Mitogen-activated Protein Kinase Activation Research Articles

Oncoprotein LAMTOR5-mediated CHOP silence via DNA hypermethylation and miR-182/miR-769 in promotion of liver cancer growth.

C/EBP homologous protein (CHOP) triggers the death of multiple cancers via endoplasmic reticulum (ER) stress. However, the function and regulatory mechanism of CHOP in liver cancer remain elusive. We have reported that late endosomal/lysosomal adapter, mitogen-activated protein kinase and mTOR activator 5 (LAMTOR5) suppresses apoptosis in various cancers. Here, we show that the transcriptional and posttranscriptional inactivation of CHOP mediated by LAMTOR5 accelerates liver cancer growth. Clinical bioinformatic analysis revealed that the expression of CHOP was low in liver cancer tissues and that its increased expression predicted a good prognosis. Elevated CHOP contributed to destruction of LAMTOR5-induced apoptotic suppression and proliferation. Mechanistically, LAMTOR5-recruited DNA methyltransferase 1 (DNMT1) to the CpG3 region (-559/-429) of the CHOP promoter and potentiated its hypermethylation to block its interaction with general transcription factor IIi (TFII-I), resulting in its inactivation. Moreover, LAMTOR5-enhanced miR-182/miR-769 reduced CHOP expression by targeting its 3'UTR. Notably, lenvatinib, a first-line targeted therapy for liver cancer, could target the LAMTOR5/CHOP axis to prevent liver cancer progression. Accordingly, LAMTOR5-mediated silencing of CHOP via the regulation of ER stress-related apoptosis promotes liver cancer growth, providing a theoretical basis for the use of lenvatinib for the treatment of liver cancer.

Differential TLR-ERK1/2 Activity Promotes Viral ssRNA and dsRNA Mimic-Induced Dysregulated Immunity in Macrophages

RNA virus-induced excessive inflammation and impaired antiviral interferon (IFN-I) responses are associated with severe disease. This innate immune response, also referred to as “dysregulated immunity” is caused by viral single-stranded RNA (ssRNA)- and double-stranded-RNA (dsRNA)-mediated exuberant inflammation and viral protein-induced IFN antagonism. However, key host factors and the underlying mechanism driving viral RNA-mediated dysregulated immunity are poorly defined. Here, using viral ssRNA and dsRNA mimics, which activate toll-like receptor 7 (TLR7) and TLR3, respectively, we evaluated the role of viral RNAs in causing dysregulated immunity. We observed that murine bone marrow-derived macrophages (BMDMs), when stimulated with TLR3 and TLR7 agonists, induced differential inflammatory and antiviral cytokine response. TLR7 activation triggered a robust inflammatory cytokine/chemokine induction compared to TLR3 activation, whereas TLR3 stimulation induced significantly increased IFN/IFN stimulated gene (ISG) response relative to TLR7 activation. To define the mechanistic basis for dysregulated immunity, we examined cell-surface and endosomal TLR levels and downstream mitogen-activated protein kinase (MAPK) and nuclear factor kappa B (NF-kB) activation. We identified significantly higher cell-surface and endosomal TLR7 levels compared to TLR3, which were associated with early and robust MAPK (p-ERK1/2, p-P38, and p-JNK) and NF-kB activation in TLR7-stimulated macrophages. Furthermore, blocking EKR1/2 and NF-kB activity reduced TLR3/7-induced inflammatory cytokine/chemokine levels, whereas only ERK1/2 inhibition enhanced viral RNA mimic-induced IFN/ISG responses. Collectively, our results illustrate that high cell-surface and endosomal TLR7 expression and robust ERK1/2 activation drive viral ssRNA mimic-induced excessive inflammatory and reduced IFN/ISG response and blocking ERK1/2 activity would likely mitigate viral-RNA/TLR-induced dysregulated immunity.

The OXI1 kinase regulates plant immunity by linking microbial pattern-induced ROS burst to MAPK activation.

