Regulation Of Signaling Research Articles

Knockdown of OsPHP1 Leads to Improved Yield Under Salinity and Drought in Rice via Regulating the Complex Set of TCS Members and Cytokinin Signalling.

Plant two-component system (TCS) is crucial for phytohormone signalling, stress response, and circadian rhythms, yet the precise role of most of the family members in rice remain poorly understood. In this study, we investigated the function of OsPHP1, a pseudo-histidine phosphotransfer protein in rice, using a functional genomics approach. OsPHP1 is localised in the nucleus and cytosol, and it exhibits strong interactions with all sensory histidine kinase proteins (OsHK1-6) and cytokinin catabolism genes. Our results demonstrate that OsPHP1 functions as a negative regulator of cytokinin signalling. Knockdown of OsPHP1 enhanced the expression of positive cytokinin signalling regulators, such as OsHKs and OsAHPs (authentic phosphotransfer proteins), while downregulating negative regulators, such as type-A response regulators (OsRRs) and cytokinin catabolism genes (CKXs). Furthermore, OsPHP1 negatively influences abiotic stress tolerance, as evidenced by the increased sensitivity of OsPHP1-OE (overexpression) lines to salinity and drought. In contrast, OsPHP1-KD (knockdown) lines showed enhanced stress resilience, with better photosynthesis, increased tiller and panicle production, higher spikelet fertility, and grain filling. The study demonstrates that OsPHP1 suppresses antioxidant and stress-responsive genes, exacerbating ion toxicity and reducing osmolyte accumulation, thereby impairing plant growth and yield under stress conditions. These findings highlight OsPHP1 as a critical modulator of plant responses to abiotic stress and suggest potential genetic targets for enhancing crop stress tolerance.

Mitochondrial unfolded protein response-dependent β-catenin signaling promotes neuroendocrine prostate cancer.

The mitochondrial unfolded protein response (UPRmt) maintains mitochondrial quality control and proteostasis under stress conditions. However, the role of UPRmt in aggressive and resistant prostate cancer is not clearly defined. We show that castration-resistant neuroendocrine prostate cancer (CRPC-NE) harbored highly dysfunctional oxidative phosphorylation (OXPHOS) Complexes. However, biochemical and protein analyses of CRPC-NE tumors showed upregulation of nuclear-encoded OXPHOS proteins and UPRmt in this lethal subset of prostate cancer suggestive of compensatory upregulation of stress signaling. Genetic deletion and pharmacological inhibition of the main chaperone of UPRmt heat shock protein 60 (HSP60) reduced neuroendocrine prostate cancer (NEPC) growth in vivo as well as reverted NEPC cells to a more epithelial-like state. HSP60-dependent aggressive NEPC phenotypes was associated with upregulation of β-catenin signaling both in cancer cells and in vivo tumors. HSP60 expression rendered enrichment of aggressive prostate cancer signatures and metastatic potential were inhibited upon suppression of UPRmt. We discovered that UPRmt promoted OXPHOS functions including mitochondrial bioenergetics in CRPC-NE via regulation of β-catenin signaling. Mitochondrial biogenesis facilitated cisplatin resistance and inhibition of UPRmt resensitizes CRPC-NE cells to cisplatin. Together, our findings demonstrated that UPRmt promotes mitochondrial health via upregulating β-catenin signaling and UPRmt represents viable therapeutic target for NEPC.

Erratum for the Research Article "Reciprocal regulation of mTORC1 signaling and ribosomal biosynthesis determines cell cycle progression in activated T cells" by T. Rosenlehner et al.

Erratum for the Research Article "Reciprocal regulation of mTORC1 signaling and ribosomal biosynthesis determines cell cycle progression in activated T cells" by T. Rosenlehner et al.

Lack of dominant-negative activity for tumor-related ZNRF3 missense mutations at endogenous levels.

