Endogenous PPARγ Ligands Research Articles

Endothelial Cells as a Key Cell Type for Innate Immunity: A Focused Review on RIG-I Signaling Pathway.

The vascular endothelium consists of a highly heterogeneous monolayer of endothelial cells (ECs) which are the primary target for bacterial and viral infections due to EC’s constant and close contact with the bloodstream. Emerging evidence has shown that ECs are a key cell type for innate immunity. Like macrophages, ECs serve as sentinels when sensing invading pathogens or microbial infection caused by viruses and bacteria. It remains elusive how ECs senses danger signals, transduce the signal and fulfil immune functions. Retinoic acid-inducible gene-I (RIG-I, gene name also known as DDX58) is an important member of RIG-I-like receptor (RLR) family that functions as an important pathogen recognition receptor (PRR) to execute immune surveillance and confer host antiviral response. Recent studies have demonstrated that virus infection, dsRNA, dsDNA, interferons, LPS, and 25-hydroxycholesterol (25-HC) can increase RIG-1 expression in ECs and propagate anti-viral response. Of translational significance, RIG-I activation can be inhibited by Panax notoginseng saponins, endogenous PPARγ ligand 15-PGJ2, tryptanthrin and 2-animopurine. Considering the pivotal role of inflammation and innate immunity in regulating endothelial dysfunction and atherosclerosis, here we provided a concise review of the role of RIG-I in endothelial cell function and highlight future direction to elucidate the potential role of RIG-I in regulating cardiovascular diseases as well as virus infectious disease, including COVID-19. Furthered understanding of RIG-I-mediated signaling pathways is important to control disorders associated with altered immunity and inflammation in ECs.

Hormone sensitive lipase ablation promotes bone regeneration

There is an inverse relationship between the differentiation of mesenchymal stem cells (MSCs) along either an adipocyte or osteoblast lineage, with lineage differentiation known to be mediated by transcription factors PPARγ and Runx2, respectively. Endogenous ligands for PPARγ are generated during the hydrolysis of triacylglycerols to fatty acids through the actions of lipases such as hormone sensitive lipase (HSL). To examine whether reduced production of endogenous PPARγ ligands would influence bone regeneration, we examined the effects of HSL knockout on fracture repair in mice using a tibial mono-cortical defect as a model. We found an improved rate of fracture repair in HSL-ko mice documented by serial μCT and bone histomorphometry compared to wild-type (WT) mice. Similarly, accelerated rates of bone regeneration were observed with a calvarial model where implantation of bone grafts from HSL-ko mice accelerated bone regeneration at the injury site. Further analysis revealed improved MSC differentiation down osteoblast and chondrocyte lineage with inhibition of HSL. MSC recruitment to the injury site was greater in HSL-ko mice than WT. Finally, we used single cell RNAseq to understand the osteoimmunological differences between WT and HSL-ko mice and found changes in the pre-osteoclast population. Our study shows HSL-ko mice as an interesting model to study improvements to bone injury repair. Furthermore, our study highlights the potential importance of pre-osteoclasts and osteoclasts in bone repair.

Differential Effects of Estrogen Receptor Alpha and Beta on Endogenous Ligands of Peroxisome Proliferator-Activated Receptor Gamma in Papillary Thyroid Cancer.

PurposeThe inhibition of estrogen receptor alpha (ERα) or the activation of ERβ can inhibit papillary thyroid cancer (PTC), but the precise mechanism is not known. We aimed to explore the role of ERα and ERβ on the production of endogenous peroxisome proliferator-activated receptor gamma (PPARγ) ligands in PTC.Methods2 PTC cell lines, 32 pairs of PTC tissues and matched normal thyroid tissues were used in this study. The levels of endogenous PPARγ ligands 15(S)-hydroxyeicosatetraenoic acid (15(S)-HETE), 13-S-hydroxyoctadecadienoic acid (13(S)-HODE), and15-deoxy-Δ12,14-prostaglandin J2 (PGJ2) were measured by ELISA.ResultsThe levels of PGJ2 and 15(S)-HETE were significantly reduced in PTC, but 13(S)-HODE was not changed. Activation of ERα or inhibition of ERβ significantly downregulated the production of PGJ2, 15(S)-HETE and 13(S)-HODE, whereas inhibition of ERα or activation of ERβ markedly upregulated the production of these three ligands. Application of endogenous PPARγ ligands inhibited growth, induced apoptosis of cancer cells, and promoted the efficacy of chemotherapy.ConclusionThe levels of endogenous PPARγ ligands PGJ2 and 15(S)-HETE are significantly decreased in PTC. The inhibition of ERα or activation of ERβ can inhibit PTC by stimulating the production of endogenous PPARγ ligands to induce apoptosis in cancer cells.