Plant cell-surface-localized pattern recognition receptors (PRRs) recognize microbial patterns and activate pattern-triggered immunity (PTI). Typical PTI responses include reactive oxygen species (ROS) burst controlled by the NADPH oxidase RESPIRATORY BURST OXIDASE HOMOLOGUE D (RbohD) and activation of the mitogen-activated protein kinase (MAPK) cascade composed of MAPKKK3/5-MKK4/5-MPK3/6. However, the mechanisms through which PRRs regulate and coordinate these immune responses are not fully understood. Here, we showed that Arabidopsis thaliana OXIDATIVE SIGNAL-INDUCIBLE1 (OXI1), a kinase known to be activated by ROS, is involved in the LYK5-CERK1 receptor complex, which recognizes fungal cell wall-derived chitin. The oxi1 mutant exhibits enhanced susceptibility to various pathogens and reduced chitin-induced MAPK activation and ROS burst. We showed that chitin induces the phosphorylation of OXI1 in an RbohD-dependent manner. H2O2 and chitin treatment causes the oxidation of OXI1 at Cys104 and Cys205, which is essential for the kinase activity of OXI1. These oxidation sites are required for chitin-induced MAPK activation and disease resistance. Activated OXI1 directly phosphorylates MAPKKK5 to regulate MAPK activation. Additionally, OXI1 phosphorylates RbohD, suggesting that it may activate RbohD to promote ROS burst to further enhance the long-term MAPK activation. Together, our findings reveal a pathway linking PRR-mediated ROS production to MAPK activation through OXI1.

PPARβ/δ prevents inflammation and fibrosis during diabetic cardiomyopathy

Diabetic cardiomyopathy (DCM) is a specific type of myocardial disease that often develops in patients suffering from diabetes, which has become the foremost cause of death among them. It is an insidious multifactorial disease caused by complex and partially unknown mechanisms that include metabolic dysregulation, local inflammation, fibrosis, and cardiomyocyte apoptosis. Despite its severity and poor prognosis, it often goes undiagnosed, and there are currently no approved specific drugs to prevent or even treat it. Peroxisome proliferator-activated receptor (PPAR)β/δ is a key metabolic regulator that has been proposed as a potential target for DCM due to its pleiotropic anti-inflammatory properties. Diabetes was induced by multiple low-dose streptozotocin (STZ) administration in wild-type and PPARβ/δ knockout male mice treated with the PPARβ/δ agonist GW0742 or vehicle. Human cardiomyocytes (AC16) and mouse atrial myocytes (HL-1) exposed to hyperglycemia and treated with PPARβ/δ agonists were also used. PPARβ/δ deletion in mice negatively impacted cardiac morphology and function, which was accompanied by interstitial fibrosis and structural remodeling of the heart. This phenotype was further exacerbated in knockout diabetic mice. At the molecular level, PPARβ/δ suppression resulted in increased expression of pro-inflammatory and pro-fibrotic markers. Some of these markers were also induced by diabetes in wild-type mice and were exacerbated in diabetic knockout mice. The activity of the transcription factors nuclear factor κB (NF-κB) and activator protein-1 (AP-1) correlated with most of these changes. Remarkably, PPARβ/δ activation partially prevented inflammation and fibrosis in the heart, as well as cardiac atrophy, induced during diabetes in mice, and also in cultured cardiomyocytes exposed to hyperglycemia. Finally, our results suggest that the beneficial effects of PPARβ/δ activation are mediated by the inhibition of mitogen-activated protein kinases (MAPK) activity and subsequent downregulation of the transcriptional activities of NF-κB and AP-1. Overall, the data suggest that PPARβ/δ agonists might be useful in preventing inflammation and fibrosis progression in DCM.

PP2C phosphatase Pic6 suppresses MAPK activation and disease resistance in tomato.

Type 2C protein phosphatases (PP2Cs) are essential for regulating plant immune responses to pathogens. Our study focuses on the tomato PP2C-immunity associated candidate 6 (Pic6), elucidating its role in negatively regulating pattern-triggered immunity (PTI) signaling pathways in tomato. Using reverse transcription quantitative polymerase chain reaction (RT-qPCR), we observed that treatment with microbe-associated molecular patterns (MAMPs)- flg22 and flgII-28-significantly increased Pic6 mRNA levels in wild-type (RG-PtoR) tomato plants. Pic6 features a conserved N-terminal kinase-interacting motif (KIM) and a C-terminal PP2C domain. We produced variants of Pic6 with mutations in these regions, demonstrating their involvements in negatively regulating tomato immunity. Agrobacterium-mediated transient overexpression of Pic6 resulted in enhanced growth of the bacterial pathogen Pseudomonas syringae pv. tomato (Pst) strain DC3000ΔhopQ1-1 compared to a YFP control. Additionally, Pic6 overexpression inhibited mitogen-activated protein kinase (MAPK) activation in response to flg22 and flgII-28 treatments. Importantly, Pic6 exhibited phosphatase activity and interacted with tomato Mkk1/Mkk2 proteins and dephosphorylated them in a KIM-dependent manner. Furthermore, we generated RG-pic6 loss-of-function mutants by CRISPR/Cas9, revealing that the absence of Pic6 heightened MAPK activity and increased resistance to Xanthomonas euvesicatoria strain 85-10 (Xe 85-10) when compared with the wild-type (RG-PtoR) plants. Transcript analyses showed that after flg22/flgII-28 treatment, PTI-reporter genes NAC and osmotin were significantly upregulated in RG-pic6 mutants in comparison to the wild-type (RG-PtoR) plants. Overall, our findings indicate that Pic6 acts as a negative regulator of MAPK signaling and playing a pivotal role in modulating tomato immunity against bacterial pathogens.

