Wild-type PPARγ Research Articles

Cigarette Smoke Extract Modulates Functions of Peroxisome Proliferator-Activated Receptors.

Cigarette smoke extract (CSE) contains many toxicants and may derange the physiological processes, such as cholesterol metabolism. We examined the impact of CSE on transcriptional regulation mediated peroxisome proliferator-activated receptors (PPARs) and its interaction with cofactors to elucidate differences in the molecular mechanism between CSE and other agonists of PPARs. We constructed several mutant PPARs (mPPARs) with amino acid substitution in the ligand-binding domain, which according to the molecular modeling, may affect the binding of agonists. In transient expression assays, each wild-type peroxisome proliferator-activated receptor (PPAR) mediated transcription stimulated by CSE was faintly yet significantly elevated compared to the control. The CSE-induced transcriptional activation was abolished in the H323A, H323Y, S342A, and H449A mPPARγs, although the activation elevated by pioglitazone was reserved. In the mPPARγ with Y473A and mPPARβ/δs with H286Y and Y436A, the pioglitazone-induced or L165041-activated transcriptional elevations were decreased and were lower than that of CSE-induced stimulation. These results suggested that CSE activated both mutant PPARs to be selectively different from those ligands. Mammalian two-hybrid assay illustrated that CSE could mildly recruit SRC1 or GRIP1 to the wild-type PPARγ. Representative ingredients, such as acrolein and crotonaldehyde present in CSE, could stimulate PPAR isoforms even at the toxicological concentrations and might possibly contribute to stimulatory effects. CSE mildly regulates the cholesterol metabolism-related genes, such as low density lipoprotein (LDL) receptor and Liver X receptor (LXR)β. In conclusion, these CSE effects the nuclear hormone receptors and their cofactors thereby disturbing metabolic phenomena. Therefore, CSE might be involved in cholesterol metabolism.

Association of polimorphism rs1801282 with different components of the metabolic syndrome

Objective. To study the association of polymorphism rs1801282 of the PPARγ gene with different components of metabolic syndrome (MS). Materials and methods. 145 subjects with newly diagnosed MS were examined. All subjects underwent a clinical examination, including general clinical, laboratory and instrumental methods; presence of polymorphism rs1801282 of the PPARγ gene was also determined. Results. In the group of subjects with rs1801282 polymorphism, hyperglycemia was observed less frequently compared with the rest of subjects the incidence was 53.8% and 79.8% cases respectively (p = 0.005). A decrease in the risk of arterial hypertension was also observed in subjects with polymorphic allele: an increase in blood pressure was noted in 70.6% of rs1801282 group and 96.2% for wild type PPARγ (p < 0.001) When analyzing levels of triglycerides and high-density lipoproteins, an increase in these parameters was noted in the group of subjects with rs1801282 polymorphism. Conclusion. The results of the study indicate the association of rs1801282 polymorphism in subjects with metabolic syndrome with a number of clinical features of the disease, dyslipidemia and a reduction of the risk of hyperglycemia and hypertension. (For citation: Eremenko TV, Kotova SM, Matezius IYu, et al. Association of polimorphism rs1801282 with different components of the metabolic syndrome. Herald of North-Western State Medical University named after I.I. Mechnikov. 2018;10(2):39-43. doi: 10.17816/mechnikov201810239-43).

Association of polimorphism rs1801282 with different components of the metabolic syndrome

Objective. To study the association of polymorphism rs1801282 of the PPARγ gene with different components of metabolic syndrome (MS).
 Materials and methods. 145 subjects with newly diagnosed MS were examined. All subjects underwent a clinical examination, including general clinical, laboratory and instrumental methods; presence of polymorphism rs1801282 of the PPARγ gene was also determined.
 Results. In the group of subjects with rs1801282 polymorphism, hyperglycemia was observed less frequently compared with the rest of subjects the incidence was 53.8% and 79.8% cases respectively (p = 0.005). A decrease in the risk of arterial hypertension was also observed in subjects with polymorphic allele: an increase in blood pressure was noted in 70.6% of rs1801282 group and 96.2% for wild type PPARγ (p < 0.001). When analyzing levels of triglycerides and high-density lipoproteins, an increase in these parameters was noted in the group of subjects with rs1801282 polymorphism.
 Conclusion. The results of the study indicate the association of rs1801282 polymorphism in subjects with metabolic syndrome with a number of clinical features of the disease, dyslipidemia and a reduction of the risk of hyperglycemia and hypertension.

