The JNK and Hippo pathways control epithelial integrity and prevent tumor initiation by regulating an overlapping transcriptome.

Epithelial organs maintain their integrity and prevent tumor initiation by actively removing defective cells, such as those that have lost apicobasal polarity. Here, we identify how transcription factors of two key signaling pathways-Jun-N-terminal kinase (JNK) and Hippo-regulate epithelial integrity by controlling transcription of an overlapping set of target genes. Targeted DamID experiments reveal that, in proliferating cells of the Drosophila melanogaster eye, the AP-1 transcription factor Jun and the Hippo pathway transcription regulators Yorkie and Scalloped bind to a common suite of target genes that promote organ growth. In defective neoplastic cells, AP-1 transcription factors repress transcription of growth genes together with the C-terminal binding protein (CtBP) co-repressor. If gene repression by AP-1/CtBP fails, neoplastic tumor growth ensues, driven by Yorkie/Scalloped. Thus, AP-1/CtBP eliminates defective cells and prevents tumor initiation by acting in parallel to Yorkie/Scalloped to repress expression of a shared transcriptome. These findings shed new light on the maintenance of epithelial integrity and tumor suppression.

Two-polarized roles of transcription factor FOSB in lung cancer progression and prognosis: dependent on p53 status.

BACKGROUND: Activator protein-1 (AP-1) represents a transcription factor family that has garnered growing attention for its extensive involvement in tumor biology. However, the roles of the AP-1 family in the evolution of lung cancer remain poorly characterized. FBJ Murine Osteosarcoma Viral Oncogene Homolog B (FOSB), a classic AP-1 family member, was previously reported to play bewilderingly two-polarized roles in non-small cell lung cancer (NSCLC) as an enigmatic double-edged sword, for which the reasons and significance warrant further elucidation. METHODS AND RESULTS: Based on the bioinformatics analysis of a large NSCLC cohort from the TCGA database, our current work found the well-known tumor suppressor gene TP53 served as a key code to decipher the two sides of FOSB - its expression indicated a positive prognosis in NSCLC patients harboring wild-type TP53 while a negative one in those harboring mutant TP53. By constructing a panel of syngeneically derived NSCLC cells expressing p53 in different statuses, the radically opposite prognostic effects of FOSB expression in NSCLC population were validated, with the TP53-R248Q mutation site emerging as particularly meaningful. Transcriptome sequencing showed that FOSB overexpression elicited diversifying transcriptomic landscapes across NSCLC cells with varying genetic backgrounds of TP53 and, combined with the validation by RT-qPCR, PREX1 (TP53-Null), IGFBP5 (TP53-WT), AKR1C3, and ALDH3A1 (TP53-R248Q) were respectively identified as p53-dependent transcriptional targets of FOSB. Subsequently, the heterogenous impacts of FOSB on the tumor biology in NSCLC cells via the above selective transcriptional targets were confirmed in vitro and in vivo. Mechanistic investigations revealed that wild-type or mutant p53 might guide FOSB to recognize and bind to distinct promoter sequences via protein-protein interactions to transcriptionally activate specific target genes, thereby creating disparate influences on the progression and prognosis in NSCLC. CONCLUSIONS: FOSB expression holds promise as a novel prognostic biomarker for NSCLC in combination with a given genetic background of TP53, and the unique interactions between FOSB and p53 may serve as underlying intervention targets for NSCLC.

Interpreting the pharmacological mechanisms of Juanbi recipe on rheumatoid arthritis through network pharmacology, molecular docking.

