Succinyl-CoA ligase ADP-forming subunit beta promotes stress granule assembly to regulate redox and drive cancer metastasis.

Although recent studies demonstrate active mitochondrial metabolism in cancers, the precise mechanisms through which mitochondrial factors contribute to cancer metastasis remain elusive. Through a customized mitochondrion RNAi screen, we identified succinyl-CoA ligase ADP-forming subunit beta (SUCLA2) as a critical anoikis resistance and metastasis driver in human cancers. Mechanistically, SUCLA2, but not the alpha subunit of its enzyme complex, relocates from mitochondria to the cytosol upon cell detachment where SUCLA2 then binds to and promotes the formation of stress granules. SUCLA2-mediated stress granules facilitate the protein translation of antioxidant enzymes including catalase, which mitigates oxidative stress and renders cancer cells resistant to anoikis. We provide clinical evidence that SUCLA2 expression correlates with catalase levels as well as metastatic potential in lung and breast cancer patients. These findings not only implicate SUCLA2 as an anticancer target, but also provide insight into a unique, noncanonical function of SUCLA2 that cancer cells co-opt to metastasize.

Rosiglitazone-dependent dissociation of HuR from PPAR-γ regulates adiponectin expression at the posttranscriptional level.

Peroxisome proliferator-activated receptor (PPAR)-γ has been implicated as a key player in the regulation of adiponectin levels via both transcriptional and posttranscriptional mechanisms. Herein, we show that PPAR-γ interacts with human antigen R (HuR) and that the PPAR-γ-HuR complex dissociates following activation of PPAR-γ by rosiglitazone, a specific ligand of PPAR-γ. This rosiglitazone-dependent dissociation of HuR from PPAR-γ leads to nucleocytoplasmic shuttling of HuR and its binding to the 3'-UTR of adiponectin mRNA. PPAR-γ with H321A and H447A double mutation (PPAR-γH321/447A), a mutant lacking ligand-binding activity, impaired HuR dissociation from the PPAR-γ-HuR complex, resulting in reduced nucleocytoplasmic shuttling, even in the presence of rosiglitazone. Consequently, rosiglitazone up-regulated adiponectin levels by modulating the stability of adiponectin mRNA, whereas these effects were abolished by HuR ablation or blocked in cells expressing the PPAR-γH321/447A mutant, indicating that the interaction of PPAR-γ and HuR is a critical event during adiponectin expression. Taken together, the findings demonstrate a novel mechanism for regulating adiponectin expression at the posttranscriptional level and suggest that ligand-mediated activation of PPAR-γ to interfere with interaction of HuR could offer a therapeutic strategy for inflammation-associated diseases that involve decreased adiponectin mRNA stability.-Hwang, J. S., Lee, W. J., Hur, J., Lee, H. G., Kim, E., Lee, G. H., Choi, M.-J., Lim, D.-S., Paek, K. S., Seo, H. G. Rosiglitazone-dependent dissociation of HuR from PPAR-γ regulates adiponectin expression at the posttranscriptional level.

Activation of peroxisome proliferator-activated receptor delta suppresses BACE1 expression by up-regulating SOCS1 in a JAK2/STAT1-dependent manner.

Neuronal expression of beta-secretase 1 (BACE1) has been implicated in the progression of Alzheimer's disease. However, the mechanisms that regulate BACE1 expression are unclear. Here, we show that peroxisome proliferator-activated receptor delta (PPARδ) decreases BACE1 expression by up-regulating suppressor of cytokine signaling 1 (SOCS1) in SH-SY5Y neuroblastoma cells. The activation of PPARδ by GW501516, a specific PPARδ agonist, inhibited expression of BACE1. This effect was abrogated by shRNA-mediated knockdown of PPARδ and by treatment with the PPARδ antagonist GSK0660, indicating that PPARδ is involved in GW501516-mediated suppression of BACE1 expression. On the other hand, GW501516-activated PPARδ induced expression of SOCS1, which is a negative regulator of cytokine signal transduction, at the transcriptional level by binding to a PPAR response element in its promoter. This GW501516-mediated induction of SOCS1 expression led to down-regulation of BACE1 expression via inactivation of signal transducer and activator of transcription 1. GW501516-activated PPARδ suppressed the generation of neurotoxic amyloid beta (Aß) in accordance with the decrease in BACE1 expression. Taken together, these results indicate that PPARδ attenuates BACE1 expression via SOCS1-mediated inhibition of signal transducer and activator of transcription 1 signaling, thereby suppressing BACE1-associated generation of neurotoxic Aß.

