IL-4 Downregulates PD-L1 Level Via SOCS1 Upregulation-Induced JNK Deactivation to Enhance Antitumor Immunity in In Vitro Colorectal Cancer.

Interleukin-4 (IL-4) controls cell growth and immune system regulation in tumorigenesis and can inhibit the growth of colon cancer cell lines, but the possible mechanism is unclear. In this study, we investigated the possible mechanism of IL-4 in colorectal cancer (CRC) through in vitro experiments. CRC cells received treatment with IL-4 (50 ng/mL), investigating the suppressor of cytokine signaling 1 (SOCS1)-related mechanism underlying the role of IL-4 in the progression and immunosuppression of CRC. The malignant processes of CRC cells and CD8+T cell-mediated immune response in CRC cells were determined by CCK-8, Transwell, wound healing, and flow cytometry assays. Programmed death ligand 1 (PD-L1), SOCS1 expressions, and c-Jun N-terminal kinase (JNK) activation in CRC cells were analyzed by quantitative reverse transcription polymerase chain reaction and/or Western blot. IL-4 repressed the malignant processes, yet promoted the apoptosis of CRC cells. Besides, IL-4 downregulated PD-L1 level, upregulated SOCS1 level, and restrained JNK activation in CRC cells, while enhancing CRC cell-killing effect of CD8+T cells. IL-4-induced effects on the aforementioned malignant processes of CRC cells and the killing effect of CD8+T cells toward CRC cells were all reversed when SOCS1 was knocked down in the CRC cells. IL-4 downregulates PD-L1 level via SOCS1 upregulation-induced JNK deactivation to enhance antitumor immunity in in vitro CRC. The study provides a theoretical basis for the clinical application of IL-4 in antitumor immunity in CRC.

MMP7, Regulated by c-Jun, is Involved in Oral Squamous Cell Carcinoma and Associated with Cancer-Related Fibroblasts Infiltration.

OBJECTIVE: This study aimed to analyze the expression of Matrix Metalloproteinase 7 (MMP7) and molecular mechanism at the Transcription Factor (TF) level in Oral Squamous Cell Carcinoma (OSCC). METHODS: MMP7 expression was preliminarily explored in Head and Neck Squamous Cell Car-cinoma (HNSCC) in the online database, followed by functional analysis and prediction of TF of MMP7. IHC was employed to detect MMP7 levels in OSCC samples. SCC9 and 293T cells were used to explore the transcriptional and regulatory effects of predicted TF on MMP7 by reporter double luciferase assay, RT-qPCR, western blotting, and cellular immunofluorescence. Transwell and TUNEL were employed to detect the migration and apoptosis. RESULTS: MMP7 was significantly up-regulated in HNSCC and OSCC tissues. Moreover, MMP7 was positively correlated with CAFs and significantly enriched in the signaling pathway of RNA degradation. The c-Jun pathway was also up-regulated in OSCC tissues, and predicted to be optimal TF of MMP7 with positive regulatory relationship. In OSCC, silencing and over-expression of c-Jun significantly decreased and increased the level of MMP7. Meanwhile, c-Jun affected the behavior of SCC9 cells, which showed that after c-Jun gene silencing, the ability of cell migration was weakened, while apoptosis was enhanced. When c-Jun gene was overexpressed, the migration ability was enhanced, but apoptosis was not significantly affected. CONCLUSION: MMP7 has been proven to be a key protein in the development of OSCC, and has the potential to become a biological marker and therapeutic target. It has been found that c-Jun could bind to the MMP7 promoter region, and the silencing or overexpression of c-Jun can positively regulate the expression of MMP7.

Astrocytes initiate autophagic flux and maintain cell viability after internalizing non-active native extracellular α-synuclein.

Astrocytes are tasked with regulating the synaptic environment. Early stages of various neurodegenerative diseases are characterized by synapse loss, and astrocytic atrophy and dysfunction has been proposed as a possible cause. α-Synuclein (αS) is a highly expressed neuronal protein located in the synapse that can be released in the extracellular space. Evidence points to astrocytes as being responsible for uptake and degradation of extracellular αS. Therefore, misfolded active fibrillized αS resulting in protein inclusions and aggregates could be due to astrocytic dysfunction. Despite these pathological hallmarks and lines of evidence, the autophagic function of astrocytes in response to monomeric non-active αS to model healthy conditions has not been investigated. Human primary cortical astrocytes were treated with 100 nM of extracellular monomeric non-active αS alone, and in combination with N-terminal binding monomeric γ-synuclein (γS) as a control. Western blot analysis and super resolution imaging of HiLyte-488 labeled αS confirmed successful internalization of αS at 12, 24 and 48 h after treatment, while αS dimers were only observed at 48 h. Western blot analysis also confirmed αS's ability to induce autophagic flux by 48 h. Annexin V/PI flow cytometry results revealed increased early apoptosis at 24 h, but which resolved itself by 48 h, indicating no cell death in cortical astrocytes at all time points, suggesting astrocytes can manage the protein degradation demand of monomeric αS in healthy physiological conditions. Likewise, astrocytes reduced secretion of apolipoprotein (ApoE), a protein involved in pro-inflammatory pathways, synapse regulation, and autophagy by 12 h. Similarly, total c-JUN protein levels, a transcription factor involved in pro-inflammatory pathways increased by 12 h in the nuclear fraction. Therefore, astrocytes are able to respond and degrade αS in healthy physiological conditions, and astrocyte dysfunction could precede detrimental αS accumulation.

