PPARγ Response Element Research Articles

Transcriptional regulation of adipogenin expression in liver steatosis by hepatic peroxisome proliferator-activated receptor gamma.

The nuclear receptors peroxisome proliferator-activated receptor gamma (PPARγ) and adipogenin (ADIG) play vital roles in lipid metabolism. However, the interaction between PPARγ and ADIG during liver steatosis remains unclear. In this study, we aimed to investigate the role of PPARγ in the transcriptional regulation of hepatic ADIG expression. Adig was found to be highly expressed in various fatty liver mouse models. Although hepatic Adig was expressed at high levels in the fatty liver of type 2 diabetic ob/ob mice and was upregulated by PPARγ agonist treatment, it was expressed at significantly low levels in liver-specific Pparg-knockout mice. Moreover, hepatic Adig expression was observed in other mouse models of liver steatosis, such as the leptin receptor mutant db/db and alcohol-fed mice. Adig was also highly expressed in the white and brown adipose tissues, skeletal muscles, and heart of ob/ob mice. Reporter and electromobility shift assays showed that PPARγ positively regulates Adig transcriptional activity by directly binding to a functional PPARγ-responsive element in the promoter region. Our results indicate that Adig is a novel target gene of hepatic PPARγ in liver steatosis.

Thiamine: A Natural Peroxisome Proliferator-Activated Receptor Gamma (PPAR-γ) Activator

Background: There has been increasing evidence of the correlation between thiamine deficiency and type 2 diabetes (T2D). T2D is a condition in which an individual’s insulin sensitivity is highly compromised. Peroxisome proliferator-activated receptor gamma (PPAR-γ) is a ligand-activated transcription factor etiologically relevant to T2D. We hypothesized that thiamine could be a PPAR-γ ligand and thus activate PPAR-γ and ameliorate T2D. Objective: This study aims to establish thiamine as a PPAR-γ ligand via molecular docking and dynamics simulations (MDS) and thiamine’s ability to induce adipogenesis while upregulating PPAR-γ and AP-2 genes using in vitro assays. Methods: Thiamine/PPAR-γ binding was studied using Schrödinger’s Glide. The bound complex was simulated in the OPLS 2005 force field using Desmond. 3T3-L1 preadipocyte cells were differentiated in the presence of thiamine and rosiglitazone and stained with Oil Red O. Nuclear protein from the differentiated cells was used to study the binding of the PPAR-γ response element (PPRE) using an ELISA-based assay. mRNA from differentiated cells was used to study the expression of genes using quantitative RTPCR. Results: In silico docking shows that thiamine binds with PPAR-γ. MDS indicate that the interactions between thiamine and PPAR-γ are stable over a significant period. Thiamine induces the differentiation of 3T3-L1 preadipocytes in a dose-dependent manner and enhances the PPRE-binding activity of PPAR-γ. Thiamine treatment significantly increases the mRNA levels of PPAR-γ and AP-2 genes. Conclusion: Our results show that thiamine is a PPAR-γ ligand. Animal studies and clinical trials are required to corroborate the results obtained.

Identification of a novel interplaying loop of PPARγ and respective lncRNAs are involved in colorectal cancer progress

Long noncoding RNAs (lncRNAs) as regulatory molecules play important roles in early treatment and diagnosis of cancers. Considering the role of PPARγ in colorectal cancer (CRC) as a tumor suppressor, the GEO database was used to identify candidate genes that affect the activation of PPARγ protein in CRC cell lines. Then were selected 5 genes containing PPARγ response element (PPRE) in up to 4000 bp upstream and were affected by PPARγ protein activation in HT-29 colon cancer cell line using UCSC database. Expression meta-analysis was applied to map the expression network between candidate genes and all known lncRNAs through expression correlation and lncRNAs that correlated with a greater number of candidate genes (R > 0.5, P.value < 0.001). Moreover, were selected 3 lncRNAs as lncRNAs affected by PPARγ protein activation. Next, the expression levels of candidate genes and lncRNAs were evaluated using RT-qPCR in HT-29 cell line. Results showed a significant increase (FDR <0.05) in the expression level of 5 candidate genes and lncRNAs LINC01133, MBNL1-AS, LOC100288911 after treatment with pioglitazone as PPARγ ligand compared to the untreated group in HT-29 cells. Although additional tests are needed to confirm bioinformatics predictions, it can be concluded that increased expression of PPARγ may increase genes and lncRNAs expression. In summary, this study could be suggested identifying lncRNAs affected by PPARγ activation could be a new strategy in understanding the function and activity of PPARγ in colon cancer.

Dlx5 Represses the Transcriptional Activity of PPARγ.

