The potential signaling pathway between peroxisome proliferator-activated receptor gamma and retinoic acid receptor alpha in renal interstitial fibrosis disease

Abstract

Peroxisome proliferator-activated receptorγ (PPARγ) can regulate the process of cell apoptosis and is related to the progression of renal disorders. Retinoic acid receptor alpha (RARα) is one of the nuclear receptors involved in a variety of kidney diseases. Renal interstitial fibrosis (RIF) is a common denominator of chronic kidney disease (CKD). This study investigated whether a potential signaling pathway existed between PPARγ and RARα in RIF rats with unilateral ureteral obstruction (UUO). The rats were randomly divided into four groups: a model group subjected to UUO (GU), and three other groups treated with rosiglitazone sodium (GRS), GW9662 and dimethyl sulfoxide (DMSO), n?=?40, respectively. Renal tissues were collected two and four weeks after post-surgery. The relevant indicators were detected. In comparison with the GU group, the expressions of PPARγ and RARα (protein and mRNA) were increased in the GRS group, and decreased in the GW9662 group (all p?<?0.01). The RIF index, mRNA and protein expression of transforming growth factor-β1 (TGF-β1), and the protein expressions of collagen-IV (Col-IV) and fibronectin (FN) in the GRS group were more markedly reduced than those in the GU group; their levels in the GW9662 group were elevated (all p?<?0.01). PPARγ or RARα was negatively correlated to the RIF index, TGF-β1, Col-IV and FN. PPARγ was positively correlated with RARα (all p?<?0.01). In conclusion, PPARγ agonist can elevate the expression of PPARγ or RARα in RIF rats. There might be a potential signaling pathway between PPARγ and RARα in RIF disease.     

视黄酸核受体γ重组腺病毒构建及其鉴定

目的构建携带大鼠视黄酸核受体γ（retinoic acid receptor γ,RARγ）的重组腺病毒,为研究RAR3,在骨髓间充质干细胞（mesenchymal stem cells,MSCs）成神经分化中的作用奠定基础。方法体外扩增大鼠础研基因,将其定向克隆至腺病毒穿梭质粒pAd Trace—TOX构建重组质粒pAdTrace—RARγ,并在BJ5183菌中与骨架质粒pAd Easy-1重组获得腺病毒载体pad—RARγ,Pac I酶切后转染HEK293细胞包装腺病毒。腺病毒Ad—RARγ感染大鼠MSCs,Real—time PCR和Western印迹检测RARγ的表达。结果PCR,酶切及测序均证实础研正确克隆至腺病毒质粒载体中,Ad—RARγ对MSCs感染率达60％～70％,并明显增强础研基因和蛋白的表达。结论成功构建携带RARγ的重组腺病毒,并具有上调大鼠MSCsRA脚基因和蛋白表达的功能。     

Retinoic acid receptors are required for skeletal growth, matrix homeostasis and growth plate function in postnatal mouse

The retinoic acid receptors α, β and γ (RARα, RARβ and RARγ) are nuclear hormone receptors that regulate fundamental processes during embryogenesis, but their roles in skeletal development and growth remain unclear. To study skeletal-specific RAR function, we created conditional mouse mutants deficient in RAR expression in cartilage. We find that mice deficient in RARα and RARγ (or RARβ and RARγ) exhibit severe growth retardation obvious by about 3 weeks postnatally. Their growth plates are defective and, importantly, display a major drop in aggrecan expression and content. Mice deficient in RARα and RARβ, however, are virtually normal, suggesting that RARγ is essential. In good correlation, we find that RARγ is the most strongly expressed RAR in mouse growth plate and its expression characterizes the proliferative and pre-hypertrophic zones where aggrecan is strongly expressed also. By being avascular, those zones lack endogenous retinoids as indicated by previous RARE reporter mice and our direct biochemical measurements and thus, RARγ is likely to exert ligand-less repressor function. Indeed, our data indicate that: aggrecan production is enhanced by RARγ over-expression in chondrocytes under retinoid-free culture conditions; production is further boosted by co-repressor Zac1 or pharmacologic agents that enhance RAR repressor function; and RAR/Zac1 function on aggrecan expression may involve Sox proteins. In sum, our data reveal that RARs, and RARγ in particular, exert previously unappreciated roles in growth plate function and skeletal growth and regulate aggrecan expression and content. Since aggrecan is critical for growth plate function, its deficiency in RAR-mutant mice is likely to have contributed directly to their growth retardation.     