Genome-wide characterization and comparative expression profiling of dual-specificity phosphatase genes in yellow catfish (Pelteobagrus fulvidraco) after infection with exogenous Aeromonas hydrophila.

Dual-specificity phosphatases (DUSPs) are crucial regulators in many mammals, managing dephosphorylation and inactivation of mitogen-activated protein kinases (MAPKs) and playing essential roles in immune responses. However, their presence and functions in teleosts, like the yellow catfish (Pelteobagrus fulvidraco), remain unexplored. In this study, eight pfDusp genes (pfDusp1-7 and pfDusp10) were identified in yellow catfish. We characterized their molecular features, conserved protein sequences, and chromosomal localization through genome-wide analyses, and we examined their expression patterns in immune responses. Our findings reveal two conserved motifs, Leu-Phe-Leu-Gly and Ala-Tyr-Leu-Met, within the DSPc domain of DUSP proteins. The genes were mapped across seven chromosomes without evidence of duplication. Comparative analysis showed high conservation of Dusp genes across vertebrates, with evolutionary analysis suggesting Dusp3 as a potential intermediate form. Dusp transcripts were significantly upregulated in the kidney post-A. hydrophila infection. These results suggest the involvement of Dusp genes in the immune response of yellow catfish to bacterial pathogens, providing insights into their evolutionary significance and potential applications in aquaculture and molecular breeding.

Antimicrobial Peptide Pro10-1D Exhibits Anti-Allergic Activity: A Promising Therapeutic Candidate.

Although antimicrobial peptides (AMPs) exhibit a range of biological functions, reports on AMPs with therapeutic effects in allergic disorders are limited. In this study, we investigated the anti-allergic effects of Pro10-1D, a 10-meric AMP derived from insect defensin protaetiamycine. Our findings demonstrate that Pro10-1D effectively inhibits antigen-induced degranulation of mast cells (MCs) with IC50 values of approximately 11.6 μM for RBL-2H3 cells and 2.7 μM for bone marrow-derived MCs. Furthermore, Pro10-1D suppressed the secretion of cytokines with IC50 values of approximately 2.8 μM for IL-4 and approximately 8.6 μM for TNF-α. Mechanistically, Pro10-1D inhibited the Syk-LAT-PLCγ1 signaling pathway in MCs and decreased the activation of mitogen-activated protein kinases (MAPKs). Pro10-1D demonstrated a dose-dependent reduction in IgE-mediated passive cutaneous anaphylaxis in mice with an ED50 value of approximately 7.6 mg/kg. Further investigation revealed that Pro10-1D significantly reduced the activity of key kinases Fyn and Lyn, which are critical in the initial phase of the FcεRI-mediated signaling pathway, with IC50 values of approximately 22.6 μM for Fyn and approximately 1.5 μM for Lyn. Collectively, these findings suggest that Pro10-1D represents a novel therapeutic candidate for the treatment of IgE-mediated allergic disorders by targeting the Lyn/Fyn Src family kinases in MCs.

Inhibition of p38-MK2 pathway enhances the efficacy of microtubule inhibitors in breast cancer cells.