Prostaglandin 15d-PGJ2 targets PPARγ and opioid receptors to prevent muscle hyperalgesia in rats.

Pharmacological agents directed to either opioid receptors or peroxisome proliferator-activated receptor gamma (PPARγ) at peripheral tissues reduce behavioral signs of persistent pain. Both receptors are expressed in muscle tissue, but the contribution of PPARγ activation to muscle pain and its modulation by opioid receptors remains unknown. To address this question, we first tested whether the endogenous PPARγ ligand 15d-PGJ2 would decrease mechanical hyperalgesia induced by carrageenan administration into the gastrocnemius muscle of rats. Next, we used receptor antagonists to determine whether the antihyperalgesic effect of 15-deoxyΔ-12,14-prostaglandin J2 (15d-PGJ2) was PPARγ- or opioid receptor-dependent. Three hours after carrageenan, muscle hyperalgesia was quantified with the Randall-Selitto test. 15d-PGJ2 prevented carrageenan-induced muscle hyperalgesia in a dose-dependent manner. The antihyperalgesic effect of 15d-PGJ2 was dose-dependently inhibited by either the PPARγ antagonist, 2-chloro-5-nitro-N-phenylbenzamide, or by the opioid receptor antagonist, naloxone. We conclude that 15d-PGJ2 targets PPARγ and opioid receptors to prevent muscle hyperalgesia. We suggest that local PPARγ receptors are important pharmacological targets for inflammatory muscle pain.

Human adipose tissue microvascular endothelial cells secrete PPARγ ligands and regulate adipose tissue lipid uptake.

Human adipose cells cannot secrete endogenous PPARγ ligands and are dependent on unknown exogenous sources. We postulated that the adipose tissue microvascular endothelial cells (aMVECs) cross-talk with the adipose cells for fatty acid (FA) transport and storage and also may secrete PPARγ ligands. We isolated aMVECs from human subcutaneous adipose tissue and showed that in these cells, but not in (pre)adipocytes from the same donors, exogenous FAs increased cellular PPARγ activation and markedly increased FA transport and the transporters FABP4 and CD36. Importantly, aMVECs only accumulated small lipid droplets and could not be differentiated to adipose cells and are not adipose precursor cells. FA exchange between aMVECs and adipose cells was bidirectional, and FA-induced PPARγ activation in aMVECs was dependent on functional adipose triglyceride lipase (ATGL) protein while deleting hormone-sensitive lipase in aMVECs had no effect. aMVECs also released lipids to the medium, which activated PPARγ in reporter cells as well as in adipose cells in coculture experiments, and this positive cross-talk was also dependent on functional ATGL in aMVECs. In sum, aMVECs are highly specialized endothelial cells, cannot be differentiated to adipose cells, are adapted to regulating lipid transport and secreting lipids that activate PPARγ, and thus, regulate adipose cell function.

Cooperative cobinding of synthetic and natural ligands to the nuclear receptor PPARγ.