Background: Traditional Chinese medicine (TCM) offers the advantage of effectively relieving rheumatoid arthritis (RA) with minimal side effects. The Juanbi recipe is a commonly utilized TCM treatment for RA, yet its pharmacological mechanism remains unclear. Network pharmacology serves as an effective tool for identifying pharmaceutical ingredients and potential therapeutic targets of TCM, thereby uncovering its mechanisms. This study aimed to identify the core target genes and explore the mechanisms underlying the treatment of RA with the Juanbi recipe. Methods: This study adopted the method of network pharmacology to filter key gene targets of Juanbi recipe in RA treatment. Single-cell ribonucleic acid (RNA) sequencing data was used to screen the key genes to form the core genes of Juanbi recipe in RA treatment. The molecular docking technique was used to verify the core target genes and explore the mechanisms of Juanbi recipe in RA treatment. The RA model of mice was induced by the collagen-induced arthritis and the effect of Juanbi recipe was evaluated by intragastric administrating of extraction of Juanbi recipe. Enzyme linked immunosorbent assay was used to analysis serum inflammatory factors. Hematoxylin and eosin staining was used to evaluate inflammation and immunohistochemical (IHC) staining was used to evaluate core target genes and pathways in synovium of ankle. Results: This study screened out 281 active molecules in Juanbi recipe, found 105 key target genes of Juanbi recipe in RA treatment, and drew an "ingredient - molecule - gene" diagram. Juanbi recipe reduced the levels of serum interleukin (IL)-1 and IL-6, the inflammatory infiltration in synovium, demonstration that Juanbi recipe reduced both systemic and synovial inflammatory response. Single cell RNA sequencing data were used to select six core target genes and six core active molecules of Juanbi recipe in RA treatment. The pathways of Juanbi recipe in RA treatment involved in activator protein-1 (AP-1) and nuclear factor kappa B (NF-κB) pathway. Results of western blot and IHC staining showed that Juanbi recipe decreased the expressions of c-jun and p65, which demonstrated that Juanbi recipe inhibited the expression of AP-1 and NF-κB pathway in RA. Conclusions: The core active molecules of Juanbi recipe could inhibit key factors of AP-1 and NF-κB pathway to inhibit the inflammation, which played a protective role in RA.

CRKL Enhances YAP Signaling through Binding and JNK/JUN Pathway Activation in Liver Cancer.

The Hippo pathway transducers yes-associated protein (YAP) and WW-domain containing transcription regulator 1 (WWTR1/TAZ) are key regulators of liver tumorigenesis, promoting tumor formation and progression. Although the first inhibitors are in clinical trials, targeting the relevant upstream regulators of YAP/TAZ activity could prove equally beneficial. To identify regulators of YAP/TAZ activity in hepatocarcinoma (HCC) cells, we carried out a proximity labelling approach (BioID) coupled with mass spectrometry. We verified CRK-like proto-oncogene adaptor protein (CRKL) as a new YAP-exclusive interaction partner. CRKL is highly expressed in HCC patients, and its expression is associated with YAP activity as well as poor survival prognosis. In vitro experiments demonstrated CRKL-dependent cell survival and the loss of YAP binding induced through actin disruption. Moreover, we delineated the activation of the JNK/JUN pathway by CRKL, which promoted YAP transcription. Our data illustrate that CRKL not only promoted YAP activity through its binding but also through the induction of YAP transcription by JNK/JUN activation. This emphasizes the potential use of targeting the JNK/JUN pathway to suppress YAP expression in HCC patients.

p-Hydroxy benzaldehyde, a phenolic compound from Nostoc commune, ameliorates DSS-induced colitis against oxidative stress via the Nrf2/HO-1/NQO-1/NF-κB/AP-1 pathway.