Microtubule-targeting agents (MTAs) have been successfully translated from basic research into clinical therapies and have been widely used as first- and second-line chemotherapy drugs for various cancers. However, current MTAs exhibit positive responses only in subsets of patients and are often accompanied by side effects due to their impact on normal cells. This underscores an urgent need to develop novel therapeutic strategies that enhance MTA efficacy while minimizing toxicity to normal tissues. In this study, we demonstrate that inhibition of the p38-MK2 (MAP kinase-activated protein kinase 2) pathway sensitizes cancer cells to MTA treatment. We utilize CMPD1, a dual-target inhibitor, to concurrently suppress the p38-MK2 pathway and microtubule dynamicity. In addition to established role as an MK2 inhibitor, we find that CMPD1 rapidly induces microtubule depolymerization, preferentially at the microtubule plus-end, leading to the inhibition of tumor growth and cancer cell invasion in both in vitro and in vivo models. Notably, 10 nM CMPD1 is sufficient to induce irreversible mitotic defects in cancer cells, but not in non-transformed RPE1 cells, highlighting its high specificity to cancer cells. We further validate that a specific p38-MK2 inhibitor significantly potentiates the efficacy of sub-clinical concentrations of MTA. In summary, our findings suggest that the p38-MK2 pathway presents a promising therapeutic target in combination with MTAs in cancer treatment.

Pim1 is Critical in T-cell-independent B-cell Response and MAPK Activation in B Cells

The Pim family consists of three members that encode a distinct class of highly conserved serine/threonine kinases. In this study, we generated and examined mice with hematopoiesis-specific deletion of Pim1 and bone marrow (BM) chimeric mice with B-cell-specific targeted deletion of Pim1. Pim1 was expressed at all stages of B-cell development and hematopoietic-specific deletion of Pim1 altered B-cell development in BM, spleen and peritoneal. However, Pim1 deficiency did not affect T-cell development. Studies of BM chimeric mice showed that Pim1 is required in a cell-intrinsic manner to maintain normal B-cell development. Pim1 deficiency led to significant changes in B cell antibody responses. Additionally, Pim1 deficiency resulted in reduced B cell receptor (BCR)-induced cell proliferation and cell cycle progression. Examination of the various BCR-activated signaling pathways in Pim1-deficient B cells reveals defective activation of mitogen-activated protein kinases (MAPKs), which are known to regulate genes involved in cell proliferation and survival. qRT-PCR analysis of BCR-engaged B cells from Pim1-deficient B cells revealed reduced expression of cyclin-dependent kinase (CDK) and cyclin genes, including CDK2, CCNB1 and CCNE1. In conclusion, Pim1 plays a crucial role in B-cell development and B cell activation.

Sinulariolide Suppresses Inflammation of Fibroblast-Like Synoviocytes in Rheumatoid Arthritis and Mitigates Collagen-Induced Arthritis Symptoms in Mice.

Rheumatoid arthritis (RA) is a systemic inflammatory disease characterized by active polyarthritis, which leads to functional loss and joint deformities. Natural compounds derived from marine organisms are considered valuable immune-modulating agents. This study aimed to assess the anti-inflammatory effect of sinulariolide, a soft coral-derived compound, on RA fibroblast-like synoviocytes and its therapeutic efficacy against collagen-induced arthritis (CIA). To determine the effects of sinulariolide on tumor necrosis factor-alpha (TNF-α)-induced inflammation, MH7A cells pre-treated with 10 ng/mL TNF-α for 24h were treated with sinulariolide. The effect of sinulariolide on proinflammatory cytokine expressions at both the mRNA and protein levels in the MH7A cells was assessed using real-time-polymerase chain reaction and enzyme-linked immunosorbent assay (ELISA). Further, we analyzed the effect of sinulariolide on the activation of mitogen-activated protein kinase (MAPK) and nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) pathways using Western blotting and the TransAM NF-κB p65 kit. To comprehensively evaluate the potential application of sinulariolide in the treatment of inflammatory diseases, we used a well-established collagen-induced arthritis (CIA) mouse model. We examined the tissue sections of the ankle joints of the mice, assessed synovial hyperplasia, inflammatory cell infiltration, and cartilage damage, and used ELISA to analyze changes in cytokine expression in the hind paw tissues. MH7A cells treated with sinulariolide showed a notable reduction in the expression of proinflammatory cytokines, which could be due to decreased activation of the MAPK and NF-kB pathways. Additionally, sinulariolide-treated mice showed significantly reduced joint swelling and lower clinical arthritis scores than those in the normal and control groups. Significant reductions in synovial hyperplasia, inflammatory cell infiltration, and cartilage damage were observed in the tissue sections of the ankle joints of the mice treated with sinulariolide. Furthermore, the expression of inflammatory cytokines in the hind paw tissue of the mice treated with sinulariolide was significantly decreased. Sinulariolide inhibited the progression of inflammation in MH7A cells. Sinulariolide treatment significantly reduced clinical arthritis symptoms and histological inflammatory responses in mice with CIA. Sinulariolide may serve as a potential therapeutic agent for RA.