ZNRF3, a negative regulator of β-catenin signaling, removes Wnt receptors from the membrane. Currently, it is unknown which tumor-associated variants can be considered driver mutations and through which mechanisms they contribute to cancer. Here we show that all truncating mutations analyzed at endogenous levels exhibit loss-of-function, with longer variants retaining partial activity. Regarding missense mutations, we show that 27/82 ZNRF3 variants in the RING and R-Spondin domain structures, lead to (partial) loss-of-function/hyperactivation. Mechanistically, defective R-Spondin domain variants appear to undergo endoplasmic-reticulum-associated degradation due to protein misfolding, leading to reduced protein levels. They fail to reach the membrane correctly, which can be partially restored for several variants by culturing cells at 27 °C. Although RING and R-Spondin domain mutations in RNF43/ZNRF3 are often considered to possess dominant-negative oncogene-like activity in cancers, our findings challenge this notion. When representative variants are heterozygously introduced into endogenous ZNRF3, their impact on β-catenin signaling mirrors that of heterozygous knockout, suggesting that the supposed dominant-negative effect is non-existent. In other words, so-called "hyperactivating" ZNRF3/RNF43 mutations behave as classical loss-of-function mutations at endogenous levels.

Inflammation-driven NFκB signaling represses Ferroportin transcription in macrophages via HDAC 1 and 3.

Anemia of Inflammation is a prevalent co-morbidity in patients with chronic inflammatory disorders. Inflammation causes hypoferremia and iron-restricted erythropoiesis by limiting Ferroportin (FPN)-mediated iron export from macrophages that recycle senescent erythrocytes. Macrophage cell surface expression of FPN is reduced by hepcidin-induced degradation and/or by repression of FPN (Slc40a1) transcription via cytokine and Toll-like receptor (TLR) stimulation. While the mechanisms underlying hepcidin-mediated control of FPN have been extensively studied, those inhibiting Slc40a1 mRNA expression remain unknown. We applied targeted RNA interference and pharmacological screens in macrophages stimulated with the TLR2/6 ligand FSL1 and identified critical signalling regulators of Slc40a1 mRNA repression downstream of TLRs and NFкB signaling. Interestingly, the NFкB regulatory hub is equally relevant for Slc40a1 mRNA repression driven by the TLR4 ligand LPS, the cytokine TNFβ/LTA and heat-killed bacteria. Mechanistically, macrophage stimulation with heat-killed Staphylococcus aureus recruits the Histone deacetylases (HDAC) 1 and 3 to the antioxidant response element (ARE) located in the Slc40a1 promoter. Accordingly, pre-treatment with a pan-HDAC inhibitor abrogates Slc40a1 mRNA repression in response to inflammatory cues, suggesting that HDACs act downstream of NFкB to repress Slc40a1 transcription. Consistently, recruitment of HDAC 1 and 3 to the Slc40a1 ARE following stimulation with heat-killed Staphylococcus aureus is dependent on NFκB signaling. These results support a model in which the ARE integrates the transcriptional responses of Slc40a1 triggered by signals from redox, metabolic and inflammatory pathways. This work identifies the long-sought mechanism of Slc40a1 transcriptional downregulation upon inflammation, paving the way for therapeutic interventions at this critical juncture.

A novel regulatory mechanism of the MAPK signaling pathway: the negative regulatory role of MKP-1 and its pathophysiological significance

he increasing number of diseases associated with aberrant MAPK signaling underscores the need for a deeper understanding of its regulatory mechanisms: the MAPK pathway is closely associated with cellular responses to various stimuli, and its dysregulation can cause pathologies. This study focuses on the regulatory mechanisms of the MAPK signaling pathway and the role of MAPK phosphatase (MKP) in regulating its activity. The goal of this study is to elucidate the specific function of MKP in the regulation of MAPK signaling and to identify potential therapeutic targets for diseases associated with MAPK dysregulation. In this study, the interaction between MKP and MAPK signaling was investigated using cellular models and biochemical experiments. The primary targets were cells expressing MAPK and MKP, and protein-protein interactions and signaling dynamics were analyzed using methods such as co-immunoprecipitation, confocal microscopy, and flow cytometry. According to this study, MKP is crucial for the exact temporal and spatial modulation of MAPK signaling through its negative regulation. An imbalance in this balance can lead to pathological conditions. This study highlights the importance of MKP in cellular homeostasis and highlights its potential as a therapeutic agent.

