PPARgamma Protein Research Articles

A Widely Used Retinoic Acid Receptor Antagonist Induces Peroxisome Proliferator-Activated Receptor-γ Activity

Nuclear receptors (NRs) are transcription factors whose activity is regulated by the binding of small lipophilic ligands, including hormones, vitamins, and metabolites. Pharmacological NR ligands serve as important therapeutic agents; for example, all-trans retinoic acid, an activating ligand for retinoic acid receptor alpha (RARalpha), is used to treat leukemia. Another RARalpha ligand, (E)-S,S-dioxide-4-(2-(7-(heptyloxy)-3,4-dihydro-4,4-dimethyl-2H-1-benzothiopyran-6-yl)-1-propenyl)-benzoic acid (Ro 41-5253), is a potent antagonist that has been a useful and purportedly specific probe of RARalpha function. Here, we report that Ro 41-5253 also activates the peroxisome proliferator-activated receptor gamma (PPARgamma), a master regulator of adipocyte differentiation and target of widely prescribed antidiabetic thiazolidinediones (TZDs). Ro 41-5253 enhanced differentiation of mouse and human preadipocytes and activated PPARgamma target genes in mature adipocytes. Like the TZDs, Ro 41-5253 also down-regulated PPARgamma protein expression in adipocytes. In addition, Ro 41-5253 activated the PPARgamma-ligand binding domain in transiently transfected HEK293T cells. These effects were not prevented by a potent RARalpha agonist or by depleting cells of RARalpha, indicating that PPARgamma activation was not related to RARalpha antagonism. Indeed, Ro 41-5253 was able to compete with TZD ligands for binding to PPARgamma, suggesting that Ro 41-5253 directly affects PPAR activity. These results vividly demonstrate that pharmacological NR ligands may have "off-target" effects on other NRs. Ro 41-5253 is a PPARgamma agonist as well as an RARalpha antagonist whose pleiotropic effects on NRs may signify a unique spectrum of biological responses.

The clinicopathological and prognostic significance of membrane type 1 matrix metalloproteinase (MT1‐MMP) and MMP‐9 according to their localization in invasive breast carcinoma

To investigate the clinicopathological and prognostic significance of membrane type 1 matrix metalloproteinase (MT1-MMP) and MMP-9 proteins expression in invasive breast carcinoma and their relationship to tumour proliferation and expression of c-erbB2 and peroxisome proliferator-activated receptor (PPAR) gamma. Immunohistochemistry was carried out on 175 paraffin-embedded breast tissue specimens to detect MT1-MMP, MMP-9, oestrogen receptor (ER), progesterone receptor, c-erbB-2, Ki67, topoisomerase IIalpha (topo IIalpha) and PPARgamma protein expression. Both MT1-MMP and MMP-9 were expressed in the cytoplasm of the malignant cells and the peritumoral stroma. Cytoplasmic MT1-MMP was more often observed in ER+ tumours (P = 0.022), of a lower nuclear grade (P = 0.020) and with reduced expression of Ki67 and topo IIalpha (P = 0.027 and P = 0.006, respectively). Moreover, cytoplasmic MT1-MMP was positively associated with MMP-9 (P = 0.010) and PPARgamma (P < 0.0001). Cytoplasmic MMP-9 was inversely associated with Ki67 (P = 0.034) and topo IIalpha (P = 0.004), whereas its relationship with MT1-MMP (P = 0.034) and PPARgamma (P = 0.024) was found to be positive. Stromal MMP-9 was more often observed in c-erbB2+ tumours (P = 0.043) and had an unfavourable impact on overall and relapse-free survival in both univariate (P = 0.0157 and P = 0.0274, respectively) and multivariate analyses (P = 0.007 and P = 0.024, respectively). Cytoplasmic MT1-MMP and MMP-9 seem to be related to well-differentiated tumours, with a low proliferation potential, while stromal MMP-9 is associated with an aggressive tumour phenotype and is recognized as an independent poor prognostic indicator.