Peroxisome proliferator-activated receptor gamma (PPARγ) is a master transcription factor in adipocyte differentiation, while distal-less homeobox 5 (Dlx5) is essential for initiating osteoblast differentiation by driving Runt-related transcription factor 2 expression. Considering that adipocytes and osteoblasts share common progenitors, there is a reciprocal correlation between bone and fat formation. However, the mechanism by which Dlx5 controls PPARγ remains unclear. We elucidated that Dlx5 physically binds to PPARγ during immunoprecipitation; in particular, the ligand-binding and DNA-binding domains of PPARγ were involved in the interaction. Transcriptional activity of PPARγ was significantly decreased by Dlx5 overexpression, whereas the opposite results were detected with Dlx5 knockdown. Rosiglitazone, a PPARγ agonist, further enhanced the PPARγ-induced transcriptional activity; however, Dlx5 overexpression effectively repressed the rosiglitazone-mediated increase in activity. Finally, DNA-binding affinity assay revealed that Dlx5 interrupts the interaction of PPARγ with the PPARγ response element promoter. In conclusion, our findings indicate that Dlx5 impedes PPARγ-induced activity, and it may be useful for managing diabetes drug-mediated obesity.

Abstract 2569: miR520a inhibits Fzd9 in bronchial epithelial cells and NSCLC

Abstract Non-small cell lung cancer (NSCLC) continues to be the most diagnosed cancer type worldwide and has the highest mortality rate. Treatment is highly researched, but 5 year survival remains around 20%. Chemoprevention could combat the development of lung cancer in individuals who are at high risk, such as former cigarette smokers. Iloprost is a prostacyclin analogue demonstrated to reduce the presence of endobronchial dysplasia in former smokers. Increased Frizzled-9 (Fzd9) expression is induced by Iloprost, resulting in activation of peroxisome proliferator activated receptor γ (PPARγ) and inhibition of transformed growth. The goal of this study is to elucidate the relationship of cigarette smoke, NSCLC, and miRNA regulation of Fzd9 expression. We hypothesized that miRNA regulate Fzd9 expression when NSCLC cells are exposed to both cigarette smoke condensate (CSC) and Iloprost. miR520a5p was predicted in silico to bind Fzd9 and we validated binding with a miR520a5p mimic and Fzd9 3'UTR luciferase in an immortalized human bronchial epithelial cell line (HBEC) and NSCLC cell lines. A549 and H322 NSCLC cells and HBEC were exposed to cigarette smoke condensate (CSC) and Iloprost. CSC increased miR520a expression, while the opposite was observed in Iloprost treated cells. We also measured Fzd9 3'UTR activity and demonstrated that CSC decreases both Fzd9 3'UTR and PPRE activity, while Iloprost does the opposite. We investigated the effect of miR520a5p on targets downstream of Fzd9, including PPARγ response element (PPRE), a direct measure of PPARγ activity, and epithelial to mesenchymal transition (EMT) gene expression, including Cox2, E-cadherin, and CRB3. We found that when Fzd9 is inhibited by miR520a5p, PPRE activity and epithelial gene expression are inhibited, while mesenchymal gene expression is stimulated. Rescue experiments combining CSC with a miR520a5p inhibitor or iloprost with a miR520a5p mimic supported a role for miR-520a-5p in inhibition of Fzd9 in a CSC carcinogenic pathway in lung epithelial cells that is reversed with iloprost treatment. Overall, these findings suggest a mechanism for miRNA regulation of Fzd9 in lung epithelial cells exposed to cigarette smoke and a potential chemoprevention target. Citation Format: Alex J. Smith, Paulina Do, Kayla M. Sompel, Alamelu Elango. miR520a inhibits Fzd9 in bronchial epithelial cells and NSCLC [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2569.

PPARγ Transcription Deficiency Exacerbates High-Fat Diet-Induced Adipocyte Hypertrophy and Insulin Resistance in Mice.

BackgroundThe transcriptional factor peroxisome proliferator–activated receptor γ (PPARγ) is an important therapeutic target for the treatment of type 2 diabetes. However, the role of the PPARγ transcriptional activity remains ambiguous in its metabolic regulation.MethodsBased on the crystal structure of PPARγ bound with the DNA target of PPARγ response element (PPRE), Arg134, Arg135, and Arg138, three crucial DNA binding sites for PPARγ, were mutated to alanine (3RA), respectively. In vitro AlphaScreen assay and cell-based reporter assay validated that PPARγ 3RA mutant cannot bind with PPRE and lost transcriptional activity, while can still bind ligand (rosiglitazone) and cofactors (SRC1, SRC2, and NCoR). By using CRISPR/Cas9, we created mice that were heterozygous for PPARγ-3RA (PPARγ3RA/+). The phenotypes of chow diet and high-fat diet fed PPARγ3RA/+ mice were investigated, and the molecular mechanism were analyzed by assessing the PPARγ transcriptional activity.ResultsHomozygous PPARγ-3RA mutant mice are embryonically lethal. The mRNA levels of PPARγ target genes were significantly decreased in PPARγ3RA/+ mice. PPARγ3RA/+ mice showed more severe adipocyte hypertrophy, insulin resistance, and hepatic steatosis than wild type mice when fed with high-fat diet. These phenotypes were ameliorated after the transcription activity of PPARγ was restored by rosiglitazone, a PPARγ agonist.ConclusionThe current report presents a novel mouse model for investigating the role of PPARγ transcription in physiological functions. The data demonstrate that the transcriptional activity plays an indispensable role for PPARγ in metabolic regulation.