Activation of PPARδ attenuates neurotoxicity by inhibiting lipopolysaccharide-triggered glutamate release in BV-2 microglial cells.

Neuroinflammation-associated release of glutamate from activated microglia has been implicated in the progression of neurodegenerative diseases. However, the regulatory mechanisms underlying this glutamate release are poorly understood. Here, we show that peroxisome proliferator-activated receptor delta (PPARδ) modulates neurotoxicity by inhibiting glutamate release in lipopolysaccharide (LPS)-activated BV-2 microglial cells. Activation of PPARδ by GW501516, a specific PPARδ agonist, inhibited glutamate release in BV-2 cells. This effect of GW501516 was significantly blocked by shRNA-mediated knockdown of PPARδ and by treatment with GSK0660, a specific PPARδ antagonist, indicating that PPARδ is associated with blockade of glutamate release. Additionally, GW501516-activated PPARδ suppressed generation of reactive oxygen species and expression of gp91phox, a functional subunit of NADPH oxidase 2, in BV-2 cells stimulated with LPS. The inhibitory effect of GW501516 on gp91phox expression and glutamate release was further potentiated in the presence of AG490, a specific inhibitor of janus kinase 2 (JAK2), leading to the inhibition of signal transducer and activator of transcription 1 (STAT1). By contrast, GW501516 upregulated the expression of suppressor of cytokine signaling 1 (SOCS1), an endogenous inhibitor of JAK2. Furthermore, neurotoxicity induced by conditioned media from LPS-stimulated BV-2 cells was significantly reduced when conditioned media from BV-2 cells treated with both LPS and GW501516 were used. These results indicate that PPARδ attenuates LPS-triggered neuroinflammation by enhancing SOCS1-mediated inhibition of JAK2/STAT1 signaling, thereby inhibiting neurotoxicity associated with glutamate release.

Ligand-activated interaction of PPARδ with c-Myc governs the tumorigenicity of breast cancer.

Peroxisome proliferator-activated receptor (PPAR) δ is a promising therapeutic target in metabolic and inflammatory disorders. However, its role in oncogenesis is controversial, and its therapeutic potential remains to be determined. In our study, we show that ligand-activated PPARδ forms a complex with the proto-oncogene product c-Myc. The interaction of PPARδ with c-Myc affected the transcriptional activity of c-Myc and the expression of its target genes. The PPARδ-dependent regulation of c-Myc activity was associated with decreased tumorigenicity in breast cancer cells. Administration of the PPARδ ligand GW501516 inhibited tumor growth in xenograft model mice bearing MDA-MB-231 cells stably expressing wild-type PPARδ, but not those expressing dominant-negative PPARδ, by interfering with c-Myc function through protein-protein interaction. Our results indicating that PPARδ forms an antitumorigenic complex with c-Myc in the presence of ligand suggest a potential role of PPARδ in breast cancer development.

Ligand-Activated Peroxisome Proliferator-Activated Receptor δ Attenuates Vascular Oxidative Stress by Inhibiting Thrombospondin-1 Expression.

Thrombospondin-1 (TSP-1) is implicated in vascular diseases associated with oxidative stress, such as abdominal aortic aneurysms, ischemia-reperfusion injury, and atherosclerosis. However, the regulatory mechanisms underlying TSP-1 expression are not fully elucidated. In this study, we found that peroxisome proliferator-activated receptor δ (PPARδ) inhibited oxidative stress-induced TSP-1 expression and migration in vascular smooth muscle cells (VSMCs). Activation of PPARδ by GW501516, a specific ligand for PPARδ, significantly attenuated hydrogen peroxide (H2O2)-induced expression of TSP-1 in VSMCs. Small interfering RNA-mediated knockdown of PPARδ and treatment with GSK0660, a selective PPARδ antagonist, reversed the effect of GW501516 on H2O2-induced expression of TSP-1, suggesting that PPARδ is associated with GW501516 activity. Furthermore, JNK (c-Jun N-terminal kinase), but not p38 and ERK (extracellular signal-regulated kinase), mediated PPARδ-dependent inhibition of TSP-1 expression in VSMCs exposed to H2O2. GW501516- activated PPARδ also reduced the H2O2-induced generation of reactive oxygen species, concomitant with inhibition of VSMC migration. In particular, TSP-1 contributed to the action of PPARδ in the regulation of H2O2-induced interleukin-1ß expression. These results suggest that PPARδ-modulated downregulation of TSP-1 is associated with reduced cellular oxidative stress, thereby inhibiting H2O2-induced pheno-typic changes in vascular cells.