Effects of peroxisome proliferator-activated receptor gamma and its ligands on cytotrophoblast invasion in first trimester of pregnancy and mechanism thereof

To investigate effects of peroxisome proliferator-activated receptor gamma (PPARgamma) and its ligands on cytotrophoblast invasion and its influence on matrix metalloproteinase (MMP)-2 and MMP-9 activities on cytotrophoblast cells. Samples of fresh placental trophoblast tissue were obtained from the pregnant women in first trimester. Cytotrophoblasts were isolated, cultured, and added with PPARgamma. Immunocytochemistry and inverted microscopy were used to examine the protein expression of PPARgamma in the cytotrophoblasts. Isolated cytotrophoblasts were inoculated in the Transwell chamber The natural stimulatory ligand of PPARgamma15-deoxy-delta (12, 14)-prostaglandin J2 (15d-PGJ2) and synthesized stimulatory ligand of PPARgamma troglitazone (TGZ) of the concentrations of 0, 1, and 10 micromol/L respectively were added respectively to examine the invasion ability of the cytotrophoblasts. Immunofluorochemistry and confocal technique and RT-PCR were used to examine the expression of MMP-2 and MMP-9. PPARgamma protein expression was detectable in the cytotrophoblasts, mainly in the nuclei. The invasion indexes of the cytotrophoblasts stimulated by 15d-PGJ2 and TGZ of different concentrations were significant lowered in a concentration-dependent manner, the effect of 15d-PGJ2 being stronger than that of TGZ (P < 0.05). After stimulated by 15d-PGJ2 and TGZ of different concentrations the mRNA expression and protein expression of MMP-9 and MMP-2 in the cytotrophoblasts were all inhibited significantly (all P < 0.05). PPARgamma plays an important role in the modulation of trophoblast invasion, and PPARgamma ligands can inhibit the trophoblast invasion through downregulating MMP-2 and MMP-9 expressions.

Induction of PPAR Gamma mRNA and Protein Expression by Rosiglitazone in Chronic Cyclosporine Nephropathy in the Rat

PurposeWe recently reported that rosiglitazone (RGTZ), a peroxisome proliferator-activated receptor gamma (PPARγ) agonist, has a protective effect against cyclosporine (CsA)-induced renal injury. Here we report the effect of RGTZ on peroxisome proliferator-activated receptor gamma (PPARγ) expression in an experimental model of chronic cyclosporine (CsA) nephropathy.Materials and MethodsChronic CsA nephropathy was induced in Sprague-Dawley rats by administering CsA (15 mg/kg per day) for 28 days, and control rats were treated with vehicle (VH group, olive oil 1 mL/kg per day) for 28 days. RGTZ (3 mg/kg) was concurrently administered via gavage to the CsA and VH groups. Expression of PPARγ mRNA and protein was evaluated with RT-PCR, immunohistochemistry, and immunoblotting.ResultsPPARγ mRNA expression was similar to the level of PPARγ protein constitutively expressed in the kidneys of the VH treated rats, with expression in the glomerular epithelial, distal tubular cells, and collecting tubular cells. PPARγ protein expression in CsA-treated rat kidneys was significantly less than in the VH group. However, concomitant administration of RGTZ restored PPARγ protein expression in the kidneys of the CsA-reated rats.ConclusionExogenous administration of RGTZ treatment upregulates PPARγ expression and that this mechanism may play a role in protecting against CsA-induced renal injury.

Effects of rosiglitazone on the liver histology and mitochondrial function in ob/ob mice†