Crystal structures of peroxisome proliferator-activated receptor gamma (PPARγ) have revealed overlapping binding modes for synthetic and natural/endogenous ligands, indicating competition for the orthosteric pocket. Here we show that cobinding of a synthetic ligand to the orthosteric pocket can push natural and endogenous PPARγ ligands (fatty acids) out of the orthosteric pocket towards an alternate ligand-binding site near the functionally important omega (Ω)-loop. X-ray crystallography, NMR spectroscopy, all-atom molecular dynamics simulations, and mutagenesis coupled to quantitative biochemical functional and cellular assays reveal that synthetic ligand and fatty acid cobinding can form a 'ligand link' to the Ω-loop and synergistically affect the structure and function of PPARγ. These findings contribute to a growing body of evidence indicating ligand binding to nuclear receptors can be more complex than the classical one-for-one orthosteric exchange of a natural or endogenous ligand with a synthetic ligand.

Lanthionine synthetase C-like protein 2 (LanCL2) is important for adipogenic differentiation

Adipogenic differentiation is a highly regulated process that is necessary for metabolic homeostasis and nutrient sensing. The expression of PPARγ and the subsequent activation of adipogenic genes is critical for the process. In this study, we identified lanthionine synthetase C-like protein 2 (LanCL2) as a positive regulator of adipogenesis in 3T3-L1 cells. Knockdown of LanCL2, but not LanCL1, inhibited adipogenic differentiation, and this effect was not mediated through cAMP or Akt signaling pathways. The expression of early adipogenic markers CCAAT enhancer binding protein β (C/EBPβ) and C/EBPδ remained intact in LanCL2 knockdown cells, but levels of late adipogenic markers PPARγ and C/EBPα were suppressed. The addition of the naturally occurring PPARγ activator 15-deoxy-Δ12,14-prostaglandin J2 or conditioned medium from differentiating cells did not restore differentiation, implying that LanCL2 may not be involved in the production of a secreted endogenous PPARγ ligand. Pulldown assays demonstrated a direct physical interaction between LanCL2 and PPARγ. Consistent with a regulatory role of LanCL2, luciferase reporter assays revealed that full transcriptional activation by PPARγ was dependent on LanCL2. Taken together, our study reveals a novel role of LanCL2 in adipogenesis, specifically involved in PPARγ-mediated transactivation of downstream adipogenic genes.

Z-ligustilide and anti-inflammatory prostaglandins have common biological properties in macrophages and leukocytes

During inflammation, immune cells produce cytokines, chemokines and prostaglandins. This results in acute or chronic inflammation, which favor the development of degenerative diseases such as diabetes, obesity or cardiovascular diseases. Inflammatory processes are modulated by intrinsic and external factors. External factors are supposed to act via similar modes of action as do endogenous molecules and mediators. Both endogenous ligands and nutrient-derived metabolites might modify the extent and status of the cellular and systemic response during inflammation. Therefore, the biological activity of endogenous mediators was compared with nutrition-derived substances. Murine macrophages (RAW264.7 cells), in vitro differentiated human promyeloid THP-1 cells and peripheral blood leukocytes (PBL) were stimulated with LPS in the presence of z-ligustilide (LIG) or the endogenous PPARγ ligand 15deoxyΔ12,14-prostaglandin J2 (15d–PGJ2). Secretion of mediators of inflammation was measured by EIA, the Griess reaction and multiplex ELISA (Luminex®). Gene expression was quantified by real-time PCR. Nuclear translocation of NF-κB was measured by cytometric techniques. LPS-activated RAW264.7 cells produced nitric oxide (NO), COX2-dependent prostaglandin E2 (PGE2), interleukins and chemokines. LIG concentration-dependently reduced the production of nitric oxide (NO) and PGE2, although it did not match the inhibitory potential of 15d–PGJ2. LIG inhibited the secretion of cytokines (IL-1α, IL-6, TNF-α) and differentiation factors (GM-CSF) in murine macrophages. It blunted the production of CCL2/MCP-1, but did not alter the secretion of CCL5/RANTES. LIG reduced mRNA levels of pro-inflammatory cytokines (e.g. TNF-α, IL-1α, IL-6), chemokines (CCL4/MIP-1β), and pro-inflammatory enzymes (iNOS). Similarly, LIG robustly impaired inflammatory mediators (e.g. CCL2/MCP-1, CCL3-MIP-1α, CCL4/MIP-1β, CXCL10/IP-10, IL-12p70, TNF-α) of LPS-activated human THP-1 cells and PBLs. Unexpectedly, it augmented the production of IL-1β, IL-6 and GM-CSF in PBLs. LIG diminished the extent of the inflammatory response measured by the production of different mediators or metabolites (NO, PGE2, interleukins, cytokines, chemokines). LIG acted at the transcriptional level and targeted the NF-κB signaling pathway. Since LIG and the anti-inflammatory prostaglandin 15d–PGJ2 share most of the analyzed biological features, we infer that they have similar modes of action. Hence, LIG acts as an anti-inflammatory prostaglandin and modulates cytokine- and chemokine-dependent inflammatory responses.