Exploiting synergy of dopamine and stressful conditions in enhancing Haematococcus lacustris biomass and astaxanthin yield.

Dopamine (DA) has attracted attention because of its effects on Haematococcus lacustris biomass, astaxanthin production, and physiological responses. The alga treated with 25 μM DA combined with 1 g L-1 sodium chloride exhibited 7.63 %, 41.25 %, and 52.04 % increases in biomass (1.41 g L-1), astaxanthin content (32.37 mg/g), and astaxanthin productivity (3.51 mg L-1 d-1) respectively, compared with the salinity stress and high light. Exogenous DA treatment promoted lipid synthesis while reducing carbohydrate and protein contents. Moreover, carotenogenesis and lipogenesis-associated genes were upregulated under DA induction. Inhibition of reactive oxygen species and autophagy, along with mitogen-activated protein kinase activation, promoted astaxanthin accumulation under DA. Furthermore, DA application boosted astaxanthin biosynthesis by regulating the levels of respiratory metabolic intermediates, the γ-aminobutyric acid shunt, and important phytohormones. These findings present a potential and successful biotechnological approach for enhancing biomass and astaxanthin production in H. lacustris under stressful conditions.

RIPK2 Is Crucial for the Microglial Inflammatory Response to Bacterial Muramyl Dipeptide but Not to Lipopolysaccharide.

Receptor-interacting serine/threonine protein kinase 2 (RIPK2) is a kinase that is essential in modulating innate and adaptive immune responses. As a downstream signaling molecule for nucleotide-binding oligomerization domain 1 (NOD1), NOD2, and Toll-like receptors (TLRs), it is implicated in the signaling triggered by recognition of microbe-associated molecular patterns by NOD1/2 and TLRs. Upon activation of these innate immune receptors, RIPK2 mediates the release of pro-inflammatory factors by activating mitogen-activated protein kinases (MAPKs) and nuclear factor-kappa B (NF-κB). However, whether RIPK2 is essential for downstream inflammatory signaling following the activation of NOD1/2, TLRs, or both remains controversial. In this study, we examined the role of RIPK2 in NOD2- and TLR4-dependent signaling cascades following stimulation of microglial cells with bacterial muramyl dipeptide (MDP), a NOD2 agonist, or lipopolysaccharide (LPS), a TLR4 agonist. We utilized a highly specific proteolysis targeting chimera (PROTAC) molecule, GSK3728857A, and found dramatic degradation of RIPK2 in a concentration- and time-dependent manner. Importantly, the PROTAC completely abolished MDP-induced increases in iNOS and COX-2 protein levels and pro-inflammatory gene transcription of Nos2, Ptgs2, Il-1β, Tnfα, Il6, Ccl2, and Mmp9. However, increases in iNOS and COX-2 proteins and pro-inflammatory gene transcription induced by the TLR4 agonist, LPS, were only slightly attenuated with the GSK3728857A pretreatment. Further findings revealed that the RIPK2 PROTAC completely blocked the phosphorylation and activation of p65 NF-κB and p38 MAPK induced by MDP, but it had no effects on the phosphorylation of these two mediators triggered by LPS. Collectively, our findings strongly suggest that RIPK2 plays an essential role in the inflammatory responses of microglia to bacterial MDP but not to LPS.

Lysosomal damage triggers a p38 MAPK-dependent phosphorylation cascade to promote lysophagy via the small heat shock protein HSP27.

