c-Jun N-terminal Kinase Pathway Research Articles

Abstract PD9-07: Role of immunosuppressive JNK pathway in the tumor microenvironment among Triple Negative Breast Cancer subtypes in IBCSG Trial 22-00

Abstract BACKGROUND: Although the triple-negative breast cancer (TNBC) tumor microenvironment (TME) has been deeply characterized, much remains unknown about pathways attributing to an immunosuppressive TME in this disease. The phosphorylation of JNK (c-Jun N-terminal kinase) pathway has been associated with promoting an immunosuppressive phenotype, enhancing TNBC aggressiveness. Here, we aimed to explore the role of the JNK pathway in TNBC using a gene signature inferring the level of JNK phosphorylation. For this purpose, we used the TNBC cohort from the phase III adjuvant IBCSG 22-00 trial, which evaluated low-dose cyclophosphamide and methotrexate (CM) chemotherapy showing no clinical benefit in the overall population. METHODS: JNK gene signature was developed in TNBC samples by integrating RNA-seq gene expression data and phospho-JNK-targeted proteomic data in The Cancer Genome Atlas. Stochastic subsampling was performed to select the candidate markers. LASSO regression was performed to determine the final transcriptional signature to reflect JNK phosphorylation. The signature was then applied in a cohort composed of 498 TNBCs selected using stratified 1:3 relapse cases and non-relapse subcohort ratio from the IBCSG 22-00 trial. RNA-seq data from FFPE tumor samples were available for 347 patients. The JNK signature was calculated as the mean of the products between gene expression and signature coefficients, defining high and low levels using the median cut-off. Immune hot tumors were defined according to TNBC molecular subtypes or tumor-infiltrating lymphocytes (TILs) levels higher than 30%. Multivariable Cox models were used for disease-free survival (DFS) analysis. Wilcoxon test was used to evaluate the association between gene signatures and the levels of JNK. RESULTS: Tumors with either immunomodulatory (IM) phenotype or high TILs showed better DFS when presenting low levels of JNK compared to high levels (HR = 0.75, 95% CI, 0.57 to 0.99; P-value = 0.024 and HR = 0.62, 95% CI, 0.43 to 0.89, P-value = 0.0013). No significant differences in DFS were observed in other TNBC subtypes or tumors with low TILs, further highlighting the relevance of JNK signaling pathway in tumors presenting immune infiltration. Moreover, immune hot tumors with high levels of JNK were enriched for angiogenesis and eosinophils signatures and linked to immunosuppression. The immune targets B7-H3, CSF1R, GITR, and GARP were significantly associated with high levels of JNK. Of note, these genes are involved in the immune escape, activation of macrophages, and regulation of Tregs population. On the other hand, low levels of JNK were associated with higher levels of activated CD8+ T cells, pointing to an anti-tumor immune response, as well as with higher levels of the immune targets PDL-1, CTLA4, CD47, DCIR, and TIGIT. Of interest, a significant DFS benefit was found in IM tumors with high levels of JNK when treated with low-dose CM, compared to those who were not (HR = 0.52, 95% CI, 0.28 to 0.99; P interaction = 0.045). CONCLUSIONS: We developed a JNK gene signature to estimate the phosphorylation level of JNK from gene expression data in TNBC, and validated the associated biological and prognostic value in the IBCSG 22-00 trial. Our results are in line with the immunosuppressive effect described by the JNK gene signature and highlight the heterogeneity of immune response in immune hot TNBCs. Of note, high levels of JNK were associated with worse DFS, as well as with a benefit from low-dose CM potentially related to the immunomodulatory effect described for metronomic regimens. Overall, our findings suggest a potential role of the JNK signature in identifying TNBCs with an immunosuppressive TME and provide the rationale to explore its role as a biomarker for immunotherapy. Further validation of these findings is required. Citation Format: Andrea Joaquin Garcia, Takashi Semba, Mattia Rediti, Daniel J. McGrail, Xuemei Xie, Xiaoping Wang, Dileep R. Rampa, David Venet, Samira Majjaj, Roswitha Kammler, Marco Colleoni, Sherene Loi, Giuseppe Viale, Meredith Regan, Françoise Rothé, Christos Sotiriou, Naoto T. Ueno. Role of immunosuppressive JNK pathway in the tumor microenvironment among Triple Negative Breast Cancer subtypes in IBCSG Trial 22-00 [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr PD9-07.

