PPARγ antagonist GW9662 induces functional estrogen receptor in mouse mammary organ culture: potential translational significance

The nuclear receptor peroxisome proliferator-activated receptor gamma (PPARγ) plays a central role in regulating metabolism, including interaction with the estrogen receptor-α (ERα). Significantly, PPARγ activity can be modulated by small molecules to control cancer both in vitro and in vivo (Yin et al., Cancer Res 69:687–694, 2009). Here, we evaluated the effects of the PPARγ agonist GW7845 and the PPARγ antagonist GW9662 on DMBA-induced mammary alveolar lesions (MAL) in a mouse mammary organ culture. The results were as follows: (a) the incidence of MAL development was significantly inhibited by GW 7845 and GW 9662; (b) GW9662 but not GW7845, in the presence of estradiol, induced ER and PR expression in mammary glands and functional ERα in MAL; (c) while GW9662 inhibited expression of adipsin and ap2, GW 7845 enhanced expression of these PPARγ-response genes; and (d) Tamoxifen caused significant inhibition of GW9662 treated MAL, suggesting that GW9662 sensitizes MAL to antiestrogen treatment, presumably through rendering functional ERα and induction of PR. The induction of ERα by GW9662, including newer analogs, may permit use of anti-ER strategies to inhibit breast cancer in ER? patients.     

Peroxisome proliferator-activated receptor γ (PPARγ) plays a critical role in the development of TGFβ resistance of H460 cell

The primary goal of the study was to investigate how peroxisome proliferator-activated receptor γ (PPARγ) played a critical role in the protection of H460 cell, one of the non-small cell lung cancer (NSCLC) cells with multidrug resistance, from transforming growth factor β (TGFβ)-mediated mitoinhibition. In the study, TGFβ resistance of H460 cell was first confirmed by analyses of PPARγ expression, its interaction with TGFβ-induced Smad3 and phospho-Smad3 (p-Smad3) and survival of H460. Results showed that enable to escape from G2/M phase arrest, H460 cell had higher resistance to TGFβ-mediated mitoinhibition than CH27 (a drug sensitive control). TGFβ significantly increased PPARγ expression of H460 but not of CH27 cell whereas nuclear accumulation of p-Smad3 was only limited to CH27, the latter was believed to contribute to the induction of P^{21 waf1/cip1} and cyclin B1, cell cycle arrest at G2/M phase and TGFβ-mediated mitoinhibition of CH27 cell. TGFβ-induced PPARγ of H460 cell was further demonstrated to bind to Smad3 and p-Smad3, and GW9662 (PPARγ inhibitor) or PPARγ-specific shRNA could disrupt the binding. GW9662 also increased the nuclear accumulation of p-Smad3 that eventually led to the reduction of TGFβ resistance of H460. A transient knockdown of PPARγ with shRNA revealed a similar effect as GW9662. In addition, activation of P³⁸ instead of ERK played a critical role in TGFβ-induced expression of PPARγ, which subsequently activated RhoA in H460 cell.     

Antagonist of peroxisome proliferator-activated receptor γ induces cerebellar amyloid-β levels and motor dysfunction in APP/PS1 transgenic mice

Recent evidences show that peroxisome proliferator-activated receptor γ (PPARγ) is involved in the modulation of the amyloid-β (Aβ) cascade causing Alzheimer’s disease (AD) and treatment with PPARγ agonists protects against AD pathology. However, the function of PPARγ steady-state activity in Aβ cascade and AD pathology remains unclear. In this study, an antagonist of PPARγ, GW9662, was injected into the fourth ventricle of APP/PS1 transgenic mice to inhibit PPARγ activity in cerebellum. The results show that inhibition of PPARγ significantly induced Aβ levels in cerebellum and caused cerebellar motor dysfunction in APP/PS1 transgenic mice. Moreover, GW9662 treatment markedly decreased the cerebellar levels of insulin-degrading enzyme (IDE), which is responsible for the cellular degradation of Aβ. Since cerebellum is spared from significant Aβ accumulation and neurotoxicity in AD patients and animal models, these findings suggest a crucial role of PPARγ steady-state activity in protection of cerebellum against AD pathology.     