Abstract 001: Mutation in the PPARγ Ligand Binding Domain Impairs the Anti-inflammatory Action of PPARγ

Peroxisome proliferator-activated receptor gamma (PPARγ) has been proposed to antagonize the activities of nuclear factor kappa B (NFκB) to regulate inflammation. Transgenic mice expressing dominant negative (DN) PPARγ specifically in vascular smooth muscle cells (SMC) exhibited exacerbated atherosclerosis but the mechanism remains unknown. We hypothesized that DN PPARγ promotes NFκB-induced inflammation in SMC. To test this, we cultured mesenteric SMCs from transgenic mice that would conditionally express wild-type (WT) or DN PPARγ (P467L) in response to adenovirus expressing Cre-recombinase (AdCRE). PPARγ expression remained silent in control SMC infected with AdGFP. TNF-α (0.05 ng/ml, 6 hr) induced NFκB target gene (MCP-1, iNOS and MMP9) expression to a greater extent in P467L-Cre compared to P467L-GFP. For example, MMP9 expression was induced 6.3±0.2-fold in P467L-Cre vs 3.2±0.5-fold in P467L-GFP (p<0.01). The NFκB subunit, p65, mRNA level was not altered in these cells. There was no induction of the PPARγ target aP2 in P467L-Cre, but it was induced 6.5±1.7-fold in WT-SMC infected with AdCRE (WT-Cre). The ability of TNF-α to induce NFκB target gene expression was blunted or abrogated in WT-Cre cells, and their expression was significantly reduced in TNF-α-treated WT-Cre compared to WT-GFP (MMP9: 0.7±1.2 vs 6.0±0.3, p<0.01). To examine mechanisms in vivo, we crossbred transgenic mice expressing WT PPARγ specifically in SMC (S-WT) with mice expressing luciferase under control of a NFκB-responsive promoter. TNF-α (500 ng/ml, 24 hr)-induced NFκB activity was decreased in aorta and carotid artery from S-WT mice compared to control mice (aorta: 4.7±1.1 v s 6.7±0.7, p<0.05, carotid artery: 4.0±0.6 vs 8.7±1.2, p<0.01). Finally, to assess the mechanism preventing anti-inflammatory activity by DN PPARγ, we assessed its interaction with p65 protein when co-expressed in HEK293T cells. WT PPARγ co-precipitated with p65, but the interaction between p65 and P467L PPARγ was severely impaired (n=6). Other mutants (R165T, V290M) could bind to p65, suggesting that loss of the ability is specific to P467L PPARγ. We conclude that SMC-PPARγ has anti-inflammatory effects mediated through inhibition of NFκB activity, which is abolished by P467L mutation.

PPARγ controls pregnancy outcome through activation of EG-VEGF: new insights into the mechanism of placental development.

PPARγ-deficient mice die at E9.5 due to placental abnormalities. The mechanism by which this occurs is unknown. We demonstrated that the new endocrine factor EG-VEGF controls the same processes as those described for PPARγ, suggesting potential regulation of EG-VEGF by PPARγ. EG-VEGF exerts its functions via prokineticin receptor 1 (PROKR1) and 2 (PROKR2). This study sought to investigate whether EG-VEGF mediates part of PPARγ effects on placental development. Three approaches were used: 1) in vitro, using human primary isolated cytotrophoblasts and the extravillous trophoblast cell line (HTR-8/SVneo); 2) ex vivo, using human placental explants (n = 46 placentas); and 3) in vivo, using gravid wild-type PPARγ(+/-) and PPARγ(-/-) mice. Major processes of placental development that are known to be controlled by PPARγ, such as trophoblast proliferation, migration, and invasion, were assessed in the absence or presence of PROKR1 and PROKR2 antagonists. In both human trophoblast cell and placental explants, we demonstrated that rosiglitazone, a PPARγ agonist, 1) increased EG-VEGF secretion, 2) increased EG-VEGF and its receptors mRNA and protein expression, 3) increased placental vascularization via PROKR1 and PROKR2, and 4) inhibited trophoblast migration and invasion via PROKR2. In the PPARγ(-/-) mouse placentas, EG-VEGF levels were significantly decreased, supporting an in vivo control of EG-VEGF/PROKRs system during pregnancy. The present data reveal EG-VEGF as a new mediator of PPARγ effects during pregnancy and bring new insights into the fine mechanism of trophoblast invasion.