Modification of the Orthosteric PPARγ Covalent Antagonist Scaffold Yields an Improved Dual-Site Allosteric Inhibitor.

GW9662 and T0070907 are widely used commercially available irreversible antagonists of peroxisome proliferator-activated receptor gamma (PPARγ). These antagonists covalently modify Cys285 located in an orthosteric ligand-binding pocket embedded in the PPARγ ligand-binding domain and are used to block binding of other ligands. However, we recently identified an alternate/allosteric ligand-binding site in the PPARγ LBD to which ligand binding is not inhibited by these orthosteric covalent antagonists. Here, we developed a series of analogs based on the orthosteric covalent antagonist scaffold with the goal of inhibiting both orthosteric and allosteric cellular activation of PPARγ by MRL20, an orthosteric agonist that also binds to an allosteric site. Our efforts resulted in the identification of SR16832 (compound 22), which functions as a dual-site covalent inhibitor of PPARγ transcription by PPARγ-binding ligands. Molecular modeling, protein NMR spectroscopy structural analysis, and biochemical assays indicate the inhibition of allosteric activation occurs in part through expansion of the 2-chloro-5-nitrobenzamidyl orthosteric covalent antagonist toward the allosteric site, weakening of allosteric ligand binding affinity, and inducing conformational changes not competent for cellular PPARγ activation. Furthermore, SR16832 better inhibits binding of rosiglitazone, a thiazolidinedione (TZD) that weakly activates PPARγ when cotreated with orthosteric covalent antagonists, and may better inhibit binding of endogenous PPARγ ligands such as docosahexaenoic acid (DHA) compared to orthosteric covalent antagonists. Compounds such as SR16832 may be useful chemical tools to use as a dual-site bitopic orthosteric and allosteric covalent inhibitor of ligand binding to PPARγ.

Ineffective correction of PPARγ signaling in cystic fibrosis airway epithelial cells undergoing repair

Peroxisome proliferator-activated receptor gamma (PPARγ) represents a potential target to treat airway mucus hypersecretion in cystic fibrosis (CF). We aimed to determine if PPARγ is altered in CF human airway epithelial cells (HAECs), if PPARγ contributes to mucin expression and HAEC differentiation, and if PPARγ ligand therapy corrects the CF phenotype. To this end, well-differentiated CF and NCF HAEC primary cultures were wounded to monitor the expression of key genes involved in PPARγ activation and mucus homeostasis, and to evaluate the effect of a PPARγ agonist, at different times of repair. Hydroxyprostaglandin dehydrogenase (HPGD) converts prostaglandin E2 to 15-keto PGE2 (15kPGE2), an endogenous PPARγ ligand. Interestingly, PPARγ and HPGD expression dramatically decreased in CF HAECs. These changes were accompanied by an increase in the expression of MUC5B. The correlation between PPARγ and MUC5B was confirmed in an airway epithelial cell line after CFTR knock-down. Exposure of HAECs to 15kPGE2 did not correct the CF phenotype but revealed a defect in the process of basal cell (BC) differentiation. The HPGD/PPARγ axis is deregulated in primary HAEC cultures from CF patients, which may impact the maturation of BCs to differentiated luminal cells. Importantly, PPARγ therapy was inefficient in correcting the CF defect.