A Dalbergia odorifera extract improves the survival of endotoxemia model mice by inhibiting HMGB1 release.

BACKGROUND: Dalbergia odorifera T. Chen (Leguminosae) is an indigenous medicinal herb that is widely used as a popular remedy in northern and eastern Asia. However, the cellular mechanisms underlying the biological activity of D. odorifera are not fully elucidated. METHODS: Anti-inflammatory effect of D. odorifera extract (DOE) was determined through intraperitoneal injection in a mouse model of endotoxemia induced by lipopolysaccharide (LPS). RAW 264.7 cells, a murine macrophage, were also treated with LPS to generate a cellular model of inflammation, and investigated the anti-inflammatory activity and underlying mechanisms of DOE and its constituent isoliquiritigenin. RESULTS: DOE dose-dependently inhibited LPS-induced release of high mobility group box 1 (HMGB1), a late proinflammatory cytokine, and decreased cytosolic translocation of HMGB1 in RAW264.7 cells. This inhibitory effect of DOE on HMGB1 release was observed in cells treated with DOE before or after LPS treatment, suggesting that DOE is effective for both treatment and prevention. In addition, DOE significantly inhibited LPS-induced formation of nitric oxide (NO) and expression of inducible NO synthase (iNOS) in a dose-dependent manner. These effects of DOE were accompanied by suppression of HMGB1 release triggered by LPS, suggesting a possible mechanism by which DOE modulates HMGB1 release through NO signaling. Isoriquiritigenin, a constituent of DOE, also attenuated LPS-triggered NO formation and HMGB1 release in RAW264.7 cells, indicating that isoriquiritigenin is an indexing molecule for the anti-inflammatory properties of DOE. Furthermore, c-Jun N-terminal kinase, but not extracellular signal-regulated kinase and p38, mediated DOE-dependent inhibition of HMGB1 release and NO/iNOS induction in RAW 264.7 cells exposed to LPS. Notably, administration of DOE ameliorated survival rates in a mouse model of endotoxemia induced by LPS, where decreased level of circulating HMGB1 was observed. CONCLUSION: These results suggest that DOE confers resistance to LPS-triggered inflammation through NO-mediated inhibitory effects on HMGB1 release.

Sirtuin 1 Mediates the Actions of Peroxisome Proliferator-Activated Receptor δ on the Oxidized Low-Density Lipoprotein-Triggered Migration and Proliferation of Vascular Smooth Muscle Cells.

Peroxisome proliferator-activated receptor δ (PPARδ) has been implicated in vascular pathophysiology. However, its functions in atherogenic changes of the vascular wall have not been fully elucidated. PPARδ activated by GW501516 (2-[2-methyl-4-[[4-methyl-2-[4-(trifluoromethyl)phenyl]-1,3-thiazol-5-yl]methylsulfanyl]phenoxy]acetic acid) significantly inhibited the migration and proliferation of vascular smooth muscle cells (VSMCs) triggered by oxidized low-density lipoprotein (oxLDL). These GW501516-mediated effects were significantly reversed by PPARδ-targeting small-interfering RNA (siRNA), indicating that PPARδ is involved in the action of GW501516. The antiproliferative effect of GW501516 was directly linked to cell cycle arrest at the G0/G1 to S phase transition, which was followed by the down-regulation of cyclin-dependent kinase 4 along with increased levels of p21 and p53. In VSMCs treated with GW501516, the expression of sirtuin 1 (SIRT1) mRNA and protein was time-dependently increased. This GW501516-mediated up-regulation of SIRT1 expression was also demonstrated even in the presence of oxLDL. In addition, GW501516-dependent inhibition of oxLDL-triggered migration and proliferation of VSMCs was almost completely abolished in the presence of SIRT1-targeting siRNA. These effects of GW501516 on oxLDL-triggered phenotypic changes of VSMCs were also demonstrated via activation or inhibition of SIRT1 activity by resveratrol or sirtinol, respectively. Finally, gain or loss of SIRT1 function imitated the action of PPARδ on oxLDL-triggered migration and proliferation of VSMCs. Taken together, these observations indicate that PPARδ-dependent up-regulation of SIRT1 contributes to the antiatherogenic activities of PPARδ by suppressing the migration and proliferation of VSMCs linked to vascular diseases such as restenosis and atherosclerosis.