Role of endothelial PPARγ: Protection against vascular dysfunction induced by IL‐1β

Peroxisome proliferator‐activated receptor gamma (PPARγ) has been proposed to mediate anti‐inflammatory actions by promoting the degradation of the p65 subunit of nuclear factor kappa B (NFκB). We hypothesize that PPARγ protects against NFκB‐induced vascular dysfunction by promoting p65 degradation in vascular endothelial cells (EC). We used a model of interleukin‐1β (IL‐1β)‐induced aortic dysfunction. Aorta was treated with IL‐1β (24 hr) in vitro and then EC function was measured in an organ chamber. IL‐1β dose‐dependently decreased IκBα expression, increased phospho‐p65, and increased luciferase activity in NFκB‐Luc transgenic mice. IL‐1β dose‐dependently reduced EC‐dependent relaxation by acetylcholine (ACh). The loss of ACh responsiveness was partially improved by pretreating vessels with the PPARγ agonist, rosiglitazone, or in transgenic mice over‐expressing wildtype PPARγ specifically in EC (E‐WT). Consistent with this, low dose IL‐1β was sufficient to cause EC dysfunction in aorta from transgenic mice expressing dominant negative PPARγ specifically in EC (E‐V290M). A p65 inhibitor peptide did not alleviate EC dysfunction induced by IL‐1β, but Tempol, a superoxide dismutase mimetic partially restored ACh responsiveness in both control and E‐WT aorta treated with IL‐1β. TUDCA, a chemical chaperone which reduces endoplasmic reticulum (ER) stress significantly improved EC dysfunction induced by IL‐1β in control aorta, but not aorta from E‐WT mice. Aorta isolated from CHOP‐deficient mice exhibited less IL‐1β‐induced EC dysfunction than control mice. These data suggest that EC PPARγ protects against IL‐1β‐mediated EC dysfunction, possibly through the reduction of reactive oxygen species and blunting IL‐1β‐mediated ER stress.

Role of peroxisome proliferator-activated receptor-γ in vascular muscle in the cerebral circulation.

Although peroxisome proliferator-activated receptor-γ (PPARγ) is thought to play a protective role in the vasculature, its cell-specific effect, particularly in resistance vessels, is poorly defined. Nitric oxide (NO) plays a major role in vascular biology in the brain. We examined the hypothesis that selective interference with PPARγ in vascular muscle would impair NO-dependent responses and augment vasoconstrictor responses in the cerebral circulation. We studied mice expressing a dominant negative mutation in human PPARγ (P467L) under the control of the smooth muscle myosin heavy chain promoter (S-P467L). In S-P467L mice, dilator responses to exogenously applied or endogenously produced NO were greatly impaired in cerebral arteries in vitro and in small cerebral arterioles in vivo. Select NO-independent responses, including vasodilation to low concentrations of potassium, were also impaired in S-P467L mice. In contrast, increased expression of wild-type PPARγ in smooth muscle had little effect on vasomotor responses. Mechanisms underlying impairment of both NO-dependent and NO-independent vasodilator responses after interference with PPARγ involved Rho kinase with no apparent contribution by oxidative stress-related mechanisms. These findings support the concept that via effects on Rho kinase-dependent signaling, PPARγ in vascular muscle is a major determinant of vascular tone in resistance vessels and, in particular, NO-mediated signaling in cerebral arteries and brain microvessels. Considering the importance of NO and Rho kinase, these findings have implications for regulation of cerebral blood flow and the pathogenesis of large and small vessel disease in brain.