Inhibition of Prostaglandin Reductase 2, a Putative Oncogene Overexpressed in Human Pancreatic Adenocarcinoma, Induces Oxidative Stress-Mediated Cell Death Involving xCT and CTH Gene Expressions through 15-Keto-PGE2.

Prostaglandin reductase 2 (PTGR2) is the enzyme that catalyzes 15-keto-PGE2, an endogenous PPARγ ligand, into 13,14-dihydro-15-keto-PGE2. Previously, we have reported a novel oncogenic role of PTGR2 in gastric cancer, where PTGR2 was discovered to modulate ROS-mediated cell death and tumor transformation. In the present study, we demonstrated the oncogenic potency of PTGR2 in pancreatic cancer. First, we observed that the majority of the human pancreatic ductal adenocarcinoma tissues was stained positive for PTGR2 expression but not in the adjacent normal parts. In vitro analyses showed that silencing of PTGR2 expression enhanced ROS production, suppressed pancreatic cell proliferation, and promoted cell death through increasing 15-keto-PGE2. Mechanistically, silencing of PTGR2 or addition of 15-keto-PGE2 suppressed the expressions of solute carrier family 7 member 11 (xCT) and cystathionine gamma-lyase (CTH), two important providers of intracellular cysteine for the generation of glutathione (GSH), which is widely accepted as the first-line antioxidative defense. The oxidative stress-mediated cell death after silencing of PTGR2 or addition of 15-keto-PGE2 was further abolished after restoring intracellular GSH concentrations and cysteine supply by N-acetyl-L-cysteine and 2-Mercaptomethanol. Our data highlight the therapeutic potential of targeting PTGR2/15-keto-PGE2 for pancreatic cancer.

ARTD1-induced poly-ADP-ribose formation enhances PPARγ ligand binding and co-factor exchange.

PPARγ-dependent gene expression during adipogenesis is facilitated by ADP-ribosyltransferase D-type 1 (ARTD1; PARP1)-catalyzed poly-ADP-ribose (PAR) formation. Adipogenesis is accompanied by a dynamic modulation of the chromatin landscape at PPARγ target genes by ligand-dependent co-factor exchange. However, how endogenous PPARγ ligands, which have a low affinity for the receptor and are present at low levels in the cell, can induce sufficient co-factor exchange is unknown. Moreover, the significance of PAR formation in PPARγ-regulated adipose tissue function is also unknown. Here, we show that inhibition of PAR formation in mice on a high-fat diet reduces weight gain and cell size of adipocytes, as well as PPARγ target gene expression in white adipose tissue. Mechanistically, topoisomerase II activity induces ARTD1 recruitment to PPARγ target genes, and ARTD1 automodification enhances ligand binding to PPARγ, thus promoting sufficient transcriptional co-factor exchange in adipocytes. Thus, ARTD1-mediated PAR formation during adipogenesis is necessary to adequately convey the low signal of endogenous PPARγ ligand to effective gene expression. These results uncover a new regulatory mechanism of ARTD1-induced ADP-ribosylation and highlight its importance for nuclear factor-regulated gene expression.

Transcriptional regulation in adipogenesis through PPARγ-dependent and -independent mechanisms by prostaglandins.

Adipogenesis is controlled by complex mechanisms, and transcription factors are involved in its regulation. PPARγ is a ligand-dependent transcription factor and the most important one for adipogenesis. Although prostaglandin (PG) D2 metabolites have been reported as being the ligands of PPARγ, the endogenous PPARγ ligand in adipocytes remains unclear. Here, we show the methods for the general analysis of adipocyte differentiation and the protocols for promoter analysis, fluorescence EMSA, and chromatin immunoprecipitation assay for the transcriptional regulation of the SREBP-1c-activated lipocalin-type PGD synthase gene in adipocytes. Moreover, we describe that PGD2 and its metabolites are involved in the regulation of adipogenesis through PPARγ-dependent and -independent mechanisms.

