TCR Transgenic Mice Reveal Stepwise, Multi-site Acquisition of the Distinctive Fat-Treg Phenotype.

Visceral adipose tissue (VAT) hosts a population of regulatory T (Treg) cells, with a unique phenotype, that controls local and systemic inflammation and metabolism. Generation of a T cell receptor transgenic mouse line, wherein VAT Tregs are highly enriched, facilitated study of their provenance, dependencies, and activities. We definitively established a role for T cell receptor specificity, uncovered an unexpected function for the primordial Treg transcription-factor, Foxp3, evidenced a cell-intrinsic role for interleukin-33 receptor, and ordered these dependencies within a coherent scenario. Genesis of the VAT-Treg phenotype entailed a priming step in the spleen, permitting them to exit the lymphoid organs and surveil nonlymphoid tissues, and a final diversification process within VAT, in response to microenvironmental cues. Understanding the principles of tissue-Treg biology is a prerequisite for precision-targeting strategies.

Obesity-induced dysregulation of skin-resident PPARγ+ Treg cells promotes IL-17A-mediated psoriatic inflammation.

Obesity is a major risk factor for psoriasis, but how obesity disrupts the regulatory mechanisms that keep skin inflammation in check is unclear. Here, we found that skin was enriched with a unique population of CD4+Foxp3+ regulatory T (Treg) cells expressing the nuclear receptor peroxisome proliferation-activated receptor gamma (PPARγ). PPARγ drove a distinctive transcriptional program and functional suppression of IL-17A+ γδ T cell-mediated psoriatic inflammation. Diet-induced obesity, however, resulted in a reduction of PPARγ+ skin Treg cells and a corresponding loss of control over IL-17A+ γδ T cell-mediated inflammation. Mechanistically, PPARγ+ skin Treg cells preferentially took up elevated levels of long-chain free fatty acids in obese mice, which led to cellular lipotoxicity, oxidative stress, and mitochondrial dysfunction. Harnessing the anti-inflammatory properties of these PPARγ+ skin Treg cells could have therapeutic potential for obesity-associated inflammatory skin diseases.

Adipose Tissue Macrophage-Derived Exosomal miRNAs Can Modulate In Vivo and In Vitro Insulin Sensitivity.

MiRNAs are regulatory molecules that can be packaged into exosomes and secreted from cells. Here, we show that adipose tissue macrophages (ATMs) in obese mice secrete miRNA-containing exosomes (Exos), which cause glucose intolerance and insulin resistance when administered to lean mice. Conversely, ATM Exos obtained from lean mice improve glucose tolerance and insulin sensitivity when administered to obese recipients. miR-155 is one of the miRNAs overexpressed in obese ATM Exos, and earlier studies have shown that PPARγ is a miR-155 target. Our results show that miR-155KO animals are insulin sensitive and glucose tolerant compared to controls. Furthermore, transplantation of WT bone marrow into miR-155KO mice mitigated this phenotype. Taken together, these studies show that ATMs secrete exosomes containing miRNA cargo. These miRNAs can be transferred to insulin target cell types through mechanisms of paracrine or endocrine regulation with robust effects on cellular insulin action, in vivo insulin sensitivity, and overall glucose homeostasis.

Isoform-specific functions of PPARγ in gene regulation and metabolism.

Peroxisome proliferator-activated receptor γ (PPARγ) is a nuclear receptor that is a vital regulator of adipogenesis, insulin sensitivity, and lipid metabolism. Activation of PPARγ by antidiabetic thiazolidinediones (TZD) reverses insulin resistance but also leads to weight gain that limits the use of these drugs. There are two main PPARγ isoforms, but the specific functions of each are not established. Here we generated mouse lines in which endogenous PPARγ1 and PPARγ2 were epitope-tagged to interrogate isoform-specific genomic binding, and mice deficient in either PPARγ1 or PPARγ2 to assess isoform-specific gene regulation. Strikingly, although PPARγ1 and PPARγ2 contain identical DNA binding domains, we uncovered isoform-specific genomic binding sites in addition to shared sites. Moreover, PPARγ1 and PPARγ2 regulated a different set of genes in adipose tissue depots, suggesting distinct roles in adipocyte biology. Indeed, mice with selective deficiency of PPARγ1 maintained body temperature better than wild-type or PPARγ2-deficient mice. Most remarkably, although TZD treatment improved glucose tolerance in mice lacking either PPARγ1 or PPARγ2, the PPARγ1-deficient mice were protected from TZD-induced body weight gain compared with PPARγ2-deficient mice. Thus, PPARγ isoforms have specific and separable metabolic functions that may be targeted to improve therapy for insulin resistance and diabetes.