Abstract 331: Mutation in the PPARG Ligand Binding Domain Impairs PPARG-Mediated Turnover of the p65 Subunit of NF-kB

Peroxisome proliferator-activated receptor gamma (PPARγ) is a ligand activated transcription factor regulating metabolic and vascular function. PPARγ exerts anti-inflammatory actions, and recent data suggest this may be mediated by promoting the degradation of the p65 subunit of nuclear factor kappa B (NFκB). Transgenic mice expressing dominant negative PPARγ specifically in vascular smooth muscle or endothelium exhibited exacerbated atherosclerosis but the mechanism remains unknown. We hypothesized that PPARγ mutants promote inflammation because PPARγ-mediated p65 degradation is impaired. We tested this by co-transfection of HEK293T cells with vectors encoding p65, wildtype (WT) PPARγ, or various PPARγ mutants. The level of p65 protein expression was decreased by co-expression with WT-PPARγ (0.53±0.09 vs control, n=8). Whereas, the P467L PPARγ exhibited impaired degradation of p65 (1.0±0.06, n=10), the V290M (0.36±0.1), S273A (0.37±0.06), or K268R/K293R (0.41±0.03) mutations in PPARγ preserved p65 degradation. WT PPARγ was co-precipitated with p65 in co-transfected cells suggesting the mechanism of PPARγ-mediated p65 degradation involves a direct interaction between them. Consistent with this, the interaction between p65 and P467L PPARγ was severely impaired. To assess functional interactions between PPARγ and NFκB, we employed a model of interleukin-1β (IL-1β) mediated dysfunction in aortic rings. IL-1β dose-dependently induced NFκB activity as measured by increased phospho-p65 and decreased IκBα in aorta cultured for 2 hours with IL-1β. IL-1β dose-dependently reduced acetylcholine (Ach)-induced endothelial-dependent relaxation of aortic rings (80±12 vs 39±16, 20 pg/mL vs 11±3, 100 pg/mL, %). IL-1β-mediated loss of Ach vasodilation was reduced by the PPARγ agonist rosiglitazone (1 μM, 25 hr, n=3, p<0.05), or by transgenic over-expression of WT-PPARγ specifically in endothelium (n=6, p<0.05). We conclude that 1) p65 turnover may be regulated by PPARγ and that its mutation can result in impaired p65 degradation, 2) PPARγ activity can protect against vascular dysfunction associated with NFκB activation, and 3) loss of PPARγ-mediated p65 degradation may contribute to inflammation in hypertension and atherosclerosis.

Pax-8-PPAR-γ fusion protein in thyroid carcinoma.

Thyroid carcinoma is the most common endocrine malignancy, and its incidence is continuing to increase. Most thyroid carcinomas contain one of several known driver mutations, such as the Val600Glu substitution in B-Raf, Ras mutations, RET gene fusions, or PAX8-PPARG gene fusions. The PAX8-PPARG gene fusion results in the production of a Pax-8-PPAR-γ fusion protein (PPFP), which is found in approximately one-third of follicular thyroid carcinomas, as well as some follicular-variant papillary thyroid carcinomas. In vitro and in vivo evidence indicates that PPFP is an oncoprotein. Although specific mechanisms of action remain to be defined, PPFP is considered to act as a dominant-negative inhibitor of wild-type PPAR-γ and/or as a unique transcriptional activator of subsets of PPAR-γ-responsive and Pax-8-responsive genes. Detection of the fusion transcript in thyroid nodule biopsy specimens can aid clinical decision-making when cytological findings are indeterminate. The PPAR-γ agonist pioglitazone is highly therapeutic in a transgenic mouse model of PPFP-positive thyroid carcinoma, suggesting that PPAR-γ agonists might be beneficial in patients with PPFP-positive thyroid carcinomas.

A Clinical Link Between Peroxisome Proliferator-Activated Receptor γ and the Renin–Angiotensin System

A mechanistic link between peroxisome proliferator-activated receptor γ (PPARγ) and the renin–angiotensin system (RAS) has been previously proposed, but clinical evidence supporting the relationship is incomplete. In the current issue of Arteriosclerosis, Thrombosis, and Vascular Biology , Caron-Debarle et al show that 4 patients with familial partial lipodystrophy associated with early-onset severe hypertension carry mutations in PPARγ that impair its ability to act as a ligand-activated transcription factor. Cells isolated from these patients, and cells transfected with the same mutations in PPARγ, exhibit activation of the cellular RAS, increased production of reactive oxygen species (ROS), and markers of inflammation, all of which are dependent on the angiotensin-II (Ang-II) AT1 receptor (AT1R). This translational study further supports a role for PPARγ as a regulator of blood pressure through its ability to modulate the RAS. See accompanying article on page 829 PPARγ is a ligand-activated transcription factor and target of the thiazolidinedione (TZD) class of antidiabetes mellitus medications. PPARγ is best recognized for its role in adipogenesis, but is also a regulator of systemic metabolism as evidenced by the pleiotropic abnormalities (lipodystrophy, insulin-resistance, and metabolic syndrome) caused by PPARγ mutations.1–3 Clinical studies of TZD use in type 2 diabetes mellitus, including the PROactive (PROspective pioglitAzone Clinical Trial In macroVascular Events) trial, documented improved endothelial function and modest, but significant reductions in blood pressure.4 Some of the same mutations that cause lipodystrophy and diabetes mellitus also cause severe hypertension and preeclampsia in human patients3 and in knock-in mice.5,6 Evidence suggests that PPARγ activity in the vascular endothelium and smooth muscle are important regulators of endothelial function, smooth muscle contraction, and systemic blood pressure.7,8 Data suggesting a role for PPARγ in regulating blood pressure led many to search for downstream …