Insulin resistance is present in almost all patients with nonalcoholic steatohepatitis (NAFLD), and mitochondrial dysfunction likely plays a critical role in the progression of fatty liver into nonalcoholic steatohepatitis. Rosiglitazone, a selective ligand of peroxisome proliferator-activated receptor gamma (PPARgamma), is an insulin sensitizer drug that has been used in a number of insulin-resistant conditions, including NAFLD. The aim of this study was to analyze the effects of rosiglitazone on the liver histology and mitochondrial function in a model of NAFLD. All studies were carried out in wild-type and leptin-deficient (ob/ob) C57BL/6J mice. Ob/ob mice were treated with 1 mg/kg/day, and activity of mitochondrial respiratory chain (MRC), beta-oxidation, lipid peroxidation, glutathione content in mitochondria, and 3-tyrosine-nitrated proteins in mitochondria were measured. In addition, histological and ultrastructural changes induced by rosiglitazone were also noted. Rosiglitazone treatment increased liver steatosis, particularly microvesicular steatosis. In these animals, mitochondria were markedly swollen with cristae peripherally placed. In ob/ob mice, this drug increased PPARgamma protein expression and lipid peroxide content in liver tissue and decreased glutathione concentration in mitochondria. Rosiglitazone suppressed the activity of complex I of the MRC in ob/ob mice, but did not affect beta-oxidation. 3-Tyrosine nitrated mitochondrial proteins, significantly increased in ob/ob mice, were not modified by rosiglitazone treatment. Treatment of ob/ob mice with rosiglitazone did not reverse histological lesions of NAFLD or improve MRC activity. On the contrary, rosiglitazone reduced activity of complex I and increased oxidative stress and liver steatosis.

Reversal of Systemic Hypertension-Associated Cardiac Remodeling in Chronic Pressure Overload Myocardium by Ciglitazone

Elevated oxidative stress has been characterized in numerous disorders including systemic hypertension, arterial stiffness, left ventricular hypertrophy (LVH) and heart failure. The peroxisome proliferator activated receptor gamma (PPARgamma) ameliorates oxidative stress and LVH. To test the hypothesis that PPARgamma decreased LVH and cardiac fibrosis in chronic pressure overload, in part, by increasing SOD, eNOS and elastin and decreasing NOX4, MMP and collagen synthesis and degradation, chronic pressure overload analogous to systemic hypertension was created in C57BL/6J mice by occluding the abdominal aorta above the kidneys (aortic stenosis-AS). The sham surgery was used as controls. Ciglitazone (CZ, a PPARgamma agonist, 4 microg/ml) was administered in drinking water. LV function was measured by M-Mode Echocardiography. We found that PPARgamma protein levels were increased by CZ. NOX-4 expression was increased by pressure-overload and such an increase was attenuated by CZ. SOD expression was not affected by CZ. Expression of iNOS was induced by pressure-overload, and such an increase was inhibited by CZ. Protein levels for MMP2, MMP-9, MMP-13 were induced and TIMP levels were decreased by pressure-overload. The CZ mitigated these levels. Collagen synthesis was increased and elastin levels were decreased by pressure-overload and CZ ameliorated these changes. Histochemistry showed that CZ inhibited interstitial and perivascular fibrosis. Echocardiography showed that CZ attenuated the systolic and diastolic LV dysfunction induced by pressure-overload. These observations suggested that CZ inhibited pressure-overlaod-induced cardiac remodeling, and inhibition of an induction of NOX4, iNOS, MMP-2/MMP-13 expression and collagen synthesis/degradation may play a role in pressure-overload induced cardiac remodeling.

Peroxisome proliferator-activated receptor γ1 expression is diminished in human osteoarthritic cartilage and is downregulated by interleukin-1β in articular chondrocytes

Peroxisome proliferator-activated receptor γ (PPARγ) is a nuclear receptor involved in the regulation of many cellular processes. We and others have previously shown that PPARγ activators display anti-inflammatory and chondroprotective properties in vitro and improve the clinical course and histopathological features in an experimental animal model of osteoarthritis (OA). However, the expression and regulation of PPARγ expression in cartilage are poorly defined. This study was undertaken to investigate the quantitative expression and distribution of PPARγ in normal and OA cartilage and to evaluate the effect of IL-1β, a prominent cytokine in OA, on PPARγ expression in cultured chondrocytes. Immunohistochemical analysis revealed that the levels of PPARγ protein expression were significantly lower in OA cartilage than in normal cartilage. Using real-time RT-PCR, we demonstrated that PPARγ1 mRNA levels were about 10-fold higher than PPARγ2 mRNA levels, and that only PPARγ1 was differentially expressed: its levels in OA cartilage was 2.4-fold lower than in normal cartilage (p < 0.001). IL-1 treatment of OA chondrocytes downregulated PPARγ1 expression in a dose- and time-dependent manner. This effect probably occurred at the transcriptional level, because IL-1 decreases both PPARγ1 mRNA expression and PPARγ1 promoter activity. TNF-α, IL-17, and prostaglandin E2 (PGE2), which are involved in the pathogenesis of OA, also downregulated PPARγ1 expression. Specific inhibitors of the mitogen-activated protein kinases (MAPKs) p38 (SB203580) and c-Jun N-terminal kinase (SP600125), but not of extracellular signal-regulated kinase (PD98059), prevented IL-1-induced downregulation of PPARγ1 expression. Similarly, inhibitors of NF-κB signaling (pyrrolidine dithiocarbamate, MG-132, and SN-50) abolished the suppressive effect of IL-1. Thus, our study demonstrated that PPARγ1 is downregulated in OA cartilage. The pro-inflammatory cytokine IL-1 may be responsible for this downregulation via a mechanism involving activation of the MAPKs (p38 and JNK) and NF-κB signaling pathways. The IL-1-induced downregulation of PPARγ expression might be a new and additional important process by which IL-1 promotes articular inflammation and cartilage degradation.