BACKGROUND: Ulcerative colitis (UC), a chronic idiopathic inflammatory bowel disease (IBD), presents with limited current drug treatment options. Consequently, the search for safe and effective drug for UC prevention and treatment is imperative. Our prior studies have demonstrated that the phenolic compound p-Hydroxybenzaldehyde (HD) from Nostoc commune, effectively mitigates intestinal inflammation. However, the mechanisms underlying HD's anti-inflammatory effects remain unclear. PURPOSE: This study delved into the pharmacodynamics of HD and its underlying anti-inflammation mechanisms. METHODS: For in vivo experiments, dextran sodium sulfate (DSS)-induced colitis mouse model was established. In vitro inflammation model was established using lipopolysaccharide (LPS)-induced RAW264.7 and bone marrow-derived macrophages (BMDMs). The protective effect of HD against colitis was determined by monitoring clinical symptoms and histological morphology in mice. The levels of inflammatory factors and oxidative stress markers were subsequently analyzed with enzyme-linked immunosorbent assay (ELISA) and biochemical kits. Furthermore, western blotting (WB), immunofluorescence (IF), luciferase reporter gene, drug affinity reaction target stability (DARTS) assay, molecular docking, and molecular dynamics (MD) simulation were used to determine the potential target and molecular mechanism of HD. RESULTS: Our findings indicate that HD significantly alleviated the clinical symptoms and histological morphology of colitis in mice, and curtailed the production of pro-inflammatory cytokines, including TNF-α, IL-6, IFN-γ, COX-2, and iNOS. Furthermore, HD stimulated the production of SOD, CAT, and GSH-px, enhanced total antioxidant capacity (T-AOC), and reduced MDA levels. Mechanically, HD augmented the expression of Nrf2, HO-1, and NQO-1, while concurrently downregulating the phosphorylation of p65, IκBα, c-Jun, and c-Fos. ML385 and siNrf2 largely attenuated the protective effect of HD in enteritis mice and RAW 264.7 cells, as well as the promotion of HO-1 expression levels. ZnPP-mediated HO-1 knockdown reversed HD-induced inhibition of colonic inflammation. Luciferase reporter assay and IF assay confirmed the transcriptional activation of Nrf2 by HD. DARTS analysis, molecular docking, and MD results showed high binding strength, interaction efficiency and remarkable stability between Nrf2 and HD. CONCLUSION: These outcomes extend our previous research results that HD can combat oxidative stress through the Nrf2/HO-1/NQO-1/NF-κB/AP-1 pathways, effectively alleviating colitis, and propose new targets for HD to protect against intestinal barrier damage.

Lnc NBAT1 Inhibits the Proliferation and Migration of Liver Cancer Cells Through the miR-21/PDCD4/AP-1 Signaling Axis.

Long non-coding RNAs (Lnc RNAs) are proven to participate in liver cancer (LC) regulation. The regulation of miR-21 by lnc NBAT1 has been studied in other cancers. However, the effect of this regulation on LC and its specific mechanism remains unclear. Lnc NBAT1 and miR-21 expressions in clinical tissues were measured by RT-qPCR. PDCD4, AP-1, p-c-Fos, p-c-Jun, and cyclin D1 expressions were analyzed by Western blot. Overexpression of lnc NBAT1 was studied to explore its influence on malignant behaviors of Bel7402 cells and the development of LC in the xenograft mouse model (XMM). The regulation mechanism of lnc NBAT1 in LC was explored by lnc NBAT1 overexpression, miR-21 mimic treatment, or PDCD4 silencing in Bel7402 cells. Lnc NBAT1 expression was downregulated while miR-21 expression was upregulated in LC tissues and cell lines. In comparison with LX-2 cells, the expressions of PDCD4 and AP-1 were downregulated in Bel7402 cells, while those of p-c-Fos, p-c-Jun, and cyclin D1 were upregulated. Further, lnc NBAT1 was found to localize primarily in the cytoplasm of Bel7402 cells. Overexpression of lnc NBAT1 enhanced cell apoptosis, blocked the cell cycle, suppressed malignant behaviors of Bel7402 cells, and inhibited tumor progression in the XMM. Mechanistically, lnc NBAT1 functioned as a competing endogenous RNA (ceRNA) by binding to the downstream target miR-21 to stabilize the expressions of PDCD4 and AP-1, thereby inhibiting malignant behaviors of Bel7402 cells. Lnc NBAT1 suppressed malignant behaviors of LC cells through the miR-21/PDCD4/AP-1 axis. Lnc NBAT1 might be a promising biomarker for LC treatment.

Assessing the therapeutic potential of long-chain isomaltooligosaccharides in diabetic and hyperlipidemic rats.

BACKGROUND: The global rise in diabetes prevalence necessitates effective treatments. Rats, mimicking physiological changes seen in Type 2 diabetes, serve as valuable models for studying metabolic disorders. Natural health supplements, especially prebiotics, are gaining interest for improving metabolic health. Isomaltooligosaccharides (IMOs), classified as functional oligosaccharides and prebiotics, have attracted attention due to their beneficial effects on gut microbiota balance and cholesterol reduction. However, commercial IMOs often contain undesirable sugars, leading to the development of long-chain IMOs with enhanced prebiotic properties. METHODS: This study assessed the therapeutic potential of long-chain IMOs derived from Bacillus subtilis strain AP-1 compared to inulin, a widely recognized prebiotic, in addressing hyperglycemia and hyperlipidemia in rats. RESULTS: IMOs treatment effectively reduced blood sugar and triglyceride levels similarly to inulin supplementation. Proteomic analysis revealed changes in hepatic protein profiles, with upregulated pathways including glutathione metabolism, oxidative phosphorylation, and pentose and glucuronate interconversion, while pathways related to fatty acid and amino acid biosynthesis exhibited downregulation. These results suggest promising therapeutic effects of IMOs treatment on diabetes and hyperlipidemia by influencing key metabolic pathways. CONCLUSIONS: Our findings highlight the potential of long-chain IMOs as targeted interventions for metabolic disorders, warranting further investigation into their clinical applicability and mechanisms of action.