Chronic alcohol consumption aggravates acute kidney injury through integrin ß1/JNK signaling.

Alcohol abuse is one of the major public health problems in the world and is associated with various health conditions. However, little is known about the effect of alcohol consumption on acute kidney injury (AKI). In this study, we demonstrate that chronic and binge alcohol feeding with a Lieber-DeCarli diet containing 5 % ethanol for 10 days, followed by a single dose of 31.5 % ethanol by gavage, aggravated AKI after ischemia-reperfusion injury (IRI) in female, but not in male, mice. Kidney dysfunction, histopathology and tubular cell apoptosis were more severe in EtOH-fed female mice after IRI, compared to pair-fed controls. RNA sequencing and experimental validation uncovered that activation of integrin ß1 and its downstream c-Jun NH2-terminal kinase (JNK) aggravated AKI in EtOH-fed mice. Knockdown of integrin ß1 inhibited JNK phosphorylation and alleviated AKI in EtOH-fed mice, whereas activation of integrin ß1 by agonist antibody increased JNK phosphorylation, worsened renal histological injury and tubular cell apoptosis, and aggravated kidney dysfunction. In vitro, activation of integrin ß1 increased JNK phosphorylation and induced tubular epithelial cell apoptosis. The detrimental effect of EtOH feeding was primarily mediated by acetaldehyde, as its levels were increased in the blood, liver and kidney of female mice fed with ethanol. Acetaldehyde per se activated integrin ß1/JNK signaling and induced tubular cell apoptosis in vitro. These findings suggest that alcohol consumption increases vulnerability to AKI in female mice, which is probably mediated by acetaldehyde/integrin ß1/JNK signaling cascade.

Neuroprotective effects of Gastrodia elata Blume on promoting M2 microglial polarization by inhibiting JNK/TLR4/T3JAM/NF-κB signaling after transient ischemic stroke in rats.

Background: Gastrodia elata Blume, also called Tian Ma (TM), has been used to treat stroke for centuries. However, its effects on inflammation in acute cerebral ischemic injury and underlying mechanisms involved in microglial polarization remain unknown. The present study explored the effects of the TM extract on the modulation of microglial M1/M2 polarization 2 days after transient cerebral ischemia. Methods: Male Sprague Dawley rats were intracerebroventricularly administered with 1% dimethyl sulfoxide 25 min before cerebral ischemia and subsequently intraperitoneally administered 0.25 g/kg (DO + TM-0.25 g), 0.5 g/kg (DO + TM-0.5 g), or 1 g/kg (DO + TM-1 g) of the TM extract after cerebral ischemia onset. Results: DO + TM-0.5 g and DO + TM-1 g treatments downregulated the following: phospho-c-Jun N-terminal kinase (p-JNK)/JNK, tumor necrosis factor (TNF) receptor-associated factor 3 (TRAF3), TRAF3-interacting JNK-activating modulator (T3JAM), p-nuclear factor-kappa B p65 (p-NF-κB p65)/NF-κB p65, ionized calcium-binding adapter molecule 1 (Iba1), CD86, TNF-α, interleukin (IL)-1ß, and IL-6 expression and toll-like receptor 4 (TLR4)/Iba1, CD86/Iba1, and p-NF-κB p65/Iba1 coexpression. These treatments also upregulated IL-10, nerve growth factor, and vascular endothelial growth factor A expression and YM-1/2/Iba1 and IL-10/neuronal nuclei coexpression in the cortical ischemic rim. The JNK inhibitor SP600125 exerted similar treatment effects as the DO + TM-0.5 g and DO + TM-1 g treatments. Conclusion: DO + TM-0.5 g and DO + TM-1 g/kg treatments attenuate cerebral infarction by inhibiting JNK-mediated signaling. TM likely exerts the neuroprotective effects of promoting M1 to M2 microglial polarization by inhibiting JNK/TLR4/T3JAM/NF-κB-mediated signaling in the cortical ischemic rim 2 days after transient cerebral ischemia.

Alda-1 attenuation of binge alcohol-caused atrial arrhythmias through a novel mechanism of suppressed c-Jun N-terminal Kinase-2 activity.

