Target genes of peroxisome proliferator-activated receptor gamma in colorectal cancer cells

Activation of the nuclear hormone peroxisome proliferator-activated receptor gamma (PPARgamma) inhibits cell growth and promotes differentiation in a broad spectrum of epithelial derived tumor cell lines. Here we utilized microarray technology to identify PPARgamma gene targets in intestinal epithelial cells. For each gene, the induction or repression was seen with two structurally distinct PPARgamma agonists, and the change in expression could be blocked by co-treatment with a specific PPARgamma antagonist. A majority of the genes could be regulated independently by a retinoid X receptor specific agonist. Genes implicated in lipid transport or storage (adipophilin and liver fatty acid-binding protein) were also activated by agonists of PPAR subtypes alpha and/or delta. In contrast, PPARgamma-selective targets included genes linked to growth regulatory pathways (regenerating gene IA), colon epithelial cell maturation (GOB-4 and keratin 20), and immune modulation (neutrophil-gelatinase-associated lipocalin). Additionally, three different genes of the carcinoembryonic antigen family were induced by PPARgamma. Cultured cells treated with PPARgamma ligands demonstrated an increase in Ca(2+)-independent, carcinoembryonic antigen-dependent homotypic aggregation, suggesting a potential role for PPARgamma in regulating intercellular adhesion. Collectively, these results will help define the mechanisms by which PPARgamma regulates intestinal epithelial cell biology.     

Functional interaction between peroxisome proliferator-activated receptor gamma and beta-catenin

Studies have demonstrated cross talk between beta-catenin and peroxisome proliferator-activated receptor gamma (PPARgamma) signaling pathways. Specifically, activation of PPARgamma induces the proteasomal degradation of beta-catenin in cells that express an adenomatous polyposis coli-containing destruction complex. In contrast, oncogenic beta-catenin is resistant to such degradation and inhibits the expression of PPARgamma target genes. In the present studies, we demonstrate a functional interaction between beta-catenin and PPARgamma that involves the T-cell factor (TCF)/lymphocyte enhancer factor (LEF) binding domain of beta-catenin and a catenin binding domain (CBD) within PPARgamma. Mutation of K312 and K435 in the TCF/LEF binding domain of an oncogenic beta-catenin (S37A) significantly reduces its ability to interact with and inhibit the activity of PPARgamma. Furthermore, these mutations render S37A beta-catenin susceptible to proteasomal degradation in response to activation of PPARgamma. Mutation of F372 within the CBD (helices 7 and 8) of PPARgamma disrupts its binding to beta-catenin and significantly reduces the ability of PPARgamma to induce the proteasomal degradation of beta-catenin. We suggest that in normal cells, PPARgamma can function to suppress tumorigenesis and/or Wnt signaling by targeting phosphorylated beta-catenin to the proteasome through a process involving its CBD. In contrast, oncogenic beta-catenin resists proteasomal degradation by inhibiting PPARgamma activity, which requires its TCF/LEF binding domain.     

Attenuation of peroxisome proliferator-activated receptor gamma (PPARgamma) mediates gastrin-stimulated colorectal cancer cell proliferation

Peroxisome proliferators-activated receptor gamma (PPARgamma) has been shown to suppress cell proliferation and tumorigenesis, whereas the gastrointestinal regulatory peptide gastrin stimulates the growth of neoplastic cells. The present studies were directed to determine whether changes in PPARgamma expression might mediate the effects of gastrin on the proliferation of colorectal cancer (CRC). Initially, using growth assays, we determined that the human CRC cell line DLD-1 expressed both functional PPARgamma and gastrin receptors. Amidated gastrin (G-17) attenuated the growth suppressing effects of PPARgamma by decreasing PPARgamma activity and total protein expression, in part through an increase in the rate of proteasomal degradation. G-17-induced degradation of PPARgamma appeared to be mediated through phosphorylation of PPARgamma at serine 84 by a process involving the biphasic phosphorylation of ERK1/2 and activation of the epidermal growth factor receptor (EGFR). These results were confirmed through the use of EGFR antagonist AG1478 and MEK1 inhibitor PD98059. Furthermore, mutation of PPARgamma at serine 84 reduced the effects of G-17, as evident by inability of G-17 to attenuate PPARgamma promoter activity, degrade PPARgamma, or inhibit the growth suppressing effects of PPARgamma. The results of these studies demonstrate that the trophic properties of gastrin in CRC may be mediated in part by transactivation of the EGFR and phosphorylation of ERK1/2, leading to degradation of PPARgamma protein and a decrease in PPARgamma activation.     