Peroxisome proliferator-activated receptor α and γ agonists together with TGF-β convert human CD4+CD25- T cells into functional Foxp3+ regulatory T cells

Human peripheral CD4(+)CD25(-) T cells can be induced to express Foxp3 when activated in vitro by TCR stimulation with TGF-β and IL-2. However, these TGF-β-induced Foxp3(+) regulatory T cells (iTregs) lack a regulatory phenotype. From libraries of nuclear receptor ligands and bioactive lipids, we screened three peroxisome proliferator-activated receptor (PPAR)α (bezafibrate, GW7647, and 5,8,11,14-eicosatetraynoic acid) and two PPARγ agonists (ciglitazone and 15-deoxy-Δ-(12,14)-PG J(2)) as molecules that increased Foxp3 expression in human iTregs significantly compared with that in DMSO-treated iTregs (control). These PPARα and PPARγ agonist-treated iTregs maintained a high level of Foxp3 expression and had suppressive properties. There were no significant differences in the suppressive properties of iTregs treated with the three PPARα and two PPARγ agonists, and all of the treated iTregs increased demethylation levels of the Foxp3 promoter and intronic conserved noncoding sequence 3 regions. Furthermore, PPARα and PPARγ agonists, together with TGF-β, more strongly inhibited the expression of all three DNA methyltransferases (DNMTs) (DNMT1, DNMT3a, and DNMT3b) in activated CD4(+) T cells. These results demonstrate that PPARα and PPARγ agonists together with TGF-β elicit Foxp3 DNA demethylation through potent downregulation of DNMTs and induce potent and stable Foxp3 expression, resulting in the generation of functional iTregs. Moreover, trichostatin A and retinoic acid enhanced the generation of iTregs synergistically with PPARα and PPARγ agonists.     

Potential signal pathway of all-trans retinoic acid for MMP-2 and MMP-9 expression in injury podocyte induced by adriamycin

Abstract

All-trans-retinoic acid (ATRA) can regulate some specific genes expression in various tissue and cells via nuclear retinoic acid receptors (RARs), including three subtypes: retinoic acid receptor-alpha (RAR-α), retinoic acid receptor-beta (RAR-β) and retinoic acid receptor-gamma (RAR-γ). Podocyte injury plays a pivotal role in the progression of glomerulosclerosis (GS). This study was performed to study the potential signal pathway of ATRA in the expression of matrix metalloproteinases-2 (MMP-2) and matrix metalloproteinases-9 (MMP-9) in injury podocyte. Cells were divided into three groups: group of negative control (NC), group of injury podocyte induced by adriamycin (ADR) (AI) and group of ADR inducing podocyte injury model treated with ATRA (AA). The cells morphology changes were detected using microscope and scanning electron microscopy. MMP-2 and MMP-9 enzymic activity was detected using the gelatin zymography method. Protein and mRNA expressions of MMP-2, MMP-9, RAR-α, RAR-β and RAR-γ were measured by western-blot and real-time RT-PCR. Enzymatic activity of MMP-2 and MMP-9 in group AA was significantly enhanced compared to AI group after ATRA-treated 24?h (p?<?0.05). The protein and mRNA expressions of MMP-2/MMP-9 in group AA were significantly increased than those in group AI at both 12 and 24?h time points (p?<?0.05). Compared to group AI, RAR-α and RAR-γ protein/mRNA expressions of group AA were significantly increased at both 12 and 24?h time points (p?<?0.05). There was no difference for the expression of RAR-β between group AI and group AA (p?>?0.05). RAR-α protein level was positively correlated with MMP-2 or MMP-9 protein expression (p?<?0.05), and RAR-γ protein level was also positively correlated with MMP-2 or MMP-9 protein expression (p?<?0.05). In conclusion, ATRA may increase expression of MMP-2 and MMP-9 by the potential signal pathway of RAR-α and RAR-γ in injury podocyte induced by adriamycin, but not RAR-β.     

The Intrinsic PEDF is Regulated by PPARγ in Permanent Focal Cerebral Ischemia of Rat

Inflammatory damage plays a pivotal role in cerebral ischemia and may represent a target for treatment. Pigment epithelium-derived factor (PEDF) is proven to possess neuroprotective property. But there is little known about the intrinsic PEDF after cerebral ischemia. This study evaluated the time course expression of the intrinsic PEDF and its underlying regulation mechanisms after cerebral ischemia. Male Sprague-Dawley rats were subjected to permanent middle cerebral artery occlusion. Telmisartan (PPARγ agonist) and GW9662 (PPARγ antagonist) were systemically administered to explore the effect on PPARγ, PEDF, NF-κB and MMP-9 expression at 24?h after cerebral ischemia by western blot and qRT-PCR. The neurological deficits, brain water content and infarct volume were measured. Compared with normal group, the expressions of PEDF and PPARγ decreased, and the expression of NF-κB and MMP-9 increased at early stage after ischemia (P?相似文献   