Holiday Heart Syndrome (HHS) is caused by excessive binge alcohol consumption, and atrial fibrillation (AF) is the most common arrhythmia among HHS patients. AF is associated with substantial morbidity and mortality, making its prevention and treatment of high clinical interest. This study defines the anti-AF action of Alda-1 (an established cardioprotective agent) and the underlying mechanisms of the action in our well-characterized HHS and cellular models. We found that Alda-1 effectively eliminated binge alcohol-evoked Ca2+ triggered activities (Ca2+ waves, prolonged Ca2+ transient diastolic decay) and arrhythmia inducibility in intact mouse atria. We then demonstrated that alcohol impaired human RyR2 channels (isolated from organ donors' hearts). The functional role of alcohol-caused RyR2 channel dysfunction in Ca2+ triggered arrhythmic activities was evidenced in a unique transgenic mouse model with a loss-of-function mutation (RyR2E4872Q+/-). Alda-1 is known to activate aldehyde dehydrogenase 2 (ALDH2), a key enzyme in alcohol detoxification. However, we found an increased level of ALDH2 and a preserved normal balance of pro- vs anti-apoptotic signaling in binge alcohol exposed hearts and H9c2 differentiated myocytes, which suggests that the link of alcohol-ALDH2-apoptosis is unlikely to be a key factor leading to binge alcohol-evoked arrhythmogenicity. We have previously reported that binge alcohol-activated stress response kinase JNK2 causatively drives Ca2+-triggered atrial arrhythmogenicity. Here, we found that JNK2-specific inhibition in either isolated human RyR2 channels or intact mouse atria abolished alcohol-evoked RyR2 channel dysfunction and Ca2+ triggered arrhythmic activities, suggesting a strong alcohol-JNK2-RyR2 interaction in atrial arrhythmogenicity. Furthermore, we revealed, for the first time, that Alda-1 suppresses JNK2 (but not JNK1) enzyme activity independently of ALDH2, which in turn alleviates binge alcohol-evoked Ca2+ triggered atrial arrhythmogenesis. Our findings provide novel mechanistic insights into the anti-arrhythmic action of Alda-1 and suggest that Alda-1 represents a potential preventative agent for AF management for HHS patients.

Saikosaponin A Recovers Impaired Filaggrin Levels in Inflamed Skin by Downregulating the Expression of FRA1 and c-Jun.

Filaggrin (FLG) is an essential structural protein expressed in differentiated keratinocytes. Insufficient FLG expression contributes to the pathogenesis of chronic inflammatory skin diseases. Saikosaponin A (SSA), a bioactive oleanane-type triterpenoid, exerts anti-inflammatory activity. However, the effects of topically applied SSA on FLG expression in inflamed skin remain unclear. This study aimed to evaluate the biological activity of SSA in restoring reduced FLG expression. The effect of SSA on FLG expression in HaCaT cells was assessed through various biological methods, including reverse transcription PCR, quantitative real-time PCR, immunoblotting, and immunofluorescence staining. TNFα and IFNγ decreased FLG mRNA, cytoplasmic FLG protein levels, and FLG gene promoter-reporter activity compared to the control groups. However, the presence of SSA restored these effects. A series of FLG promoter-reporter constructs were generated to investigate the underlying mechanism of the effect of SSA on FLG expression. Mutation of the AP1-binding site (mtAP1) in the -343/+25 FLG promoter-reporter abrogated the decrease in reporter activities caused by TNFα + IFNγ, suggesting the importance of the AP1-binding site in reducing FLG expression. The SSA treatment restored FLG expression by inhibiting the expression and nuclear localization of FRA1 and c-Jun, components of AP1, triggered by TNFα + IFNγ stimulation. The ERK1/2 mitogen-activated protein kinase signaling pathway upregulates FRA1 and c-Jun expression, thereby reducing FLG levels. The SSA treatment inhibited ERK1/2 activation caused by TNFα + IFNγ stimulation and reduced the levels of FRA1 and c-Jun proteins in the nucleus, leading to a decrease in the binding of FRA1, c-Jun, p-STAT1, and HDAC1 to the AP1-binding site in the FLG promoter. The effect of SSA was evaluated in an animal study using a BALB/c mouse model, which induces human atopic-dermatitis-like skin lesions via the topical application of dinitrochlorobenzene. Topically applied SSA significantly reduced skin thickening, immune cell infiltration, and the expression of FRA1, c-Jun, and p-ERK1/2 compared to the vehicle-treated group. These results suggest that SSA can effectively recover impaired FLG levels in inflamed skin by preventing the formation of the repressor complex consisting of FRA1, c-Jun, HDAC1, and STAT1.

Dysregulation of c-Jun (JUN) and FBJ murine osteosarcoma viral oncogene homolog B (FOSB) in obese people and their predictive values for metabolic syndrome.

