stimulated CHON-001 cell inflammation and ECM degradation by activating the MAPK signaling pathway.

MicroRNA-141-3p inhibits retinal neovascularization and retinal ganglion cell apoptosis in glaucoma mice through the inactivation of Docking protein 5-dependent mitogen-activated protein kinase signaling pathway.

Retinal neovascularization occurs in various ocular disorders including proliferative diabetic retinopathy and secondary neovascular glaucoma, resulting in blindness. This paper aims to investigate the effect of microRNA-141-3p (miR-141-3p) on retinal neovascularization and retinal ganglion cells (RGCs) in glaucoma mice through the Docking protein 5 (DOK5)-mediated mitogen-activated protein kinase (MAPK) signaling pathway. Chip retrieval and difference analysis were used for the potential mechanism of miR-141-3p on glaucoma. All modeled mice were transfected with different expression of mimic or inhibitor. The expressions of miR-141-3p, DOK5, and related genes and proteins of the MAPK signaling pathway were detected by Reverse transcription quantitative polymerase chain reaction and western blot analysis. Cell proliferation, lumen formation, and apoptosis in the retinal vascular epithelial cells and RGCs were detected using Matrigel angiogenesis and terminal deoxynucleotidyl transferase mediated dUTP nick-end labeling assays. Moreover, a total of 63 and 294 differentially expressed genes were obtained in GSE2378 and GSE9944 chips, and 4 genes were within the intersection of the chips. In addition, the results showed that miR-141-3p was found to inhibit the DOK5 gene and activate the MAPK pathway. The number of RGCs, the expression of p38, extracellular-signal-regulated kinases (ERK), Jun N-terminal kinase (JNK), IGF-1, VEGF, HIF1-α, Bax, caspase-3, and the extent of p38, ERK, and JNK phosphorylated were decreased with miR-141-3p upregulation. Lastly, the results obtained showed that miR-141-3p inhibited the proliferation of retinal vascular epithelial cells and inhibited angiogenesis, as well as promoted apoptosis of RGCs. The study suggests that miR-141-3p inhibits retinal neovascularization in glaucoma mice by impeding the activation of the DOK5-mediated MAPK signaling pathway.

microRNA-542-5p protects against acute lung injury in mice with severe acute pancreatitis by suppressing the mitogen-activated protein kinase signaling pathway through the negative regulation of P21-activated kinase 1.

Severe acute pancreatitis (SAP) is a condition associated with high rates of mortality and lengthy hospital stays. In the current study, SAP mouse models were established in BALB/c wild-type and P21-activated kinase 1 (PAK1) knockdown mice with the objective of determining the expression of microRNA-542-5p (miR-542-5p) and the subsequent elucidation of the mechanism by which it influences acute lung injury (ALI) by mediating mitogen-activated protein kinase (MAPK) signaling and binding to PAK1. The targeting relationship between miR-542-5p and PAK1 was verified using the bioinformatics prediction website and by the means of a dual-luciferase reporter assay. Following the SAP model establishment, the mice were assigned into various groups with the introduction of different mimic and inhibitors in an attempt to investigate the effects involved with miR-542-5p on inflammatory reactions among mice with SAP-associated ALI. Our results indicated that PAK1 was targeted and negatively mediated by miR-542-5p. Mice with SAP-associated ALI exhibited an increased wet-to-dry weight ratio, myeloperoxidase activity, serum amylase activity, TNF-α, interleukin-1 beta (IL-1ß), and intercellular adhesion molecule-1 (ICAM-1) contents, p-p38MAPK, p-ERK1/2, and p-JNK protein levels as well as PAK1 positive expression, while decreased miR-542-5p levels were observed. Functionally, overexpression of miR-542-5p improves ALI in mice with SAP via inhibition of the MAPK signaling pathway by binding to PAK1.Based on the evidence from experimental models, miR-542-5p was shown to improve ALI among mice with SAP, while suggesting that the effect may be related to the inactivation of the MAPK signaling pathway and downregulation of PAK1 gene. Thus, miR-542-5p could serve as a promising target for ALI treatment.

Silencing CTGF/CCN2 inactivates the MAPK signaling pathway to alleviate myocardial fibrosis and left ventricular hypertrophy in rats with dilated cardiomyopathy.