Telmisartan prevented cognitive decline partly due to PPAR-gamma activation

Telmisartan is a unique angiotensin receptor blocker (ARB) and partial agonist of peroxisome proliferator-activated receptor (PPAR)-γ. Here, we investigated the preventive effect of telmisartan on cognitive decline in Alzheimer disease. In ddY mice, intracerebroventricular injection of Aβ 1-40 significantly attenuated their cognitive function evaluated by shuttle avoidance test. Pretreatment with a non-hypotensive dose of telmisartan significantly inhibited such cognitive decline. Interestingly, co-treatment with GW9662, a PPAR-γ antagonist, partially inhibited this improvement of cognitive decline. Another ARB, losartan, which has less PPAR-γ agonistic effect, also inhibited Aβ-injection-induced cognitive decline; however the effect was smaller than that of telmisartan and was not affected by GW9662. Immunohistochemical staining for Aβ showed the reduced Aβ deposition in telmisartan-treated mice. However, this reduction was not observed in mice co-administered GW9662. These findings suggest that ARB has a preventive effect on cognitive impairment in Alzheimer disease, and telmisartan, with PPAR-γ activation, could exert a stronger effect.     

Evodiamine inhibits the proliferation of leukemia cell line K562 by regulating peroxisome proliferators-activated receptor gamma (PPARγ) pathway

Evodiamine, a quinolone alkaloid, is one of the major bioactive compounds of Evodia rutaecarpa Bentham (Rutaceae). It exhibits excellent biological activities, especially the anticancer activity. This study aims to investigate the effect of evodiamine on the proliferation of leukemia cell line K562 and to explore the underlying mechanism. The effect of evodiamine on K562 cells proliferation was analyzed by trypan blue dye exclusion assay and MTT assay. The expression levels of peroxisome proliferators-activated receptor gamma (PPARγ), cyclin D1, and p21 were detected by western blot assay. The results demonstrated that evodiamine inhibited the proliferation and decreased the viability of K562 cells in a dose- and time-dependent manner. 2-Chloro-5-nitro-N-phenylbenzamide (GW9662) and/or PPARγ-siRNA pretreatment alleviated the cell growth suppression triggered by evodiamine. Meanwhile, evodiamine intervention elevated the expression of PPARγ in K562 cells, while pretreatment with GW9662 attenuated the enhanced upregulation of PPARγ expression induced by evodiamine. In addition, GW9662 and PPARγ-siRNA pretreatment also significantly attenuated the downregulation of the cell cycle control protein cyclin D1 and the upregulation of cyclin-dependent kinase inhibitor p21 induced by evodiamine. In conclusion, PPARγ signaling pathway may involve in the proliferation inhibition of evodiamine on K562 cells via inhibiting cylcin D1 and stimulating of p21.     

Inhibitory effect of memantine, an NMDA-receptor antagonist, on electroporation-induced inward currents in pituitary GH3 cells

Experimental and clinical data support the notion that peroxisome proliferator-activated receptor γ (PPARγ) activation is associated with anti-atherosclerosis as well as anti-diabetic effect. Telmisartan, an angiotensin receptor blocker (ARB), acts as a partial PPARγ agonist. We hypothesized that telmisartan protects against diabetic vascular complications, through PPARγ activation. We compared the effects of telmisartan, telmisartan combined with GW9662 (a PPARγ antagonist), and losartan with no PPARγ activity on vascular injury in obese type 2 diabetic db/db mice. Compared to losartan, telmisartan significantly ameliorated vascular endothelial dysfunction, downregulation of phospho-eNOS, and coronary arterial remodeling in db/db mice. More vascular protective effects of telmisartan than losartan were associated with greater anti-inflammatory effects of telmisartan, as shown by attenuation of vascular nuclear factor kappa B (NFκB) activation and tumor necrosis factor α. Coadministration of GW9662 with telmisartan abolished the above mentioned greater protective effects of telmisartan against vascular injury than losartan in db/db mice. Thus, PPARγ activity appears to be involved in the vascular protective effects of telmisartan in db/db mice. Moreover, telmisartan, but not losartan, prevented the downregulation of vascular PPARγ in db/db mice and this effect of telmisartan was cancelled by the coadministration of GW9662. Our data provided the first evidence indicating that PPARγ activity of telmisartan contributed to the protective effects of telmisartan against diabetic vascular complication. PPARγ activity of telmisartan was involved in the normalization of vascular PPARγ downregulation in diabetic mice. Thus, telmisartan seems to exert vascular protective effects in hypertensive patients with diabetes.     