New Insights into the PPARγAgonists for the Treatment of Diabetic Nephropathy

Diabetic nephropathy (DN) is a severe complication of diabetes and serves as the leading cause of chronic renal failure. In the past decades, angiotensin-converting enzyme inhibitors (ACEIs)/angiotensin II receptor blockers (ARBs) based first-line therapy can slow but cannot stop the progression of DN, which urgently requests the innovation of therapeutic strategies. Thiazolidinediones (TZDs), the synthetic exogenous ligands of nuclear receptor peroxisome proliferator-activated receptor-γ (PPARγ), had been thought to be a promising candidate for strengthening the therapy of DN. However, the severe adverse effects including fluid retention, cardiovascular complications, and bone loss greatly limited their use in clinic. Recently, numerous novel PPARγ agonists involving the endogenous PPARγ ligands and selective PPARγ modulators (SPPARMs) are emerging as the promising candidates of the next generation of antidiabetic drugs instead of TZDs. Due to the higher selectivity of these novel PPARγ agonists on the regulation of the antidiabetes-associated genes than that of the side effect-associated genes, they present fewer adverse effects than TZDs. The present review was undertaken to address the advancements and the therapeutic potential of these newly developed PPARγ agonists in dealing with diabetic kidney disease. At the same time, the new insights into the therapeutic strategies of DN based on the PPARγ agonists were fully addressed.

Prostaglandin reductase-3 negatively modulates adipogenesis through regulation of PPARγ activity

Adipocyte differentiation is a multistep program under regulation by several factors. Peroxisome proliferator-activated receptor γ (PPARγ) serves as a master regulator of adipogenesis. However, the endogenous ligand for PPARγ remained elusive until 15-keto-PGE2 was identified recently as an endogenous PPARγ ligand. In this study, we demonstrate that zinc-containing alcohol dehydrogenase 2 (ZADH2; here termed prostaglandin reductase-3, PTGR-3) is a new member of prostaglandin reductase family that converts 15-keto-PGE2 to 13,14-dihydro-15-keto-PGE2. Adipogenesis is accelerated when endogenous PTGR-3 is silenced in 3T3-L1 preadipocytes, whereas forced expression of PTGR-3 significantly decreases adipogenesis. PTGR-3 expression decreased during adipocyte differentiation, accompanied by an increased level of 15-keto-PGE2. 15-keto-PGE2 exerts a potent proadipogenic effect by enhancing PPARγ activity, whereas overexpression of PTGR-3 in 3T3-L1 preadipocytes markedly suppressed the proadipogenic effect of 15-keto-PGE2 by repressing PPARγ activity. Taken together, these findings demonstrate for the first time that PTGR-3 is a novel 15-oxoprostaglandin-Δ(13)-reductase and plays a critical role in modulation of normal adipocyte differentiation via regulation of PPARγ activity. Thus, modulation of PTGR-3 might provide a novel avenue for treating obesity and related metabolic disorders.

Eicosanoids and Docosanoids in Plasma and Aorta of Healthy and Atherosclerotic Rabbits

Eicosanoids and docosanoids have been shown to be involved in atherosclerosis. Polyunsaturated fatty acids (PUFAs) are important nutrients that are metabolized by lipoxygenases and cyclooxygenases to various mono-hydroxy metabolites which can be further metabolized by specific enzymes to more complex eicosanoids and docosanoids. In this study a high-performance liquid chromatography methodology was established and rabbits were fed with a control or a high-cholesterol diet to induce atherosclerotic lesions to determine pro- or anti-inflammatory lipid mediators in atherosclerotic vessels. In aortic samples from atherosclerotic rabbits we determined for the first time various eicosanoids/docosanoids and observed an increased concentration of 12-lipoxygenase metabolites. Increased levels of 12-hydroxyeicosatetraenoic acid (12-HETE) in high-cholesterol versus control animals as well as increased ratios of 12-HETE/arachidonic acid ratios indicate that 12-lipoxygenase metabolites may have importance in atherosclerosis. In addition, decreased concentrations of the 5-lipoxygenase metabolite leukotriene B4 levels were detected in high-cholesterol animals. A positive correlation of total plaque area with plasma levels of 12-HETE and a negative correlation with aortic levels of endogenous PPARγ-ligand 13-oxo-octadecadienoic acid were found. This study let us conclude that the cholesterol content in the diet might influence atherosclerosis via increased 12-lipoxygenase- and cyclooxygenase-mediated pathways and reduced 5-lipoxygenase pathways.