Dilated cardiomyopathy (DCM) is characterized by left ventricular dilation associated with systolic dysfunction. The purpose of the current study is to clarify the effect of connective tissue growth factor (CTGF/CCN2) on myocardial fibrosis and left ventricular hypertrophy (LVH) of rats with DCM through the mitogen-activated protein kinase (MAPK) signaling pathway. First, DCM rat models were established and sh-CTGF/CCN2 lentiviral expression vectors were constructed. Then, by observing the pathological changes and myocardial ultrastructure as well as detecting cardiac functions, myocardial fibrosis, and LVH of rats, the effect of CTGF/CCN2 gene silencing on rats with DCM was investigated. To further explore how CTGF/CCN2 gene silencing affects rats with DCM, the expression of CTGF/CCN2 and the related genes of the MAPK signaling pathway was detected. Sh-CTGF/CCN2-2 and sh-CTGF/CCN2-3 with lower CTGF/CCN2 expression were selected for further experimentation. CTGF/CCN2 gene silencing improved cardiac function and alleviated myocardial fibrosis and LVH of rats with DCM. It was also verified that CTGF/CCN2 gene silencing could relieve the pathology of rats with DCM by inactivation of the MAPK signaling pathway. We conclude that CTGF/CCN2 gene silencing inhibits the activation of the MAPK signaling pathway, thus decreases myocardial fibrosis and LVH, and then improves the pathological symptoms of DCM in rats.

Short communication: Camel milk ameliorates inflammatory responses and oxidative stress and downregulates mitogen-activated protein kinase signaling pathways in lipopolysaccharide-induced acute respiratory distress syndrome in rats.

Acute respiratory distress syndrome (ARDS) is a complex syndrome disorder with high mortality rate. Camel milk (CM) contains antiinflammatory and antioxidant properties and protects against numerous diseases. This study aimed to demonstrate the function of CM in lipopolysaccharide (LPS)-induced ARDS in rats. Camel milk reduced the lung wet:dry weight ratio and significantly reduced LPS-induced increases in neutrophil infiltration, interstitial and intra-alveolar edema, thickness of the alveolar wall, and lung injury scores of lung tissues. It also had antiinflammatory and antioxidant effects on LPS-induced ARDS. After LPS stimulation, the levels of proinflammatory cytokines (tumor necrosis factor-α, IL-10, and IL-1ß) in serum and oxidative stress markers (malondialdehyde, myeloperoxidase, and total antioxidant capacity) in lung tissue were notably attenuated by CM. Camel milk also downregulated mitogen-activated protein kinase signaling pathways. Given these results, CM is a potential complementary food for ARDS treatment.

The transmembrane protein FgSho1 regulates fungal development and pathogenicity via the MAPK module Ste50-Ste11-Ste7 in Fusarium graminearum.

The mitogen-activated protein kinase (MAPK) signaling pathways have been characterized in Fusarium graminearum. Currently, the upstream sensors of these pathways are unknown. Biological functions of a transmembrane protein FgSho1 were investigated using a target gene deletion strategy. The relationship between FgSho1 and the MAPK cassette FgSte50-Ste11-Ste7 was analyzed in depth. The transmembrane protein FgSho1 is required for conidiation, full virulence, and deoxynivalenol (DON) biosynthesis in F. graminearum. Furthermore, FgSho1 and FgSln1 have an additive effect on virulence of F. graminearum. The yeast two-hybrid, coimmunoprecipitation, colocalization and affinity capture-mass spectrometry analyses strongly indicated that FgSho1 physically interacts with the MAPK module FgSte50-Ste11-Ste7. Similar to the FgSho1 mutant, the mutants of FgSte50, FgSte11, and FgSte7 were defective in conidiation, pathogenicity, and DON biosynthesis. In addition, FgSho1 plays a minor role in the response to osmotic stress but it is involved in the cell wall integrity pathway, which is independent of the module FgSte50-Ste11-Ste7 in F. graminearum. Collectively, results of this study strongly indicate that FgSho1 regulates fungal development and pathogenicity via the MAPK module FgSte50-Ste11-Ste7 in F. graminearum, which is different from what is known in the budding yeast Saccharomyces cerevisiae.

Immunostimulatory Effect of Ovomucin Hydrolysates by Pancreatin in RAW 264.7 Macrophages via Mitogen-Activated Protein Kinase (MAPK) Signaling Pathway.