The incidences of metabolic syndrome (MetS), denoting insulin resistance-associated various metabolic disorders, are increasing. This study aimed to identify new biomarkers for predicting MetS and provide a novel diagnostic approach. Herein, the expression profiles of c-Jun (JUN) and FBJ murine osteosarcoma viral oncogene homolog B (FOSB) in individuals with obesity and patients with MetS from the Gene Expression Omnibus database. Quantitative reverse transcription polymerase chain reaction (RT-qPCR) was used to evaluate the messenger RNA levels of JUN and FOSB in the peripheral blood of healthy volunteers (lean and obese) and patients with MetS (lean and obese), along with that in the adipose tissue and peripheral blood of obese mouse model. Furthermore, receiver operating characteristic (ROC) curve and logistic regression analyses were performed to determine the diagnostic value of JUN and FOSB in MetS. The expression profiles and RT-qPCR results showed that JUN and FOSB were highly expressed in individuals with obesity, obese mouse models, and patients with MetS. The ROC analysis results showed an area under the curve values of 0.872 and 0.879 for JUN, 0.802 and 0.962 for FOSB, and 0.946 and 0.979 for JUN-FOSB in the lean group and the group with obesity, respectively, in predicting MetS. Logistic regression analysis showed that the p-values of both JUN and FOSB as MetS-affecting factors were <0.05. Altogether, the findings of this study indicate that both JUN and FOSB, abnormally expressed in individuals with obesity, are good biomarkers of MetS.

Galangin alleviated Doxorubicin-induced cardiotoxicity by inhibiting ferroptosis through GSTP1/JNK pathway.

BACKGROUND: Doxorubicin (DOX) is a potent anticancer medication, but its significant cardiotoxicity poses a challenge in clinical practice. Galangin (Gal), a flavonoid compound with diverse pharmacological activities, has shown potential in exerting cardioprotective effects. However, the related molecular mechanism has not been fully elucidated. PURPOSE: Combined with bioinformatics and experimental verification methods to investigate Gal's potential role and underlying mechanisms in mitigating DOX-induced cardiotoxicity (DIC). METHODS: C57BL/6 mice received a single dose of DOX via intraperitoneal injection 4 days before the end of the gavage period with Gal. Myocardial injury was evaluated using echocardiography, myocardial injury biomarkers, Sirius Red and H&E staining. H9c2 cells were stimulated with DOX to mimic DIC in vitro. The potential therapeutic target of Gal was identified through network pharmacology, molecular docking and cellular thermal shift assay (CETSA), complemented by an in-depth exploration of the GSTP1/JNK signaling pathway using immunofluorescence. Subsequently, the GSTP1 inhibitor Ezatiostat (Eza) substantiated the signaling pathway. RESULTS: Gal administration considerably raised DOX-inhibited the left ventricular ejection fractions (LVEF), reduced levels of myocardial injury markers (c-TnI, c-TnT, CKMB, LDH, and AST), and alleviated DOX-induced myocardial histopathological injury and fibrosis in mice, thereby improving cardiac dysfunction. The ferroptosis induced by DOX was inhibited by Gal treatment. Gal remarkably ameliorated the DOX-induced lipid peroxidation, accumulation of iron and Ptgs2 expression both in H9c2 cells and cardiac tissue. Furthermore, Gal effectively rescued the DOX-inhibited crucial regulators of ferroptosis such as Gpx4, Nrf2, Fpn, and Slc7a11. The mechanistic investigations revealed that Glutathione S-transferase P1 (GSTP1) may be a potential target for Gal in attenuating DIC. Gal act on GSTP1 by stimulating its expression, thereby enhancing the interaction between GSTP1 and c-Jun N-terminal kinase (JNK), leading to the deactivation of JNK/c-Jun pathway. Furthermore, interference of GSTP1 with inhibitor Eza abrogated the cardioprotective and anti-ferroptotic effects of Gal, as evidenced by decreased cell viability, reduced expression of GSTP1 and Gpx4, elevated MDA levels, and promoted phosphorylation of JNK and c-Jun compared with Gal treatment. CONCLUSION: Gal could inhibit ferroptosis and protect against DIC through regulating the GSTP1/JNK pathway. Our research has identified a novel pathway through which Gal regulates DIC, providing valuable insights into the potential therapeutic efficacy of Gal in mitigating cardiotoxic effects.

NF-κB p105-mediated nuclear translocation of ERK is required for full activation of IFNγ-induced iNOS expression.

Inducible nitric oxidase (iNOS) encoded by Nos2 is a representative IFNγ-inducible effector molecule that plays an important role in both innate and adaptive immunity. In the present study, we demonstrated that full-length NF-κB p105 (p105), which is a precursor of NF-κB p50 (p50), is required for full activation of IFNγ-induced iNOS expression in the RAW264.7 mouse macrophage cell line. In comparison to wild-type (WT) RAW264.7 cells, p105 KO RAW264.7 (p105 KO) cells completely lost IFNγ-induced iNOS expression. Despite the limited effect of exogenous expression of p50 in p105 KO cells on IFNγ-induced Nos2 promoter activity, p105 expression fully restored IFNγ-induced Nos2 promoter activity to a level comparable to that of WT cells, suggesting an important role for full-length p105 in IFNγ-induced iNOS expression. While the expression and phosphorylation of JAK1 and STAT1 were rather enhanced in p105 KO cells, the phosphorylation of c-Jun downstream of MAPK signaling was decreased. IFNγ-induced phosphorylation of ERK, a kinase for IFNγ-induced c-Jun phosphorylation, was not significantly reduced in p105 KO cells, although the nuclear activity of ERK was significantly decreased due to its reduced translocation to the nucleus. Expression of iNOS, nuclear translocation of ERK, and phosphorylation of c-Jun were restored by stable supplementation of p105 in p105 KO cells. These results suggest that p105 is required for the nuclear translocation of ERK and the subsequent phosphorylation of c-Jun, which are necessary for full activation of IFNγ-induced iNOS expression. Reduced nuclear translocation of ERK in p105 KO cells was also observed in the activation of ERK following serum starvation, further suggesting that the involvement of p105 in ERK nuclear translocation is not limited to IFNγ-stimulated cells but is a more general function of p105.