Dominant negative PPARγ promotes atherosclerosis, vascular dysfunction, and hypertension through distinct effects in endothelium and vascular muscle

Agonists of the nuclear hormone receptor peroxisome proliferator-activated receptor γ (PPARγ) have potent insulin-sensitizing effects and inhibit atherosclerosis progression in patients with Type II diabetes. Conversely, missense mutations in the ligand-binding domain of PPARγ that render the transcription factor dominant negative (DN) cause early-onset hypertension and Type II diabetes. We tested the hypothesis that DN PPARγ-mediated interference of endogenous wild-type PPARγ in the endothelium and vascular smooth muscle exacerbates atherosclerosis in apolipoprotein E-deficient (ApoE(-/-)) mice. Endothelium-specific expression of DN PPARγ on the ApoE(-/-) background unmasked significant impairment of endothelium-dependent relaxation in aortic rings, increased systolic blood pressure, altered expression of atherogenic markers (e.g., Cd36, Mcp1, Catalase), and enhanced diet-induced atherosclerotic lesion formation in aorta. Smooth muscle-specific expression of DN PPARγ, which induces aortic dysfunction and increased systolic blood pressure at baseline, also resulted in enhanced diet-induced atherosclerotic lesion formation in aorta on the ApoE(-/-) background that was associated with altered expression of a shared, yet distinct, set of atherogenic markers (e.g., Cd36, Mcp1, Osteopontin, Vcam1). In particular, induction of Osteopontin expression by smooth muscle-specific DN PPARγ correlated with increased plaque calcification. These data demonstrate that inhibition of PPARγ function specifically in the vascular endothelium or smooth muscle may contribute to cardiovascular disease.

Peroxisome Proliferator-Activated Receptor-γ Mutations Responsible for Lipodystrophy With Severe Hypertension Activate the Cellular Renin–Angiotensin System

Inactivating peroxisome proliferator-activated receptor-γ (PPARγ) mutations lead to a syndrome of familial partial lipodystrophy (FPLD3) associated with early-onset severe hypertension. PPARγ can repress the vascular renin-angiotensin system (RAS) and angiotensin II receptor 1 expression. We evaluated the relationships between PPARγ inactivation and cellular RAS using FPLD3 patients' cells and human vascular smooth muscle cells expressing mutant or wild-type PPARγ. Approach and Results- We identified 2 novel PPARG mutations, R165T and L339X, located in the DNA and ligand-binding domains of PPARγ, respectively in 4 patients from 2 FPLD3 families. In cultured skin fibroblasts and peripheral blood mononuclear cells from the 4 patients and healthy controls, we compared markers of RAS activation, oxidative stress, and inflammation, and tested the effect of modulators of PPARγ and angiotensin II receptor 1. We studied the impact of the 2 mutations on the transcriptional activity of PPARγ and on the vascular RAS in transfected human vascular smooth muscle cells. Systemic RAS was not altered in patients. However, RAS markers were overexpressed in patients' fibroblasts and peripheral blood mononuclear cells, as in vascular cells expressing mutant PPARγ. Angiotensin II-mediated mitogen-activated protein kinase activity increased in patients' fibroblasts, consistent with RAS constitutive activation. Patients' cells also displayed oxidative stress and inflammation. PPARγ activation and angiotensin II receptor 1 mRNA silencing reversed RAS overactivation, oxidative stress, and inflammation, arguing for a role of angiotensin II receptor 1 in these processes. Two novel FPLD3-linked PPARG mutations are associated with a defective transrepression of cellular RAS leading to cellular dysfunction, which might contribute to the specific FPLD3-linked severe hypertension.