Effects of Pioglitazone on Glucose-Dependent Insulinotropic Polypeptide-Mediated Insulin Secretion and Adipocyte Receptor Expression in Patients With Type 2 Diabetes.

Incretin hormone dysregulation contributes to reduced insulin secretion and hyperglycemia in patients with type 2 diabetes mellitus (T2DM). Resistance to glucose-dependent insulinotropic polypeptide (GIP) action may occur through desensitization or downregulation of β-cell GIP receptors (GIP-R). Studies in rodents and cell lines show GIP-R expression can be regulated through peroxisome proliferator-activated receptor γ (PPARγ) response elements (PPREs). Whether this occurs in humans is unknown. To test this, we conducted a randomized, double-blind, placebo-controlled trial of pioglitazone therapy on GIP-mediated insulin secretion and adipocyte GIP-R expression in subjects with well-controlled T2DM. Insulin sensitivity improved, but the insulinotropic effect of infused GIP was unchanged following 12 weeks of pioglitazone treatment. In parallel, we observed increased GIP-R mRNA expression in subcutaneous abdominal adipocytes from subjects treated with pioglitazone. Treatment of cultured human adipocytes with troglitazone increased PPARγ binding to GIP-R PPREs. These results show PPARγ agonists regulate GIP-R expression through PPREs in human adipocytes, but suggest this mechanism is not important for regulation of the insulinotropic effect of GIP in subjects with T2DM. Because GIP has antilipolytic and lipogenic effects in adipocytes, the increased GIP-R expression may mediate accretion of fat in patients with T2DM treated with PPARγ agonists.

Identification of Nogo-B as a new molecular target of peroxisome proliferator-activated receptor gamma

Nonalcoholic fatty liver disease (NAFLD) is a fast-growing chronic liver disease worldwide which can lead to liver cirrhosis. Peroxisome proliferator-activated receptor γ (PPARγ), a ligand-activated transcription factor, plays an important role in lipogenesis. Increased Nogo-B expression can be determined in the liver of cirrhosis patients. However, the effect of PPARγ activation on hepatic Nogo-B expression remains unknown. In this study, we found PPARγ activation by rosiglitazone or dephosphorylation increased Nogo-B expression at mRNA and protein levels in HepG2 cells and mouse primary hepatocytes. Furthermore, we identified a PPARγ response element (PPRE) in Nogo-B promoter and found PPARγ enhanced Nogo-B transcription in a PPRE-dependent manner. ChIP assay further confirms rosiglitazone enhanced the binding of PPARγ to Nogo-B promoter. Using a liver specific PPARγ deficient mice, we determined the critical role of PPARγ expression in regulating hepatic Nogo-B expression. Increased glucose and palmitate in culture medium activated Nogo-B and PPARγ expression in mouse primary hepatocytes, and corresponding, high-fat diet (HFD) induced fatty liver associated with increased hepatic Nogo-B and PPARγ expression in mice. Similarly, serum Nogo-B levels in patients with NAFLD were increased. However, rosiglitazone treatment reduced HFD-induced fatty liver and Nogo-B expression. In summary, our study identifies Nogo-B as a new molecular target of PPARγ, and suggests increased Nogo-B might be a potential indicator for NAFLD.

Curcumin inhibits the TGF-\u03b21-dependent differentiation of lung fibroblasts via PPAR\u03b3-driven upregulation of cathepsins B and L

Pulmonary fibrosis is a progressive disease characterized by a widespread accumulation of myofibroblasts and extracellular matrix components. Growing evidences support that cysteine cathepsins, embracing cathepsin B (CatB) that affects TGF-β1-driven Smad pathway, along with their extracellular inhibitor cystatin C, participate in myofibrogenesis. Here we established that curcumin, a potent antifibrotic drug used in traditional Asian medicine, impaired the expression of both α-smooth muscle actin and mature TGF-β1 and inhibited the differentiation of human lung fibroblasts (CCD-19Lu cells). Curcumin induced a compelling upregulation of CatB and CatL. Conversely cystatin C was downregulated, which allowed the recovery of the peptidase activity of secreted cathepsins and the restoration of the proteolytic balance. Consistently, the amount of both insoluble and soluble type I collagen decreased, reaching levels similar to those observed for undifferentiated fibroblasts. The signaling pathways activated by curcumin were further examined. Curcumin triggered the expression of nuclear peroxisome proliferator-activated receptor γ (PPARγ). Contrariwise PPARγ inhibition, either by an antagonist (2-chloro-5-nitro-N-4-pyridinyl-benzamide) or by RNA silencing, restored TGF-β1-driven differentiation of curcumin-treated CCD-19Lu cells. PPARγ response element (PPRE)-like sequences were identified in the promoter regions of both CatB and CatL. Finally, we established that the transcriptional induction of CatB and CatL depends on the binding of PPARγ to PPRE sequences as a PPARγ/Retinoid X Receptor-α heterodimer.