Intestinal epithelium dysfunctions cause IgA deposition in the kidney glomeruli of intestine-specific Ap1m2-deficient mice.

BACKGROUND: Intestinal epithelial cells (IECs) serve as robust barriers against potentially hostile luminal antigens and commensal microbiota. Epithelial barrier dysfunction enhances intestinal permeability, leading to leaky gut syndrome (LGS) associated with autoimmune and chronic inflammatory disorders. However, a causal relationship between LGS and systemic disorders remains unclear. Ap1m2 encodes clathrin adaptor protein complex 1 subunit mu 2, which facilitates polarized protein trafficking toward the basolateral membrane and contributes to the establishment of epithelial barrier functions. METHODS: We generated IEC-specific Ap1m2-deficient (Ap1m2ΔIEC) mice with low intestinal barrier integrity as an LSG model and examined the systemic impact. FINDINGS: Ap1m2ΔIEC mice spontaneously developed IgA nephropathy (IgAN)-like features characterized by the deposition of IgA-IgG immune complexes and complement factors in the kidney glomeruli. Ap1m2 deficiency markedly enhanced aberrantly glycosylated IgA in the serum owing to downregulation and mis-sorting of polymeric immunoglobulin receptors in IECs. Furthermore, Ap1m2 deficiency caused intestinal dysbiosis by attenuating IL-22-STAT3 signaling. Intestinal dysbiosis contributed to the pathogenesis of IgAN because antibiotic treatment reduced aberrantly glycosylated IgA production and renal IgA deposition in Ap1m2ΔIEC mice. INTERPRETATION: IEC barrier dysfunction and subsequent dysbiosis by AP-1B deficiency provoke IgA deposition in the mouse kidney. Our findings provide experimental evidence of a pathological link between LGS and IgAN. FUNDING: AMED, AMED-CREST, JSPS Grants-in-Aid for Scientific Research, JST CREST, Fuji Foundation for Protein Research, and Keio University Program for the Advancement of Next Generation Research Projects.

The activity of early-life gene regulatory elements is hijacked in aging through pervasive AP-1-linked chromatin opening.

A mechanistic connection between aging and development is largely unexplored. Through profiling age-related chromatin and transcriptional changes across 22 murine cell types, analyzed alongside previous mouse and human organismal maturation datasets, we uncovered a transcription factor binding site (TFBS) signature common to both processes. Early-life candidate cis-regulatory elements (cCREs), progressively losing accessibility during maturation and aging, are enriched for cell-type identity TFBSs. Conversely, cCREs gaining accessibility throughout life have a lower abundance of cell identity TFBSs but elevated activator protein 1 (AP-1) levels. We implicate TF redistribution toward these AP-1 TFBS-rich cCREs, in synergy with mild downregulation of cell identity TFs, as driving early-life cCRE accessibility loss and altering developmental and metabolic gene expression. Such remodeling can be triggered by elevating AP-1 or depleting repressive H3K27me3. We propose that AP-1-linked chromatin opening drives organismal maturation by disrupting cell identity TFBS-rich cCREs, thereby reprogramming transcriptome and cell function, a mechanism hijacked in aging through ongoing chromatin opening.

A multi-omics approach to reveal critical mechanisms of activator protein 1 (AP-1).