PPARγ的翻译后修饰

过氧化物酶体增殖物激活受体γ(peroxisome proliferator-activated receptor gamma,PPARγ)是一种配体依赖性核转录因子,它具有调控细胞分化、脂肪代谢、糖代谢及炎症等多种生物学功能.机体对PPARγ转录活性的调控方式是多种多样的,包括蛋白表达水平、配体以及转录辅助因子等不同层次上的调控.近年来众多证据揭示,蛋白翻译后修饰(posttranslational modifications,PTMs)是机体调节PPARγ转录活性的另一重要方式.目前,已报道的PPARγ翻译后修饰包括磷酸化、泛素化、SUMO化和亚硝基化等,它们能够改变蛋白构象、调控蛋白相互作用、改变受体与配体间的亲和力,从而调控PPARγ下游基因的转录.重要的是,PPARγ的翻译后修饰与一些疾病如糖尿病、动脉粥样硬化、肿瘤等密切相关.本文将主要围绕PPARγ的各种翻译后修饰及其在疾病的发生、发展和治疗中的意义作一综述.     

PPARγ and mitophagy are involved in hypoxia/reoxygenation-induced renal tubular epithelial cells injury

Abstract

Renal tubular epithelial cell (RTEC) injury is the main cause and common pathological process of various renal diseases. Mitochondrial dysfunction (MtD) is a pathological process after renal injury. Mitophagy is vital for mitochondrial function. Hypoxia is a common cause of RTEC injury. Peroxisome proliferator-activated receptor γ (PPARγ) is involved in cell proliferation, apoptosis, and inflammation. Previous studies have shown that the low expression of PPARγ might be involved in hypoxia-induced RTEC injury. The present study aimed to investigate the correlation between PPARγ and mitophagy in damaged RTEC in the hypoxia/reoxygenation (HR) model. The results showed that HR inhibited the expression of PPARγ, but increased the expression of LC3II, Atg5, SQSTM1/P62, and PINK1 in a time-dependent manner. Moreover, mitochondrial DNA (mt DNA) copy number, mitochondria membrane potential (MMP) levels, ATP content, and cell viability were decreased in hypoxic RTECs, the expression of SQSTM1/P62 and PINK1, the release of cytochrome c (cyt C), and production of reactive oxygen species (ROS) were increased. Mitochondrial-containing autophagosomes (APs) were detected using transmission election microscope (TEM) and laser scanning confocal microscope (LSCM). Furthermore, PPARγ protein expression was negatively correlated with that of LC3II, PINK1, and the positive rate of RTEC-containing mitochondrial-containing APs (all p?<?.05), but positively correlated with cell viability, MMP level, and ATP content (all p?<?.05). These data suggested that PPARγ and mitophagy are involved in the RTEC injury process. Thus, a close association could be detected between PPARγ and mitophagy in HR-induced RTEC injury, albeit additional investigation is imperative.     

Upregulation of transient receptor potential melastatin 6 channel expression by rosiglitazone and all-trans-retinoic acid in erlotinib-treated renal tubular epithelial cells

Anti-epidermal growth factor receptor (EGFR) drugs including erlotinib cause a side effect of hypomagnesemia. In lung adenocarcinoma A549 cells, anticancer agents such as cisplatin and doxorubicin dose-dependently increased toxicity, but the effects were significantly suppressed by culturing the cells in low Mg²⁺-containing media. To obtain the maximum effect in cancer chemotherapy, it should be necessary to prevent the reduction of body Mg ²⁺ content. Anti-EGFR drugs inhibit EGF-induced elevation of transient receptor potential melastatin 6 (TRPM6) Mg ²⁺ channel in renal tubular epithelial NRK-52E cells. Here, we found that rosiglitazone, an antidiabetic drug, and all- trans-retinoic acid (ATRA), a vitamin A derivative, increase the messenger RNA (mRNA) level of TRPM6 in the presence of erlotinib. The rosiglitazone- and ATRA-induced elevation of mRNA level, Mg ²⁺ influx, and promoter activity of TRPM6 were inhibited by GW-9662, a potent antagonist of peroxisome proliferator-activated receptor (PPAR)γ, and LE135, a retinoic acid receptor (RAR) antagonist, respectively. Rosiglitazone increased the phosphorylation and nuclear localization levels of PPARγ, which were inhibited by GW-9662. In contrast, RAR was mainly distributed in the nuclei under control conditions, which was unchanged by ATRA and LE135. The promoter activity of TRPM6 was inhibited by a mutation in the peroxisome proliferator hormone response element (PPRE). A chromatin immunoprecipitation assay revealed that PPARγ and RAR bind to the PPRE, which was blocked by GW-9662 and LE135, respectively. These results suggest that rosiglitazone and ATRA reverse the reduction in Mg ²⁺ reabsorption caused by anti-EGFR drugs.     