ZFP423 controls EBF2 coactivator recruitment and PPARγ occupancy to determine the thermogenic plasticity of adipocytes.

Energy-storing white adipocytes maintain their identity by suppressing the energy-burning thermogenic gene program of brown and beige adipocytes. Here, we reveal that the protein-protein interaction between the transcriptional coregulator ZFP423 and brown fat determination factor EBF2 is essential for restraining the thermogenic phenotype of white adipose tissue (WAT). Disruption of the ZFP423-EBF2 protein interaction through CRISPR-Cas9 gene editing triggers widespread "browning" of WAT in adult mice. Mechanistically, ZFP423 recruits the NuRD corepressor complex to EBF2-bound thermogenic gene enhancers. Loss of adipocyte Zfp423 induces an EBF2 NuRD-to-BAF coregulator switch and a shift in PPARγ occupancy to thermogenic genes. This shift in PPARγ occupancy increases the antidiabetic efficacy of the PPARγ agonist rosiglitazone in obesity while diminishing the unwanted weight-gaining effect of the drug. These data indicate that ZFP423 controls EBF2 coactivator recruitment and PPARγ occupancy to determine the thermogenic plasticity of adipocytes and highlight the potential of therapeutically targeting transcriptional brakes to induce beige adipocyte biogenesis in obesity.

Unraveling the complexity of thermogenic remodeling of white fat reveals potential antiobesity therapies.

Adipose tissue is a complex organ consisting of a mixture of mature adipocytes and stromal vascular cells. It displays a remarkable ability to adapt to environmental and dietary cues by changing its morphology and metabolic capacity. This plasticity is demonstrated by the emergence of interspersed thermogenic beige adipocytes within white depots in response to catecholamines secretion. Coordinated cellular interaction between different cell types within the tissue and a fine-tuned transcriptional program synergistically take place to promote beige remodeling. However, both cell-cell interactions and molecular mechanisms governing beige adipocyte appearance and maintenance are poorly understood. In this and the previous issue of Genes & Development, Shao and colleagues (pp. 1461-1474) and Shan and colleagues (pp. 1333-1338) advance our understanding of these issues and, in doing so, highlight potential therapeutic strategies to combat obesity-associated diseases.

The Nuclear Receptor PPARγ Controls Progressive Macrophage Polarization as a Ligand-Insensitive Epigenomic Ratchet of Transcriptional Memory.

Macrophages polarize into distinct phenotypes in response to complex environmental cues. We found that the nuclear receptor PPARγ drove robust phenotypic changes in macrophages upon repeated stimulation with interleukin (IL)-4. The functions of PPARγ on macrophage polarization in this setting were independent of ligand binding. Ligand-insensitive PPARγ bound DNA and recruited the coactivator P300 and the architectural protein RAD21. This established a permissive chromatin environment that conferred transcriptional memory by facilitating the binding of the transcriptional regulator STAT6 and RNA polymerase II, leading to robust production of enhancer and mRNAs upon IL-4 re-stimulation. Ligand-insensitive PPARγ binding controlled the expression of an extracellular matrix remodeling-related gene network in macrophages. Expression of these genes increased during muscle regeneration in a mouse model of injury, and this increase coincided with the detection of IL-4 and PPARγ in the affected tissue. Thus, a predominantly ligand-insensitive PPARγ:RXR cistrome regulates progressive and/or reinforcing macrophage polarization.

Naa10p Inhibits Beige Adipocyte-Mediated Thermogenesis through N-α-acetylation of Pgc1α.

Diet-induced obesity can be caused by impaired thermogenesis of beige adipocytes, the brown-like adipocytes in white adipose tissue (WAT). Promoting brown-like features in WAT has been an attractive therapeutic approach for obesity. However, the mechanism underlying beige adipocyte formation is largely unknown. N-α-acetyltransferase 10 protein (Naa10p) catalyzes N-α-acetylation of nascent proteins, and overexpression of human Naa10p is linked to cancer development. Here, we report that both conventional and adipose-specific Naa10p deletions in mice result in increased energy expenditure, thermogenesis, and beige adipocyte differentiation. Mechanistically, Naa10p acetylates the N terminus of Pgc1α, which prevents Pgc1α from interacting with Pparγ to activate key genes, such as Ucp1, involved in beige adipocyte function. Consistently, fat tissues of obese human individuals show higher NAA10 expression. Thus, Naa10p-mediated N-terminal acetylation of Pgc1α downregulates thermogenic gene expression, making inhibition of Naa10p enzymatic activity a potential strategy for treating obesity.