MHZ3: a key regulator of ethylene signaling in rice

MHZ3: a key regulator of ethylene signaling in rice

Identification of common core genes and pathways in childhood sepsis and cancer by bioinformatics analysis

IntroductionSepsis and cancer are both leading causes of death worldwide, and they share several pathophysiological characteristics. Some studies have suggested a possible association between sepsis and cancer; however, few have investigated the core genes involved in both diseases.MethodsCore genes common to both sepsis and cancer were identified using pediatric sepsis datasets (GEO: GSE26378, GSE4607, GSE8121 and GSE13904) and cancer databases (TCGA: BRCA, COADREAD, ESCA, KIRC, LIHC, LUAD, STAD). Gene Ontology (GO) and Reactome enrichment analyses, along with a protein–protein interaction (PPI) network analysis, were performed. Pharmacophore screening was applied to predict the targets of oxymatrine and ulinastatin, and potential target genes shared by both cancer and sepsis were identified. Survival analysis was performed. The association between the target genes and tumor size and number of positive lymph nodes was investigated by Pearson correlation analysis. The association between the target genes and tumor stage was investigated by Fisher’s exact test. Molecular docking analysis was performed to evaluate the affinity of the candidate drugs for their targets.ResultsA total of 641 common genes were identified. GO enrichment analysis showed that common genes were enriched in neutrophil degranulation, inflammatory response and innate immune response. Reactome enrichment analysis showed that common genes were enriched in neutrophil degranulation, interleukin-4 and interleukin-13 signaling, transcriptional regulation of granulopoiesis and interleukin-10 signaling. The PPI network showed that the top 10 core genes were TLR4, IL1B, IL10, ITGAM, TLR2, PTPRC, CDK1, FOS, MMP9 and ITGB2. The survival analysis showed that the high expression of BCAT1, CSAD, G6PD, GM2A, MMP9, PYGL and TOP2A was associated with poorer prognosis in several cancers. Molecular docking showed that oxymatrine and ulinastatin can bind to protein targets with highly stable binding.ConclusionsWe identified genes with common effects on both childhood sepsis and cancer, which provides new insights into the association between sepsis and cancer. In addition, two drugs with potential clinical application value were identified. Further studies are required to validate the role of these common core genes in sepsis and cancer and to evaluate the potential utility of these drugs.

Regulation of ROS signaling by TIGAR induces cancer-modulating responses in the tumor microenvironment

The consequences of reactive oxygen species (ROS) in cancer cells are complex and have been shown to both promote and retard tumorigenesis in different models. In mouse models of pancreatic ductal adenocarcinoma (PDAC), loss of the antioxidant defense gene Tigar results in both a reduction in the development of early pancreatic intraepithelial neoplasia and an increase in invasive and metastatic capacity, accompanied by decreased survival of mice lacking pancreatic TIGAR. We previously demonstrated that increased ROS following loss of TIGAR promotes various cancer cell-intrinsic changes that contribute to metastatic capacity, including epithelial to mesenchymal transition, enhanced migration and invasion, and an increase in ERK signaling. In this study, we show that pancreatic overexpression of TIGAR decreases metastatic capacity and migratory phenotypes in an aggressive model of PDAC, consistent with the concept that dynamic modulation of TIGAR in PDAC contributes to the development and progression of these tumors. Using TIGAR deficient and overexpressing mouse models, we find that the impact of modulation of TIGAR and ROS in PDAC cells also has a profound effect on the normal stromal cells surrounding the tumor. Loss of TIGAR promotes the production of cytokines by cancer cells that induce changes in the surrounding fibroblasts to adopt a tumor-supportive phenotype. Furthermore, these cytokines also attract macrophages that support PDAC dissemination and metastasis. Taken together our work shows that TIGAR-modulated ROS in PDAC can control cell intrinsic and extrinsic changes to impact tumor aggression.