Gbb Regulates Blood Cell Proliferation and Differentiation through JNK and EGFR Signaling Pathways in the Drosophila Lymph Gland.

The Drosophila lymph gland is an ideal model for studying hematopoiesis, and unraveling the mechanisms of Drosophila hematopoiesis can improve our understanding of the pathogenesis of human hematopoietic malignancies. Bone morphogenetic protein (BMP) signaling is involved in a variety of biological processes and is highly conserved between Drosophila and mammals. Decapentaplegic (Dpp)/BMP signaling is known to limit posterior signaling center (PSC) cell proliferation by repressing the protooncogene dmyc. However, the role of two other TGF-β family ligands, Glass bottom boat (Gbb) and Screw (Scw), in Drosophila hematopoiesis is currently largely unknown. Here, we showed that the loss of Gbb in the cortical zone (CZ) induced lamellocyte differentiation by overactivation of the EGFR and JNK pathways and caused excessive differentiation of plasmatocytes, mainly by the hyperactivation of EGFR. Furthermore, we found that Gbb was also required for preventing the hyperproliferation of the lymph glands by inhibiting the overactivation of the Epidermal Growth Factor Receptor (EGFR) and c-Jun N-terminal Kinase (JNK) pathways. These results further advance our understanding of the roles of Gbb protein and the BMP signaling in Drosophila hematopoiesis and the regulatory relationship between the BMP, EGFR, and JNK pathways in the proliferation and differentiation of lymph gland hemocytes.

ZFP36L1 Promotes Gastric Cancer Progression via Regulating JNK and p38 MAPK Signaling Pathways.

The RNA-binding protein Zinc Finger Protein 36 like 1(ZFP36L1) plays an important role in regulating the AU-rich elements (AREs) in the 3' untranslated region (3' UTR) of mRNAs, indicating a potential link between its expression and cancers. However, the role and mechanism of ZFP36L1 in gastric cancer (GC) are unclear. This study aimed to explore the role and mechanism of ZFP36L1 in gastric cancer. GC tissue samples and matched normal gastric tissues were collected, and the ZFP36L1 expression in these samples was evaluated by immunohistochemistry analysis. GC cells with different differentiation were selected for in vitro experiments. The ZFP36L1 expression in GC cells was examined by quantitative real-time polymerase chain reaction (qRTPCR) and Western blot analysis. The viability and invasiveness of GC cells were assayed by 5- Ethynyl-2-deoxyuridine (EdU) and Transwell assays, respectively. Western blot assay was used to detect the expression of epithelial-to-mesenchymal transition (EMT) related proteins and proteins of the c-Jun N-terminal kinase (JNK) and p38 Mitogen-Activated Protein Kinase (MAPK) signaling pathways. ZFP36L1 is overexpressed in GC tissues. Patients with high ZFP36L1 expression have a poor prognosis. Moreover, ZFP36L1 is overexpressed in the cell lines with a high degree of malignancy. ZFP36L1 increases cell proliferation, invasion, and migration in vitro. Furthermore, ZFP36L1 induces EMT. The JNK inhibitor and p38 inhibitor alone or in combination affect the biological function of GC cells. Furthermore, ZFP36L1 promotes GC progression by inhibiting JNK and p38 MAPK signaling pathways. RNA-binding protein ZFP36L1 exerts a role in the occurrence of gastric cancer by the regulation of the JNK and p38 MAPK signaling pathways. The combination of inhibitors of the JNK and p38 MAPK signaling pathways could be a novel treatment strategy for gastric cancer.

Astragaloside IV ameliorates insulin induced insulin resistance in HepG2 cells through reactive oxygen species mediated c-Jun N-terminal kinase pathway.