15-PGDH inhibits hepatocellular carcinoma growth through 15-keto-PGE2/PPARγ-mediated activation of p21WAF1/Cip1

15-hydroxyprostaglandin dehydrogenase (15-PGDH) is a key enzyme in prostaglandin metabolism. This study provides important evidence for inhibition of hepatocellular carcinoma (HCC) growth by 15-PGDH through the 15-keto-PGE2/PPARγ/p21WAF1/Cip1 signaling pathway. Forced overexpression of 15-PGDH inhibited HCC cell growth in vitro, whereas knockdown of 15-PGDH enhanced tumor growth parameters. In a tumor xenograft model in SCID mice, inoculation of human HCC cells (Huh7) with overexpression of 15-PGDH led to significant inhibition of tumor growth, while knockdown of 15-PGDH enhanced tumor growth. In a separate tumor xenograft model in which mouse HCC cells (Hepa1-6) were inoculated into syngeneic C57BL/6 mice, intratumoral injection of adenovirus vector expressing 15-PGDH (pAd-15-PGDH) significantly inhibited xenograft tumor growth. The anti-tumor effect of 15-PGDH is mediated through its enzymatic product, 15-keto-PGE2, which serves as an endogenous PPARγ ligand. Activation of PPARγ by 15-PGDH-derived 15-keto-PGE2 enhanced the association of PPARγ with the p21WAF1/Cip1 promoter and increased p21 expression and association with CDK2, CDK4 and PCNA. Depletion of p21 by shRNA reversed 15-PGDH-induced inhibition of HCC cell growth; overexpression of p21 prevented 15-PGDH knockdown-induced tumor cell growth. These results demonstrate a key 15-PGDH/15-keto-PGE2-mediated activation of PPARγ and p21WAF1/Cip1 signaling cascade that regulates hepatocarcinogenesis and tumor progression.

Transcriptional Regulation of IL-10 Expression by Peroxisome Proliferator-Activated Receptor (Pparγ) in Human Intestinal Epithelial Cells

IL-10 is an important immunomodulatory cytokine in the regulation of intestinal inflammation in inflammatory bowel disease (IBD). PPARγ is a nuclear receptor known to modulate transcriptional activities of many cytokines and is highly expressed in intestinal epithelial cells (IECs). Human IECs have been shown to express IL-10, but how its expression is regulated remains obscure especially in the setting of inflammation. We have demonstrated that intestinal 15d-PGJ2 (an endogenous PPARγ ligand) is predominantly induced by infiltrated macrophages (Mφs) through toll-like receptor 4 (TLR4)-mediated Cox-2 induction during colitis. In this study, we addressed whether PPARγ activation regulates expression of IL-10 in IECs. Human IEC cell line SW480-APC expressing TLR4 (top) and mouse peritoneal Mφs (bottom) were co-cultured in transwells. 15d-PGJ2 production and IL-10 mRNA expression were measured by enzyme linked assay and real-time PCR in the presence or absence of lipopolysaccharide (LPS). In order to examine whether the IL-10 promoter has PPARγ responsive elements, a 2,000 bp of IL-10 promoter was cloned from SW480-APC genomic DNA into the luciferase reporter vector. Expression and activity of PPARγ were blocked by siRNA and a specific inhibitor SR202, respectively. The IL-10 promoter activity was analyzed by luciferase reporter assay. PPARγ activity was confirmed using a reporter vector PPRE X3-TK-luc. Co-culturing SW480-APC with Mφs increased IL-10 expression in IECs within 24 hours, which was significantly enhanced by LPS stimulation in later time point (72 hours). LPS stimulation of SW480-APC resulted in PPARγ activation peaked at 48 hours. During SW480-APC/Mφ co-culture, LPS stimulation induced 15d-PGJ2 production, which is abolished when TLR4-deficient Mφs were used. Knocking down the expression of PPARγ suppressed IL-10 promoter activity and mRNA expression in SW480-APC in baseline as well as after LPS stimulation. These results suggest that PPARγ positively regulates IL-10 expression in IECs at the transcriptional level. During inflammation, mucosal Mφs induce PPARγ ligand (15d-PGJ2) in a TLR4-dependent manner that contributes to PPARγ activation in IECs and thus IL-10 expression. This may be an intrinsic immunoregulatory program that is mediated through host-bacterial interactions, which dampens on-going inflammation during intestinal inflammation and thus can be a novel therapeutic target for IBD.