Maintenance of lysosomal integrity is essential for cell viability. Upon injury, lysosomes may be targeted for degradation via a selective form of autophagy known as lysophagy. The engulfment of a damaged lysosome by an autophagosome is mediated by the recruitment of adaptor proteins, including SQSTM1/p62. p62 promotes lysophagy via the formation of phase-separated condensates in a mechanism that is regulated by the heat shock protein HSP27. Here, we demonstrate a direct interaction between HSP27 and p62. We used structural modeling to predict the binding interface between HSP27 and p62 and identify several disease-associated mutations that map to this interface. We used proteomics to identify post-translational modifications of HSP27 that regulate HSP27 recruitment to stressed lysosomes, finding robust phosphorylation at several serine residues. Next, we characterized the upstream signaling mechanism leading to HSP27 phosphorylation and found that p38 mitogen-activated protein kinase (MAPK) and its effector kinase MAP kinase-activated protein kinase 2 (MK2) are activated upon lysosomal damage by the kinase mTOR and the production of intracellular reactive oxygen species (ROS). Increased ROS activates p38 MAPK, which in turn allows MK2-dependent phosphorylation of HSP27. Depletion of HSP27 or the inhibition of HSP27 phosphorylation alters the dynamics of p62 condensates on stressed lysosomes, significantly inhibiting p62-dependent lysophagy. Thus, we define a novel lysosomal quality control mechanism in which lysosomal injury triggers a p38 MAPK/MK2 signaling cascade promoting p62-dependent lysophagy. Further, this signaling cascade is activated by many cellular stressors, including oxidative and heat stress, suggesting that other forms of selective autophagy may be regulated by p38 MAPK/MK2/HSP27.

A receptor kinase senses sterol by coupling with elicitins in auxotrophic Phytophthora

Sterols are vital nutrients and signals for eukaryotic organisms. Mammalian cells are known to sense and respond to sterol status changes to maintain them within strict limits, a process associated with various human diseases. However, this process is not understood in oomycete pathogens, most of which are sterol auxotrophic and must obtain sterols from host plants. Here, we report that Phytophthora sojae SSRK1 (sterol-sensing receptor kinase 1) detects host sterols by coupling with elicitins, thereby controlling signaling and sterol absorption. Sterols are recruited by extracellular soluble elicitins, and these complexes then bind to SSRK1 to form trimolecular complexes. These complexes subsequently trigger downstream calcium influx, activation of mitogen-activated protein kinase, and transcriptome reprogramming through the receptor's kinase activity. Our data demonstrate a unique sterol sensing pathway where elicitins and a transmembrane receptor kinase SSRK1 act as coreceptors for extracellular sterols. These findings also portray a sterol-based war between oomycetes and plants, providing a unique perspective on how a pathogen detects host signals during infection.

Dapagliflozin mitigates cellular stress and inflammation through PI3K/AKT pathway modulation in cardiomyocytes, aortic endothelial cells, and stem cell-derived β cells

Dapagliflozin (DAPA), a sodium-glucose cotransporter 2 (SGLT2) inhibitor, is well-recognized for its therapeutic benefits in type 2 diabetes (T2D) and cardiovascular diseases. In this comprehensive in vitro study, we investigated DAPA’s effects on cardiomyocytes, aortic endothelial cells (AECs), and stem cell-derived beta cells (SC-β), focusing on its impact on hypertrophy, inflammation, and cellular stress. Our results demonstrate that DAPA effectively attenuates isoproterenol (ISO)-induced hypertrophy in cardiomyocytes, reducing cell size and improving cellular structure. Mechanistically, DAPA mitigates reactive oxygen species (ROS) production and inflammation by activating the AKT pathway, which influences downstream markers of fibrosis, hypertrophy, and inflammation. Additionally, DAPA’s modulation of SGLT2, the Na+/H + exchanger 1 (NHE1), and glucose transporter (GLUT 1) type 1 highlights its critical role in maintaining cellular ion balance and glucose metabolism, providing insights into its cardioprotective mechanisms. In aortic endothelial cells (AECs), DAPA exhibited notable anti-inflammatory properties by restoring AKT and phosphoinositide 3-kinase (PI3K) expression, enhancing mitogen-activated protein kinase (MAPK) activation, and downregulating inflammatory cytokines at both the gene and protein levels. Furthermore, DAPA alleviated tumor necrosis factor (TNFα)-induced inflammation and stress responses while enhancing endothelial nitric oxide synthase (eNOS) expression, suggesting its potential to preserve vascular function and improve endothelial health. Investigating SC-β cells, we found that DAPA enhances insulin functionality without altering cell identity, indicating potential benefits for diabetes management. DAPA also upregulated MAFA, PI3K, and NRF2 expression, positively influencing β-cell function and stress response. Additionally, it attenuated NLRP3 activation in inflammation and reduced NHE1 and glucose-regulated protein GRP78 expression, offering novel insights into its anti-inflammatory and stress-modulating effects. Overall, our findings elucidate the multifaceted therapeutic potential of DAPA across various cellular models, emphasizing its role in mitigating hypertrophy, inflammation, and cellular stress through the activation of the AKT pathway and other signaling cascades. These mechanisms may not only contribute to enhanced cardiac and endothelial function but also underscore DAPA’s potential to address metabolic dysregulation in T2D.Graphical abstract