The ARK2N (C18ORF25) Genetic Variant Is Associated with Muscle Fiber Size and Strength Athlete Status

Background: Data on the genetic factors contributing to inter-individual variability in muscle fiber size are limited. Recent research has demonstrated that mice lacking the Arkadia (RNF111) N-terminal-like PKA signaling regulator 2N (Ark2n; also known as C18orf25) gene exhibit reduced muscle fiber size, contraction force, and exercise capacity, along with defects in calcium handling within fast-twitch muscle fibers. However, the role of the ARK2N gene in human muscle physiology, and particularly in athletic populations, remains poorly understood. The aim of this study was threefold: (a) to compare ARK2N gene expression between power and endurance athletes; (b) to analyze the relationship between ARK2N gene expression and muscle fiber composition; and (c) to investigate the association between the functional variant of the ARK2N gene, muscle fiber size, and sport-related phenotypes. Results: We found that ARK2N gene expression was significantly higher in power athletes compared to endurance athletes (p = 0.042) and was positively associated with the proportion of oxidative fast-twitch (type IIA) muscle fibers in untrained subjects (p = 0.017, adjusted for age and sex). Additionally, we observed that the ARK2N rs6507691 T allele, which predicts high ARK2N gene expression (p = 3.8 × 10−12), was associated with a greater cross-sectional area of fast-twitch muscle fibers in strength athletes (p = 0.015) and was over-represented in world-class strength athletes (38.6%; OR = 2.2, p = 0.023) and wrestlers (33.8%; OR = 1.8, p = 0.044) compared to controls (22.0%). Conclusions: In conclusion, ARK2N appears to be a gene specific to oxidative fast-twitch myofibers, with its functional variant being associated with muscle fiber size and strength-athlete status.

Identification of new salicylic acid signaling regulators for root development and microbiota composition in plants.

Besides playing a crucial role in plant immunity via the nonexpressor of pathogenesis-related (NPR) proteins, increasing evidence shows that salicylic acid (SA) can also regulate plant root growth. However, the transcriptional regulatory network controlling this SA response in plant roots is still unclear. Here, we found that NPR1 and WRKY45, the central regulators of SA response in rice leaves, control only a reduced sector of the root SA signaling network. We demonstrated that SA attenuates root growth via a novel NPR1/WRKY45-independent pathway. Furthermore, using regulatory network analysis and mutant characterization, we identified a set of new NPR1/WRKY45-independent regulators that conservedly modulate the root development and root-associated microbiota composition in both Oryza sativa (monocot) and Arabidopsis thaliana (dicot) in response to SA. Our results established the SA signaling as a central element regulating plant root functions under ecologically relevant conditions. These results provide new insights to understand how regulatory networks control plant responses to abiotic and biotic stresses.

Developmental and Adult Striatal Patterning of Nociceptin Ligand Marks Striosomal Population With Direct Dopamine Projections.

Circuit influences on the midbrain dopamine system are crucial to adaptive behavior and cognition. Recent developments in the study of neuropeptide systems have enabled high-resolution investigations of the intersection of neuromodulatory signals with basal ganglia circuitry, identifying the nociceptin/orphanin FQ (N/OFQ) endogenous opioid peptide system as a prospective regulator of striatal dopamine signaling. Using a prepronociceptin-Cre reporter mouse line, we characterized highly selective striosomal patterning of Pnoc mRNA expression in mouse dorsal striatum, reflecting the early developmental expression of Pnoc. In the ventral striatum, Pnoc expression in the nucleus accumbens core was grouped in clusters akin to the distribution found in striosomes. We found that PnoctdTomato reporter cells largely comprise a population of dopamine receptor D1 (Drd1) expressing medium spiny projection neurons localized in dorsal striosomes, known to be unique among striatal projection neurons for their direct innervation of midbrain dopamine neurons. These findings provide a new understanding of the intersection of the N/OFQ system among basal ganglia circuits with particular implications for developmental regulation or wiring of striato-nigral circuits.