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.     

Activation of peroxisome proliferator-activated receptor gamma inhibits osteoprotegerin gene expression in human aortic smooth muscle cells

Increasing evidence indicates an important role of PPAR gamma activation in modulating the development and progression of atherosclerosis, however, the mechanisms involved in these effects are not well understood since the PPAR gamma-regulated genes in vascular smooth muscle cells (VSMC) are poorly defined. Here we reported that PPAR gamma ligands, GW7845, ciglitazone and troglitazone had the effect of inhibiting osteoprotegerin (OPG) expression in human aortic smooth muscle cells (HASMC). The effect of GW7845 and ciglitazone on OPG expression was completely abolished by GW9662, a PPAR gamma antagonist. Overexpression of PPAR gamma in HASMC by the infection of a PPAR gamma adenovirus dramatically decreased OPG expression. In addition, PPAR gamma activation inhibited OPG promoter activity. Taken together, our data suggest that OPG expression is a novel PPAR gamma target gene in VSMC and downregulation of OPG expression by PPAR gamma activation provides a new insight into the understanding of the role of PPAR gamma in atheroscelrosis and hypertension.     

Glutathione peroxidase 3 mediates the antioxidant effect of peroxisome proliferator-activated receptor gamma in human skeletal muscle cells

Oxidative stress plays an important role in the pathogenesis of insulin resistance and type 2 diabetes mellitus and in diabetic vascular complications. Thiazolidinediones (TZDs), a class of peroxisome proliferator-activated receptor gamma (PPARgamma) agonists, improve insulin sensitivity and are currently used for the treatment of type 2 diabetes mellitus. Here, we show that TZD prevents oxidative stress-induced insulin resistance in human skeletal muscle cells, as indicated by the increase in insulin-stimulated glucose uptake and insulin signaling. Importantly, TZD-mediated activation of PPARgamma induces gene expression of glutathione peroxidase 3 (GPx3), which reduces extracellular H(2)O(2) levels causing insulin resistance in skeletal muscle cells. Inhibition of GPx3 expression prevents the antioxidant effects of TZDs on insulin action in oxidative stress-induced insulin-resistant cells, suggesting that GPx3 is required for the regulation of PPARgamma-mediated antioxidant effects. Furthermore, reduced plasma GPx3 levels were found in patients with type 2 diabetes mellitus and in db/db/DIO mice. Collectively, these results suggest that the antioxidant effect of PPARgamma is exclusively mediated by GPx3 and further imply that GPx3 may be a therapeutic target for insulin resistance and diabetes mellitus.     

Peroxisome proliferator-activated receptor gamma overexpression suppresses proliferation of human colon cancer cells

Peroxisome proliferator-activated receptor gamma (PPARγ) plays an important role in the differentiation of intestinal cells and tissues. Our previous reports indicate that PPARγ is expressed at considerable levels in human colon cancer cells. This suggests that PPARγ expression may be an important factor for cell growth regulation in colon cancer. In this study, we investigated PPARγ expression in 4 human colon cancer cell lines, HT-29, LOVO, DLD-1, and Caco-2. Real-time polymerase chain reaction (PCR) and Western blot analysis revealed that the relative levels of PPARγ mRNA and protein in these cells were in the order HT-29>LOVO>Caco-2>DLD-1. We also found that PPARγ overexpression promoted cell growth inhibition in PPARγ lower-expressing cell lines (Caco-2 and DLD-1), but not in higher-expressing cells (HT-29 and LOVO). We observed a correlation between the level of PPARγ expression and the cells' sensitivity for proliferation.     