To investigate the effects and elucidate the mechanism of Astragaloside IV (AS-IV) for insulin resistance (IR) and type 2 diabet es mellitus (T2DM). CCK8 kit was used to detect cell viability, glucose detection kit was used to detect the concentration of glucose in cell supernatant, reactive oxygen species (ROS) detection kit and Western blot were used to explore the mechanism of Astragaloside IV (AS-IV) in improving IR. A diabetic rat model was also established by feeding high sugar and fat diet and streptozotocin (STZ) injection. After treatment with AS-IV, rosiglitazone (ROZ), or normal saline, the fasting blood glucose (FBG), C peptide (C-P), tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6) and the glucose tolerance were assessed. AS-IV could effectively reduce the content of ROS and increase the glucose uptake in high insulin-treated IR-type HepG2 cells. The results of molecular mechanisms indicated that AS-IV could improve insulin resistance by reducing JNK phosphorylation and regulating c-Jun N-terminal kinase (JNK) downstream protein expression. Additionally, AS-IV could significantly reduce the levels of FBG, TNF-α, IL-6 and the glucose tolerance in diabetic rats ( < 0.05 or < 0.01). The high and medium dose groups of AS-IV could significantly increase the C-P levels in diabetic rats ( < 0.05 or < 0.01). Our results indicated that AS-IV improve liver IR through the JNK pathway and ROS, which meant a new molecular target for the treatment of diabetes. The AS-IV also helped to prevent and improved the insulin resistance of rats.

Baicalin suppresses interleukin-1β-induced apoptosis, inflammatory response, oxidative stress, and extracellular matrix degradation in human nucleus pulposus cells

Objective To explore the effect of baicalin on human nucleus pulposus cells (NPCs) in response to interleukin (IL)-1β stimulation. Methods Viability of NPCs was measured by cell counting kit-8 (CCK-8) assay. TUNEL staining assay and flow cytometry were performed to detect apoptotic cell death of NPCs. Western blot analysis was conducted to detect the expression levels of proteins. Enzyme-linked immunosorbent assay (ELISA) was applied for the determination of inducible nitric oxide synthase (iNOS), cyclooxygenase-2 (COX-2), tumor necrosis factor alpha (TNF-α), and IL-6. Oxidative stress indicators including reactive oxygen species (ROS) production, malondialdehyde (MDA) level, and superoxide dismutase (SOD) activity were measured. Results Baicalin attenuated IL-1β-caused cell viability reduction and apoptosis in NPCs. IL-1β-induced increase in Bax expression and decrease in Bcl-2 expression were attenuated by baicalin treatment. IL-1β-induced production of iNOS, COX-2, IL-6, and TNF-α in NPCs was inhibited by baicalin treatment. Baicalin treatment reversed IL-1β-induced increase in ROS production and MDA level, as well as decrease in SOD activity. Furthermore, baicalin treatment elevated the expression levels of Col II and Aggrecan and downregulated the expression levels of MMP3, MMP13, and ADAMTS5 in IL-1β-induced NPCs. A total of 402 related targets of baicalin and 134 related targets of intervertebral disk degeneration were found, and nine intersection targets were screened out. The Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis showed that mitogen-activated protein kinase (MAPK) pathway was found to be involved in the effects of baicalin. Conclusions Baicalin exhibited protective effects on IL-1β-caused cell viability reduction, apoptosis, oxidative stress, inflammation, and extracellular matrix degradation in NPCs. In addition, we found c-Jun N-terminal kinase (JNK) and p38 MAPK pathways as targets of baicalin through bioinformatic analysis.

Chondrogenic medium in combination with a c-Jun N-terminal kinase inhibitor mediates engineered cartilage regeneration by regulating matrix metabolism and cell proliferation.

Cartilage tissue engineering is a promising strategy for repairing cartilage defects. However, achieving satisfactory cartilage regeneration in vitro and maintaining its stability in vivo remains a challenge. The key to achieving this goal is establishing an efficient cartilage regeneration culture system to retain sufficient active cells with physiological functions, generate abundant cartilage extracellular matrix (ECM) and maintain a low level of cartilage ECM degradation. The current chondrogenic medium (CM) can effectively promote cartilage ECM production; however, it has a negative effect on cell proliferation. Meanwhile, the specific c-Jun N-terminal kinase pathway inhibitor SP600125 promotes chondrocyte proliferation but inhibits ECM synthesis. Here, we aimed to construct a three-dimensional cartilage regeneration model using a polyglycolic acid/polylactic acid scaffold in combination with chondrocytes to investigate the effect of different culture modes with CM and SP600125 on in vitro cartilage regeneration and their long-term outcomes in vivo systematically. Our results demonstrate that the long-term combination of CM and SP600125 made up for each other and maximized their respective advantages to obtain optimal cartilage regeneration in vitro. Moreover, the long-term combination achieved stable cartilage regeneration after implantation in vivo with a relatively low initial cell-seeding concentration. Therefore, the long-term combination of CM and SP600125 enhanced in vitro and in vivo cartilage regeneration stability with fewer initial seeding cells and thus optimized the cartilage regeneration culture system.