Characterization of tripartite motif containing 59 (TRIM59) in Epinephelus akaara: Insights into its immune involvement and functional properties in viral pathogenesis, macrophage polarization, and apoptosis regulation

The tripartite motif-containing (TRIM) superfamily is the largest family of RING-type E3 ubiquitin ligases that is conserved across the metazoan kingdom. Previous studies in mammals have demonstrated that TRIM59 possesses ubiquitin-protein ligase activity and acts as a negative regulator of NF-κB signaling. However, TRIM59 has rarely been characterized in fish. This study aimed to characterize TRIM59 from Epinephelus akaara (Eatrim59) and elucidate its structural features, expression patterns, and functional properties in innate immune responses and in the regulation of apoptosis. Eatrim59 is composed of 406 amino acids with a molecular weight of 45.84 kDa and a theoretical isoelectric point of 5.25. It comprises a conserved RING domain, a B-box motif, and a coiled-coil region. Subcellular localization analysis revealed that Eatrim59 was localized in the endoplasmic reticulum. Eatrim59 was ubiquitously expressed in all tissues examined, with the highest relative expression detected in the blood, followed by the brain and spleen. Temporal expression of Eatrim59 was dynamically regulated in response to in vivo immune stimulation by Toll-like receptor ligands and nervous necrosis virus infection. In FHM cells overexpressing Eatrim59, an increase in viral replication was observed upon infection with the Viral hemorrhagic septicemia virus. This phenomenon is attributed to Eatrim59-mediated downregulation of interferon, pro-inflammatory cytokines, and other antiviral pathways. Moreover, macrophages stably overexpressing Eatrim59 exhibited a decrease in nitric oxide production and the formation of a filamentous actin structure upon lipopolysaccharide stimulation, indicating dampened M1 polarization. Furthermore, a decrease in apoptosis was observed in Eatrim59-overexpressing FHM cells under oxidative stress induced by H2O2. In conclusion, these findings demonstrate the multifaceted role of Eatrim59 as a regulator of innate immune response and apoptosis in E. akaara.

Fluoxetine treatment reverses chronic stress-induced promotion on Fk506-binding protein 5 expression and multiple effects on glucocorticoid receptor phosphorylation in the paraventricular nucleus of mice

BackgroundFluoxetine is widely used as a first-line antidepressant. However, the molecular mechanisms for its antidepressant effects are still not fully understood. Hyperactivation of the hypothalamic-pituitary-adrenal (HPA) axis is a core pathogenic mechanism contributing to depression, and fluoxetine treatment prevents this dysfunction. The glucocorticoid receptor (GR) is a major negative feedback regulator of the HPA axis, while Fk506-binding protein 5 (Fkbp5) is a negative regulator of the GR signaling. Therefore, we examined the effects of fluoxetine on Fkbp5 and the GR signaling in the hypothalamic paraventricular nucleus (PVN) of depressed mice. MethodsMice were exposed to chronic social defeat stress (CSDS), chronic unpredictable mild stress (CUMS), or chronic restraint stress (CRS) with or without fluoxetine treatment (intraperitoneally injected, 20 mg/kg) and examined for changes in depression-like behaviors and HPA axis activity as well as Fkbp5 expression and GR phosphorylation in the PVN. We then examined if adeno-associated virus (AAV)-mediated Fkbp5 overexpression in the PVN affected the antidepressant actions of fluoxetine in mice. ResultsFluoxetine treatment significantly mitigated CSDS-, CUMS-, and CRS-induced depression-like behaviors and HPA axis hyperactivity in mice. Subsequent western blotting analyses showed that fluoxetine treatment fully reversed not only chronic stress-induced upregulation of Fkbp5 and CRH but also chronic stress-induced increase in Ser203 phosphorylation and decrease in Ser211 and Ser234 phosphorylation in GR in the PVN. Moreover, quantitative real-time reverse transcription PCR (qRT-PCR) analyses revealed that the enhanced mRNA levels of Fkbp5 and CRH in PVN neurons of mice subjected to CSDS/CUMS/CRS were also notably reversed by fluoxetine administration. Conversely, Fkbp5 overexpression in the PVN significantly eliminated the antidepressant effects of fluoxetine in mice without affecting their locomotor activity. ConclusionThese results together suggest that fluoxetine treatment reverses chronic stress-induced promotion on Fkbp5 expression and multiple effects on GR phosphorylation in the PVN of mice. Significance statementThe selective serotonin reuptake inhibitor fluoxetine (sold as Prozac) is a widely used treatment for depression, but the full spectrum of therapeutic mechanisms is still debated. Recent evidence suggests that these therapeutic mechanisms include suppression of chronic stress-activated hypothalamus–pituitary–adrenal (HPA) axis. The current study presents the first in vivo evidence showing that suppression of HPA axis hyperactivity by fluoxetine treatment involves reversal of glucocorticoid receptor (GR) phosphorylation via modulation of the GR negative regulator Fk506-binding protein 5 (Fkbp5) in the hypothalamic paraventricular nucleus (PVN). Fluoxetine treatment not only inhibited chronic stress-induced depression-like behaviors and HPA axis hyperactivity but also reversed Fkbp5 upregulation and GR phosphorylation changes in the PVN, while adeno-associated virus (AAV)-based Fkbp5 overexpression in the PVN eliminated the antidepressant effects of fluoxetine. These findings may expand our understanding of the pharmacological effects of fluoxetine, and further identify Fkbp5 as a possible target for novel antidepressants.