NRF2 is a spatiotemporal metabolic hub essential for the polyfunctionality of Th2 cells.

Upon encountering allergens, CD4+ T cells differentiate into IL-4-producing Th2 cells in lymph nodes, which later transform into polyfunctional Th2 cells producing IL-5 and IL-13 in inflamed tissues. However, the precise mechanism underlying their polyfunctionality remains elusive. In this study, we elucidate the pivotal role of NRF2 in polyfunctional Th2 cells in murine models of allergic asthma and in human Th2 cells. We found that an increase in reactive oxygen species (ROS) in immune cells infiltrating the lungs is necessary for the development of eosinophilic asthma and polyfunctional Th2 cells in vivo. Deletion of the ROS sensor NRF2 specifically in T cells, but not in dendritic cells, significantly abolished eosinophilia and polyfunctional Th2 cells in the airway. Mechanistically, NRF2 intrinsic to T cells is essential for inducing optimal oxidative phosphorylation and glycolysis capacity, thereby driving Th2 cell polyfunctionality independently of IL-33, partially by inducing PPARγ. Treatment with an NRF2 inhibitor leads to a substantial decrease in polyfunctional Th2 cells and subsequent eosinophilia in mice and a reduction in the production of Th2 cytokines from peripheral blood mononuclear cells in asthmatic patients. These findings highlight the critical role of Nrf2 as a spatial and temporal metabolic hub that is essential for polyfunctional Th2 cells, suggesting potential therapeutic implications for allergic diseases.

DIDO is necessary for the adipogenesis that promotes diet-induced obesity.

The prevalence of overweight and obesity continues to rise in the population worldwide. Because it is an important predisposing factor for cancer, cardiovascular diseases, diabetes mellitus, and COVID-19, obesity reduces life expectancy. Adipose tissue (AT), the main fat storage organ with endocrine capacity, plays fundamental roles in systemic metabolism and obesity-related diseases. Dysfunctional AT can induce excess or reduced body fat (lipodystrophy). Dido1 is a marker gene for stemness; gene-targeting experiments compromised several functions ranging from cell division to embryonic stem cell differentiation, both in vivo and in vitro. We report that mutant mice lacking the DIDO N terminus show a lean phenotype. This consists of reduced AT and hypolipidemia, even when mice are fed a high-nutrient diet. DIDO mutation caused hypothermia due to lipoatrophy of white adipose tissue (WAT) and dermal fat thinning. Deep sequencing of the epididymal white fat (Epi WAT) transcriptome supported Dido1 control of the cellular lipid metabolic process. We found that, by controlling the expression of transcription factors such as C/EBPα or PPARγ, Dido1 is necessary for adipocyte differentiation, and that restoring their expression reestablished adipogenesis capacity in Dido1 mutants. Our model differs from other lipodystrophic mice and could constitute a new system for the development of therapeutic intervention in obesity.

Epigenomic regulation of macrophage polarization: Where do the nuclear receptors belong?

Our laboratory has a long-standing research interest in understanding how lipid-activated transcription factors, nuclear hormone receptors, contribute to dendritic cell and macrophage gene expression regulation, subtype specification, and responses to a changing extra and intracellular milieu. This journey in the last more than two decades took us from identifying target genes for various RXR heterodimers to systematically mapping nuclear receptor-mediated pathways in dendritic cells to identifying hierarchies of transcription factors in alternative polarization in macrophages to broaden the role of nuclear receptors beyond strictly ligand-regulated gene expression. We detail here the milestones of the road traveled and draw conclusions regarding the unexpectedly broad role of nuclear hormone receptors as epigenomic components of dendritic cell and macrophage gene regulation as we are getting ready for the next challenges.

Loss of PPARγ activity characterizes early protumorigenic stromal reprogramming and dictates the therapeutic window of opportunity.