Regulation of inflammatory and lipid metabolism genes by eicosapentaenoic acid-rich oil

Omega-3-PUFAs, eicosapentaenoic acid (EPA), and docosahexaenoic acid (DHA), are associated with prevention of various aspects of metabolic syndrome. In the present studies, the effects of oil rich in EPA on gene expression and activation of nuclear receptors was examined and compared with other ω3-PUFAs. The EPA-rich oil (EO) altered the expression of FA metabolism genes in THP-1 cells, including stearoyl CoA desaturase (SCD) and FA desaturase-1 and -2 (FASDS1 and -2). Other ω3-PUFAs resulted in a similar gene expression response for a subset of genes involved in lipid metabolism and inflammation. In reporter assays, EO activated human peroxisome proliferator-activated receptor α (PPARα) and PPARβ/γ with minimal effects on PPARγ, liver X receptor, retinoid X receptor, farnesoid X receptor, and retinoid acid receptor γ (RARγ); these effects were similar to that observed for purified EPA. When serum from a 6 week clinical intervention with dietary supplements containing olive oil (control), DHA, or two levels of EPA were applied to THP-1 cells, the expression of SCD and FADS2 decreased in the cells treated with serum from the ω3-PUFA-supplemented individuals. Taken together, these studies indicate regulation of gene expression by EO that is consistent with treating aspects of dyslipidemia and inflammation.     

Lack of PPARγ in myeloid cells confers resistance to Listeria monocytogenes infection

The peroxisomal proliferator-activated receptor γ (PPARγ) is a nuclear receptor that controls inflammation and immunity. Innate immune defense against bacterial infection appears to be compromised by PPARγ. The relevance of PPARγ in myeloid cells, that organize anti-bacterial immunity, for the outcome of immune responses against intracellular bacteria such as Listeria monocytogenes in vivo is unknown. We found that Listeria monocytogenes infection of macrophages rapidly led to increased expression of PPARγ. This prompted us to investigate whether PPARγ in myeloid cells influences innate immunity against Listeria monocytogenes infection by using transgenic mice with myeloid-cell specific ablation of PPARγ (LysMCre×PPARγ(flox/flox)). Loss of PPARγ in myeloid cells results in enhanced innate immune defense against Listeria monocytogenes infection both, in vitro and in vivo. This increased resistance against infection was characterized by augmented levels of bactericidal factors and inflammatory cytokines: ROS, NO, IFNγ TNF IL-6 and IL-12. Moreover, myeloid cell-specific loss of PPARγ enhanced chemokine and adhesion molecule expression leading to improved recruitment of inflammatory Ly6C(hi) monocytes to sites of infection. Importantly, increased resistance against Listeria infection in the absence of PPARγ was not accompanied by enhanced immunopathology. Our results elucidate a yet unknown regulatory network in myeloid cells that is governed by PPARγ and restrains both listeriocidal activity and recruitment of inflammatory monocytes during Listeria infection, which may contribute to bacterial immune escape. Pharmacological interference with PPARγ activity in myeloid cells might represent a novel strategy to overcome intracellular bacterial infection.     

Oncogenic retinoic acid receptor γ knockdown reverses multi-drug resistance of human colorectal cancer via Wnt/β-catenin pathway

Retinoic acid receptor γ (RARγ), a unique member of the nuclear receptor superfamily, plays an important role in the progression of several cancers such as hepatocellular carcinoma, esophageal cancer, and cholangiocarcinoma. However, little is known about the regulatory mechanism of the RARγ expression in colorectal cancer (CRC) progression. In the present study, we found that RARγ was frequently overexpressed in human CRC specimens and CRC cell lines, and it mainly resided in the cytoplasm in CRC specimens. Tissue microarrays showed that RARγ indicated vital clinical significance in CRC. RARγ knockdown neither affected CRC cell proliferation nor blocked the cell cycle of CRC cells. However, RARγ knockdown increased the sensitivity of CRC cells to chemotherapeutics through downregulation of multi-drug resistance 1(MDR1). Further studies suggested that RARγ knockdown resulted in downregulation of MDR1, in parallel with suppression of the Wnt/β-catenin pathway. Moreover, a significantly positive association between RARγ and MDR1 was demonstrated in CRC tissue microarrays. Collectively, these results suggested that overexpression of RARγ contributed to the multidrug chemoresistance of CRC cells, at least in part due to upregulation of MDR1 via activation of the Wnt/β-catenin pathway, indicating that RARγ might serve as a potential therapeutic target for chemoresistant CRC patients.     