Upregulation of MKP-7 in response to rosiglitazone treatment ameliorates lipopolysaccharide-induced destabilization of SIRT1 by inactivating JNK.

Silent mating type information regulation 2 homolog 1 (SIRT1), a NAD-dependent deacetylase, mediates cellular processes involved in gene silencing and aging. The regulation of lifespan by SIRT1 has been extensively investigated, but less is known about the mechanisms associated with its cellular turnover during inflammatory responses. In this study, we found that peroxisome proliferator-activated receptor (PPAR) γ is associated with SIRT1 stability in murine macrophage RAW 264.7 cells exposed to lipopolysaccharide (LPS). Activation of PPARγ by rosiglitazone, a specific ligand of PPARγ, rescues LPS-induced destabilization of SIRT1, with a concomitant decrease in phosphorylation of residue Ser-46, which is targeted by JNK-1 to promote proteasome-mediated degradation of SIRT1. The rosiglitazone-mediated increase in SIRT1 stability is accompanied by upregulation of mitogen-activated protein kinase phosphatase (MKP)-7, a JNK-specific phosphatase. These effects are significantly influenced by ablation or ectopic expression of PPARγ, indicating that PPARγ is directly involved in the regulation of SIRT1 stability. Furthermore, gain of MKP-7 function mimicked the effect of rosiglitazone on LPS-induced destabilization and ubiquitination of SIRT1. These results indicate that PPARγ-dependent upregulation of MKP-7 improves the stability of SIRT1 by inactivating JNK during inflammatory responses of LPS-activated macrophages.

Ethanol extract of Dalbergia odorifera protects skin keratinocytes against ultraviolet B-induced photoaging by suppressing production of reactive oxygen species.

Dalbergia odorifera T. Chen (Leguminosae), an indigenous medicinal herb, has been widely used in northern and eastern Asia to treat diverse diseases. Here, we investigated the anti-senescent effects of ethanolic extracts of Dalbergia odorifera (EEDO) in ultraviolet (UV) B-irradiated skin cells. EEDO significantly inhibited UVB-induced senescence of human keratinocytes in a concentration-dependent manner, concomitant with inhibition of reactive oxygen species (ROS) generation. UVB-induced increases in the levels of p53 and p21, biomarkers of cellular senescence, were almost completely abolished in the presence of EEDO. Sativanone, a major constituent of EEDO, also attenuated UVB-induced senescence and ROS generation in keratinocytes, indicating that sativanone is an indexing (marker) molecule for the anti-senescence properties of EEDO. Finally, treatment of EEDO to mice exposed to UVB significantly reduced ROS levels and the number of senescent cells in the skin. Thus, EEDO confers resistance to UVB-induced cellular senescence by inhibiting ROS generation in skin cells.

Dalbergia odorifera Extract Ameliorates UVB-Induced Wrinkle Formation by Modulating Expression of Extracellular Matrix Proteins.

Preclinical Research Emerging evidence suggests that Dalbergia odorifera T. Chen (Leguminosae), an indigenous medicinal herb, has therapeutic potential. This study examined the antiwrinkle effects of ethanol extracts of D. odorifera in UVB-irradiated human skin cells. Ethanol extracts of D. odorifera and thier constituents, dalbergin and sativanone, induced expression of collagen type I and transforming growth factor (TGF)-ß1 in human dermal fibroblasts. In HR-1 hairless mice exposed to UVB, the ethanol extract reduced wrinkle formation and skin thickness. This inhibitory effect of ethanol extract was associated with the restoration of collagen type I, TGF-ß1, and elastin to levels approaching those in skin tissues not exposed to UVB, which was accompanied by the reduction of matrix metalloproteinase-2 and upregulation of tissue inhibitors of metalloproteinase (TIMP)-2 and TIMP-3 in skin tissue exposed to UVB. These results suggest that the ethanol extracts prevent some effects of photoaging and maintain skin integrity by regulating the degradation of the extracellular matrix proteins. © 2015 Wiley Periodicals, Inc.

Activation of peroxisome proliferator-activated receptor δ inhibits angiotensin II-induced activation of matrix metalloproteinase-2 in vascular smooth muscle cells.