Although robustly expressed in the disease-free (DF) breast stroma, CD36 is consistently absent from the stroma surrounding invasive breast cancers (IBCs). In this study, we primarily observed CD36 expression in adipocytes and intralobular capillaries within the DF breast. Larger vessels concentrated in interlobular regions lacked CD36 and were instead marked by the expression of CD31. When evaluated in perilesional capillaries surrounding ductal carcinoma in situ, a nonobligate IBC precursor, CD36 loss was more commonly observed in lesions associated with subsequent IBC. Peroxisome proliferator-activated receptor γ (PPARγ) governs the expression of CD36 and genes involved in differentiation, metabolism, angiogenesis, and inflammation. Coincident with CD36 loss, we observed a dramatic suppression of PPARγ and its target genes in capillary endothelial cells (ECs) and pericytes, which typically surround and support the stability of the capillary endothelium. Factors present in conditioned media from malignant cells repressed PPARγ and its target genes not only in cultured ECs and pericytes but also in adipocytes, which require PPARγ for proper differentiation. In addition, we identified a role for PPARγ in opposing the transition of pericytes toward a tumor-supportive myofibroblast phenotype. In mouse xenograft models, early intervention with rosiglitazone, a PPARγ agonist, demonstrated significant antitumor effects; however, following the development of a palpable tumor, the antitumor effects of rosiglitazone were negated by the repression of PPARγ in the mouse stroma. In summary, PPARγ activity in healthy tissues places several stromal cell types in an antitumorigenic state, directly inhibiting EC proliferation, maintaining adipocyte differentiation, and suppressing the transition of pericytes into tumor-supportive myofibroblasts.

PPARγ is a nexus controlling alternative activation of macrophages via glutamine metabolism.

The nuclear receptor peroxisome proliferator-activated receptor γ (PPARγ) is known to regulate lipid metabolism in many tissues, including macrophages. Here we report that peritoneal macrophage respiration is enhanced by rosiglitazone, an activating PPARγ ligand, in a PPARγ-dependent manner. Moreover, PPARγ is required for macrophage respiration even in the absence of exogenous ligand. Unexpectedly, the absence of PPARγ dramatically affects the oxidation of glutamine. Both glutamine and PPARγ have been implicated in alternative activation (AA) of macrophages, and PPARγ was required for interleukin 4 (IL4)-dependent gene expression and stimulation of macrophage respiration. Indeed, unstimulated macrophages lacking PPARγ contained elevated levels of the inflammation-associated metabolite itaconate and express a proinflammatory transcriptome that, remarkably, phenocopied that of macrophages depleted of glutamine. Thus, PPARγ functions as a checkpoint, guarding against inflammation, and is permissive for AA by facilitating glutamine metabolism. However, PPARγ expression is itself markedly increased by IL4. This suggests that PPARγ functions at the center of a feed-forward loop that is central to AA of macrophages.

Testicular ACE regulates sperm metabolism and fertilization through the transcription factor PPARγ.

Testis angiotensin-converting enzyme (tACE) plays a critical role in male fertility, but the mechanism is unknown. By using ACE C-domain KO (CKO) mice which lack tACE activity, we found that ATP in CKO sperm was 9.4-fold lower than WT sperm. Similarly, an ACE inhibitor (ACEi) reduced ATP production in mouse sperm by 72%. Metabolic profiling showed that tACE inactivation severely affects oxidative metabolism with decreases in several Krebs cycle intermediates including citric acid, cis-aconitic acid, NAD, α-ketoglutaric acid, succinate, and L-malic acid. We found that sperms lacking tACE activity displayed lower levels of oxidative enzymes (CISY, ODO1, MDHM, QCR2, SDHA, FUMH, CPT2, and ATPA) leading to a decreased mitochondrial respiration rate. The reduced energy production in CKO sperms leads to defects in their physiological functions including motility, acrosine activity, and fertilization in vitro and in vivo. Male mice treated with ACEi show severe impairment in reproductive capacity when mated with female mice. In contrast, an angiotensin II receptor blocker (ARB) had no effect. CKO sperms express significantly less peroxisome proliferators-activated receptor gamma (PPARγ) transcription factor, and its blockade eliminates the functional differences between CKO and WT sperms, indicating PPARγ might mediate the effects of tACE on sperm metabolism. Finally, in a cohort of human volunteers, in vitro treatment with the ramipril or a PPARγ inhibitor reduced ATP production in human sperm and hence its motility and acrosine activity. These findings may have clinical significance since millions of people take ACEi daily, including men who are reproductively active.

The anti-tumor effects of cosmosiin through regulating AhR/CYP1A1-PPARγ in breast cancer.