Endothelial Cell Peroxisome Proliferator–Activated Receptor γ Reduces Endotoxemic Pulmonary Inflammation and Injury

Bacterial endotoxin (LPS)-mediated sepsis involves severe, dysregulated inflammation that injures the lungs and other organs, often fatally. Vascular endothelial cells are both key mediators and targets of LPS-induced inflammatory responses. The nuclear hormone receptor peroxisome proliferator-activated receptor γ (PPARγ) exerts anti-inflammatory actions in various cells, but it is unknown whether it modulates inflammation through actions within endothelial cells. To determine whether PPARγ acts within endothelial cells to diminish endotoxemic lung inflammation and injury, we measured inflammatory responses and mediators in mice with endothelial-targeted deletion of PPARγ. Endothelial cell PPARγ (ePPARγ) knockout exacerbated LPS-induced pulmonary inflammation and injury as shown by several measures, including infiltration of inflammatory cells, edema, and production of reactive oxygen species and proinflammatory cytokines, along with upregulation of the LPS receptor TLR4 in lung tissue and increased activation of its downstream signaling pathways. In isolated LPS-stimulated endothelial cells in vitro, absence of PPARγ enhanced the production of numerous inflammatory markers. We hypothesized that the observed in vivo activity of the ligand-activated ePPARγ may arise, in part, from nitrated fatty acids (NFAs), a novel class of endogenous PPARγ ligands. Supporting this idea, we found that treating isolated endothelial cells with physiologically relevant concentrations of the endogenous NFA 10-nitro-oleate reduced LPS-induced expression of a wide range of inflammatory markers in the presence of PPARγ, but not in its absence, and also inhibited neutrophil mobility in a PPARγ-dependent manner. Our results demonstrate a key protective role of ePPARγ against endotoxemic injury and a potential ePPARγ-mediated anti-inflammatory role for NFAs.

Identification of novel markers of alternative activation and potential endogenous PPARγ ligand production mechanisms in human IL-4 stimulated differentiating macrophages

We analyzed global gene expression profiles of IL-4 induced alternatively activated as well as IFNγ+TNFα stimulated classically activated human monocyte derived macrophages and identified novel IL-4 regulated alternative activation marker genes including MS4A4A, SLA, CD180, and ENPP2. Transcription factor prediction analysis of IL-4 regulated genes suggested that the regulated genes are involved in a complex regulation of lipid metabolism, defense against cell metabolism derived reactive oxygen species, and basal expression of inflammation linked genes. Both an in silico transcription activation prediction as well as experimental data suggested the presence of alternative macrophage activation specific endogenous PPARγ ligand producing mechanisms. We found the induction of three enzymes whose activity can potentially generate endogenous PPARγ ligands in an IL-4 dependent manner. These are MAOA, ENPP2, and ALOX15 producing 5-methoxy-indole acetate, lysophosphatidic acid (LPA) and 13-hydroxyoctadienoic acid (13-HODE), and/or 15-hydroxyeicosatetraenoic acid (15-HETE), respectively. Our data suggest that global gene expression profiling, combined with computational transcription activity prediction, can lead to identification of transcriptional networks that underpin cellular subtype specification.