Estrogen Receptor-α Exacerbates EGF-Inducing Airway Remodeling and Mucus Production in Bronchial Epithelium of Asthmatics.

Although estrogen receptors (ERs) signal pathways are involved in the pathogenesis and development of asthma, their expressions and effects remain controversial. This study aimed to investigate the expressions of ERα and ERβ as well as their mechanisms in airway remodeling and mucus production in asthma. The expressions of ERα and ERβ in the airway epithelial cells of bronchial biopsies and induced sputum cells were examined by immunohistochemistry. The associations of ERs expressions with airway inflammation and remodeling were evaluated in asthmatic patients. In vitro, the regulations of ERs expressions in human bronchial epithelial cell lines were examined using western blot analysis. The epidermal growth factor (EGF)-mediated ligand-independent activation of ERα and its effect on epithelial-mesenchymal transitions (EMTs) were investigated in asthmatic epithelial cells by western blot, immunofluorescent staining, and quantitative real-time polymerase chain reaction. ERα and ERβ were expressed on both bronchial epithelial cells and induced sputum cells, and the expressions showed no sex difference. Compared to controls, male asthmatic patients had higher levels of ERα on the bronchial epithelium, and there were cell-specific expressions of ERα and ERβ in induced sputum. The expression of ERα in the airway epithelium was inversely correlated to forced expiratory volume in 1 second (FEV1) % and FEV1/forced vital capacity. Severe asthmatic patients had significantly greater levels of ERα in the airway epithelium than mild-moderate patients. ERα level was positively correlated with the thickness of the subepithelial basement membrane and airway epithelium. In vitro, co-stimulation of interleukin (IL)-4 and EGF increased the expression of ERα and promoted its nuclear translocation. EGF activated the phosphorylation of ERα via extracellular signal-regulated kinase and c-Jun N-terminal kinase pathways. ERα knockdown alleviated EGF-mediated EMTs and mucus production in airway epithelial cells of asthma. ERα contributes to asthmatic airway remodeling and mucus production through the EGF-mediated ligand-independent pathway.

Synaptic alterations as a common phase in neurological and neurodevelopmental diseases: JNK is a key mediator in synaptic changes.

Synaptic alterations as a common phase in neurological and neurodevelopmental diseases: JNK is a key mediator in synaptic changes.

Transmembrane p24 trafficking protein 10 (TMED10) inhibits mitochondrial damage and protects neurons in ischemic stroke via the c-Jun N-terminal kinase (JNK) signaling pathway.

Stroke, a type of acute cerebrovascular disease, is a global disease with high mortality. Neuronal ischemia and hypoxia are closely related to occurrence and development of cognitive impairment. Transmembrane p24 trafficking protein 10 (TMED10) as a transmembrane protein involves in vesicle protein transport in the secretory pathways. However, the function and mechanism of TMED10 on ischemic stroke and cognitive impairments remain unclear. In current study, TMED10 was highly expressed in cerebral ischemic penumbra of middle cerebral artery occlusion (MCAO) mouse model. Downregulation of TMED10 suppressed cell survival and facilitated apoptosis in primary cortical neurons, which were grown under oxygen glucose deprivation/reoxygenation (OGD/R) condition. Upregulation of TMED10 protected neurons form apoptosis induced by OGD/R. Further research indicated that the decrease of TMED10 resulted in neuronal mitochondrial injury through increasing reactive oxygen species (ROS) production. Meanwhile, TMED10 reduction induced neuronal apoptosis and mitochondrial damage through activating the c-Jun N-terminal kinase (JNK) pathway. Moreover, the knockdown of TMED10 increased cerebral infarction area, aggravated neuronal injury and promoted neuronal apoptosis through activating the JNK pathway in the cerebral ischemic penumbra of MCAO mouse model. Additionally, Morris water maze test verified that the severity of cognitive impairment increased with the decline of TMED10. Collectively, this study reveals that TMED10 inhibits mitochondrial damage, and protects neurons from apoptosis in MCAO-induced ischemic stroke and cognitive impairment via blocking the JNK pathway.