Peroxisome Proliferator-activated Receptor γ Agonists Induce Cell Cycle Arrest through Transcriptional Regulation of Krüppel-like Factor 4 (KLF4)

Peroxisome proliferator-activated receptor γ (PPARγ), a subgroup of ligand-activated nuclear receptors, plays critical roles in cell cycle regulation, differentiation, apoptosis, and invasion. PPARγ is involved in tumorigenesis and is a potent target for cancer therapy. PPARγ transactivation of KLF4 has been demonstrated in various studies; however, how PPARγ regulates KLF4 expression is not clear. In this study, we reveal that PPARγ regulates the expression of KLF4 by binding directly to the PPAR response element (PPRE) within the KLF4 promoter. The PPRE resides at -1657 to -1669 bp upstream of the KLF4 ATG codon, which is essential for the transactivation of troglitazone-induced KLF4 expression. Furthermore, we found that stable silencing of KLF4 obviously suppressed the G(1)/S arrest and anti-proliferation effects induced by PPARγ ligands. Taken together, our data indicate that up-regulation of KLF4 upon PPARγ activation is mediated through the PPRE in the KLF4 promoter, thus providing further insights into the PPARγ signal transduction pathway as well as a novel cancer therapeutic strategy.

Characterisation of non-obese diabetic patients with marked insulin resistance identifies a novel familial partial lipodystrophy-associated PPARγ mutation (Y151C)

Aims/hypothesisFamilial partial lipodystrophy (FPLD) is a rare metabolic disorder with clinical features that may not be readily recognised. As FPLD patients require a specific therapeutic approach, early identification is warranted. In the present study we aimed to identify cases of FPLD among non-obese patients with type 2 diabetes mellitus and marked insulin resistance.MethodsWe searched the databases of three diabetic outpatient clinics for patients with marked insulin resistance, arbitrarily defined as the use of ≥100 U insulin/day, and BMI ≤ 27 kg/m2. In all patients, metabolic variables and anthropomorphic measurements were evaluated and DNA was sequenced for mutations in the genes encoding lamin A/C (LMNA), peroxisome proliferator-activated receptor γ (PPARγ) and cell death-inducing DFFA-like effector c (CIDEC).ResultsOut of 5,221 diabetic individuals, 24 patients fulfilled all criteria. Twelve patients were willing to participate, of whom five showed clinical features of lipodystrophy. In three of these patients the clinical diagnosis of FPLD was confirmed by the presence of mutations in LMNA or PPARG; one patient harboured a novel heterozygous mutation (Y151C) in PPARG. The Y151C mutant displayed impaired DNA-binding capacity and hence reduced transcriptional activity compared with wild-type PPARγ. Dominant-negative activity was absent.Conclusion/interpretationThe combination of BMI ≤ 27 kg/m2 and the use of >100 U insulin/day increases the chance of identifying lipodystrophy. Thus careful assessment of clinical features of FPLD should be considered in these patients, allowing earlier therapeutic interventions.Electronic supplementary materialThe online version of this article (doi:10.1007/s00125-011-2142-4) contains supplementary material, which is available to authorised users.

Abstract 1720: Activating peroxisome proliferator-activated receptor γ mutant promotes tumor growth in vivo by enhancing angiogenesis

Abstract PPARγ, originally cloned as a transcription factor involved in fat cell differentiation, is activated by endogenous ligands, such as fatty acids produced by sterol response element binding protein 1 (SREBP1) and fatty acids synthase (FASN) pathways and synthetic ligands including the thiazolidinediones (TZD). PPARγ is expressed in a variety of cancer cells. Independent lines of evidence support a role for PPARγ to either inhibit or promote tumorigenesis. Human breast cancer is associated with ErbB2 amplification or overexpression in >30% of patients. Although PPARγ is reduced in ErbB2 induced tumor, the role of PPARγ in growth control of ErbB2-induced tumorigenesis is poorly understood. The addition of ligand activates the receptor by inducing a conformational change in the receptor, which can be recapitulated by mutation. To investigate the role of activated PPARγ signaling in breast cancer, we compared the function of a constitutively active PPARγ (PγCA) mutant with the wild-type PPARγ in ErbB2-induced mammary tumorigenesis in vivo. Tumor cells transduced with either PPARγ or PγCA were implanted into immune competent FVB mice. Enhanced tumor growth was observed in PγCA-transduced cells, which was associated with increased angiogenesis and endothelial stem cells as evidenced by increased number of cells stained with von Willebrand factor (vWF), c-Kit, CD133 and CD31. Genome-wide expression profiling identified a group of genes within the angiogenesis pathway, including Angptl4, as targets of activated PPARγ. PγCA also induced Angptl4 protein secretion in ErbB2-transformed mammary epithelial cells. Angptl4 promoted vascular endothelial cell migration and conversely immuno-depletion of Angptl4 reduced PγCA-mediated cellular migration. Collectively, these studies suggest that activated PPARγ induces Angptl4 to promote tumor growth through enhanced angiogenesis in vivo. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 1720.