Sex Differences in PPAR.GAMMA. Expressions in Rat Adipose Tissues

Clinical studies had suggested that there were sex differences in pharmacological and side effects of pioglitazone. However, there are few studies on the sex differences of peroxisome proliferator-activated receptor (PPAR) gamma expressions in rat adipose tissues. We investigated the sex differences in peroxisome proliferator-activated receptor (PPAR) gamma expressions in rat adipose tissues. Subcutaneous abdominal adipose tissues and perigonadal adipose tissues were obtained from male and female Wistar rats (12 weeks of age). Expressions of PPARgamma protein were determined by Western blot analysis with anti-PPARgamma antibody. Vaginal smear check was performed in female rats. We obtained adipose tissues from females, according to the different phases of the estrous cycle. Both PPARgamma1 and gamma2 subtypes were expressed in subcutaneous adipose tissues. Expressions of PPARgamma2 in subcutaneous adipose tissues were significantly higher in males than in females. On the other hand, expressions of PPARgamma2 in perigonadal adipose tissues were significantly higher in females than in males. Expressions of PPARgamma2 in perigonadal adipose tissues in females significantly decreased during diestrus. It can be suggested that the sex hormones might affect the expressions of PPARgamma2 in adipose tissues.

Aberrant Hepatic Expression of PPARγ2 Stimulates Hepatic Lipogenesis in a Mouse Model of Obesity, Insulin Resistance, Dyslipidemia, and Hepatic Steatosis

Insulin-resistant apoB/BATless mice have hypertriglyceridemia because of increased assembly and secretion of very low density apolipoprotein B (apoB) and triglycerides compared with mice expressing only apoB (Siri, P., Candela, N., Ko, C., Zhang, Y., Eusufzai, S., Ginsberg, H. N., and Huang, L. S. (2001) J. Biol. Chem. 276, 46064-46072). Despite increased very low density lipoprotein secretion, apoB/BATless mice have fatty livers. We found that hepatic mRNA levels of key lipogenic enzymes, acetyl-CoA carboxylase, fatty-acid synthase, and stearoyl-CoA desaturase-1 were increased in apoB/BATless mice compared with levels in apoB mice, suggesting increased lipogenesis in apoB/BATless mice. This was confirmed by determining incorporation of tritiated water into fatty acids. Neither the hepatic mRNA of the lipogenic transcription factor, SREBP-1c (sterol-response element-binding protein 1c), nor the nuclear levels of the mature form of SREBP-1 protein were elevated in apoB/BATless mice. By contrast, hepatic levels of peroxisomal proliferator-activated receptor 2 (PPARgamma2) mRNA and protein were specifically increased in apoB/BATless mice, as were hepatic mRNA levels of two targets of PPARgamma, CD36 and aP2. Treatment of apoB/BATless mice for 4 weeks with intraperitoneal injections of a PPARgamma antisense oligonucleotide resulted in dramatic reductions of both PPARgamma1 and PPARgamma2 mRNA, PPARgamma2 protein, and mRNA levels of fatty-acid synthase and acetyl-CoA carboxylase. These changes were associated with decreased hepatic de novo lipogenesis and hepatic triglyceride concentrations. We conclude that hepatic steatosis in apoB/BATless mice is associated with elevated rates of hepatic lipogenesis that are linked directly to increased hepatic expression of PPARgamma2. The mechanism whereby hepatic Ppargamma2 gene expression is increased and how PPARgamma2 stimulates lipogenesis is under investigation.