A weekly 4-methylpyrazole treatment attenuates the development of non-obese metabolic dysfunction-associated steatotic liver disease (MASLD) in male mice: Role of JNK.

BACKGROUND: 4-methylpyrazole (4MP, fomepizole) is a competitive inhibitor of alcohol dehydrogenase (ADH) preventing the metabolism of ethylene glycol and methanol, respectively, into their toxic metabolites. 4MP seems also to possess a potential in the treatment of intoxication from other substance, for example, acetaminophen, and to modulate JNK-dependent signalling. Here, we determined if a treatment with 4MP once weekly affects the development of diet-induced non-obese metabolic dysfunction-associated steatotic liver disease (MASLD) in C57BL/6 mice. METHODS: Male C57BL/6 mice (6-8 weeks old, n = 7-8/group) were pair-fed either a liquid control diet (C) or a liquid sucrose-, fat- and cholesterol-rich diet (SFC) for 8 weeks while being concomitantly treated with 4MP (50 mg/kg bw i.p.) or vehicle once a week. Liver damage, inflammatory markers and glucose tolerance were assessed. Moreover, in endotoxin-challenged J774A.1 cells pretreated with 4MP, pro-inflammatory markers were assessed. RESULTS: The concomitant treatment of SFC-fed mice with 4MP attenuated the increase in JNK phosphorylation and pro-inflammatory markers like IFNγ, IL-6 and 3-nitrotyrosine protein adducts in liver tissue found in vehicle-treated SFC-fed mice, while not affecting impairments of glucose tolerance or the increase in portal endotoxin levels. Moreover, a pretreatment of endotoxin-stimulated J774A.1 cells with 4MP significantly attenuated the increases in JNK phosphorylation and pro-inflammatory mediators like IL-6 and Mcp1. CONCLUSIONS: Taken together, our results suggest that a treatment with 4MP once weekly attenuates the activation of JNK and dampens the development of non-obese MASLD in mice.

Update on JNK inhibitor patents: 2015 to present.

INTRODUCTION: c-Jun N-terminal kinase (JNK) regulates various biological processes through the phosphorylation cascade and is closely associated with numerous diseases, including inflammation, cardiovascular diseases, and neurological disorders. Therefore, JNKs have emerged as potential targets for disease treatment. AREAS COVERED: This review compiles the patents and literatures concerning JNK inhibitors through retrieving relevant information from the SciFinder, Google Patents databases, and PubMed from 2015 to the present. It summarizes the structure-activity relationship (SAR) and biological activity profiles of JNK inhibitors, offering valuable perspectives on their potential therapeutic applications. EXPERT OPINION: The JNK kinase serves as a novel target for the treatment of neurodegenerative disorders, pulmonary fibrosis, and other illnesses. A variety of small-molecule inhibitors targeting JNKs have demonstrated promising therapeutic potential in preclinical studies, which act upon JNK kinases via distinct mechanisms, encompassing traditional ATP competitive inhibition, covalent inhibition, and bidentate inhibition. Among them, several JNK inhibitors from PregLem SA, Celegene SA, and Xigen SA have accomplished the early stage of clinical trials, and their results will guide the development and indications of future JNK inhibitors.

A novel biomarker GATM suppresses proliferation and malignancy of cholangiocarcinoma cells by modulating the JNK/c-Jun signalling pathways.

Background: Cholangiocarcinoma (CCA) is the second most common primary malignancy of the liver and is associated with poor prognosis. Despite the emerging role of glycine amidinotransferase (GATM) in cancer development, its function in CCA remains elusive. This study investigated the biological significance and molecular mechanisms of GATM in CCA. Method: GATM expression was measured using immunohistochemistry and western blotting. Cell proliferation, migration, and invasion were assessed through CCK-8, EdU, clone formation, wound healing, and Transwell assays. Rescue experiments were performed to determine whether the JNK/c-Jun pathway is involved in GATM-mediated CCA development. Immunoprecipitation and mass spectrometry were performed to screen for proteins that interact with GATM. The role of GATM in vivo was investigated according to the xenograft experiment. Result: GATM expression was downregulated in CCA tissues and cells (p < 0.05) and had a significant suppressive effect on CCA cell proliferation, migration, and invasion in vitro as well as on tumour growth in vivo (p < 0.05); conversely, GATM knockdown promoted these phenotypes (p < 0.05). Notably, GATM inhibited the JNK/c-Jun pathway, and JNK activation abrogated GATM's antitumor effects (p < 0.05). Isocitrate dehydrogenase 1 (IDH1) interacts with GATM, and IDH1 knockdown significantly attenuated GATM protein degradation. Overexpression of IDH1 restored the biological function of CCA by reversing the inhibition of JNK/c-Jun pathway phosphorylation by GATM (p < 0.05). Conclusion: GATM acts as a tumour suppressor in CCA by regulating the phosphorylation of the JNK/c-Jun pathway. IDH1 interacted with GATM to regulate CCA progression.