Endothelial and Vascular Muscle PPARγ in Arterial Pressure Regulation

Peroxisome proliferator-activated receptor gamma (PPARγ, NR1C3) is a ligand-activated transcription factor belonging to the retinoic acid receptor and thyroid hormone receptor family of nuclear receptors. Classically, these transcription factors regulate expression of target genes by binding to PPAR response elements (PPRE) in the 5′ flanking region of target genes as a heterodimer with retinoid X receptors (RXR). PPARγ first gained prominence when it was discovered that it was expressed specifically in adipocytes and its expression induced their differentiation.1 It has now become clear that although expression of PPARγ may be the highest in adipose tissue, it is expressed in many, if not all, cell types and tissues. Among the many roles ascribed to PPARγ are adipogenesis, insulin action, glucose homeostasis, and lipid metabolism. Other tissue-specific functions for PPARγ in liver, muscle, heart, macrophages, bone, and, more recently, the vasculature have been proposed, ascribed mainly from the analysis of cultured cells and a large and rapidly expanding set of tissue-specific PPARγ knockouts. The standing of PPARγ as a clinically important molecule emerged when it was discovered that PPARγ is the molecular target of the thiazolidinediones (TZD) class of antidiabetic agents.2 Although of understandable interest to diabetes researchers and clinicians, interest among “hypertensionists” increased when it became clear that TZD, in addition to improving glycemic control, tended to lower blood pressure. Many of these studies involved high-risk patients with diabetes or metabolic syndrome and an average decrease of ≈5 mmHg systolic blood pressure and 3 mmHg (diastolic blood pressure [DBP]) was typically reported. According to guidelines from the American Heart Association, 65 million Americans have hypertension and millions of others have prehypertension. Therefore, an analysis of any drug effective at lowering arterial pressure or physiological pathway targeted by that drug involved in regulating arterial pressure deserves serious attention. In this …

Activating Peroxisome Proliferator-Activated Receptor γ Mutant Promotes Tumor Growth In vivo by Enhancing Angiogenesis

Peroxisome proliferator-activated receptor gamma (PPARgamma) is expressed in a variety of cancer cells. The addition of ligand activates the receptor by inducing a conformational change in the receptor, which can be recapitulated by mutation. To investigate the role of activated PPARgamma signaling in breast cancer, we compared the function of a constitutively active PPARgamma (PgammaCA) mutant with the wild-type PPARgamma in ErbB2-induced mammary tumorigenesis in vivo. Tumor cells transduced with either PPARgamma or PgammaCA were implanted into immunocompetent FVB mice. Enhanced tumor growth was observed in PgammaCA-transduced cells, which was associated with increased angiogenesis and endothelial stem cells as evidenced by increased number of cells stained with von Willebrand factor, c-Kit, CD133, and CD31. Genome-wide expression profiling identified a group of genes within the angiogenesis pathway, including Angptl4, as targets of activated PPARgamma; PgammaCA also induced Angptl4 protein secretion in ErbB2-transformed mammary epithelial cells. Angptl4 promoted vascular endothelial cell migration; conversely, immunodepletion of Angptl4 reduced PgammaCA-mediated cellular migration. Collectively, these studies suggest that activated PPARgamma induces Angptl4 to promote tumor growth through enhanced angiogenesis in vivo.