Breast cancer is one of the threatening malignant tumors with the highest mortality and incidence rate over the world. There are a lot of breast cancer patients dying every year due to the lack of effective and safe therapeutic drugs. Therefore, it is highly necessary to develop more effective drugs to overcome breast cancer. As a glycoside derivative of apigenin, cosmosiin is characterized by low toxicity, high water solubility, and wide distribution in nature. Additionally, cosmosiin has been shown to perform anti-tumor effects in cervical cancer, hepatocellular carcinoma and melanoma. However, its pharmacological effects on breast cancer and its mechanisms are still unknown. In our study, the anti-breast cancer effect and mechanism of cosmosiin were investigated by using breast cancer models in vivo and in vitro. The results showed that cosmosiin inhibited the proliferation, migration, and adhesion of breast cancer cells in vitro and suppressed the growth of tumor in vivo through binding with AhR and inhibiting it, thus regulating the downstream CYP1A1/AMPK/mTOR and PPARγ/Wnt/ß-catenin signaling pathways. Collectively, our findings have made contribution to the development of novel drugs against breast cancer by targeting AhR and provided a new direction for the research in the field of anti-breast cancer therapy.

Functional role of Ash2l in oxLDL induced endothelial dysfunction and atherosclerosis.

Endothelial injury and dysfunction in the artery wall fuel the process of atherosclerosis. As a key epigenetic regulator, Ash2l (Absent, small, or homeotic-Like 2) is involved in regulating vascular injury and its complications. However, the role of Ash2l in atherosclerosis has not yet been fully elucidated. Here, we found increased Ash2l expression in high-cholesterol diet-fed ApoE-/- mice and oxidized LDL (oxLDL) treated endothelial cells (ECs). Furthermore, Ash2l promoted the scavenger receptors transcription by catalyzing histone H3 lysine 4 (H3K4) trimethylation at the promoter region of transcription factor peroxisome proliferator-activated receptor-γ (PPARγ) and triggered the activation of the pro-inflammatory nuclear factor-kappa B (NF-κB) by enhancing interaction between CD36 and toll-like receptor 4 (TLR4). Meanwhile, enhanced expression of scavenger receptors drove more oxLDL uptake by ECs. In vivo studies revealed that ECs-specific Ash2l knockdown reduced atherosclerotic lesion formation and promoted fibrous cap stability in the aorta of ApoE-/- mice, which was partly associated with a reduced endothelial activation by suppressing scavenger receptors and the uptake of lipids by ECs. Collectively, our findings identify Ash2l as a novel regulator that mediates endothelial injury and atherosclerosis. Targeting Ash2l may provide valuable insights for developing novel therapeutic candidates for atherosclerosis.

DNMT3a-mediated methylation of PPARγ promote intervertebral disc degeneration by regulating the NF-κB pathway.

Intervertebral disc degeneration (IVDD) is a common chronic musculoskeletal disease that causes chronic low back pain and imposes an immense financial strain on patients. The pathological mechanisms underlying IVDD have not been fully elucidated. The development of IVDD is closely associated with abnormal epigenetic changes, suggesting that IVDD progression may be controlled by epigenetic mechanisms. Consequently, this study aimed to investigate the role of epigenetic regulation, including DNA methyltransferase 3a (DNMT3a)-mediated methylation and peroxisome proliferator-activated receptor γ (PPARγ) inhibition, in IVDD development. The expression of DNMT3a and PPARγ in early and late IVDD of nucleus pulposus (NP) tissues was detected using immunohistochemistry and western blotting analyses. Cellularly, DNMT3a inhibition significantly inhibited IL-1ß-induced apoptosis and extracellular matrix (ECM) degradation in rat NP cells. Pretreatment with T0070907, a specific inhibitor of PPARγ, significantly reversed the anti-apoptotic and ECM degradation effects of DNMT3a inhibition. Mechanistically, DNMT3a modified PPARγ promoter hypermethylation to activate the nuclear factor-κB (NF-κB) pathway. DNMT3a inhibition alleviated IVDD progression. Conclusively, the results of this study show that DNMT3a activates the NF-κB pathway by modifying PPARγ promoter hypermethylation to promote apoptosis and ECM degradation. Therefore, we believe that the ability of DNMT3a to mediate the PPARγ/NF-κB axis may provide new ideas for the potential pathogenesis of IVDD and may become an attractive target for the treatment of IVDD.

Selective PPARγ modulator diosmin improves insulin sensitivity and promotes browning of white fat.