Up-regulation of Peroxisome Proliferator-Activated Receptor γ in Perennial Allergic Rhinitis

To investigate the expression of peroxisome proliferator-activated receptor gamma (PPAR-gamma) messenger RNA and protein and to localize the PPAR-gamma protein in the nasal mucosa of patients with allergic rhinitis and control subjects. Prospective study. Tertiary academic institution. Patients Twenty patients with perennial allergic rhinitis and 20 matched nonallergic patients. Inferior turbinate mucosa samples were obtained from 20 patients with perennial allergic rhinitis and 20 matched nonallegic patients. Peroxisome proliferator-activated receptor gamma messenger RNA was extracted from the inferior turbinate mucosae, and then reverse transcription-polymerase chain reaction was performed. Western blot testing was used to analyze differences in PPAR-gamma protein expression levels between patients with allergic rhinitis and normal controls, and the PPAR-gamma protein was localized immunohistochemically. The expression levels of PPAR-gamma messenger RNA and protein in the nasal mucosa was significantly increased in patients with perennial allergic rhinitis compared with controls. Peroxisome proliferator-activated receptor gamma protein was expressed in the epithelium, infiltrating inflammatory cells, and submucosal glands. Peroxisome proliferator-activated receptor gamma is expressed in the human nasal mucosa and is up-regulated in perennial allergic rhinitis. These results suggest a possible contribution for PPAR-gamma in chronic inflammation of the nasal mucosa in perennial allergic rhinitis.

Molecular determinants of FGF‐21 activity—synergy and cross‐talk with PPARγ signaling

Fibroblast growth factor (FGF)-21 is a novel regulator of insulin-independent glucose transport in 3T3-L1 adipocytes and has glucose and triglyceride lowering effects in rodent models of diabetes. The precise mechanisms whereby FGF-21 regulates metabolism remain to be determined. Here we describe the early signaling events triggered by FGF-21 treatment of 3T3-L1 adipocytes and reveal a functional interplay between FGF-21 and peroxisome proliferator-activated receptor gamma (PPARgamma) pathways that leads to a marked stimulation of glucose transport. While the early actions of FGF-21 on 3T3-L1 adipocytes involve rapid accumulation of intracellular calcium and phosphorylation of Akt, GSK-3, p70(S6K), SHP-2, MEK1/2, and Stat3, continuous treatment for 72 h induces an increase in PPARgamma protein expression. Moreover, chronic activation of the PPARgamma pathway in 3T3-L1 adipocytes with the PPARgamma agonist and anti-diabetic agent, rosiglitazone (BRL 49653), enhances FGF-21 action to induce tyrosine phosphorylation of FGF receptor-2. Strikingly, treatment of cells with FGF-21 and rosiglitazone in combination leads to a pronounced increase in expression of the GLUT1 glucose transporter and a marked synergy in stimulation of glucose transport. Together these results reveal a novel synergy between two regulators of glucose homeostasis, FGF-21 and PPARgamma, and further define FGF-21 mechanism of action.

PPAR? phosphorylation mediated by JNK MAPK: a potential role in mac-rophage-derived foam cell formation

To investigate whether oxidized low-density lipoprotein (ox-LDL) modulates peroxisome proliferator-activated receptor gamma (PPARgamma) activity through phosphorylation in macrophages, and the effect of PPARgamma phosphorylation on macrophages-derived foam cell formation. After exposing the cultured THP-1 cells to ox-LDL in the presence or absence of different mitogen-activated protein kinase (MAPK) inhibitors, PPARgamma and phosphorylated PPARgamma protein levels were detected by Western blot. MAPK activity was analyzed using MAP Kinase Assay Kit. Intracellular cholesterol accumulation was assessed by Oil red O staining and cholesterol oxidase enzymatic method. The mRNA level of PPARgamma target gene was determined by reverse transcription-polymerase chain reaction (RT-PCR). ox-LDL evaluated PPARgamma phosphorylation status and subsequently decreased PPAR gamma target gene expression in a dose-dependent manner. ox-LDL also induced MAPK activation. Treatment of THP-1 cells with c-Jun N-terminal kinase-, but not p38- or extracellular signal-regulated kinase-MAPK inhibitor, significantly suppressed PPARgamma phosphorylation induced by ox-LDL, which in turn inhibited foam cell formation. In addition to its ligand-dependent activation, ox-LDL modulates PPAR gamma activity through phosphorylation, which is mediated by MAPK activation. PPARgamma phosphorylation mediated by MAPK facilitates foam cell formation from macrophages exposed to ox-LDL.