We investigated the role of peroxisome proliferator-activated receptor (PPAR) δ on angiotensin (Ang) II-induced activation of matrix metalloproteinase (MMP)-2 in vascular smooth muscle cells (VSMCs). Activation of PPARδ by GW501516, a specific ligand for PPARδ, attenuated Ang II-induced activation of MMP-2 in a concentration-dependent manner. GW501516 also inhibited the generation of reactive oxygen species in VSMCs treated with Ang II. A marked increase in the mRNA levels of tissue inhibitor of metalloproteinase (TIMP)-2 and -3, endogenous antagonists of MMPs, was also observed in GW501516-treated VSMCs. These effects were markedly reduced in the presence of siRNAs against PPARδ, indicating that the effects of GW501516 are PPARδ dependent. Among the protein kinases inhibited by GW501516, suppression of phosphatidylinositol 3-kinase/Akt signaling was shown to have the greatest effect on activation of MMP-2 in VSMCs treated with Ang II. Concomitantly, GW501516-mediated inhibition of MMP-2 activation in VSMCs treated with Ang II was associated with the suppression of cell migration to levels approaching those in cells not exposed to Ang II. Thus, activation of PPARδ confers resistance to Ang II-induced degradation of the extracellular matrix by upregulating expression of its endogenous inhibitor TIMP and thereby modulating cellular responses to Ang II in vascular cells.

Ligand-activated PPARδ inhibits UVB-induced senescence of human keratinocytes via PTEN-mediated inhibition of superoxide production.

UV radiation-mediated photodamage to the skin has been implicated in premature aging and photoaging-related skin cancer and melanoma. Little is known about the cellular events that underlie premature senescence, or how to impede these events. In the present study we demonstrate that PPARδ (peroxisome-proliferator-activated receptor δ) regulates UVB-induced premature senescence of normal keratinocytes. Activation of PPARδ by GW501516, a specific ligand of PPARδ, significantly attenuated UVB-mediated generation of ROS (reactive oxygen species) and suppressed senescence of human keratinocytes. Ligand-activated PPARδ up-regulated the expression of PTEN (phosphatase and tensin homologue deleted on chromosome 10) and suppressed the PI3K (phosphatidylinositol 3-kinase)/Akt pathway. Concomitantly, translocation of Rac1 to the plasma membrane, which leads to the activation of NADPH oxidases and generation of ROS, was significantly attenuated. siRNA (small interfering RNA)-mediated knockdown of PTEN abrogated the effects of PPARδ on cellular senescence, on PI3K/Akt/Rac1 signalling and on generation of ROS in keratinocytes exposed to UVB. Finally, when HR-1 hairless mice were treated with GW501516 before exposure to UVB, the number of senescent cells in the skin was significantly reduced. Thus ligand-activated PPARδ confers resistance to UVB-induced cellular senescence by up-regulating PTEN and thereby modulating PI3K/Akt/Rac1 signalling to reduce ROS generation in keratinocytes.

Zinc finger protein 251 deficiency impairs glucose metabolism by inducing adipocyte hypertrophy.

Zinc finger protein (ZFP) 251 is a member of the C2H2 ZFP family containing a Krüppel-associated box domain that might mainly act as a transcriptional repressor. However, its cellular function remains largely unknown. Here, we discovered that ZFP251 deficiency caused glucose intolerance in mice. This phenotype was associated with impaired insulin signaling due to hypertrophic changes in white adipose tissue (WAT). Gene ontology analysis revealed that ZFP251 deficiency affected the expression of genes associated with adipocyte differentiation and lipid and fatty acid metabolism. Consistent with in vivo results, hypertrophic changes were observed in Zfp251 knockdown (KD) 3T3-L1 adipocytes. In addition, Zfp251 KD 3T3-L1 preadipocytes exhibited cell cycle arrest in G0/G1 phase, leading to impaired differentiation into mature adipocytes, upon which abnormal mitotic clonal expansion and reduced expression of adipogenic markers were exhibited. These results suggest that ZFP251 deficiency causes impaired adipogenesis and adipocyte hypertrophy, leading to dysfunction of WAT.

PPARδ coordinates angiotensin II-induced senescence in vascular smooth muscle cells through PTEN-mediated inhibition of superoxide generation.