PPARgamma-mediated ALDH1A3 suppression exerts anti-proliferative effects in lung cancer by inducing lipid peroxidation

Context: The metabolic function of peroxisome proliferator-activated receptor gamma (PPARγ) in lung cancer remains unclear.Objectives: To determine the relationship of PPARγ on ALDH1A3-induced lipid peroxidation to inhibit lung cancer cell growth.Materials and methods: In silico analysis using microarray dataset was performed to screen the positive correlation between PPARγ and all ALDH isoforms. NUBIscan software and ChIP assay were used to identify the binding sites (BSs) of PPARγ on ALDH1A3 promoter. The expression of ALDH1A3 under thiazolidinedione (TZD) treatment was evaluated by QPCR and Western Blot in HBEC and H1993 cell lines. Upon treatment of TZD, colony formation assay was used to check cell growth inhibition and 4-hydroxy-2-nonenal (4HNE) production as lipid peroxidation marker was determined by Western Blot in PPARγ positive cell H1993 and PPARγ negative cell H1299.Results: Compared to other ALDH isoforms, ALDH1A3 showed the highest positive correlation to PPARγ expression. ALDH1A3 upregulated PPARγ expression while PPARγ activation suppressed ALDH1A3. Among 2 potential screened PPARγ response elements, BS 1 and 2 in the promoter of ALDH1A3 gene, PPARγ bound directly to BS2. Ligand activation of PPARγ suppressed mRNA and protein expression of ALDH1A3. Growth inhibition was observed in H1993 (PPARγ positive cell) treated with PPARγ activator and ALDH inhibitor compared to H1299 (PPARγ negative cell). PPARγ activation increased 4HNE which is known to be suppressed by ALDH1A3.Conclusions: ALDH1A3 suppression could be one of PPARγ tumor suppressive function. This study provides a better understanding of the role of PPARγ in lung cancer.

Jujuboside A promotes Aβ clearance and ameliorates cognitive deficiency in Alzheimer's disease through activating Axl/HSP90/PPARγ pathway.

Rationale: It has been reported that peroxisome proliferator activated receptor γ (PPARγ) level decreases significantly in the brains of Alzheimer's disease (AD) patients and mice models, while the mechanism is unclear. This study aims to unravel the mechanism that amyloid β (Aβ) decreases PPARγ and attempted to discover lead compound that preserves PPARγ.Methods: In APP/PS1 transgenic mice and Aβ treated microglia, the interaction between HSP90 and PPARγ were analyzed by western blot. Using a PPRE (PPARγ responsive element) containing reporter cell line, compounds that activate PPARγ activity were identified. After genetic ablation or pharmacological inhibition of potential target pathways, the target of jujuboside A (JuA) was discovered through Axl/HSP90β. After oral administration or intrathecal injection, the anti-AD activity of JuA was evaluated by Morris water maze (MWM) test and object recognition test. Soluble Aβ42 levels and plaque numbers after JuA treatment were detected by thioflavin S staining, and the activation of microglia was assayed by immunofluorescence staining against Iba-1.Results: We found that Aβ stress decreased heat shock protein 90 β (HSP90β), subsequently reduced the abundance of PPARγ, and down-regulated Aβ clearance-related genes in BV2 cells and primary microglia. We identified that JuA stimulated the expression of HSP90β, strengthened the interaction between HSP90β and PPARγ, preserved PPARγ levels, and thus effectively promoted the clearance of Aβ42. We demonstrated that JuA increased HSP90β expression through Axl/ERK pathway. JuA significantly ameliorated cognitive deficiency in APP/PS1 transgenic mice, meanwhile, JuA significantly reduced the soluble Aβ42 levels and plaque numbers in the brain. Notably, the therapeutic effects of JuA were dampened by R428, an Axl inhibitor.Conclusions: This study suggests that the up-regulation of HSP90β by JuA through Axl is a potential therapeutic strategy to facilitate Aβ42 clearance and ameliorate cognitive deficiency in AD.

Arsenic, Cadmium, and Lead Like Troglitazone Trigger PPARγ-Dependent Poly (ADP-Ribose) Polymerase Expression and Subsequent Apoptosis in Rat Brain Astrocytes.