The Expression of Peroxisome Proliferator‐Activated Receptor γ in Pig Fetal Tissue and Primary Stromal‐Vascular Cultures

Objective: This study was designed to determine when peroxisome proliferator‐activated receptor γ (PPARγ) is expressed in developing fetal adipose tissue and stromal‐vascular adipose precursor cells derived from adipose tissue. In addition we examined developing tissue for CCAAT/enhancer‐binding protein β (C/EBPβ) expression to see if it was correlated with PPARγ expression. Pituitary function and hormones involved with differentiation (dexamethasone and retinoic acid) were also tested for their effects on PPARγ expression to determine if hormones known to affect differentiation also effect PPARγ expression in vivo and in cell culture. Research Methods and Procedures: Developing subcutaneous adipose tissues from the dorsal region of the fetal pig were collected at different gestation times and assayed using Western blot analysis to determine levels of PPARγ and C/EBPβ. Hypophysectomy was performed on 75‐day pig fetuses and tissue samples were then taken at 105 days for Western blot analysis. Adipose tissue was also taken from postnatal pigs to isolate stromal‐vascular (S‐V) cells. These adipose precursor cells were grown in culture and samples were taken for Western blot analysis to determine expression levels of PPARγ. Results: Our results indicate that PPARγ is expressed as early as 50 days of fetal development in adipose tissue and continues through 105 days. Expression of PPARγ was found to be significantly enhanced in adipose tissue from hypophysectomized fetuses at 105 days of fetal development (p < 0.05). C/EBPβ was not found in 50‐ or 75‐day fetal tissues and was found only at low levels in 105‐day tissues. C/EBPβ was not found in hypophysectomized (hypoxed) 105‐day tissue where PPARγ was elevated. S‐V cells freshly isolated from adipose tissue of 5‐ to 7‐day postnatal pigs showed the expression of PPARγ1. When S‐V cells were cultured, both PPARγ1 and 2 were expressed after the first day and continued as cells differentiated. High concentrations of retinoic acid decreased PPARγ expression in early S‐V cultures (p < 0.05). Discussion: Our data indicate that PPARγ is expressed in fetal adipose tissue very early before distinct fat cells are observed and can be expressed without the expression of C/EBPβ. The increase in PPARγ expression after hypophysectomy may explain the increase in fat cell size under these conditions. Adipose precursor cells (S‐V cells) from 5‐ to 7‐day postnatal pigs also express PPARγ in the tissue before being induced to differentiate in culture. Thus S‐V cells from newborn pig adipose tissue are probably more advanced in development than the 3T3‐L1 cell model. S‐V cells may be in a state where PPARγ and C/EBPα are expressed but new signals or vascularization are needed before cells are fully committed and lipid filling begins.     

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.     

Peroxisome proliferator-activated receptors gamma reverses hepatic nutritional fibrosis in mice and suppresses activation of hepatic stellate cells in vitro

Nonalcoholic steatohepatitis with fibrosis is a more severe form of nonalcoholic fatty liver disease, one of the most common liver diseases. We have previously shown that peroxisome proliferator-activated receptors gamma (PPARγ) ligand, rosiglitazone, prevented the development of the methionine choline deficient (MCD) diet-induced fibrosing steatohepatitis. We have now tested whether overexpression of PPARγ ameliorates established steatohepatitis and fibrosis. Male C57BL6 mice fed with MCD diet for 8 weeks developed hepatic fibrosis with increased hepatic expression of collagen1α(I), inhibitors of fibrosis reversal-1, regulator involved in matrix degradation-9 and connective tissue growth factor. After 2 weeks of transduction of PPARγ through an adenovirus-expressing PPARγ (Ad-PPARγ), expression of these genes was reduced in a manner that paralleled the reduction in activated hepatic stellate cells (HSCs) and resolution of liver fibrosis. On the in vitro study, PPARγ is expressed in primary quiescent HSC, but depleted in culture activated HSC. Conversely, ectopic expression of PPARγ in activated HSC achieved the phenotypic reversal to the quiescent cell. Such induction markedly suppressed cell viability and cell proliferation, downregulated proliferating cell nuclear antigen, and caused cell cycle arrest at G0/G1 phase. Further, introduction of PPARγ in HSC increased cell apoptosis, this was confirmed by enhanced expression of FasL, cleaved caspase-3, cleaved caspase-7 and poly ADP-ribose polymerase, indicating an extrinsic apoptosis pathway. In conclusion, the present study shows that MCD diet-induced fibrosing steatohepatitis can be reversed by overexpression of PPARγ. It is likely that PPARγ reverses fibrosis by reducing HSCs proliferation, inducing cell cycle arrest and apoptosis.