Ovomucin (OM), which has insoluble fractions is a viscous glycoprotein, found in egg albumin. Enzymatic hydrolysates of OM have water solubility and bioactive properties. This study investigated that the immunostimulatory effects of OM hydrolysates (OMHs) obtained by using various proteolytic enzymes (Alcalase®, bromelain, α-chymotrypsin, Neutrase®, pancreatin, papain, Protamax®, and trypsin) in RAW 264.7 cells. The results showed that OMH prepared with pancreatin (OMPA) produced the highest levels of nitrite oxide in RAW 264.7 cells, through upregulation of inducible nitric oxide synthase mRNA expression. The production of pro-inflammatory cytokines such as tumor necrosis factor-α and interleukin-6 were increased with the cytokines mRNA expression. The effect of OMPA on mitogen-activated protein kinase signaling pathway was increased the phosphorylation of p38, c-Jun NH2-terminal kinase, and extracellular signal-regulated kinase in a concentration-dependent manner. Therefore, OMPA could be used as a potential immune-stimulating agent in the functional food industry.

Activation of MAP Kinase Pathway by Polyisoprenylated Cysteinyl Amide Inhibitors Causes Apoptosis and Disrupts Breast Cancer Cell Invasion.

Prognoses for TNBC remain poor due to its aggressive nature and the lack of therapies that target its "drivers". RASA1, a RAS-GAP or GTPase-activating protein whose activity inhibits RAS signaling, is downregulated in up to 77% of TNBC cases. As such, RAS proteins become hyperactive and similar in effect to mutant hyperactive RAS proteins with impaired GTPase activities. PCAIs are a novel class of agents designed to target and disrupt the activities of KRAS and other G-proteins that are hyperactive in various cancers. This study shows the anticancer mechanisms of the PCAIs in two breast cancer cell lines, MDA-MB-468 and MDA-MB-231. PCAIs (NSL-YHJ-2-27) treatment increased BRAF phosphorylation, whereas CRAF phosphorylation significantly decreased in both cell lines. Moreover, the PCAIs also stimulated the phosphorylation of MEK, ERK, and p90RSK by 116, 340, and 240% in MDA-MB-468 cells, respectively. However, in MDA-MB-231 cells, a significant increase of 105% was observed only in p90RSK phosphorylation. Opposing effects were observed for AKT phosphorylation, whereby an increase was detected in MDA-MB-468 cells and a decrease in MDA-MB-231 cells. The PCAIs also induced apoptosis, as observed in the increased pro-apoptotic protein BAK1, by 51%, after treatment. The proportion of live cells in PCAIs-treated spheroids decreased by 42 and 34% in MDA-MB-468 and MDA-MB-231 cells, respectively, which further explains the PCAIs-induced apoptosis. The movement of the cells through the Matrigel was also inhibited by 74% after PCAIs exposure, which could have been due to the depleted levels of F-actin and vinculin punctate, resulting in the shrinkage of the cells by 76%, thereby impeding cell movement. These results show promise for PCAIs as potential therapies for TNBC as they significantly inhibit the hallmark processes and pathways that promote cell proliferation, migration, and invasion, which result in poor prognoses for breast cancer patients.

The highly pathogenic H5N1 avian influenza virus induces the mitogen-activated protein kinase signaling pathway in the trachea of two Ri chicken lines.

OBJECTIVE: The highly pathogenic avian influenza virus (HPAIV) is a threat to the poultry industry and economy and remains a potential source of pandemic infection in humans. Antiviral genes are considered a potential factor for studies on HPAIV resistance. Therefore, in this study, we investigated gene expression related to the mitogen-activated protein kinase (MAPK) signaling pathway by comparing non-infected, HPAI-infected resistant, and susceptible Ri chicken lines. METHODS: Resistant (Mx/A; BF2/B21) and susceptible Ri chickens (Mx/G; BF2/B13) were selected by genotyping the Mx and BF2 genes. Then, the tracheal tissues of non-infected and HPAIV H5N1 infected chickens were collected for RNA sequencing. RESULTS: A gene set overlapping test between the analyzed differentially expressed genes (DEGs) and functionally categorized genes was performed, including biological processes of the gene ontology (GO) and Kyoto encyclopedia of genes and genomes (KEGG) pathways. A total of 1,794 DEGs were observed between control and H5N1-infected resistant Ri chickens, 432 DEGs between control and infected susceptible Ri chickens, and 1,202 DEGs between infected susceptible and infected resistant Ri chickens. The expression levels of MAPK signaling pathway-related genes (including MyD88, NF-κB, AP-1, c-fos, Jun, JunD, MAX, c-Myc), cytokines (IL-1ß, IL-6, IL-8), type I interferons (IFN-α, IFN-ß), and IFN-stimulated genes (Mx1, CCL19, OASL, and PRK) were higher in H5N1-infected than in non-infected resistant Ri chickens. MyD88, Jun, JunD, MAX, cytokines, chemokines, IFNs, and IFN-stimulated expressed genes were higher in resistant-infected than in susceptible-infected Ri chickens. CONCLUSION: Resistant Ri chickens showed higher antiviral activity compared to susceptible Ri chickens, and H5N1-infected resistant Ri chickens had immune responses and antiviral activity (cytokines, chemokines, interferons, and IFN-stimulated genes), which may have been induced through the MAPK signaling pathway in response to H5N1 infection.