We previously demonstrated that arsenic, cadmium, and lead mixture at environmentally relevant doses induces astrocyte apoptosis in the developing brain. Here, we investigated the mechanism and contribution of each metal in inducing the apoptosis. We hypothesized participation of transcription factor, peroxisome proliferator-activated receptor gamma (PPARγ), reported to affect astrocyte survival. We treated cultured rat astrocytes with single metals and their combinations and performed apoptosis assay and measured PPARγ expression levels. We found that cadmium demonstrated maximum increase in PPARγ as well as apoptosis, followed by arsenic and then lead. Interestingly, we observed that the metals mimicked PPARγ agonist, troglitazone, and enhanced PPARγ-transcriptional activity. Co-treatment with PPARγ-siRNA or PPARγ-antagonist, GW9662, suppressed the astrocyte apoptosis, suggesting a prominent participation of PPARγ in metal(s)-induced astrocyte loss. We explored PPARγ-transcriptional activity and identify its target gene in apoptosis, performed in silico screening. We spotted PPARγ-response elements (PPREs) within poly(ADP-ribose) polymerase (PARP) gene, and through gel-shift assay verified metal(s)-mediated increased PPARγ binding to PARP-PPREs. Chromatin-immunoprecipitation and luciferase-reporter assays followed by real-time PCR and Western blotting proved PPRE-mediated PARP expression, where cadmium contributed most and lead least, and the effects of metal mixture were comparable with troglitazone. Eventually, dose-dependent increased cleaved-PARP/PARP ratio confirmed astrocyte apoptosis. Additionally, we found that PPARγ and PARP expressions were c-Jun N-terminal kinases and cyclin-dependent kinase5-dependent. In vivo treatment of developing rats with the metals corroborated enhanced PPARγ-dependent PARP and astrocyte apoptosis, where yet again cadmium contributed most. Overall, our study enlightens a novel PPARγ-dependent mechanism of As-, Cd-, and Pb-induced astrocyte apoptosis.

Inhibition of Macrophage CD36 Expression and Cellular Oxidized Low Density Lipoprotein (oxLDL) Accumulation by Tamoxifen: APEROXISOME PROLIFERATOR-ACTIVATED RECEPTOR (PPAR)γ-DEPENDENTMECHANISM

Macrophage CD36 binds and internalizes oxidized low density lipoprotein (oxLDL) to facilitate foam cell formation. CD36 expression is activated by peroxisome proliferator-activated receptor γ (PPARγ). Tamoxifen, an anti-breast cancer medicine, has demonstrated pleiotropic functions including cardioprotection with unfully elucidated mechanisms. In this study, we determined that treatment of ApoE-deficient mice with tamoxifen reduced atherosclerosis, which was associated with decreased CD36 and PPARγ expression in lesion areas. At the cellular level, we observed that tamoxifen inhibited CD36 protein expression in human THP-1 monocytes, THP-1/PMA macrophages, and human blood monocyte-derived macrophages. Associated with decreased CD36 protein expression, tamoxifen reduced cellular oxLDL accumulation in a CD36-dependent manner. At the transcriptional level, tamoxifen decreased CD36 mRNA expression, promoter activity, and the binding of the PPARγ response element in CD36 promoter to PPARγ protein. Tamoxifen blocked ligand-induced PPARγ nuclear translocation and CD36 expression, but it increased PPARγ phosphorylation, which was due to that tamoxifen-activated ERK1/2. Furthermore, deficiency of PPARγ expression in macrophages abolished the inhibitory effect of tamoxifen on CD36 expression or cellular oxLDL accumulation both in vitro and in vivo Taken together, our study demonstrates that tamoxifen inhibits CD36 expression and cellular oxLDL accumulation by inactivating the PPARγ signaling pathway, and the inhibition of macrophage CD36 expression can be attributed to the anti-atherogenic properties of tamoxifen.

Bisphenol S- and bisphenol A-induced adipogenesis of murine preadipocytes occurs through direct peroxisome proliferator-activated receptor gamma activation.

The use of bisphenol A (BPA) in consumer products and food packaging has been associated under certain conditions with a risk of negative health outcomes. This prompted its removal from many products and replacement with structural analogs. Bisphenol S (BPS) is one such analog, but its metabolic effects have not been fully characterized. The objective of our study was to determine whether BPS functions similarly to BPA at inducing adipogenesis. Murine 3T3-L1 preadipocytes were used to evaluate and compare the adipogenic potential of BPS to BPA. Cells were treated with 0.01-50 μM BPS or 0.01-50 μM BPA and adipogenic effects were measured. Further, their ability to activate peroxisome proliferator-activated receptor gamma (PPARγ), an adipogenic transcription factor, was also determined. Our results indicate that treatment of 3T3-L1 cells with BPS induced lipid accumulation and increased mRNA and protein expression of key adipogenic markers (1-50 μM; P<0.05). BPS treatment resulted in a higher expression of adipogenic markers as well as greater lipid accumulation when compared with BPA treatment. We showed that BPS can upregulate lipoprotein lipase, adipocyte protein 2, PPARγ, perilipin, adipsin and CCAAT/enhancer-binding protein alpha mRNA expression levels. Furthermore, using transcriptional assays, we showed that BPS and BPA can modestly activate PPARγ using a PPRE (PPARγ response element)-dependent luciferase construct by 1.5-fold (P<0.05). However, BPS but not BPA was able to competitively inhibit rosiglitazone (ROSI)-activated PPARγ, suggesting that BPS interacts with PPARγ distinctly from BPA. Co-treatment of cells with the selective PPARγ antagonist GW9662 inhibits BPS-, BPA-, ROSI- but not dexamethasone-dependent adipogenic differentiation. Both BPA and BPS can enhance 3T3-L1 adipocyte differentiation in a dose-dependent manner and require PPARγ to induce adipogenesis. Through direct comparison, we show that BPS is a more potent adipogen than BPA.