Protein Kinases in Obesity, and the Kinase-Targeted Therapy.

The action of protein kinases and protein phosphatases is essential for multiple physiological responses. Each protein kinase displays its own unique substrate specificity and a regulatory mechanism that may be modulated by association with other proteins. Protein kinases are classified as dual-specificity kinases and dual-specificity phosphatases. Dual-specificity phosphatases are important signal transduction enzymes that regulate various cellular processes in coordination with protein kinases and play an important role in obesity. Impairment of insulin signaling in obesity is largely mediated by the activation of the inhibitor of kappa B-kinase beta and the c-Jun N-terminal kinase (JNK). Oxidative stress and endoplasmic reticulum (ER) stress activate the JNK pathway which suppresses insulin biosynthesis. Adenosine monophosphate (AMP)-activated protein kinase (AMPK) and mammalian target of rapamycin (mTOR) are important for proper regulation of glucose metabolism in mammals at both the hormonal and cellular levels. Additionally, obesity-activated calcium/calmodulin dependent-protein kinase II/p38 suppresses insulin-induced protein kinase B phosphorylation by activating the ER stress effector, activating transcription factor-4. To alleviate lipotoxicity and insulin resistance, promising targets are pharmacologically inhibited. Nifedipine, calcium channel blocker, stimulates lipogenesis and adipogenesis by downregulating AMPK and upregulating mTOR, which thereby enhances lipid storage. Contrary to the nifedipine, metformin activates AMPK, increases fatty acid oxidation, suppresses fatty acid synthesis and deposition, and thus alleviates lipotoxicity. Obese adults with vascular endothelial dysfunction have greater endothelial cells activation of unfolded protein response stress sensors, RNA-dependent protein kinase-like ER eukaryotic initiation factor-2 alpha kinase (PERK), and activating transcription factor-6. The transcriptional regulation of adipogenesis in obesity is influenced by AGC (protein kinase A (PKA), PKG, PKC) family signaling kinases. Obesity may induce systemic oxidative stress and increase reactive oxygen species in adipocytes. An increase in intracellular oxidative stress can promote PKC-ß activation. Activated PKC-ß induces growth factor adapter Shc phosphorylation. Shc-generated peroxides reduce mitochondrial oxygen consumption and enhance triglyceride accumulation and lipotoxicity. Liraglutide attenuates mitochondrial dysfunction and reactive oxygen species generation. Co-treatment of antiobesity and antidiabetic herbal compound, berberine with antipsychotic drug olanzapine decreases the accumulation of triglyceride. While low-dose rapamycin, metformin, amlexanox, thiazolidinediones, and saroglitazar protect against insulin resistance, glucagon-like peptide-1 analog liraglutide inhibits palmitate-induced inflammation by suppressing mTOR complex 1 (mTORC1) activity and protects against lipotoxicity.

Heterogeneous Patterns of Endothelial NF-κB p65 and MAPK c-Jun Activation, Adhesion Molecule Expression, and Leukocyte Recruitment in Lung Microvasculature of Mice with Sepsis.

BACKGROUND: Sepsis is an uncontrolled systemic inflammatory response to an infection that can result in acute failure of the function of the lung called acute respiratory distress syndrome. Leukocyte recruitment is an important hallmark of acute lung failure in patients with sepsis. Endothelial cells (EC) participate in this process by facilitating tethering, rolling, adhesion, and transmigration of leukocytes via adhesion molecules on their cell surface. In in vivo studies, endothelial nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) p65 and mitogen-activated protein kinase (MAPK) c-Jun intracellular signal transduction pathways were reported to regulate the expression of adhesion molecules. METHODS: Mice underwent cecal ligation and puncture (CLP) to induce polymicrobial sepsis and were sacrificed at different time points up to 72 h after sepsis onset. Immunohistochemistry and reverse transcription-quantitative polymerase chain reaction (RT-qPCR) analyses were used to determine the kinetics of nuclear localization of p65 and c-Jun in EC, expression and location of adhesion molecules E-selectin and vascular cell adhesion molecule 1 (VCAM-1). Furthermore, the extent and location of leukocyte recruitment were assessed based on Ly6G staining of neutrophils, cluster determinant (CD) 3 staining of T lymphocytes, and CD68 staining of macrophages. RESULTS: In all pulmonary microvascular beds, we identified p65 and c-Jun nuclear accumulation in a subset of endothelial cells within the first 24 h after CLP-sepsis initiation. E-selectin protein was expressed in a subset of microvessels at 4 and 7 h after sepsis initiation, while VCAM-1 was expressed in a scattered pattern in alveolar tissue and microvessels, without discernible changes during sepsis development. CLP-induced sepsis predominantly promoted the accumulation of neutrophils and T lymphocytes 4 and 7 h after disease onset. Neutrophil accumulation occurred in all pulmonary microvascular beds, while T lymphocytes were present in alveolar tissue and postcapillary venules. Taken together, nuclear localization of p65 and c-Jun in EC and neutrophil recruitment could be associated with induced E-selectin expression in the pulmonary microvessels in CLP-septic mice at the early stage of the disease. In alveolar capillaries, on the other hand, activation of these molecular pathways and leukocyte accumulation occurred in the absence of E-selectin or VCAM-1. CONCLUSIONS: Endothelial activation and leukocyte recruitment in sepsis-induced lung injury are regulated by multiple, heterogeneously controlled mechanisms, which vary depending on the type of microvascular bed involved.