The identification of key genes and pathways in polycystic ovary syndrome by bioinformatics analysis of next-generation sequencing data

BackgroundPolycystic ovary syndrome (PCOS) is a reproductive endocrine disorder. The specific molecular mechanism of PCOS remains unclear. The aim of this study was to apply a bioinformatics approach to reveal related pathways or genes involved in the development of PCOS.MethodsThe next-generation sequencing (NGS) dataset GSE199225 was downloaded from the gene expression omnibus (GEO) database and NGS dataset analyzed is obtained from in vitro culture of PCOS patients’ muscle cells and muscle cells of healthy lean control women. Differentially expressed gene (DEG) analysis was performed using DESeq2. The g:Profiler was utilized to analyze the gene ontology (GO) and REACTOME pathways of the differentially expressed genes. A protein–protein interaction (PPI) network was constructed and module analysis was performed using HiPPIE and cytoscape. The miRNA-hub gene regulatory network and TF-hub gene regulatory network were constructed. The hub genes were validated by using receiver operating characteristic (ROC) curve analysis.ResultsWe have identified 957 DEG in total, including 478 upregulated genes and 479 downregulated gene. GO terms and REACTOME pathways illustrated that DEG were significantly enriched in regulation of molecular function, developmental process, interferon signaling and platelet activation, signaling, and aggregation. The top 5 upregulated hub genes including HSPA5, PLK1, RIN3, DBN1, and CCDC85B and top 5 downregulated hub genes including DISC1, AR, MTUS2, LYN, and TCF4 might be associated with PCOS. The hub gens of HSPA5 and KMT2A, together with corresponding predicted miRNAs (e.g., hsa-mir-34b-5p and hsa-mir-378a-5p), and HSPA5 and TCF4 together with corresponding predicted TF (e.g., RCOR3 and TEAD4) were found to be significantly correlated with PCOS.ConclusionsThese study uses of bioinformatics analysis of NGS data to obtain hub genes and key signaling pathways related to PCOS and its associated complications. Also provides novel ideas for finding biomarkers and treatment methods for PCOS and its associated complications.

RhoGDI in RBL-2H3 cells acts as a negative regulator of Rho GTPase signaling to inhibit granule exocytosis.