Inhibition of hypoxia-induced cyclooxygenase-2 by Korean Red Ginseng is dependent on peroxisome proliferator-activated receptor gamma

BackgroundKorean Red Ginseng (KRG) is a traditional herbal medicine made by steaming and drying fresh ginseng. It strengthens the endocrine and immune systems to ameliorate various inflammatory responses. The cyclooxygenase-2 (COX-2)/prostaglandin E2 pathway has important implications for inflammation responses and tumorigenesis. Peroxisome proliferator-activated receptor gamma (PPARγ) is a transcription factor that regulates not only adipogenesis and lipid homeostasis, but also angiogenesis and inflammatory responses. MethodsThe effects of the KRG on inhibition of hypoxia-induced COX-2 via PPARγ in A549 cells were determined by luciferase assay, Western blot, and/or quantitative reverse transcription-polymerase chain reaction (qRT-PCR). The antimigration and invasive effects of KRG were evaluated on A549 cells using migration and matrigel invasion assays. Results and conclusionWe previously reported that hypoxia-induced COX-2 protein and mRNA levels were suppressed by KRG. This study examines the possibility of PPARγ as a cellular target of KRG for the suppression of hypoxia-induced COX-2. PPARγ protein levels and PPARγ-responsive element (PPRE)-driven reporter activities were increased by KRG. Reduction of hypoxia-induced COX-2 by KRG was abolished by the PPARγ inhibitor GW9662. In addition, the inhibition of PPARγ abolished the effect of KRG on hypoxia-induced cell migration and invasion. DiscussionOur results show that KRG inhibition of hypoxia-induced COX-2 expression and cell invasion is dependent on PPARγ activation, supporting the therapeutic potential for suppression of inflammation under hypoxia. Further studies are required to demonstrate whether KRG activates directly PPARγ and to identify the constituents responsible for this activity.

IGF1 Promotes Adipogenesis by a Lineage Bias of Endogenous Adipose Stem/Progenitor Cells.

Adipogenesis is essential for soft tissue reconstruction following trauma or tumor resection. We demonstrate that CD31(-)/34(+)/146(-) cells, a subpopulation of the stromal vascular fraction (SVF) of human adipose tissue, were robustly adipogenic. Insulin growth factor-1 (IGF1) promoted a lineage bias towards CD31(-)/34(+)/146(-) cells at the expense of CD31(-)/34(+)/146(+) cells. IGF1 was microencapsulated in poly(lactic-co-glycolic acid) scaffolds and implanted in the inguinal fat pad of C57Bl6 mice. Control-released IGF1 induced remarkable adipogenesis in vivo by recruiting endogenous cells. In comparison with the CD31(-)/34(+)/146(+) cells, CD31(-)/34(+)/146(-) cells had a weaker Wnt/β-catenin signal. IGF1 attenuated Wnt/β-catenin signaling by activating Axin2/PPARγ pathways in SVF cells, suggesting IGF1 promotes CD31(-)/34(+)/146(-) bias through tuning Wnt signal. PPARγ response element (PPRE) in Axin2 promoter was crucial for Axin2 upregulation, suggesting that PPARγ transcriptionally activates Axin2. Together, these findings illustrate an Axin2/PPARγ axis in adipogenesis that is particularly attributable to a lineage bias towards CD31(-)/34(+)/146(-) cells, with implications in adipose regeneration.