Cellular senescence-associated changes in blood vessels have been implicated in aging and age-related cardiovascular disorders. Here, we demonstrate that peroxisome proliferator-activated receptor (PPAR) δ coordinates angiotensin (Ang) II-induced senescence of human vascular smooth muscle cells (VSMCs). Activation of PPARδ by GW501516, a specific ligand for PPARδ, significantly attenuated Ang II-induced generation of superoxides and suppressed senescence of VSMCs. A marked increase in the levels of p53 and p21 induced by Ang II was blunted by the treatment with GW501516. Ligand-activated PPARδ up-regulated expression of phosphatase and tensin homolog deleted on chromosome 10 (PTEN) and suppressed the phosphatidylinositol 3-kinase (PI3K)/Akt pathway. Knockdown of PTEN with siRNA abrogated the effects of PPARδ on cellular senescence, on PI3K/Akt signaling, and on generation of ROS in VSMCs treated with Ang II. Finally, administration of GW501516 to apoE-deficient mice treated with Ang II significantly reduced the number of senescent cells in the aorta, where up-regulation of PTEN with reduced levels of phosphorylated Akt and ROS was demonstrated. Thus, ligand-activated PPARδ confers resistance to Ang II-induced senescence by up-regulation of PTEN and ensuing modulation of the PI3K/Akt signaling to reduce ROS generation in vascular cells.

Activation of peroxisome proliferator-activated receptor-δ attenuates glutamate-induced neurotoxicity in HT22 mouse hippocampal cells.

Glutamate-induced neurotoxicity has been implicated in the pathogenesis of neurodegenerative disorders; however, little is known about the cellular events that underlie neurotoxicity or how to impede these events. This study demonstrates that peroxisome proliferator-activated receptor (PPAR)-δ regulates glutamate-induced neurotoxicity in HT22 mouse hippocampal cells. Activation of PPARδ by GW501516, a specific ligand, significantly inhibited glutamate-induced cell death and reactive oxygen species (ROS) production in HT22 cells. The siRNA-mediated knockdown of PPARδ abrogated the effects of GW501516 in neuronal toxicity and ROS production induced by glutamate. In addition, ligand-activated PPARδ reduced the glutamate-induced level of intracellular calcium ions (Ca(2+)) by modulating the influx of Ca(2+) from the extracellular space. Similarly, glutamate-induced cell death and intracellular Ca(2+) levels were attenuated in the presence of LY83583, an inhibitor of soluble guanylyl cyclase. Taken together, these results suggest that PPARδ plays an important role in glutamate-induced neurotoxicity by modulating oxidative stress and Ca(2+) influx.

Activation of peroxisome proliferator-activated receptor γ by rosiglitazone inhibits lipopolysaccharide-induced release of high mobility group box 1.

Peroxisome proliferator-activated receptors (PPARs) are shown to modulate the pathological status of sepsis by regulating the release of high mobility group box 1 (HMGB1), a well-known late proinflammatory mediator of sepsis. Ligand-activated PPARs markedly inhibited lipopolysaccharide- (LPS) induced release of HMGB1 in RAW 264.7 cells. Among the ligands of PPAR, the effect of rosiglitazone, a specific ligand for PPARγ, was superior in the inhibition of HMGB1 release induced by LPS. This effect was observed in cells that received rosiglitazone before LPS or after LPS treatment, indicating that rosiglitazone is effective in both treatment and prevention. Ablation of PPARγ with small interfering RNA or GW9662-mediated inhibition of PPARγ abolished the effect of rosiglitazone on HMGB1 release. Furthermore, the overexpression of PPARγ markedly potentiated the inhibitory effect of rosiglitazone on HMGB1 release. In addition, rosiglitazone inhibited LPS-induced expression of Toll-like receptor 4 signal molecules, suggesting a possible mechanism by which rosiglitazone modulates HMGB1 release. Notably, the administration of rosiglitazone to mice improved survival rates in an LPS-induced animal model of endotoxemia, where reduced levels of circulating HMGB1 were demonstrated. Taken together, these results suggest that PPARs play an important role in the cellular response to inflammation by inhibiting HMGB1 release.

PPARδ Activation Mitigates 6-OHDA-Induced Neuronal Damage by Regulating Intracellular Iron Levels.