The Activator Protein 1 (AP-1) transcription factor complex plays a pivotal role in the regulation of cancer-related genes, influencing cancer cell proliferation, invasion, migration, angiogenesis, and apoptosis. Composed of multiple subunits, AP-1 has diverse roles across different cancer types and environmental contexts, but its specific mechanisms remain unclear. The advent of multi-omics approaches has shed light on a more comprehensive understanding of AP-1's role and mechanism in gene regulation. This review collates recent genome-wide data on AP-1 and provides an overview of its expression, structure, function, and interaction across different diseases. An examination of these findings can illuminate the intricate nature of AP-1 regulation and its significant involvement in the progression of different diseases. Moreover, we discuss the potential use of AP-1 as a target for individual therapy and explore the various challenges associated with such an approach. Ultimately, this review provides valuable insights into the biology of AP-1 and its potential as a therapeutic target for cancer and disease treatments.

Novel PROTAC probes targeting FOSL1 degradation to eliminate head and neck squamous cell carcinoma cancer stem cells.

Previously, we identified that AP-1 transcription factor FOSL1 is required to maintain cancer stem cells (CSCs) in HNSCC, and an AP-1 inhibitor, T-5224, can eliminate HNSCC CSCs. However, its potency is relatively low, and furthermore, whether T-5224 eradicates CSCs through targeting FOSL1 and whether FOSL1 serves as an effective target for eliminating CSCs in HNSCC, require further validation. We first found that T-5224 can bind to FOSL1 directly. As a proof-of-principle, several cereblon (CRBN)-recruiting PROTACs were designed and synthesized using T-5224 as a warhead for more effective of targeting FOSL1. The top compound can potently degrade FOSL1 in HNSCC, thereby effectively eliminating CSCs to suppress HNSCC tumorigenesis, with around 30 to 100-fold improved potency over T-5224. In summary, our study further validates FOSL1 as an effective target for eliminating CSCs in HNSCC and suggests that PROTACs may provide a unique molecular tool for the development of novel molecules for targeting FOSL1.

The WDR11 complex is a receptor for acidic-cluster-containing cargo proteins.

Vesicle trafficking is a fundamental process that allows for the sorting and transport of specific proteins (i.e., "cargoes") to different compartments of eukaryotic cells. Cargo recognition primarily occurs through coats and the associated proteins at the donor membrane. However, it remains unclear whether cargoes can also be selected at other stages of vesicle trafficking to further enhance the fidelity of the process. The WDR11-FAM91A1 complex functions downstream of the clathrin-associated AP-1 complex to facilitate protein transport from endosomes to the TGN. Here, we report the cryo-EM structure of human WDR11-FAM91A1 complex. WDR11 directly and specifically recognizes a subset of acidic clusters, which we term super acidic clusters (SACs). WDR11 complex assembly and its binding to SAC-containing proteins are indispensable for the trafficking of SAC-containing proteins and proper neuronal development in zebrafish. Our studies thus uncover that cargo proteins could be recognized in a sequence-specific manner downstream of a protein coat.

Jun and JunB members of the AP-1 complex are potential therapeutic targets for silicosis.

Silicosis is a systemic disease with predominantly diffuse fibrosis of the lungs due to prolonged inhalation of free SiO2 dust during the manufacturing process, for which there is no effective treatment. In this study, we used a combined epigenetic and transcriptomic approach to reveal the chromatin-opening features of silicosis and identify the key transcription factor activator protein 1 (AP-1) that responds to silicosis fibrosis. Therapeutic administration of an AP-1 inhibitor inhibits the PI3K/AKT signaling pathway, reduces fibrosis marker proteins, and significantly ameliorates lung fibrosis in a mouse model of silicosis. In addition, it was observed that the expression of Jun and JunB was significantly up-regulated in a TGF-ß1-induced in vitro transdifferentiation model of NIH/3T3 cells, and Co-IP confirmed that a protein complex could be formed between Jun and JunB. Mechanistically, silencing of Jun and JunB expression reversed the activation of the PI3K/AKT signaling pathway and the upregulation of fibrosis marker proteins in NIH/3 T3 cells after TGF-ß1 stimulation. Taken together, Jun/JunB is expected to be a potential therapeutic target for silicosis fibrosis.

The natural sesquiterpene lactone inulicin suppresses the production of pro-inflammatory mediators via inhibiting NF-κB and AP-1 pathways in LPS-activated macrophages.