Two homoisoflavonoids act as peroxisome proliferator-activated receptor agonists

Polygonatum odoratum (Mill.) Druce is a well-known traditional Chinese medicine exerting multi-biological functions like removing dryness, promoting secretion of fluid, and quenching thirst, but its molecular mechanism is not fully understood. Peroxisome proliferator-activated receptor (PPAR) γ is a member of nuclear receptor superfamily, and its agonists are prescribed as anti-hyperglycemic drugs. In this study, homoisoflavonoids isolated from P. odoratum were evaluated for their activity against PPARγ by the fluorescence polarization competitive binding assay and transient transfection reporter assays. Primarily, the fluorescence polarization competitive binding assay results showed that 5,7-dihydroxy-6, 8-dimethyl-3-(4′-hydroxybenzyl)chroman-4-one (IC50 = 7 μM) and 5,7-dihydroxy-6-methyl-8-methoxy-3-(4′-hydroxybenzyl)chroman-4-one (IC50 = 12 μM) bound to PPARγ ligand-binding domain with fair binding affinity. Moreover, both homoisoflavonoids transactivated PPARγ-dependent promoters including PPRE (PPARγ response element), SHP, and ABCA1 gene promoters in dose-dependent manner. Further molecular modeling studies demonstrated that they interacted with PPARγ ligand-binding pocket in a similar manner as indeglitazar, a novel PPARγ agonist. All these results highly indicated that 5,7-dihydroxy-6, 8-dimethyl-3-(4′-hydroxybenzyl)chroman-4-one and 5,7-dihydroxy-6-methyl-8-methoxy-3-(4′-hydroxybenzyl)chroman-4-one are the active constituents of P. odoratum and act as PPARγ agonists.

ATF3 inhibits PPARγ-stimulated transactivation in adipocyte cells

Previously, we reported that activating transcription factor 3 (ATF3) downregulates peroxisome proliferator activated receptor (PPARγ) gene expression and inhibits adipocyte differentiation in 3T3-L1 cells. Here, we investigated another role of ATF3 on the regulation of PPARγ activity. ATF3 inhibited PPARγ-stimulated transactivation of PPARγ responsive element (PPRE)-containing reporter or GAL4/PPARγ chimeric reporter. Thus, ATF3 effectively repressed rosiglitazone-stimulated expression of adipocyte fatty acid binding protein (aP2), PPARγ target gene, in 3T3-L1 cells. Coimmunoprecipitation and GST pulldown assay demonstrated that ATF3 interacted with PPARγ. Accordingly, ATF3 prevented PPARγ from binding to PPRE on the aP2 promoter. Furthermore, ATF3 suppressed p300-mediated transcriptional coactivation of PPRE-containing reporter. Chromatin immunoprecipitation assay showed that overexpression of ATF3 blocked both binding of PPARγ and recruitment of p300 to PPRE on aP2 promoter induced by rosiglitazone treatment in 3T3-L1 cells. Taken together, these results suggest that ATF3 interacts with PPARγ and represses PPARγ-mediated transactivation through suppression of p300-stimulated coactivation in 3T3-L1 cells, which may play a role in inhibition of adipocyte differentiation.

CDK5-induced p-PPARγ(Ser 112) downregulates GFAP via PPREs in developing rat brain: effect of metal mixture and troglitazone in astrocytes

The peroxisome proliferator-activated receptor gamma (PPARγ), a group of ligand-activated transcriptional factors, is expressed in glial fibrillary acidic protein (GFAP)-immunoreactive astrocytes. Here, we investigated the role of PPARγ in regulating GFAP using a mixture of As, Cd and Pb (metal mixture, MM) that induces apoptosis and aberrant morphology in rat brain astrocytes. We observed a phospho PPARγ (serine 112 (S112)) (p-PPARγ (S112))-mediated downregulation of GFAP in the MM-exposed astrocytes. We validated this using pure PPARγ agonist, troglitazone (TZ). As reported with MM, TZ induced astrocyte damage owing to reduced GFAP. In silico analysis in the non-coding region of GFAP gene revealed two PPARγ response elements (PPREs); inverted repeat 10 and direct repeat 1 sequences. Gel shift and chromatin immunoprecipitation assays demonstrated enhancement in binding of p-PPARγ (S112) to the sequences, and luciferase reporter assay revealed strong repression of GFAP via PPREs, in response to both MM and TZ. This indicated that suppression in GFAP indeed occurs through direct regulation of these elements by p-PPARγ (S112). Signaling studies proved that MM, as well as TZ, activated the cyclin-dependent kinase 5 (CDK5) and enhanced its interaction with PPARγ resulting into increased p-PPARγ (S112). The p-CDK5 levels were dependent on proximal activation of extracellular signal-regulated protein kinase 1/2 and downstream Jun N-terminal kinase. Taken together, these results are the first to delineate downregulation of GFAP through genomic and non-genomic signaling of PPARγ. It also brings forth a resemblance of TZ with MM in terms of astrocyte disarray in developing brain.