Peroxisome Proliferator‐Activated Receptor α, δ, γ1 and γ2 Expressions are Present in Human Monocyte‐Derived Dendritic Cells and Modulate Dendritic Cell Maturation by Addition of Subtype‐Specific Ligands

It has recently been shown by Chang et al. (J Immunol 2000;165:3584-91) that the maturation of dendritic cells (DC) in the presence of long-chain fatty acids redirects DC into Th0/Th2-inducing cells suggesting the involvement of a receptor for long-chain fatty acids like members of the peroxisome proliferator-activated receptors (PPAR) superfamily. Here, we show that immature and mature monocyte-derived DC (Mo-DC) express PPARalpha, PPARdelta, PPARgamma1 and PPARgamma2 mRNA with the highest level of PPARgamma1 mRNA. We were only able to observe the expression of PPARgamma1 protein by Western blotting probably because the protein level of the other subtypes is below the detection limit. Synthetic ligands specific for PPARalpha, PPARdelta or PPARgamma added at day 0-6 have similar effect on the maturation of Mo-DC driving the maturation of Mo-DC with atypical phenotype, reduced expression of IL-10, IL-12 p35 and IL-12 p40 mRNA and with reduced stimulatory effects in mixed leucocyte reaction (MLR). Our data suggest that naturally occurring PPAR ligands like fatty acids and fatty acid derivates have anti-inflammatory effects by redirecting DC into a less stimulatory mode.

Peroxisome proliferator‐activated receptor γ ligands stimulate myeloid differentiation and lipogenensis in human leukemia NB4 cells

Peroxisome proliferator-activated receptor gamma (PPARgamma) plays a central role in adipocyte and macrophage differentiation. Pioglitazone (Actos, AD4833), an antidiabetic drug, and 15-deoxy-Delta(12,14)-prostaglandin J2 (PGJ2) have recently been identified as synthetic and natural ligands for PPARgamma, respectively. In this study, we examined the effects of PPARgamma ligands on differentiation and lipogenesis in promyelocytic leukemia NB4 cells, in which PPARgamma protein was expressed and ligand-stimulated PPARgamma-specific transcription of adipocyte fatty-acid binding protein was confirmed. Treatment with PPARgamma ligand (AD4833 or PGJ2) alone markedly suppressed proliferation but did not induce differentiation. The combined treatment of the cells with PPARgamma ligand and all-trans retinoic acid (ATRA) synergistically induced myelocytic differentiation, as determined by nitroblue tetrazolium reducing ability and cell morphology. During these processes of differentiation, we observed marked accumulation of lipid droplets in the cytoplasm. The cellular triacylglycerol levels increased 2.7-fold after treatment with the inducers. Simultaneously, BODIPY-fatty acid was incorporated into the cytosol and concentrated in lipid droplets. The biosynthesis of triacylglycerol-containing BODIPY-fatty acids was increased twofold in differentiated cells. These findings clearly demonstrate that treatment with PPARgamma ligands not only induced differentiation but also stimulated lipogenesis in NB4 cells, indicating a close association between differentiation and lipogenesis in PPARgamma-stimulated human myeloid cells.