Intracellular iron accumulation in dopaminergic neurons contributes to neuronal cell death in progressive neurodegenerative disorders such as Parkinson's disease. However, the mechanisms of iron homeostasis in this context remain incompletely understood. In the present study, we assessed the role of the nuclear receptor peroxisome proliferator-activated receptor δ (PPARδ) in cellular iron homeostasis. We identified that PPARδ inhibited 6-hydroxydopamine (6-OHDA)-triggered neurotoxicity in SH-SY5Y neuroblastoma cells. PPARδ activation with GW501516, a specific PPARδ agonist, mitigated 6-OHDA-induced neuronal damage. Further, PPARδ activation also suppressed iron accumulation, which contributes to 6-OHDA-induced neuronal damage. PPARδ activation attenuated 6-OHDA-induced neuronal damage in a similar manner to that of the iron chelator deferoxamine. We further elucidated that PPARδ modulated cellular iron homeostasis by regulating expression of divalent metal transporter 1, ferroportin 1, and ferritin, but not transferrin receptor 1, through iron regulatory protein 1 in 6-OHDA-treated cells. Interestingly, PPARδ activation suppressed 6-OHDA-triggered generation of reactive oxygen species and lipid peroxides. The effects of GW501516 were abrogated by shRNA knockdown of PPARδ, indicating that the effects of GW501516 were PPARδ-dependent. Taken together, these findings suggest that PPARδ attenuates 6-OHDA-induced neurotoxicity by preventing intracellular iron accumulation, thereby suppressing iron overload-associated generation of reactive oxygen species and lipid peroxides, key mediators of ferroptotic cell death.

PPARδ Inhibits Hyperglycemia-Triggered Senescence of Retinal Pigment Epithelial Cells by Upregulating SIRT1.

Emerging evidence shows that peroxisome proliferator-activated receptor delta (PPARδ) plays a pivotal role in cellular aging. However, its function in retinal disease processes such as hyperglycemia-associated diabetic retinopathy is unclear. Here, we demonstrate that PPARδ inhibits premature senescence of retinal pigment epithelial (RPE) cells induced by high glucose (HG) through SIRT1 upregulation. A specific ligand GW501516-activation of PPARδ suppressed premature senescence and production of reactive oxygen species induced by HG in ARPE-19 cells, a spontaneously arising human RPE cell line. These effects were accompanied by the regulation of the premature senescence-associated genes p53, p21, and SMP-30. Furthermore, GW501516-activated PPARδ almost completely abolished the effects of HG treatment on the formation of phosphorylated H2A histone family member X (γ-H2A.X) foci, a molecular marker of aging. These inhibitory effects of GW501516 were significantly reversed in ARPE-19 cells stably expressing small hairpin RNA targeting PPARδ. Notably, GW501516 significantly increased the mRNA and protein levels of SIRT1, indicating that GW501516-activated PPARδ exerted its beneficial effects through SIRT1. In addition, GW501516 restored HG-suppressed SIRT1 expression, corroborating the role of SIRT1 in the anti-senescence function of PPARδ. The effects of PPARδ on HG-induced premature senescence and the expression of the senescence-associated genes p53, p21, and SMP-30 were mimicked by the SIRT1 activator resveratrol, but blocked by the SIRT1 inhibitor sirtinol. Collectively, these results indicate that GW501516-activated PPARδ inhibits HG-triggered premature senescence of RPE cells by modulating SIRT1 signaling.

Metabolic inhibitor screening identifies dihydrofolate reductase as an inducer of the tumor immune escape mediator CD24.

Immune checkpoint inhibitors have improved the clinical management of some cancer cases, yet patients still fail to respond to immunotherapy. Dysregulated metabolism is a common feature of many cancers, and metabolites are known to modulate functions in cancer cells. To identify potential metabolic pathways involved in anti-tumor immune response, we employed a metabolic inhibitor-based drug screen in human lung cancer cell lines and examined expression changes in a panel of immune regulator genes. Notably, pharmacologic inhibition of dihydrofolate reductase (DHFR) downregulated cancer cell expression of cluster of differentiation 24 (CD24), an anti-phagocytic surface protein. Genetic modulation of DHFR resulted in decrease of CD24 expression, whereas tetrahydrofolate, the product of DHFR, enhanced CD24 expression. DHFR inhibition and the consequent CD24 decrease enhanced T cell-mediated tumor cell killing, whereas replenishment of DHFR or CD24 partially mitigated the immune-mediated tumor cell killing that resulted from methotrexate treatment in cancer cells. Moreover, publicly available clinical data analyses further revealed the link between DHFR, CD24, and the antitumor immune response in lung cancer patients. Our study highlights a novel connection between folate metabolism and the anti-tumor immune response and partially interprets how DHFR inhibitors lead to clinical benefits when combined with cancer immunotherapy agents.