Evaluation of Nitric Oxide-Donating Properties of 11H-indeno[1,2-b]quinoxalin-11-one Oxime (IQ-1) by Electron Paramagnetic Resonance Spectroscopy.

IQ-1 (11H-indeno[1,2-b]quinoxalin-11-one oxime) is a specific c-Jun N-terminal kinase (JNK) inhibitor with anticancer and neuro- and cardioprotective properties. Because aryloxime derivatives undergo cytochrome P450-catalyzed oxidation to nitric oxide (NO) and ketones in liver microsomes, NO formation may be an additional mechanism of IQ-1 pharmacological action. In the present study, electron paramagnetic resonance (EPR) of the Fe2+ complex with diethyldithiocarbamate (DETC) as a spin trap and hemoglobin (Hb) was used to detect NO formation from IQ-1 in the liver and blood of rats, respectively, after IQ-1 intraperitoneal administration (50 mg/kg). Introducing the spin trap and IQ-1 led to signal characteristics of the complex (DETC)2-Fe2+-NO in rat liver. Similarly, the introduction of the spin trap components and IQ-1 resulted in an increase in the Hb-NO signal for both the R- and the T-conformers in blood samples. The density functional theory (DFT) calculations were in accordance with the experimental data and indicated that the NO formation of IQ-1 through the action of superoxide anion radical is thermodynamically favorable. We conclude that the administration of IQ-1 releases NO during its oxidoreductive bioconversion in vivo.

Loss of prohibitin 2 in Schwann cells dysregulates key transcription factors controlling developmental myelination.

Schwann cells are critical for the proper development and function of the peripheral nervous system (PNS), where they form a collaborative relationship with axons. Past studies highlighted that a pair of proteins called the prohibitins play major roles in Schwann cell biology. Prohibitins are ubiquitously expressed and versatile proteins. We have previously shown that while prohibitins play a crucial role in Schwann cell mitochondria for long-term myelin maintenance and axon health, they may also be present at the Schwann cell-axon interface during development. Here, we expand on this, showing that drug-mediated modulation of prohibitins in vitro disrupts myelination and confirming that Schwann cell-specific ablation of prohibitin 2 (Phb2) in vivo results in severe defects in radial sorting and myelination. We show in vivo that Phb2-null Schwann cells cannot effectively proliferate and the transcription factors EGR2 (KROX20), POU3F1 (OCT6), and POU3F2 (BRN2), necessary for proper Schwann cell maturation, are dysregulated. Schwann cell-specific deletion of Jun, a transcription factor associated with negative regulation of myelination, confers partial rescue of the developmental defect seen in mice lacking Schwann cell Phb2. Finally, we identify a pool of candidate PHB2 interactors that change their interaction with PHB2 depending on neuronal signals, and thus are potential mediators of PHB2-associated developmental defects. This work develops our understanding of Schwann cell biology, revealing that Phb2 may modulate the timely expression of transcription factors necessary for proper PNS development, and proposing candidates that may play a role in PHB2-mediated integration of axon signals in the Schwann cell.

Mucosa­associated lymphoid tissue lymphoma translocation protein 1 inhibitor, MI­2, attenuates non­small cell lung cancer cell proliferation, migration and invasion, and promotes apoptosis by suppressing the JNK/c­JUN pathway.