Role of peroxisome proliferator-activated receptor gamma in glucose-induced insulin secretion

Peroxisome proliferator-activated receptor (PPAR) isoforms (α and γ) are known to beexpressed in pancreatic islets as well as in insulin-producing cell lines.Ligands of PPAR have been shoWn toenhance glucose-induced insulin secretion in rat pancreatic islets.However,their effect on insulin secretionis still unclear.To understand the molecular mechanism by which PPAR7 exerts its effect on glucose-induced insulin secretion,we examined the endogenous activity of PPAR isoforms,and studied the PPARyfunction and its target gene expression in INS-1 cells.We found that:(1)endogenous PPARγ was activatedin a ligand-dependent manner in INS-1 cells;(2)overexpression of PPARy in the absence of PPARγ ligandsenhanced glucose-induced insulin secretion,which indicates that the increased glucose-induced insulin secretionis a PPARγ-mediated event;(3)the addition of both PPARγ and retinoid X receptor (RXR) ligands showed asynergistic effect on the augmentation of reporter activity,suggesting that the hetero-dimerization of PPAR7and RXR is required for the regulation of the target genes;(4)PPARs upregulated both the glucose transporter2 (GLUT2) and Cbl-associated protein (CAP) genes in INS-1 cells.Our findings suggest an importantmechanistic pathway in which PPARγ enhances glucose-induced insulin secretion by activating the expressionof GLUT2 and CAP genes in a ligand-dependent manner.     

Approaches to understanding the importance and clinical implications of peroxisome proliferator-activated receptor gamma (PPARgamma) signaling in prostate cancer

The development and maintenance of the prostate are dependent upon a complex series of interactions occurring between the epithelial and stromal tissues (Hayward and Cunha [2000]: Radiol. Clin. N. Am. 38:1-14). During the process of prostatic carcinogenesis, there are progressive changes in the interactions of the nascent tumor with its surrounding stroma and extracellular matrix. These include the development of a reactive stromal phenotype and the possible promotion, by stromal cells, of epithelial proliferation and loss of differentiated function (Hayward et al. [1996]: Ann. N. Y. Acad. Sci. 784:50-62; Grossfeld et al. [1998]: Endocr. Related Cancer 5:253-270; Rowley [1998]: Cancer Metastasis Rev. 17:411-419; Tuxhorn et al. [2002]: Clin. Cancer Res. 8:2912-2923). Many molecules play an as yet poorly defined role in establishing and maintaining a growth quiescent glandular structure in the adult. Peroxisome proliferator-activated receptor gamma (PPARgamma) is a candidate regulator of prostatic epithelial differentiation and may play a role in restricting epithelial proliferation. PPARgamma agonists are relatively non-toxic and have been used with limited success to treat some prostate cancer patients. We would propose that a more complete understanding of PPARgamma biology, particularly in the context of appropriate stromal-epithelial and host-tumor interactions would allow for the selection of patients most likely to benefit from this line of therapy. In particular, it seems reasonable to suggest that the patients most likely to benefit may be those with relatively indolent low stage disease for whom this line of therapy could be a useful additive to watchful waiting.     

15-lipoxygenase-1 metabolites down-regulate peroxisome proliferator-activated receptor gamma via the MAPK signaling pathway.