OBJECTIVE: Inulicin is a sesquiterpene lactone in Inulae Flos which is clinically used for the treatment of inflammatory diseases, such as cough, sputum production, and vomiting. This study aimed to demonstrate the anti-inflammatory activity and the underlying mechanism of inulicin by using lipopolysaccharide (LPS)-induced in vitro and in vivo models. METHODS: LPS-stimulated RAW264.7 macrophages and mouse peritoneal macrophages (MPMs) were used for evaluating the in vitro anti-inflammatory activity of inulicin, while endotoxemia mice were used for evaluating its in vivo action. Cytokines' levels were determined by ELISA. RT-qPCR and western blot were used for assaying the mRNA and protein levels of target genes. RAW264.7 macrophages transfected with reporter plasmid pNFκB-TA-luc or pAP1-TA-luc were used for assaying the activation of NF-κB or AP-1 signaling. RESULTS: Inulicin significantly inhibited LPS-induced production of NO, IL-6, c-c motif chemokine ligand 2 (CCL2), and IL-1ß in both RAW264.7 cells and MPMs. Mechanism study indicated that it could suppress inducible nitric oxide synthase, IL-6, CCL2, and IL-1ß mRNA levels in LPS-stimulated RAW264.7 cells. Moreover, inulicin inhibited IκBα phosphorylation and prevented the nuclear translocation of p65, thereby inactivating NF-κB signaling. Concurrently, it also inhibited AP-1 signaling by reducing the phosphorylation of C-Jun N-terminal kinase (JNK) and extracellular signal-regulated kinase (ERK). In endotoxemia mice, a single intraperitoneal administration of inulicin could decrease the production of pro-inflammatory cytokines in serum and peritoneal lavage fluid. CONCLUSIONS: The present study demonstrates that inulicin possesses anti-inflammatory effects in vitro and in vivo, which suggests that inulicin might be a promising candidate for the treatment of inflammatory diseases.

Super-enhancer-driven IRF2BP2 enhances ALK activity and promotes neuroblastoma cell proliferation.

BACKGROUND: Super-enhancers (SEs) typically govern the expression of critical oncogenes and play a fundamental role in the initiation and progression of cancer. Focusing on genes that are abnormally regulated by SE in cancer may be a new strategy for understanding pathogenesis. In the context of this investigation, we have identified a previously unreported SE-driven gene IRF2BP2 in neuroblastoma (NB). METHODS: The expression and prognostic value of IRF2BP2 were detected in public databases and clinical samples. The effect of IRF2BP2 on NB cell growth and apoptosis was evaluated through in vivo and in vitro functional loss experiments. The molecular mechanism of IRF2BP2 was investigated by the study of chromatin regulatory regions and transcriptome sequencing. RESULTS: The sustained high expression of IRF2BP2 results from the activation of a novel SE established by NB master transcription factors MYCN, MEIS2 and HAND2, and they form a new complex that regulates the gene network associated with the proliferation of NB cell populations. We also observed a significant enrichment of the AP-1 family at the binding sites of IRF2BP2. Remarkably, within NB cells, AP-1 plays a pivotal role in shaping the chromatin accessibility landscape, thereby exposing the binding site for IRF2BP2. This orchestrated action enables AP-1 and IRF2BP2 to collaboratively stimulate the expression of the NB susceptibility gene ALK, thereby upholding the highly proliferative phenotype characteristic of NB. CONCLUSION: Our findings indicate that SE-driven IRF2BP2 can bind to AP-1 to maintain the survival of tumor cells via regulating chromatin accessibility of NB susceptibility gene ALK.

Autoimmune uveitis attenuated in diabetic mice through imbalance of Th1/Th17 differentiation via suppression of AP-1 signaling pathway in Th cells.