Modulation of Cardiac and Aortic Peroxisome Proliferator-Activated Receptor-γ Expression by Oxidative Stress in Chronically Glucose-Fed Rats

The aim of the present study was to examine the chronic effects of alpha-lipoic acid on proliferator peroxisome activated receptors-gamma (PPAR-gamma) and PPAR-alpha expressions in cardiovascular tissues of chronically hypertensive insulinoresistant rats. We have also evaluated the chronic effects of high levels of insulin, glucose, or both on superoxide anion (O(2)(-)) production in aortic smooth muscle cells (SMCs) in the presence or in absence of pioglitazone, a PPAR-gamma agonist. The PPAR-gamma and PPAR-alpha expressions were measured by Western blot. The oxidative stress was evaluated by measuring the O(2)(-) production using the lucigenin method. Increases in blood pressure, in aortic O(2)(-) production, in glucose or insulin levels, and in insulin resistance, as well as the decrease in PPAR-gamma protein levels in aorta and heart tissues were prevented or attenuated in glucose-treated rats fed with lipoic acid. Chronic treatment with pioglitazone prevented the marked increase in O(2)(-) production in cultured SMCs chronically treated with high insulin combined or not with high glucose levels. The combined therapy with the antioxidant alpha-lipoic acid restored PPAR-gamma levels in cardiovascular tissues and attenuated or prevented the development of insulin resistance and hypertension in chronically glucose-fed rats. Moreover, the finding that pioglitazone was also efficient in preventing the increase in oxidative stress in SMCs treated with high insulin combined with high glucose concentrations supports the hypothesis that the activation of PPAR-gamma activity can counteract the oxidative stress that seems to be implicated in the development of hypertension and insulin resistance.

Indol-1-yl Acetic Acids as Peroxisome Proliferator-Activated Receptor Agonists: Design, Synthesis, Structural Biology, and Molecular Docking Studies

A series of novel indole-based PPAR agonists is described leading to discovery of 10k, a highly potent PPAR pan-agonist. The structural biology and molecular docking studies revealed that the distances between the acidic group and the linker, when a ligand was complexed with PPARgamma protein, were important for the potent activity. The hydrophobic tail part of 10k makes intensive hydrophobic interaction with the PPARgamma protein resulting in potent activity.

PPAR $$\upgamma$$ represses VEGF expression in human endometrial cells: Implications for uterine angiogenesis

Endometrial vasculature supports physiological uterine growth, embryonic implantation and endometrial pathology. Vascular endothelial growth factor (VEGF) is regulated by diverse developmental and hormonal signals, including eicosanoid ligands of PPARgamma. The action of natural and synthetic PPARgamma ligands on VEGF expression in primary and transformed human endometrial cell cultures was established by quantifying endogenous gene expression and transfected VEGF gene reporters. VEGF promoter-luciferase constructs were truncated and mutated to map functional sequences. Endometrial tissues and cells express PPARgamma protein. Treatment of transformed and primary endometrial cells with rosiglitazone, a synthetic PPARgamma agonist, or prostaglandin 15-deoxy-Delta12-14 J(2), a naturally occurring eicosanoid ligand, decreased VEGF protein secretion. In transiently transfected Ishikawa cells, rosiglitazone repressed VEGF gene promoter-luciferase activation with an IC(50) approximately approximately 50 nM. Truncated and mutated VEGF promoter constructs revealed that the PPARgamma-regulated domain is a direct repeat (DR)-1 motif -443 bp upstream of the transcriptional start site. PPARgamma ligands repress VEGF gene expression via a PPARgamma-responsive element (PPRE) in the VEGF gene promoter. Agonists of this nuclear receptor might be exploited pharmacologically to inhibit pathological vascularization in complications of pregnancy, endometriosis and endometrial adenocarcinoma.

Influence of acid and bile acid on ERK activity, PPARγ expression and cell proliferation in normal human esophageal epithelial cells