Hexokinase 2 senses fructose in tumor-associated macrophages to promote colorectal cancer growth

Fructose is associated with colorectal cancer tumorigenesis and metastasis through ketohexokinase-mediated metabolism in the colorectal epithelium, yet its role in the tumor immune microenvironment remains largely unknown. Here, we show that a modest amount of fructose, without affecting obesity and associated complications, promotes colorectal cancer tumorigenesis and growth by suppressing the polarization of M1-like macrophages. Fructose inhibits M1-like macrophage polarization independently of fructose-mediated metabolism. Instead, it serves as a signal molecule to promote the interaction between hexokinase 2 and inositol 1,4,5-trisphophate receptor type 3, the predominant Ca2+ channel on the endoplasmic reticulum. The interaction reduces Ca2+ levels in cytosol and mitochondria, thereby suppressing the activation of mitogen-activated protein kinase (MAPK) and signal transducer and activator of transcription 1 (STAT1) as well as NOD-, LRR- and pyrin domain-containing protein 3 (NLRP3) inflammasome activation. Consequently, this impedes M1-like macrophage polarization. Our study highlights the critical role of fructose as a signaling molecule that impairs the polarization of M1-like macrophages for tumor growth.

Hemozoin induces malaria via activation of DNA damage, p38 MAPK and neurodegenerative pathways in a human iPSC-derived neuronal model of cerebral malaria

Malaria caused by Plasmodium falciparum infection results in severe complications including cerebral malaria (CM), in which approximately 30% of patients end up with neurological sequelae. Sparse in vitro cell culture-based experimental models which recapitulate the molecular basis of CM in humans has impeded progress in our understanding of its etiology. This study employed healthy human induced pluripotent stem cells (iPSCs)-derived neuronal cultures stimulated with hemozoin (HMZ) - the malarial toxin as a model for CM. Secretome, qRT-PCR, Metascape, and KEGG pathway analyses were conducted to assess elevated proteins, genes, and pathways. Neuronal cultures treated with HMZ showed enhanced secretion of interferon-gamma (IFN-γ), interleukin (IL)1-beta (IL-1β), IL-8 and IL-16. Enrichment analysis revealed malaria, positive regulation of cytokine production and positive regulation of mitogen-activated protein kinase (MAPK) cascade which confirm inflammatory response to HMZ exposure. KEGG assessment revealed up-regulation of malaria, MAPK and neurodegenerative diseases-associated pathways which corroborates findings from previous studies. Additionally, HMZ induced DNA damage in neurons. This study has unveiled that exposure of neuronal cultures to HMZ, activates molecules and pathways similar to those observed in CM and neurodegenerative diseases. Furthermore, our model is an alternative to rodent experimental models of CM.

T lymphocyte-dependent IL-10 down-regulates a cytokine storm driven by Toxoplasma gondii GRA24.

As a model organism in the study of immunity to infection, Toxoplasma gondii has been instrumental in establishing key principles of host anti-microbial defense and its regulation. Here, we employed an attenuated uracil auxotroph strain of Type I Toxoplasma designated OMP to further untangle the early immune response to this parasitic pathogen. Experiments using αβ T cell-deficient Tcrb-/- mice unexpectedly revealed that an intact αβ T lymphocyte compartment was essential to survive infection with OMP. Subsequent antibody depletion and knockout mouse experiments demonstrated contributions from CD4+ T cells and most predominantly CD8+ T cells in resistance. Using transgenic knockout mice, we found only a partial requirement for IFN-γ and a lack of requirement for Toll-like receptor (TLR) adaptor MyD88 in resistance. In contrast to other studies on Toxoplasma, the ability to survive OMP infection did not require IL-12p40. Surprisingly, T cell-dependent IL-10 was found to be critical for survival, and deficiency of this cytokine triggered an abnormally high systemic inflammatory response. We also found that parasite molecule GRA24, a dense granule protein that triggers TLR-independent IL-12 production, acts as a virulence factor contributing to death of OMP-infected Tcrb-/- and IL-10-/- mice. Furthermore, resistance against OMP was restored in Tcrb-/- mice via monoclonal depletion of IL-12p40, suggesting that GRA24-induced IL-12 underlies the fatal immunopathology observed. Collectively, our studies provide insight into a novel and rapidly arising T lymphocyte-dependent anti-inflammatory response to T. gondii which operates independently of MyD88 and IL-12 and that depends on the function of parasite-dense granule protein GRA24.IMPORTANCEAs a model infectious microbe and an important human pathogen, the apicomplexan Toxoplasma gondii has provided many important insights into innate and adaptive immunity to infection. We show here that a low virulence uracil auxotrophic Toxoplasma strain emerges as a virulent parasite in the absence of an intact T cell compartment. Both CD4+ and CD8+ T lymphocytes are required for optimal protection, in line with previous findings in other models of Toxoplasma infection. Nevertheless, several novel aspects of the response were identified in our study. Protection occurs independently of IL-12 and MyD88 and only partially requires IFN-γ. This is noteworthy particularly because the cytokines IL-12 and IFN-γ have previously been regarded as essential for protective immunity to T. gondii. Instead, we identified the anti-inflammatory effects of T cell-dependent IL-10 as the critical factor enabling host survival. The parasite dense granule protein GRA24, a host-directed mitogen-activated protein kinase activator, was identified as a major virulence factor in T cell-deficient hosts. Collectively, our results provide new and unexpected insights into host resistance to Toxoplasma.