Purpose: Inflammation is involved in the pathogenesis of diabetes, however the impact of diabetes on organ-specific autoimmune diseases remains unexplored. Experimental autoimmune uveoretinitis (EAU) is a widely accepted animal model of human endogenous uveitis. In this study, we investigated the effects of diabetic conditions on the development of EAU using a mouse diabetes model. Methods: EAU was induced in wild-type C57BL/6 (WT) mice and Ins2Akita (Akita) mice with spontaneous diabetes by immunization with IRBP peptide. Clinical and histopathological examinations, and analysis of T cell activation state were conducted. In addition, alternations in the composition of immune cell types and gene expression profiles of relevant immune functions were identified using single-cell RNA sequencing. Results: The development of EAU was significantly attenuated in immunized Akita (Akita-EAU) mice compared with immunized WT (WT-EAU) mice, although T cells were fully activated in Akita-EAU mice, and the differentiation into Th17 cells and regulatory T (Treg) cells was promoted. However, Th1 cell differentiation was inhibited in Akita-EAU mice, and single-cell analysis indicated that gene expression associated AP-1 signaling pathway (JUN, FOS, and FOSB) was downregulated not only in Th1 cells but also in Th17, and Treg cells in Akita-EAU mice at the onset of EAU. Conclusions: In diabetic mice, EAU was significantly attenuated. This was related to selective inhibition of Th1 cell differentiation and downregulated AP-1 signaling pathway in both Th1 and Th17 cells.

Genome and RNA sequencing were essential to reveal cryptic intronic variants associated to defective ATP6AP1 mRNA processing.

The diagnosis of Mendelian disorders has notably advanced with integration of whole exome and genome sequencing (WES and WGS) in clinical practice. However, challenges in variant interpretation and uncovered variants by WES still leave a substantial percentage of patients undiagnosed. In this context, integrating RNA sequencing (RNA-seq) improves diagnostic workflows, particularly for WES inconclusive cases. Additionally, functional studies are often necessary to elucidate the impact of prioritized variants on gene expression and protein function. Our study focused on three unrelated male patients (P1-P3) with ATP6AP1-CDG (congenital disorder of glycosylation), presenting with intellectual disability and varying degrees of hepatopathy, glycosylation defects, and an initially inconclusive diagnosis through WES. Subsequent RNA-seq was pivotal in identifying the underlying genetic causes in P1 and P2, detecting ATP6AP1 underexpression and aberrant splicing. Molecular studies in fibroblasts confirmed these findings and identified the rare intronic variants c.289-233C > T and c.289-289G > A in P1 and P2, respectively. Trio-WGS also revealed the variant c.289-289G > A in P3, which was a de novo change in both patients. Functional assays expressing the mutant alleles in HAP1 cells demonstrated the pathogenic impact of these variants by reproducing the splicing alterations observed in patients. Our study underscores the role of RNA-seq and WGS in enhancing diagnostic rates for genetic diseases such as CDG, providing new insights into ATP6AP1-CDG molecular bases by identifying the first two deep intronic variants in this X-linked gene. Additionally, our study highlights the need to integrate RNA-seq and WGS, followed by functional validation, in routine diagnostics for a comprehensive evaluation of patients with an unidentified molecular etiology.

γ-Mangosteen, an autophagy enhancer, prevents skin-aging via activating KEAP1/NRF2 signaling and downregulating MAPKs/AP-1/NF-κB-mediated MMPs.

BACKGROUND: Mangosteens, a naturally occurring xanthones, found abundantly in mangosteen fruits. The anti-skin aging potential of γ-mangosteen (GM) remains unexplored; therefore, we investigated the UVB-induced anti-skin aging of GM via activation of autophagy. HYPOTHESIS: We hypothesized that GM exerts antioxidant and anti-aging capabilities both in vitro and in vivo through activation of autophagy as well as control of KEAP1/NRF2 signaling and MAPKs/AP-1/NF-κB-mediated MMPs pathways. METHODS: The anti-skin aging effects of GM were studied using HDF cells and a mice model. Various assays, such as DPPH, ABTS, CUPRAC, FRAP, and ROS generation, assessed antioxidant activities. Kits measured antioxidant enzymes, SA-ß-gal staining, collagen, MDA content, si-RNA experiments, and promoter assays. Western blotting evaluated protein levels of c-Jun, c-Fos, p-IκBα/ß, p-NF-κB, MAPK, MMPs, collagenase, elastin, KEAP1, NRF2, HO-1, and autophagy-related proteins. RESULTS: GM exhibited strong antioxidant, collagenase and elastase enzyme inhibition activity surpassing α- and ß-mangosteen. GM competitively inhibited elastase with a Ki value of 29.04 µM. GM orchestrated the KEAP1-NRF2 pathway, enhancing HO-1 expression, and suppressed UVB-induced ROS in HDF cells. NRF2 knockdown compromised GM's antioxidant efficacy, leading to uncontrolled ROS post-UVB. GM bolstered endogenous antioxidants, curbing lipid peroxidation in UVB-exposed HDF cells and BALB/c mice. GM effectively halted UVB-induced cell senescence, and reduced MMP-1/-9, while elevated TIMP-1 levels, augmented COL1A1, ELN, and HAS-2 expression in vitro and in vivo. Additionally, it suppressed UVB-induced MAPKs, AP-1, NF-κB phosphorylation. Pharmacological inhibitors synergistically enhanced GM's anti-skin aging potential. Moreover, GM inhibited UVB-induced mTOR activation, upregulated LC3-II, Atg5, Beclin 1, and reduced p62 in both UVB induced HDF cells and BALB/c mice, while blocking of autophagy successfully halt the GM effects against the UVB-induced increase of cell senescence, degradation of collagen through upregulation of MMP-1, underscoring GM's substantial anti-skin aging impact via autophagy induction in vitro and in vivo. CONCLUSION: Together, GM has potent antioxidant and anti-skin aging ingredients that can be used to formulate skin care products for both the nutraceutical and cosmeceutical industries.