A Dominant‐Negative PPARγ Mutant Promotes Cell Cycle Progression and Cell Growth in Vascular Smooth Muscle Cells

PPARγ ligands have been shown to have antiproliferative effects on many cell types. We herein report that a synthetic dominant-negative (DN) PPARγ mutant functions like a growth factor to promote cell cycle progression and cell proliferation in human coronary artery smooth muscle cells (CASMCs). In quiescent CASMCs, adenovirus-expressed DN-PPARγ promoted G1→S cell cycle progression, enhanced BrdU incorporation, and increased cell proliferation. DN-PPARγ expression also markedly enhanced positive regulators of the cell cycle, increasing Rb and CDC2 phosphorylation and the expression of cyclin A, B1, D1, and MCM7. Conversely, overexpression of wild-type (WT) or constitutively-active (CA) PPARγ inhibited cell cycle progression and the activity and expression of positive regulators of the cell cycle. DN-PPARγ expression, however, did not up-regulate positive cell cycle regulators in PPARγ-deficient cells, strongly suggesting that DN-PPARγ effects on cell cycle result from blocking the function of endogenous wild-type PPARγ. DN-PPARγ expression enhanced phosphorylation of ERK MAPKs. Furthermore, the ERK specific-inhibitor PD98059 blocked DN-PPARγ-induced phosphorylation of Rb and expression of cyclin A and MCM7. Our data thus suggest that DN-PPARγ promotes cell cycle progression and cell growth in CASMCs by modulating fundamental cell cycle regulatory proteins and MAPK mitogenic signaling pathways in vascular smooth muscle cells (VSMCs).

A Functional Peroxisome Proliferator-activated Receptor-γ Ligand-binding Domain Is Not Required for Adipogenesis

The nuclear hormone receptor peroxisome proliferator-activated receptor-gamma (PPARgamma) is the central regulator of adipogenesis. Although it is the target for several drugs that function as agonist activators, a high affinity endogenous ligand for this receptor that is involved in regulating adipogenesis has yet to be identified. Here, we investigated the requirement for ligand activation of PPARgamma in fat cell differentiation, taking advantage of a natural mutant of this receptor that does not bind or become activated by any known natural or synthetic ligand. When ectopically expressed in PPARgamma-null fibroblasts, this Q286P allele was able to strongly promote morphological adipogenesis, without any significant difference compared with wild-type PPARgamma. In addition, no significant differences were found in the expression of several adipogenic genes between the wild-type and Q286P mutant alleles. To extend our studies to an in vivo setting, we performed subcutaneous injections of PPARgamma-expressing fibroblasts into nude mice. We found that both wild-type and Q286P mutant-expressing fibroblasts were able to generate fat pads in the mice. These results suggest that the binding and activation of PPARgamma by agonist ligands may not be required for adipogenesis under physiological conditions.

Endothelium-Specific Interference With Peroxisome Proliferator Activated Receptor Gamma Causes Cerebral Vascular Dysfunction in Response to a High-Fat Diet

The ligand-activated transcription factor peroxisome proliferator activated receptor gamma (PPARgamma) is expressed in vascular endothelium where it exerts anti-inflammatory and antioxidant effects. However, its role in regulating vascular function remains undefined. We examined endothelial function in transgenic mice expressing dominant-negative mutants of PPARgamma under the control of an endothelial-specific promoter to test the hypothesis that endothelial PPARgamma plays a protective role in the vasculature. Under baseline conditions, responses to the endothelium-dependent agonist acetylcholine were not affected in either aorta or the basilar artery in vitro. In response to feeding a high-fat diet for 12 weeks, acetylcholine produced dilation that was markedly impaired in the basilar artery of mice expressing dominant-negative mutants, but not in mice expressing wild-type PPARgamma controlled by the same promoter. Unlike basilar artery, 12 weeks of a high-fat diet was not sufficient to cause endothelial dysfunction in the aorta of mice expressing dominant-negative PPARgamma, although aortic dysfunction became evident after 25 weeks. The responses to acetylcholine in basilar artery were restored to normal after treatment with a scavenger of superoxide. Baseline blood pressure was only slightly elevated in the transgenic mice, but the pressor response to angiotensin II was augmented. Thus, interference with PPARgamma in the endothelium produces endothelial dysfunction in the cerebral circulation through a mechanism involving oxidative stress. Consistent with its role as a fatty acid sensor, these findings provide genetic evidence that endothelial PPARgamma plays a critical role in protecting blood vessels in response to a high-fat diet.