EIF4F controls ERK MAPK signaling in melanomas with BRAF and NRAS mutations

The eIF4F translation initiation complex plays a critical role in melanoma resistance to clinical BRAF and MEK inhibitors. In this study, we uncover a function of eIF4F in the negative regulation of the rat sarcoma (RAS)/rapidly accelerated fibrosarcoma (RAF)/mitogen-activated protein kinase kinase (MEK)/extracellular signal-regulated kinase (ERK) mitogen-activated protein kinase (MAPK) signaling pathway. We demonstrate that eIF4F is essential for controlling ERK signaling intensity in treatment-naïve melanoma cells harboring BRAF or NRAS mutations. Specifically, the dual-specificity phosphatase DUSP6/MKP3, which acts as a negative feedback regulator of ERK activity, requires continuous production in an eIF4F-dependent manner to limit excessive ERK signaling driven by oncogenic RAF/RAS mutations. Treatment with small-molecule eIF4F inhibitors disrupts the negative feedback control of MAPK signaling, leading to ERK hyperactivation and EGR1 overexpression in melanoma cells in vitro and in vivo. Furthermore, our quantitative analyses reveal a high spare signaling capacity in the ERK pathway, suggesting that eIF4F-dependent feedback keeps the majority of ERK molecules inactive under normal conditions. Overall, our findings highlight the crucial role of eIF4F in regulating ERK signaling flux and suggest that pharmacological eIF4F inhibitors can disrupt the negative feedback control of MAPK activity in melanomas with BRAF and NRAS activating mutations.

Delphinidin or α-amyrin attenuated liver steatosis and metabolic disarrangement in rats fed a high-fat diet.

Non-alcoholic fatty liver disease (NAFLD) is a liver pathology concomitant with metabolic disarrangement. This study assessed the therapeutic impacts of delphinidin, an anthocyanin, or α-amyrin, a pentacyclic triterpenoid, on NAFLD in rats and the underlying mechanisms involved. NAFLD was established by feeding a high-fat diet (HFD) for 10 weeks, either alone or in combination with delphinidin (40 mg/kg, oral) or α-amyrin (20 mg/kg, oral). Delphinidin or α-amyrin ameliorated the metabolic and histopathological perturbations induced by HFD. These compounds markedly attenuated NAFLD-induced hepatic steatosis, as evidenced by a substantial decrease in body weight, insulin resistance, and liver and adipose tissue indices. Alongside normalization of the atherogenic index, both improved HFD-mediated abnormalities in serum lipids, liver enzymes, leptin, and ghrelin levels. Moreover, their intervention activated the NFE2 like bZIP transcription factor 2 and heme oxygenase 1 pathways and abrogated HFD-triggered activation of mitogen-activated protein kinase 1 signaling. These remedies inhibited hepatic apoptosis and modulated the gene expression of lipogenic enzymes. Furthermore, histological analysis corroborated the suppression of lipid accumulation and amelioration of hepatic architecture in the treated rats. Our findings highlight the hepatoprotective value of delphinidin or α-amyrin against NAFLD and related metabolic diseases through their insulin-sensitizing, anti-inflammatory, antioxidant, and antiapoptotic effects.