Integrated single-cell analysis defines the epigenetic basis of castration-resistant prostate luminal cells.

Understanding prostate response to castration and androgen receptor signaling inhibitors (ARSI) is critical to improving long-term prostate cancer (PCa) patient survival. Here, we use a multi-omics approach on 229,794 single cells to create a mouse single-cell reference atlas for interpreting mouse prostate biology and castration response. Our reference atlas refines single-cell annotations and provides a chromatin context, which, when coupled with mouse lineage tracing, demonstrates that castration-resistant luminal cells are distinct from the pre-existent urethra-proximal stem/progenitor cells. Molecular pathway analysis and therapeutic studies further implicate AP1 (JUN/FOS), WNT/ß-catenin, FOXQ1, NF-κB, and JAK/STAT pathways as major drivers of castration-resistant luminal populations with relevance to human PCa. Our datasets, which can be explored through an interactive portal (https://visportal.roswellpark.org/data/tang/), can aid in developing combination treatments with ARSI for advanced PCa patients.

JUN mediates the senescence associated secretory phenotype and immune cell recruitment to prevent prostate cancer progression.

BACKGROUND: Prostate cancer develops through malignant transformation of the prostate epithelium in a stepwise, mutation-driven process. Although activator protein-1 transcription factors such as JUN have been implicated as potential oncogenic drivers, the molecular programs contributing to prostate cancer progression are not fully understood. METHODS: We analyzed JUN expression in clinical prostate cancer samples across different stages and investigated its functional role in a Pten-deficient mouse model. We performed histopathological examinations, transcriptomic analyses and explored the senescence-associated secretory phenotype in the tumor microenvironment. RESULTS: Elevated JUN levels characterized early-stage prostate cancer and predicted improved survival in human and murine samples. Immune-phenotyping of Pten-deficient prostates revealed high accumulation of tumor-infiltrating leukocytes, particularly innate immune cells, neutrophils and macrophages as well as high levels of STAT3 activation and IL-1ß production. Jun depletion in a Pten-deficient background prevented immune cell attraction which was accompanied by significant reduction of active STAT3 and IL-1ß and accelerated prostate tumor growth. Comparative transcriptome profiling of prostate epithelial cells revealed a senescence-associated gene signature, upregulation of pro-inflammatory processes involved in immune cell attraction and of chemokines such as IL-1ß, TNF-α, CCL3 and CCL8 in Pten-deficient prostates. Strikingly, JUN depletion reversed both the senescence-associated secretory phenotype and senescence-associated immune cell infiltration but had no impact on cell cycle arrest. As a result, JUN depletion in Pten-deficient prostates interfered with the senescence-associated immune clearance and accelerated tumor growth. CONCLUSIONS: Our results suggest that JUN acts as tumor-suppressor and decelerates the progression of prostate cancer by transcriptional regulation of senescence- and inflammation-associated genes. This study opens avenues for novel treatment strategies that could impede disease progression and improve patient outcomes.