Amorphous selenium nanodots alleviate non-alcoholic fatty liver disease via activating VEGF receptor 1 to further inhibit phosphorylation of JNK/p38 MAPK pathways.

In clinic, there is still no unified standard for the treatment of non-alcoholic fatty liver disease (NAFLD), and the development of effective novel drugs to alleviate NAFLD remains a challenge. This study aimed to explore the effect and mechanism of amorphous selenium nanodots (A SeNDs) in alleviating NAFLD. Model rats with NAFLD were induced by the high-fat diet (HFD). Histomorphology was used to observe liver tissue, automatic biochemical analyzer was used to analyze liver function indicators, and ELISA kit was used to detect the effect of A SeNDs on oxidative stress and inflammatory factors in NAFLD rats. The results exhibited that A SeNDs could reduce hepatocyte steatosis, liver index, blood lipid level, and transaminase level in NAFLD rats. Furthermore, A SeNDs could relieve the oxidative stress and inflammatory reaction and maintain liver tissue structure in NAFLD rats. Mechanistically, A SeNDs (0.3 mg/kg/day) inhibit the phosphorylation of JNK/p38 MAPK pathways after activating vascular endothelial growth factor receptor 1 (VEGFR1) in the liver of rats with NAFLD to allay oxidative stress and inflammatory response and improves hepatic structure and liver function. Therefore, it should be an important method to mitigate NAFLD by supplementing A SeNDs to normalize hepatic structure and liver function.

Integrin-α9 and Its Corresponding Ligands Play Regulatory Roles in Chronic Periodontitis.

Integrin-α9 (ITGA9) and its corresponding ligands are involved in inflammatory and immune responses. The present study aimed to investigate whether ITGA9 participates in the development of chronic periodontitis (ChP) and to explore the underlying mechanisms. We collected gingival tissue and gingival crevicular fluid in vivo from patients to determine the levels of ITGA9 and its ligands. We cultured primary periodontal ligament cells (PDLCs) in vitro and applied small interfering RNA to knock down ITGA9 in order to analyze the changes of inflammatory cytokines and explore the related cellular signaling pathways. The expression level of ITGA9 was significantly higher in the gingiva of patients with ChP than that of healthy individuals. ITGA9 knockdown in the PDLCs inhibited the secretion of interleukin (IL)-1ß, IL-6, and IL-8. Western blot analysis indicated that this change could be attributed to the regulation of the mitogen-activated protein kinase (MAPK) signaling pathway. ITGA9 plays a regulatory role in the homeostasis of ChP. The results of the present study provide potential insights into the treatment of periodontitis. Graphical abstract.

Multikinase inhibitors sorafenib and sunitinib as radiosensitizers in head and neck cancer cell lines.

BACKGROUND: Radioresistance is a common feature of head and neck squamous cell carcinoma (HNSCC). We previously showed that the irradiation- activated vascular endothelial growth factor (VEGF)-extracellular signal-regulated kinase (ERK)-axis is fundamental for the survival of resistant tumors. In this study, we examined if treatment with potent multikinase (MK) inhibitors, sorafenib and sunitinib, could radiosensitize tumor cells. METHODS: Cultured HNSCC cell lines were treated with inhibitors and subsequently irradiated. Radiosensitizing effects were functionally assessed by annexin-V apoptosis and clonogenic assays and confirmed by Western blot. Additionally, we surveyed human HNSCC tissue microarrays (TMAs) for activated ERK expression. RESULTS: Based on combination indexes, we found that combining irradiation with both inhibitors exerted strong and supra-additive antitumor effects on clonogenic survival. Kinase inhibition enhanced irradiation-induced apoptotic rates and inhibited postradiogenic phospho-ERK-expression. Patients with recurrent HNSCC displayed significantly lower extracellular signal-regulated kinase phosphorylation (pERK) levels than relapse-free patients. CONCLUSION: We propose further evaluation of sorafenib and sunitinib as potential radiosensitizing agents in HNSCC treatment. © 2017 Wiley Periodicals, Inc. Head Neck 39: 623-632, 2017.