Repression of Alzheimer's beta-Secretase

Alzheimer's disease (AD) is one of the most devastating neuro-degenerative disorders characterized by the two pathological hallmarks of amyloid plaques and neurofibrillary tangles. Multiple environmental factors, such as diet and life style, along with genetic factors are all significant contributors. In addition, classic cardio-metabolic factors such as hypertension, stroke, diabetes and hypercholesterolemia increase the risk of AD. Epidemiological, clinical and experimental evidence strongly link metabolic defects with functional alterations associated with AD pathogenesis. Amyloid peptides (Aβ), the major constituent of plaques, are generated by sequential proteolytic cleavage of the amyloid precursor protein (APP) via β-secretase (BACE1) and the γ-secretase. The selectively affected regions in the frontal brains of AD patients are often found to not only display high accumulation of Aβ peptides but also aberrant metabolic states (impaired homeostasis of lipid, glucose and energy metabolism) and chronic neuroinflammation. Together, these events trigger ultimate neuronal loss and cognitive deficit. BACE1 is the rate-limiting enzyme in APP processing and Aβ generation and thus remains one of the most favored therapeutic targets for treating AD. BACE1 expression is tightly regulated at multiple levels including transcription, post-transcription, translation and post-translation (Vassar et al. J Neurosci. 2009; 29:12787). As was recently reviewed (Chami et al., Mol. Neurodegener. 2012; 7:52), BACE1 is a stress-inducible protease. Multiple conditions including oxidative stress, inflammation, hypoxia/ischemia and traumatic conditions induce BACE1 expression primarily via transcriptional activation. We have recently reported differential regulation of BACE1 by oxidative and nitrosative signals (Kwak et al. Mol Neurodegener. 2011; 6:17) and by metabolic stress conditions (Wang et al. Cell Metab. 2013; 17:685). The peroxisome proliferator-activated receptor-γ (PPAR γ) is a prototypic ligand-activated nuclear receptor (NR) that utilizes PPARγ coactivator (PGC-1α) as its coactivator to coordinate lipid, glucose and mitochondrial metabolism. Based on the ability of PPARγ agonists to elicit anti-amyloidogenic, anti-inflammatory and insulin-sensitizing effects, rosiglitazone and several non-steroidal anti-inflammatory drugs (NSAIDs) are being evaluated in AD clinical trials (Heneka et al. Biochim Biophys Acta. 2007; 1771:1031; Landreth et al. Neurotherapeutics._2008; 5:481). We recently investigated this PPARγ-PGC-1 axis in BACE1 regulation under various metabolic stress conditions and made several previously unappreciated observations. We discovered that 1) feeding mice high-fat-diets upregulate BACE1 transcription while fasting suppress BACE1, 2) up- or down-regulation of PGC-1α reciprocally regulate BACE1 transcription, 3) full activity of PPARγ and PGC-1α are required for suppressing BACE1 and both rely on a SIRT1-mediated deacetylation, and 4) the role of PPARγ-PGC-1α in transcriptional repression of BACE1 appears to represent a unique noncanonical mechanism which is not dependent on its ligand rosiglitazone but rather involves recruitment of a corepressor NCoR. To our knowledge, the PPARγ-PGC-1α inhibition of BACE1 gene expression is the first report of direct transcriptional repression by PGC-1α. Nuclear receptor signaling is increasingly recognized as having important roles in CNS functions (Malaspina, J Neurochem. 2008; 104:584). Classically, NRs are ligand-activated transcription factors which bind to specific responsive elements located in the promoter regions of their target genes and recruit transcriptional coactivators. In contrast, nuclear receptor-mediated repression has been reported as occurring through the recruitment of correpressor complexes (e.g., NCoR/SMRT) to unliganded receptor heterodimers such as PPARγ-RXR or RAR-RXR or LXR-RXR (Perissi et al. Nat. Rev. 2010; 11:109). Since the PPARγ-responsive element (PPRE) overlaps with retinoid acid receptor (RAR-RXR) binding motifs in the BACE1 promoter, we also investigated the potential suppressive role of retinoid acids. Indeed, we found that all-trans retinoid acid (atRA), but not the RXR ligand 9-cis RA, suppressed BACE1 transcription both in vitro and in vivo. Furthermore, we have obtained preliminary data demonstrating the beneficial effects of estrogen (E2) and vitamin D3 (1, 25-OH form) in suppressing BACE1 transcription (Wang and Liao, unpublished data). The levels of both vitamin D3 and estrogen are often decreased dramatically in menopausal women, likely contributing to the cognitive decline in a significant portion of these individuals. A correlative relationship between vitamin D receptor (VDR) and estrogen receptor (ER) gene polymorphisms and bone mineral density has been reported in some populations. We speculate that the effects with E2 and vitamin D3 on BACE1 suppression are mediated by their respective nuclear receptors (VDR and ER). We further speculate that interaction between these receptors with ER- and VDR-responsive element(s) may recruit different corepressors such as ligand dependent corepressor (LCoR) in a ligand-dependent manner. Alternatively, the ER and VDR may couple with the NCoR recruited by PPARγ-PGC-1 (Figure ​(Figure1).1). A fuller understanding of these NR-mediated regulatory mechanisms in suppressing BACE1 expression is fundamental to the future design of novel therapeutics. Continued study of the detailed interplay between the different nuclear receptor signaling pathways may provide a rationale for selective combination therapy between PPARγ agonists, atRA, vitamin D3 and estrogen to achieve maximum synergistic or additive effects. Figure 1 Complex transcriptional repression of BACE1 gene expression by various NR signaling pathways.