Mast cells are hematopoietic-derived immune cells that possess numerous cytoplasmic granules containing immune mediators such as cytokines and histamine. Antigen stimulation triggers mast cell granule exocytosis, releasing granule contents in a process known as degranulation. We have shown that Rho GTPase signaling is an essential component of granule exocytosis, however, the proteins that regulate Rho GTPases during this process are not well defined. Here we examined the role of Rho guanine-nucleotide dissociation inhibitors (RhoGDIs) in regulating Rho GTPase signaling using RBL-2H3 cells as a mast cell model. We found that RBL-2H3 cells express two RhoGDI isoforms which are primarily localized to the cytosol. Knockdown of RhoGDI1 and RhoGDI2 greatly reduced the levels of all Rho GTPases tested: RhoA, RhoG, Rac1, Rac2, and Cdc42. The reduction in Rho GTPase levels was accompanied by an increase in their membrane-localized fraction and an elevation in the levels of active Rho GTPases. All RhoGDI knockdown strains had altered resting cell morphology, although each strain was activation competent when stimulated. Live cell imaging revealed that the RhoGDI1/2 double knockdown (DKD) strain maintained its activated state for prolonged periods of time compared to the other strains. Only the RhoGDI1/2 DKD strain showed a significant increase in granule exocytosis. Conversely, RhoGDI overexpression in RBL-2H3 cells did not noticeably affect Rho GTPases or degranulation. Based on these results, RhoGDIs act as negative regulators of Rho GTPases during mast cell degranulation, and inhibit exocytosis by sequestering Rho GTPases in the cytosol.

Resolution of acute inflammation induced by monosodium urate crystals (MSU) through neutrophil extracellular trap-MSU aggregate-mediated negative signaling

BackgroundPolymorphonuclear neutrophils (PMN) activation by monosodium urate crystals (MSU) is crucial to acute gouty arthritis and subsequent spontaneous remission within 7–10 days. Activated PMNs release neutrophil extracellular traps (NETs) that entrap MSU crystals, forming NET-MSU aggregates. Whether NET-MSU aggregates contribute to the resolution of acute inflammation remains to be elucidated. This study uses a cell-based approach to unveil their molecular bases.MethodsAll-trans retinoic acid-differentiated HL-60 cells (dHL-60) served as surrogate PMNs. NET release from MSU-activated dHL-60 was confirmed by detecting DNA, neutrophil elastase, and citrullinated histone 3, forming large NET-MSU aggregates. NET area was measured with Fiji software after SYTOX Green staining. Released pro-inflammatory cytokines IL-8 and TNF-α, and the anti-inflammatory cytokine IL-1RA in culture supernatants were quantified to calculate the estimate inflammation score (EIS). Cellular redox state was determined by a FRET-based sensor. Expression of intracellular positive (ERK1/2) and negative (SHP-1 and SHIP-1) cytokine signaling regulators was detected by western blot. qPCR detected mRNA expressions of CISH and SOCS1–SOCS7. Flow cytometry measured neutrophil N1 (CD54) and N2 (CD182) surface markers after staining with fluorescent-conjugated antibodies.ResultsIncubating dHL-60 with MSU for 4 h maximized NET-MSU aggregate formation and acute inflammation with an EIS of 11.6. Prolonging the incubation of dHL-60 + MSU to 22 h gradually raised the EIS to 19.40 without increasing NET area, due to reduced cellular redox capacity. Adding both new dHL-60 and new MSU crystals to the culture, mimicking the clinical scenario, increased NET area but conversely suppressed EIS to 1.53, indicating acute inflammation resolution. The resolution of acute inflammation following prolonged incubation was attributed to decreases in P-ERK and increases in P-SHP-1, SOCS2, SOCS3, and CISH gene expressions, which may suppress pro-inflammatory and enhance anti-inflammatory cytokine production. Moreover, the large NET-MSU aggregates facilitated N1 to N2 polarization, crucial for accelerating inflammation resolution.ConclusionWe explored the potential molecular basis for the spontaneous resolution of MSU induced acute inflammation using a cell-based model in that huge NET-MSU aggregates frustrate the transformation of newly entering PMNs to the N2 phenotype, enhancing the production of the anti-inflammatory cytokine IL-1RA.

RNA N6-Methyladenosine-Binding Protein YTHDFs Redundantly Attenuate Cancer Immunity by Downregulating IFN-γ Signaling in Gastric Cancer.