Peroxisome proliferator-activated receptor γ (PPARγ) is a master regulator of adipocyte differentiation, glucolipid metabolism, and inflammation. Thiazolidinediones are PPARγ full agonists with potent insulin-sensitizing effects, whereas their oral usage is restricted because of unwanted side effects, including obesity and cardiovascular risks. Here, via virtual screening, microscale thermophoresis analysis, and molecular confirmation, we demonstrate that diosmin, a natural compound of wide and long-term clinical use, is a selective PPARγ modulator that binds to PPARγ and blocks PPARγ phosphorylation with weak transcriptional activity. Local diosmin administration in subcutaneous fat (inguinal white adipose tissue [iWAT]) improved insulin sensitivity and attenuated obesity via enhancing browning of white fat and energy expenditure. Besides, diosmin ameliorated inflammation in WAT and liver and reduced hepatic steatosis. Of note, we determined that iWAT local administration of diosmin did not exhibit obvious side effects. Taken together, the present study demonstrated that iWAT local delivery of diosmin protected mice from diet-induced insulin resistance, obesity, and fatty liver by blocking PPARγ phosphorylation, without apparent side effects, making it a potential therapeutic agent for the treatment of metabolic diseases.

PPARγ inhibition promotes osteogenic differentiation of bone marrow mesenchymal stem cells and fracture healing.

This study aimed to explore the effects of peroxisome proliferator-activated receptor γ (PPARγ) inhibition on fracture healing of nonunion and the underlying mechanisms. Bone marrow mesenchymal stem cells (BMSCs) were treated with PPARγ antagonist GW9662 (5 µM, 10 µM). Alkaline phosphatase (ALP) staining and Alizarin Red S was used to assess early stage of osteogenesis and osteogenic differentiation. GW9662 (1 mg/kg/day) were administered intraperitoneally into the rats with bone fracture. Bone healing processes in the rat femur fracture model were recorded and assessed by radiographic methods on Weeks 8, 14, and 20 postoperation. Osteogenesis and angiogenesis at the fracture sites were evaluated by radiographic and histological methods on postoperative Week 20. GW9662 treatment increased ALP activity and Alp mRNA expression in rat BMSCs. Moreover, GW9662 administration increased matrix mineralization and mRNA and protein levels of Bmp2 and Runx2 in the BMSCs. In addition, GW9662 treatment improved radiographic score in the fracture rats and increased osteogenesis-related proteins, including type I collagen, osteopontin, and osteoglycin, in the bone tissues of the fracture sites. In conclusion, PPARγ inhibition promotes osteogenic differentiation of rat BMSCs, as well as improves the fracture healing of rats through Bmp2/Runx2 signaling pathway in the rat model of bone fracture.

BRD4 promotes gouty arthritis through MDM2-mediated PPARγ degradation and pyroptosis.

BACKGROUND: Gouty arthritis (GA) is characterized by monosodium urate (MSU) crystal accumulation that instigates NLRP3-mediated pyroptosis; however, the underlying regulatory mechanisms have yet to be fully elucidated. The present research endeavors to elucidate the regulatory mechanisms underpinning this MSU-induced pyroptotic cascade in GA. METHODS: J774 cells were exposed to lipopolysaccharide and MSU crystals to establish in vitro GA models, whereas C57BL/6 J male mice received MSU crystal injections to mimic in vivo GA conditions. Gene and protein expression levels were evaluated using real-time quantitative PCR, Western blotting, and immunohistochemical assays. Inflammatory markers were quantified via enzyme-linked immunosorbent assays. Pyroptosis was evaluated using immunofluorescence staining for caspase-1 and flow cytometry with caspase-1/propidium iodide staining. The interaction between MDM2 and PPARγ was analyzed through co-immunoprecipitation assays, whereas the interaction between BRD4 and the MDM2 promoter was examined using chromatin immunoprecipitation and dual-luciferase reporter assays. Mouse joint tissues were histopathologically evaluated using hematoxylin and eosin staining. RESULTS: In GA, PPARγ was downregulated, whereas its overexpression mitigated NLRP3 inflammasome activation and pyroptosis. MDM2, which was upregulated in GA, destabilized PPARγ through the ubiquitin-proteasome degradation pathway, whereas its silencing attenuated NLRP3 activation by elevating PPARγ levels. Concurrently, BRD4 was elevated in GA and exacerbated NLRP3 activation and pyroptosis by transcriptionally upregulating MDM2, thereby promoting PPARγ degradation. In vivo experiments showed that BRD4 silencing ameliorated GA through this MDM2-PPARγ-pyroptosis axis. CONCLUSION: BRD4 promotes inflammation and pyroptosis in GA through MDM2-mediated PPARγ degradation, underscoring the therapeutic potential of targeting this pathway in GA management.