To observe the effects of acid and bile acid exposure on cell proliferation and the expression of extracellular signal-regulated protein kinase (ERK) and peroxisome proliferator-activated receptor gamma (PPARgamma) in normal human esophageal epithelial cells in vitro. In vitro cultured normal human esophageal epithelial cells were exposed to acidic media (pH 4.0-6.5), media containing different bile acid (250 mumol/L), media containing acid and bile acid, respectively. Cell proliferation was assessed using MTT and flow cytometry. The expressions of phosphorylated ERK(1/2) and PPARgamma protein were determined by the immunoblotting technique. Acid-exposed (3 min) esophageal cells exhibited a significant increase in proliferation ratio, S phase of the cell cycle (P<0.05) and the level of phosphorylated ERK(1/2) protein. When the acid-exposure period exceeded 6 min, we observed a decrease in proliferation ratio and S phase of the cell cycle, with an increased apoptosis ratio (P<0.05). Bile acid exposure (3-12 min) also produced an increase in proliferation ratio, S phase of the cell cycle (P<0.05) and phosphorylated ERK(1/2) expression. On the contrary, deoxycholic acid (DCA) exposure (>20 min) decreased proliferation ratio. Compared with bile acid exposure (pH 7.4), bile acid exposure (pH 6.5, 4) significantly decreased proliferation ratio (P<0.05). There was no expression of PPARgamma in normal human esophageal epithelial cells. The rapid stimuli of acid or bile acid increase proliferation in normal human esophageal epithelial cells by activating the ERK pathway.

Growth Inhibition and Differentiation Induced by Peroxisome Proliferator Activated Receptor Gamma Ligand Rosiglitazone in Human Melanoma Cell Line A375

Ligands of peroxisome proliferator-activated receptors (PPARs) have been demonstrated to be antitumorgenic in vitro and in vivo due to their antiproliferative, prodifferential, and antiangiogenic effects. The aim of this study is to evaluate the effects and mechanisms of PPARgamma ligand rosiglitazone (ROZ) on the growth, apoptosis, and differentiation in human melanoma cancer cell line A375. The effects of ROZ on A375 cell proliferation were measured by clonogenic assay, apoptosis and cell cycle kinetics by FACS with ROZ for 72 h, PPARgamma protein was detected by Western blot analysis and immunocytochemical staining, and PPARgamma mRNA expression by RT-PCR. The differentiation effect of ROZ was determined by measurement of melanin content and tyrosinase activity. The levels of Bcl-2, P53, p-ERK, and ERK were also detected by Western blot analysis. Inhibition of tumorigensis was observed in nude mice. ROZ inhibited colony formation and induced apoptosis in A375 cells, and the cells were arrested in G1 phase. This effect was associated with a decrease of the expression of Bcl-2 and increase of the expression of P53. ROZ also induced the differentiation in A375 cells. ROZ increased expression of PPARgamma and decreased the expression of ERK and p-ERK. Data in vivo showed that ROZ could inhibit tumorigensis in nude mice. These results demonstrated that ROZ inhibited growth of A375 cells via the induction of apoptosis, necrosis, and differentiation in a PPARgamma-dependent manner and might present a promising therapeutic approach in certain human maligancies.

Up‐Regulation of Peroxidase Proliferator‐Activated Receptorγ in Cholesteatoma

To evaluate the localization and expression of peroxidase proliferator-activated receptor (PPAR)gamma in cholesteatoma epithelium. Experimental study. Reverse-transcription polymerase chain reaction was performed on cholesteatoma tissues from 10 adult patients undergoing tympanomastoid surgery for middle ear cholesteatoma and on 10 samples of normal external auditory canal skin tissue. The expression levels of PPARgamma to glyceraldehyde-3-phosphate dehydrogenase transcripts were semiquantified by densitometry. We also characterized the cellular localization of the PPARgamma protein immunohistochemically. Ki-67 was also localized to compare the proliferative activity of cells in cholesteatoma epithelium and in normal external auditory canal skin. PPARgamma mRNA and protein were detected in normal external auditory canal skin and in cholesteatoma epithelium. The expression level of PPARgamma mRNA in cholesteatoma was significantly increased compared with that in normal external auditory canal skin. PPARgamma protein was expressed in cells mainly in the granular and prickle cell layers. However, the intensity of its expression was generally decreased in the parabasal layer of the cholesteatoma epithelium. Ki-67 was expressed in the nuclei of cells in the basal and parabasal layers, and a greater number of cells were Ki-67 immunopositive in cholesteatoma epithelium. PPARgamma is up-regulated in the cholesteatoma epithelium compared with normal external auditory canal skin. These results suggest that PPARgamma may play an important role in the pathogenesis of cholesteatoma.