Trehaloza kao potencijalni terapeutski agens u različitim bolestima

Trehalose is a natural, non-reducing disaccharide synthesized in some bacteria, fungi, plants, and insects. Due to its advantageous physical and chemical properties, trehalose can stabilize proteins and membranes, and protect cells from desiccation, heating, and freezing. Vertebrates do not synthesize trehalose, but the beneficial effects of trehalose have been demonstrated in numerous diseases as it eliminates aggregates, misfolded proteins, and damaged organelles, and reduces hyperinflammation and oxidative stress. Trehalose induces autophagy through nuclear translocation and activation of transcription factor EB (TFEB) in an mTOR-independent manner, but increases the expression of SQSTM1/p62 and has antioxidant properties in an autophagy-independent manner. Furthermore, trehalose induces apoptosis in tumor cells by increasing membrane fluidity through the activation of caspase 3, 6 and the JNK (c-Jun N-terminal kinase) pathway. Overall, in this review, previous knowledge on the therapeutic potential of trehalose in various diseases such as dry eye syndrome, neurodegenerative diseases, and tumors, was summarized, focusing on the underlying molecular mechanisms.

Signaling interactions among neurons impact cell fitness and death in Alzheimer's disease.

The pathology of Alzheimer's disease involves a long preclinical period, where the characteristic clinical symptoms of the changes in the brain are undetectable. During the preclinical period, homeostatic mechanisms may help prevent widespread cell death. Evidence has pointed towards selective cell death of diseased neurons playing a potentially protective role. As the disease progresses, dysregulation of signaling pathways that govern cell death contributes to neurodegeneration. Aberrant activation of the c-Jun N-terminal kinase pathway has been established in human and animal models of Alzheimer's disease caused by amyloid-beta 42- or tau-mediated neurodegeneration. Clonal mosaic studies in Drosophila that examine amyloid-beta 42 in a subset of neurons suggest complex interplay between amyloid-beta 42-expressing and wild-type cells. This review examines the role of c-Jun N-terminal kinase signaling in the context of cell competition and short-range signaling interactions between amyloid-beta 42-expressing and wild-type neurons. Cell competition is a conserved phenomenon regulating tissue integrity by assessing the fitness of cells relative to their neighbors and eliminating suboptimal cells. Somatic clones of amyloid-beta 42 that juxtapose genetically distinct neuronal cell populations show promise for studying neurodegeneration. Generating genetic mosaics with labeled clones of amyloid-beta 42- or tau-expressing and wild-type neurons will allow us to understand how short-range signaling alterations trigger cell death in neurons and thereby contribute to the progression of Alzheimer's disease. These approaches have the potential to uncover biomarkers for early Alzheimer's disease detection and new therapeutic targets for intervention.

Antiproliferative Evaluation of Novel 4-Imidazolidinone Derivatives as Anticancer Agent Which Triggers ROS-Dependent Apoptosis in Colorectal Cancer Cell.

Colorectal cancer (CRC) is one of the most common causes of cancer-related death worldwide, and more therapies are needed to treat CRC. To discover novel CRC chemotherapeutic molecules, we used a series of previously synthesized novel imidazolidin-4-one derivatives to study their anticancer role in several cancer cell lines. Among these compounds, compound 9r exhibited the best anticancer activity in CRC cell lines HCT116 and SW620. We further investigated the anticancer molecular mechanism of compound 9r. We found that compound 9r induced mitochondrial pathway apoptosis in HCT116 and SW620 cells by inducing reactive oxygen species (ROS) production. Moreover, the elevated ROS generation activated the c-Jun N-terminal kinase (JNK) pathway, which further accelerated apoptosis. N-acetylcysteine (NAC), an antioxidant reagent, suppressed compound 9r-induced ROS production, JNK pathway activation, and apoptosis. Collectively, this research synthesized a series of imidazolidin-4-one derivatives, evaluated their anticancer activity, and explored the molecular mechanism of compound 9r-induced apoptosis in CRC cells. The present results suggest that compound 9r has a potential therapeutic role in CRC. Hence, it deserves further exploration as a lead compound for CRC treatment.

Protective effect of astragalus membranaceus and its bioactive compounds against the intestinal inflammation in Drosophila.