Immunotherapy holds potential as a treatment for gastric cancer (GC), though immune checkpoint inhibitor (ICI) resistance remains an obstacle. One resistance mechanism involves defects in interferon-γ (IFN-γ) signaling, in which IFN-γ is linked to improved responsiveness to ICIs. Herein, the roles of RNA N6-methyladenosine (m6A) modifications in regulation of IFN-γ signaling and the responsiveness to ICIs are unveiled. The m6A-binding protein YTH N6-methyladenosine RNA-binding protein F1 (YTHDF1) is overexpressed in GC tissues, correlating with the suppression of cancer immunity and poorer survival rates. YTHDF1 overexpression impaired the responsiveness to IFN-γ in GC cells, and knockdown studies indicated the redundant effects of YTHDF2 and YTHDF3 with YTHDF1 in IFN-γ responsiveness. RNA immunoprecipitation sequencing revealed YTHDFs directly target interferon regulatory factor 1 (IRF1) mRNA, a master regulator of IFN-γ signaling, leading to reduced RNA stability and consequent downregulation of IFN-γ signaling. Furthermore, in mouse syngeneic tumor models, Ythdf1 depletion in cancer cells resulted in reduced tumor growth and increased tumor-infiltrating lymphocytes, which are attributed to the augmentation of IFN-γ signaling. Collectively, these findings highlight how YTHDFs modulate cancer immunity by influencing IFN-γ signaling through IRF1 regulation, suggesting their viability as therapeutic targets in cancer immunotherapy.

Promotion of TLR7-MyD88-dependent inflammation and autoimmunity in mice through stem-loop changes in Lnc-Atg16l1

Uncontrolled TLR signaling can cause inflammatory immunopathology and trigger autoimmune diseases. For example, TLR7 promotes pathogenesis of systemic lupus erythematosus. However, whether RNA structural changes affect nucleic acids-sensing TLRs signaling and impact disease progression is unclear. Here by iCLIP-seq we identify a TLR7-binding long non-coding RNA, Lnc-Atg16l1, and find that it promotes TLR7 and other MyD88-dependent TLRs signaling in various types of immune cells. Depletion of Lnc-Atg16l1 attenuates development of TLR7-linked autoimmune phenotypes in the mouse SLE model. Mechanistically, we find that Lnc-Atg16l1 binds to TLR7 at bases near U84 and MyD88 at bases around A129. The analysis of Lnc-Atg16l1 in situ structures show that it strengthens the interaction between TIR domain of TLR7 and MyD88 through specific stem-loop structure changes as a molecular scaffold after TLR7 activation to promote TLR7 downstream signaling. Therefore, we discover a mechanism for host RNA regulation of innate signaling and autoimmune disease through its structural changes. These findings provide insights into the pro-inflammatory function of self RNA in a structure-dependent manner and suggest a potential target for TLR-related autoimmune disorders.

The RALF1-FERONIA complex phosphorylates FREE1 to attenuate abscisic acid signaling during seedling establishment.

The receptor-like kinase FERONIA (FER), together with its ligand rapid alkalinization factor 1 (RALF1) peptide, plays a crucial role in regulating stress responses, including its involvement in modulating abscisic acid (ABA) signaling. FER has been shown to activate ABA Insensitive 2 (ABI2) in the cytoplasm, leading to the suppression of ABA signaling. However, its regulation of nucleus events in the ABA response remains unclear. FREE1, identified as a plant-specific component of the endosomal sorting complex required for transport (ESCRT) in eukaryotes, serves as an important negative regulator in ABA signaling. In this study, we elucidate that upon RALF1 treatment, FER phosphorylates FREE1, promoting the accumulation of FREE1 protein in the nucleus in Arabidopsis (Arabidopsis thaliana). Consequently, FREE1 suppresses ABA sensitivity by inhibiting the expression of ABA-response genes. Mutating the six identified phosphorylation sites on FREE1, mediated by FER, to non-phosphorylable residues results in reduced nucleus localization of FREE1 and increased hypersensitivity to ABA. Our data also show that these six phosphorylation sites are likely involved in regulating plant survival under salt stress. Collectively, our study not only unveils an additional function of FER in attenuating ABA signaling in the nucleus but also provides a possible insight into the role of the RALF1-FER-FREE1 module in coordinating plant growth and salt stress tolerance.