Cell death is induced by ciglitazone, a peroxisome proliferator-activated receptor γ (PPARγ) agonist, independently of PPARγ in human glioma cells

Peroxisome proliferator-activated receptor γ (PPARγ) regulates multiple signaling pathways, and its agonists induce apoptosis in various cancer cells. However, their role in cell death is unclear. In this study, the relationship between ciglitazone (CGZ) and PPARγ in CGZ-induced cell death was examined. At concentrations of greater than 30 μM, CGZ, a synthetic PPARγ agonist, activated caspase-3 and induced apoptosis in T98G cells. Treatment of T98G cells with less than 30 μM CGZ effectively induced cell death after pretreatment with 30 μM of the PPARγ antagonist GW9662, although GW9662 alone did not induce cell death. This cell death was also observed when cells were co-treated with CGZ and GW9662, but was not observed when cells were treated with CGZ prior to GW9662. In cells in which PPARγ was down-regulated cells by siRNA, lower concentrations of CGZ (<30 μM) were sufficient to induce cell death, although higher concentrations of CGZ (≥30 μM) were required to induce cell death in control T98G cells, indicating that CGZ effectively induces cell death in T98G cells independently of PPARγ. Treatment with GW9662 followed by CGZ resulted in a down-regulation of Akt activity and the loss of mitochondrial membrane potential (MMP), which was accompanied by a decrease in Bcl-2 expression and an increase in Bid cleavage. These data suggest that CGZ is capable of inducing apoptotic cell death independently of PPARγ in glioma cells, by down-regulating Akt activity and inducing MMP collapse.     

Stable reporter cell lines for peroxisome proliferator-activated receptor γ (PPARγ)-mediated modulation of gene expression

Activation of peroxisome proliferator-activated receptor γ (PPARγ) by ligands is associated with beneficial health effects, including anti-inflammatory and insulin-sensitizing effects. The aim of the current study was to develop luciferase reporter gene assays to enable fast and low-cost measurement of PPARγ agonist and antagonist activity. Two reporter gene assays, PPARγ1 CALUX and PPARγ2 CALUX, were developed by stable transfection of U2OS cells with an expression vector for PPARγ1 or PPARγ2 and a pGL3–3xPPRE–tata-luc or pGL4–3xPPRE–tata-luc reporter construct, respectively. PPARγ1 CALUX and PPARγ2 CALUX cells showed similar concentration-dependent luciferase induction upon exposure to the PPARγ agonists rosiglitazone, troglitazone, pioglitazone, ciglitazone, netoglitazone, and 15-deoxy-Δ^12,14-prostaglandin J₂. The potency to induce luciferase decreased in the following order: rosiglitazone > troglitazone = pioglitazone > netoglitazone > ciglitazone. A concentration-dependent decrease in the response to 50 nM rosiglitazone was observed on the addition of PPARγ antagonist GW9662 or T0070907 in both PPARγ1 CALUX and PPARγ2 CALUX cells. The PPARα agonists WY14643 and fenofibrate failed to induce luciferase activity, confirming the specificity of these cell lines for PPARγ agonists. In conclusion, PPARγ1 CALUX and PPARγ2 CALUX cells provide a reliable and useful tool to screen (bio)chemicals for PPARγ agonist or antagonist activity.     

PPARγ-dependent pathway in the growth-inhibitory effects of K562 cells by carotenoids in combination with rosiglitazone

Background

Carotenoids have been found to play roles in the prevention and therapy of some cancers which PPARγ was also discovered to be involved in. The present studies were directed to determine the inhibitory effects of carotenoids in combination with rosiglitazone, a synthetic PPARγ agonist, on K562 cell proliferation and elucidate the contribution of PPARγ-dependent pathway to cell proliferation suppression.

Methods

The effects of carotenoid and rosiglitazone combination on K562 cell proliferation were evaluated by trypan blue dye exclusion assay and MTT assay. When PPARγ has been inhibited by GW9662 and siRNA, cycle-related regulator expression in K562 cells treated with carotenoid and rosiglitazone combination was analyzed by Western blotting.

Results

Rosiglitazone inhibited K562 cell proliferation and augmented the inhibitory effects of carotenoids on the cell proliferation greatly. Specific PPARγ inhibition attenuated the cell growth suppression induced by carotenoid and rosiglitazone combination. GW9662 pre-treatment attenuated the enhanced up-regulation of PPARγ expression caused by the combination treatment. Moreover, GW9662 and PPARγ siRNA also significantly attenuated the up-regulation of p21 and down-regulation of cyclin D1 caused by carotenoids and rosiglitazone.

Conclusions

PPARγ signaling pathway, via stimulating p21 and inhibiting cyclin D1, may play an important role in the anti-proliferative effects of carotenoid and rosiglitazone combination on K562 cells.

General significance

Carotenoids in combination with rosiglitazone are hopeful to provide attractive dietary or supplementation-based and pharmaceutical strategies to treat cancer diseases.