Inflammatory bowel disease (IBD) is characterized by chronic and relapsing intestinal inflammation, which currently lacks safe and effective medicines. Astragalus membranaceus (AM), also named Huangqi, is one of the most commonly used fundamental herbs in China. Here, we aimed to investigate mechanism and bioactive compounds of AM on treating sodium dodecyl sulfate (SDS)- induced colitis in Drosophila flies. Our data showed that AM extract (AME) supplementation had no toxic effect in flies, and protected flies against SDS-induced lifespan shortening, intestinal morphological damage, and colon length shortening. Moreover, AME supplementation remarkably rescued SDS-induced intestinal stem cell (ISC) overproliferation and increased reactive oxygen species (ROS) level in the intestine. Mechanistically, AME remarkably rescued the altered expression levels of genes and proteins in c-Jun N-terminal kinase (JNK) and JAK-STAT signaling pathways induced by SDS in gut. Additionally, formononetin, isoliquiritigenin, isorhamnetin, astragaloside I, astragaloside III, vanillic acid, and caffeic acid in AM had protection against SDS-induced inflammatory damage in flies. Taken together, AME could ameliorate the intestinal inflammation partially by suppressing oxidative stress-associated JNK signaling and JAK-STAT signaling pathways. AME may provide a theoretical basis for natural medicine toward treating intestinal inflammatory disease in human.

Arsenic trioxide promotes ERK1/2-mediated phosphorylation and degradation of BIMEL to attenuate apoptosis in BEAS-2B cells

Inorganic arsenic is highly toxic, widely distributed in the human environment and may result in multisystem diseases and several types of cancers. The BCL-2-interacting mediator of cell death protein (BIM) is a key modulator of the intrinsic apoptosis pathway. Interestingly, in the present study, we found that arsenic trioxide (As2O3) decreased BIMEL levels in human bronchial epithelial cell line BEAS-2B and increased BIMEL levels in human lung carcinoma cell line A549 and mouse Sertoli cell line TM4. Mechanismly, the 26S proteasome inhibitors MG132 and bortezomib could effectively inhibit BIMEL degradation induced by As2O3 in BEAS-2B cells. As2O3 activated extracellular signal-regulated kinase (ERK) 1/2, c-Jun N-terminal kinase (JNK) and p38 mitogen-activated protein kinase (MAPK) signaling pathways, but only the ERK1/2 MAPK inhibitor PD98059 blocked BIMEL degradation induced by As2O3. Furthermore, As2O3 induced-phosphorylation of BIMEL at multiple sites was inhibited by ERK1/2 MAPK inhibitor PD98059. Inhibition of As2O3-induced ERK1/2 MAPK phosphorylation increased the levels of BIMEL and cleaved-caspase-3 proteins and decreased BEAS-2B cell viability. As2O3 also markedly mitigated tunicamycin-induced apoptosis of BEAS-2B cells by increasing ERK1/2 phosphorylation and BIMEL degradation. Our results suggest that As2O3-induced activation of the ERK1/2 MAPK pathway increases phosphorylation of BIMEL and promotes BIMEL degradation, thereby alleviating the role of apoptosis in As2O3-induced cell death. This study provides new insights into how to maintain the survival of BEAS-2B cells before malignant transformation induced by high doses of As2O3.

Delavatine A Attenuates OGD/R-Caused PC12 Cell Injury and Apoptosis through Suppressing the MKK7/JNK Signaling Pathway.

Delavatine A (DA) is an unusual isoquinoline alkaloid with a novel skeleton isolated from Chinese folk medicine Incarvillea delavayi. Studies conducted in our lab have demonstrated that DA has potential anti-inflammatory activity in lipopolysaccharide (LPS)-treated BV-2 cells. DA, however, has not been studied for its protective effect on neuronal cells yet. Thus, to explore whether DA can protect neurons, oxygen and glucose deprivation/reperfusion (OGD/R)-injured PC12 cell and middle cerebral artery occlusion/reperfusion (MCAO/R) rat model were used to assess the protective efficacy of DA against OGD/R damaged PC12 cells and MCAO/R injured rats. Our results demonstrated that DA pretreatment (0.31-2.5 µM) dose-dependently increased cell survival and mitochondrial membrane potential (MMP), whereas it lowered the leakage of lactate dehydrogenase (LDH), intracellular cumulation of Ca2+, and overproduction of reactive oxygen species (ROS), and inhibited the apoptosis rate in OGD/R-injured PC12 cells. Western blot demonstrated that DA pretreatment lowered the expression of apoptotic proteins and repressed the activation of the mitogen-activated protein kinase kinase 7 (MKK7)/c-Jun N-terminal kinase (JNK) pathway. It was also found that the neuroprotective efficacy of DA was significantly reversed by co-treatment with the JNK agonist anisomycin, suggesting that DA reduced PC12 cell injury and apoptosis by suppressing the MKK7/JNK pathway. Furthermore, DA oral administration greatly alleviated the neurological dysfunction and reduced the infarct volume of MCAO/R rats. Taken together, DA could ameliorate OGD/R-caused PC12 cell injury and improve brain ischemia/reperfusion (I/R) damage in MCAO/R rats, and its neuroprotection might be attributed to suppressing the MKK7/JNK signaling pathway.