Human colon tumors have elevated levels of 15-lipoxygenase-1 (15-LO-1), suggesting that 15-LO-1 may play a role in the development of colorectal cancer. Also, 15-LO-1 metabolites can up-regulate epidermal growth factor signaling pathways, which results in an increase in mitogenesis. However, metabolites of 15-LO-1 can serve as ligands for peroxisome proliferator-activated receptor gamma (PPARgamma), and activation of this receptor causes most colon cancer cell lines to undergo a differentiative response and reverse their malignant phenotype. Hence, the role 15-LO-1 plays in colon cancer is not clear. To clarify the role of 15-LO-1 in carcinogenesis, the effect of 15-LO-1 and its metabolites on epidermal growth factor signaling and PPARgamma was investigated. In HCT-116 cells, exogenously added 15-LO-1 metabolites, 13-(S)-hydroxyoctadecadienoic acid, 13-(R)-hydroxyoctadecadienoic acid, and 13-(S)-hydroperoxyoctadecadienoic acid, up-regulated the MAPK signaling pathway, and an increase in PPARgamma phosphorylation was observed. Furthermore, in stable overexpressing 15-LO-1 HCT-116 cells, which produce endogenous 15-LO-1 metabolites, an up-regulation in mitogen-activated protein kinase and PPARgamma phosphorylation was observed. Incubation with a MAPK inhibitor ablated MAPK and PPARgamma phosphorylation. The 15-LO-1 up-regulates MAPK activity and increases PPARgamma phosphorylation, resulting in a down-regulation of PPARgamma activity. Thus, 15-LO-1 metabolites may not only serve as ligands for PPARgamma but can down-regulate PPARgamma activity via the MAPK signaling pathway.     

Leukotriene E4 activates peroxisome proliferator-activated receptor gamma and induces prostaglandin D2 generation by human mast cells

Cysteinyl leukotrienes (cys-LTs) are potent inflammatory lipid mediators, of which leukotriene (LT) E(4) is the most stable and abundant in vivo. Although only a weak agonist of established G protein-coupled receptors (GPCRs) for cys-LTs, LTE(4) potentiates airway hyper-responsiveness (AHR) by a cyclooxygenase (COX)-dependent mechanism and induces bronchial eosinophilia. We now report that LTE(4) activates human mast cells (MCs) by a pathway involving cooperation between an MK571-sensitive GPCR and peroxisome proliferator-activated receptor (PPAR)gamma, a nuclear receptor for dietary lipids. Although LTD(4) is more potent than LTE(4) for inducing calcium flux by the human MC sarcoma line LAD2, LTE(4) is more potent for inducing proliferation and chemokine generation, and is at least as potent for upregulating COX-2 expression and causing prostaglandin D(2) (PGD(2)) generation. LTE(4) caused phosphorylation of extracellular signal-regulated kinase (ERK), p90RSK, and cyclic AMP-regulated-binding protein (CREB). ERK activation in response to LTE(4), but not to LTD(4), was resistant to inhibitors of phosphoinositol 3-kinase. LTE(4)-mediated COX-2 induction, PGD(2) generation, and ERK phosphorylation were all sensitive to interference by the PPARgamma antagonist GW9662 and to targeted knockdown of PPARgamma. Although LTE(4)-mediated PGD(2) production was also sensitive to MK571, an antagonist for the type 1 receptor for cys-LTs (CysLT(1)R), it was resistant to knockdown of this receptor. This LTE(4)-selective receptor-mediated pathway may explain the unique physiologic responses of human airways to LTE(4) in vivo.     

The protective role of peroxisome proliferator-activated receptor gamma in lipotoxic podocytes

Podocytes are specialized epithelial cells that maintain the glomerular filtration barrier. These cells are susceptible to lipotoxicity in the obese state and irreversibly lost during kidney disease leading to proteinuria and renal injury. PPARγ is a nuclear receptor whose activation can be renoprotective. This study examined the role of PPARγ in the lipotoxic podocyte using a PPARγ knockout (PPARγKO) cell line and since the activation of PPARγ by Thiazolidinediones (TZD) is limited by their side effects, it explored other alternative therapies to prevent podocyte lipotoxic damage.Wild-type and PPARγKO podocytes were exposed to the fatty acid palmitic acid (PA) and treated with the TZD (Pioglitazone) and/or the Retinoid X receptor (RXR) agonist Bexarotene (BX).It revealed that podocyte PPARγ is essential for podocyte function. PPARγ deletion reduced key podocyte proteins including podocin and nephrin while increasing basal levels of oxidative and ER stress causing apoptosis and cell death. A combination therapy of low-dose TZD and BX activated both the PPARγ and RXR receptors reducing PA-induced podocyte damage. This study confirms the crucial role of PPARγ in podocyte biology and that their activation in combination therapy of TZD and BX may be beneficial in the treatment of obesity-related kidney disease.