Mucosa-associated lymphoid tissue lymphoma translocation protein 1 (MALT1) inhibitors are effective in attenuating the progression of several types of cancer. However, their role in lung cancer requires further investigation. Therefore, the present study aimed to explore the effect of the MALT1 inhibitor, MI-2, on the behavior of non-small cell lung cancer (NSCLC) cells and to uncover their possible underlying mechanism of action. The mRNA and protein expression levels of MALT1 were detected in the human normal lung epithelial cell line BEAS-2B, and the NSCLC cell lines, NCI-H1299, NCI-H1650, HCC827, A549 and NCI-H23. Subsequently, NCI-H1650 and A549 cells were treated with MI-2. Additionally, NCI-H1650 and A549 cells were co-treated with anisomycin, a c-JUN N-terminal kinase (JNK) pathway activator, with or without MI-2. The results illustrated that the mRNA and protein expression levels of MALT1 were significantly increased in NCI-H1299, NCI-H1650, A549 and NCI-H23 cells compared with those in BEAS-2B cells. Treatment of NCI-H1650 and A549 cells with MI-2 for 72 h reduced the optical density value as determined using the Cell Counting Kit-8 assay. Consistently, the 5-ethynyl-2'-deoxyuridine assay also showed that proliferation was reduced in MI-2-treated NSCLC cells. In addition, MI-2 downregulated B-cell lymphoma 2 (BCL2), and enhanced BCL2-associated X-protein expression and apoptotic rate in NCI-H1650 and A549 cells. These findings indicated that MI-2 could inhibit NCI-H1650 and A549 cell proliferation and promote apoptosis. Furthermore, treatment of cells with MI-2 only attenuated the migration and invasion of NCI-H1650 cells. Notably, MI-2 decreased the expression levels of phosphorylated (p)-JNK and p-c-JUN in NCI-H1650 and A549 cells, thus suggesting that MI-2 could suppress the JNK/c-JUN signaling pathway. However, NSCLC cell co-treatment with anisomycin (JNK pathway activator) reversed the effect of MI-2 on the proliferation, apoptosis and activation of the JNK/c-JUN pathway in NCI-H1650 and A549 cells. In conclusion, the present study demonstrated that the MALT1 inhibitor, MI-2, could suppress NSCLC cell proliferation, migration and invasion, and induce apoptosis via inactivating the JNK/c-JUN pathway.

A Novel 3-Benzyloxychromone From Celastrus orbiculatus Thunb. Exhibits Anticancer Effects on Non-Small Cell Lung Cancer Cells via GSK-3ß-Dependent c-Jun/ATF2 Pathway.

Celastrus orbiculatus Thunb. is a vine used as a traditional Chinese medicinal herb. In this study, we focused on the anticancer cytotoxicity and underlying mechanism of previously unreported 3-oxygen-substituted isoflavone analogue (3-benzyloxychromone, 3-Boc) from the herb. Initially, we established cell line-derived xenograft mouse model using H1299 non-small cell lung cancer (NSCLC) cells and found that the ethanol crude extracts of the stem part of C. orbiculatus (200 mg/kg) could substantially suppress the growth of xenograft tumors in athymic nu/nu mice. We compared 3-Boc with three other flavonoid analogues isolated from the stem part of C. orbiculatus. Among these, 3-Boc showed the most potent cytotoxicity against H1299 and H1975 NSCLC cells. Colony formation, EdU incorporation and Annexin V-FITC/PI apoptosis assays demonstrated that 3-Boc induced growth inhibition primarily by inhibiting DNA replication and inducing apoptotic death of NSCLC cells. Structure-based target prediction and MD simulation suggested that 3-Boc potentially suppressed the activity of glycogen synthase kinase-3ß (GSK-3ß) by interacting with the ATP-binding site. Western blot analysis indicated that 3-Boc triggered the phosphorylation of Serine 21 of GSK-3α or Serine 9 of GSK-3ß in a time- and dose-dependent manner. To investigate the dependency of GSK-3ß, we established GSK-3ß knockout in H1299 cells. Depletion of GSK-3ß enhanced 3-Boc-induced cytotoxicity compared with wild-type counterparts through activated c-Jun/ATF2 signaling pathway. Altogether, our study highlights the anticancer potential of C. orbiculatus and the discovery of novel 3-oxygen-substituted chromone from the herb, which may have important implications for screening promising modulators of GSK-3ß and related signaling pathways in the treatment of cancer.

EWS-FLI1 and Activator Protein-1 (AP-1) Reciprocally Regulate Extracellular-Matrix Proteins in Ewing sarcoma Cells.

Ribonucleotide reductase (RNR) is the rate-limiting enzyme in the synthesis of deoxyribonucleotides and the target of multiple chemotherapy drugs, including gemcitabine. We previously identified that inhibition of RNR in Ewing sarcoma tumors upregulates the expression levels of multiple members of the activator protein-1 (AP-1) transcription factor family, including c-Jun and c-Fos, and downregulates the expression of c-Myc. However, the broader functions and downstream targets of AP-1, which are highly context- and cell-dependent, are unknown in Ewing sarcoma tumors. Consequently, in this work, we used genetically defined models, transcriptome profiling, and gene-set -enrichment analysis to identify that AP-1 and EWS-FLI1, the driver oncogene in most Ewing sarcoma tumors, reciprocally regulate the expression of multiple extracellular-matrix proteins, including fibronectins, integrins, and collagens. AP-1 expression in Ewing sarcoma cells also drives, concurrent with these perturbations in gene and protein expression, changes in cell morphology and phenotype. We also identified that EWS-FLI1 dysregulates the expression of multiple AP-1 proteins, aligning with previous reports demonstrating genetic and physical interactions between EWS-FLI1 and AP-1. Overall, these results provide novel insights into the distinct, EWS-FLI1-dependent features of Ewing sarcoma tumors and identify a novel, reciprocal regulation of extracellular-matrix components by EWS-FLI1 and AP-1.