Porphyromonas gingivalis Outer Membrane Vesicles Stimulate Gingival Epithelial Cells to Induce Pro-Inflammatory Cytokines via the MAPK and STING Pathways.

Porphyromonas gingivalis (Pg) is a keystone pathogen associated with chronic periodontitis and produces outer membrane vesicles (OMVs) that contain lipopolysaccharide (LPS), gingipains, and pathogen-derived DNA and RNA. Pg-OMVs are involved in the pathogenesis of periodontitis. Pg-OMV-activated pathways that induce the production of the pro-inflammatory cytokines, interleukin (IL)-6, and IL-8 in the human gingival epithelial cell line, OBA-9, were investigated. The role of mitogen-activated protein kinase (MAPK) and nuclear factor (NF)-κB in levels of Pg-OMV-induced pro-inflammatory cytokines was investigated using Western blot analysis and specific pathway inhibitors. Pg-OMVs induced IL-6 and IL-8 production via the extracellular signal-regulated kinase (Erk) 1/2, c-Jun N-terminal kinase (JNK), p38 MAPK, and NF-κB signaling pathways in OBA-9 cells. In addition, the stimulator of interferon genes (STING), an essential innate immune signaling molecule, was triggered by a cytosolic pathogen DNA. Pg-OMV-induced IL-6 and IL-8 mRNA expression and production were significantly suppressed by STING-specific small interfering RNA. Taken together, these results demonstrated that Pg-OMV-activated Erk1/2, JNK, p38 MAPK, STING, and NF-κB signaling pathways resulting in increased IL-6 and IL-8 expression in human gingival epithelial cells. These results suggest that Pg-OMVs may play important roles in periodontitis exacerbation by stimulating various pathways.

KCNH6 Enhanced Hepatic Glucose Metabolism through Mitochondrial Ca2+ Regulation and Oxidative Stress Inhibition.

KCNH6 has been proven to affect glucose metabolism and insulin secretion both in humans and mice. Further study revealed that Kcnh6 knockout (KO) mice showed impaired glucose tolerance. However, the precise function of KCNH6 in the liver remains unknown. Mitochondria have been suggested to maintain intracellular Ca2+ homeostasis; ROS generation and defective mitochondria can cause glucose metabolism disorders, including type 2 diabetes (T2D). Here, we found that Kcnh6 attenuated glucose metabolism disorders by decreasing PEPCK and G6pase abundance and induced Glut2 and IRS2 expression. Overexpression of Kcnh6 increased hepatic glucose uptake and glycogen synthesis. Kcnh6 attenuated intracellular and mitochondrial calcium levels in primary hepatocytes and reduced intracellular ROS and mitochondrial superoxide production. Kcnh6 suppressed oxidative stress by inhibiting mitochondrial pathway activation and NADPH oxidase expression. Experiments demonstrated that Kcnh6 expression improved hepatic glucose metabolism disorder through the c-Jun N-terminal kinase and p38MAPK signaling pathways. These results were confirmed by experiments evaluating the extent to which forced Kcnh6 expression rescued metabolic disorder in KO mice. In conclusion, KCNH6 enhanced hepatic glucose metabolism by regulating mitochondrial Ca2+ levels and inhibiting oxidative stress. As liver glucose metabolism is key to T2D, understanding KCNH6 functions may provide new insights into the causes of diabetes.

Extracellular sulfatase-2 is overexpressed in rheumatoid arthritis and mediates the TNF-α-induced inflammatory activation of synovial fibroblasts.

Extracellular sulfatase-2 (Sulf-2) influences receptor-ligand binding and subsequent signaling by chemokines and growth factors, yet Sulf-2 remains unexplored in inflammatory cytokine signaling in the context of rheumatoid arthritis (RA). In the present study, we characterized Sulf-2 expression in RA and investigated its potential role in TNF-α-induced synovial inflammation using primary human RA synovial fibroblasts (RASFs). Sulf-2 expression was significantly higher in serum and synovial tissues from patients with RA and in synovium and serum from hTNFtg mice. RNA sequencing analysis of TNF-α-stimulated RASFs showed that Sulf-2 siRNA modulated ~2500 genes compared to scrambled siRNA. Ingenuity Pathway Analysis of RNA sequencing data identified Sulf-2 as a primary target in fibroblasts and macrophages in RA. Western blot, ELISA, and qRT‒PCR analyses confirmed that Sulf-2 knockdown reduced the TNF-α-induced expression of ICAM1, VCAM1, CAD11, PDPN, CCL5, CX3CL1, CXCL10, and CXCL11. Signaling studies identified the protein kinase C-delta (PKCδ) and c-Jun N-terminal kinase (JNK) pathways as key in the TNF-α-mediated induction of proteins related to cellular adhesion and invasion. Knockdown of Sulf-2 abrogated TNF-α-induced RASF proliferation. Sulf-2 knockdown with siRNA and inhibition by OKN-007 suppressed the TNF-α-induced phosphorylation of PKCδ and JNK, thereby suppressing the nuclear translocation and DNA binding activity of the transcription factors AP-1 and NF-κBp65 in human RASFs. Interestingly, Sulf-2 expression positively correlated with the expression of TNF receptor 1, and coimmunoprecipitation assays demonstrated the binding of these two proteins, suggesting they exhibit crosstalk in TNF-α signaling. This study identified a novel role of Sulf-2 in TNF-α signaling and the activation of RA synoviocytes, providing the rationale for evaluating the therapeutic targeting of Sulf-2 in preclinical models of RA.

The Distinct Roles of LKB1 and AMPK in p53-Dependent Apoptosis Induced by Cisplatin.

Liver kinase B1 (LKB1) is a serine/threonine protein kinase that acts as a key tumor suppressor protein by activating its downstream kinases, such as AMP-activated protein kinase (AMPK). However, the regulatory actions of LKB1 and AMPK on DNA damage response (DDR) remain to be explored. In this study, we investigated the function of LKB1 in DDR induced by cisplatin, a representative DNA-damaging agent, and found that LKB1 stabilizes and activates p53 through the c-Jun N-terminal kinase (JNK) pathway, which promotes cisplatin-induced apoptosis in human fibrosarcoma cell line HT1080. On the other hand, we found that AMPKα1 and α2 double knockout (DKO) cells showed enhanced stabilization of p53 and increased susceptibility to apoptosis induced by cisplatin, suggesting that AMPK negatively regulates cisplatin-induced apoptosis. Moreover, the additional stabilization of p53 and subsequent apoptosis in AMPK DKO cells were clearly canceled by the treatment with the antioxidants, raising the possibility that AMPK suppresses the p53 activation mediated by oxidative stress. Thus, our findings unexpectedly demonstrate the reciprocal regulation of p53 by LKB1 and AMPK in DDR, which provides insights into the molecular mechanisms of DDR.

The immune deficiency and c-Jun N-terminal kinase pathways drive the functional integration of the immune and circulatory systems of mosquitoes.

The immune and circulatory systems of animals are functionally integrated. In mammals, the spleen and lymph nodes filter and destroy microbes circulating in the blood and lymph, respectively. In insects, immune cells that surround the heart valves (ostia), called periostial haemocytes, destroy pathogens in the areas of the body that experience the swiftest haemolymph (blood) flow. An infection recruits additional periostial haemocytes, amplifying heart-associated immune responses. Although the structural mechanics of periostial haemocyte aggregation have been defined, the genetic factors that regulate this process remain less understood. Here, we conducted RNA sequencing in the African malaria mosquito, Anopheles gambiae, and discovered that an infection upregulates multiple components of the immune deficiency (IMD) and c-Jun N-terminal kinase (JNK) pathways in the heart with periostial haemocytes. This upregulation is greater in the heart with periostial haemocytes than in the circulating haemocytes or the entire abdomen. RNA interference-based knockdown then showed that the IMD and JNK pathways drive periostial haemocyte aggregation and alter phagocytosis and melanization on the heart, thereby demonstrating that these pathways regulate the functional integration between the immune and circulatory systems. Understanding how insects fight infection lays the foundation for novel strategies that could protect beneficial insects and harm detrimental ones.