Repression of peroxisome proliferator-activated receptor gamma by mucosal ribotoxic insult-activated CCAAT/enhancer-binding protein homologous protein

CCAAT/enhancer-binding protein homologous protein (CHOP) is a crucial stress-responsive factor in various mucosal injuries, including cellular translational stress conditions. In this study, chemical ribosome-inactivating stresses were assessed for their effects on stress-inducible CHOP expression and its association with epithelial inflammatory cytokine production. Several representative ribotoxic agents (deoxynivalenol, anisomycin, and 15-acetyldeoxynivalenol) enhanced CHOP expression and its nuclear translocation in human intestinal epithelial cells. Moreover, CHOP was a strong positive regulator of IL-8 production, but CHOP-mediated IL-8 production was inversely associated with expression of the mucosal regulatory factor peroxisome proliferator-activated receptor γ (PPARγ). Based on our recent report that PPARγ is a negative regulator of mRNA stability of IL-8, PPARγ was linked to a notable mRNA stabilizing protein, HuR, since ribotoxin-induced IL-8 mRNA is stabilized by HuR protein. Expression of exogenous PPARγ suppressed ribotoxin-triggered cytoplasmic translocation of HuR. In contrast, PPARγ-regulating CHOP was a positive modulator of HuR protein export from nuclei. Taken together, the results indicate that ribotoxin-induced CHOP protein is positively associated with production of proinflammatory cytokine IL-8, but it downregulates PPARγ action, subsequently allowing the cytosolic translocation of HuR protein and stabilization of IL-8 mRNA in gut epithelial cells. CHOP and PPARγ may represent critical mechanistic links between ribotoxic stress and proinflammatory cytokine production, and they may have a broader functional significance with regard to gastrointestinal stresses by toxic mucosal insults.     

Enhancement of the aquaporin adipose gene expression by a peroxisome proliferator-activated receptor gamma.

The current study demonstrates that aquaporin adipose (AQPap), an adipose-specific glycerol channel (Kishida, K., Kuriyama, H., Funahashi, T., Shimomura, I., Kihara, S., Ouchi, N., Nishida, M., Nishizawa, H., Matsuda, M., Takahashi, M., Hotta, K., Nakamura, T., Yamashita, S., Tochino, Y., and Matsuzawa, Y. (2000) J. Biol. Chem. 275, 20896-20902), is a target gene of peroxisome proliferator-activated receptor (PPAR) gamma. The AQPap mRNA amounts increased following the induction of PPARgamma in the differentiation of 3T3-L1 adipocytes. The AQPap mRNA in the adipose tissue increased when mice were treated with pioglitazone (PGZ), a synthetic PPARgamma ligand, and decreased in PPARgamma(+/-) heterozygous knockout mice. In 3T3-L1 adipocytes, PGZ augmented the AQPap mRNA expression and its promoter activity. Serial deletion of the promoter revealed the putative peroxisome proliferator-activated receptor response element (PPRE) at -93/-77. In 3T3-L1 preadipocytes, the expression of PPARgamma by transfection and PGZ activated the luciferase activity of the promoter containing the PPRE, whereas the PPRE-deleted mutant was not affected. The gel mobility shift assay showed the direct binding of PPARgamma-retinoid X receptor alpha complex to the PPRE. DeltaPPARgamma, which we generated as the dominant negative PPARgamma lacking the activation function-2 domain, suppressed the promoter activity in 3T3-L1 cells, dose-dependently. We conclude that AQPap is a novel adipose-specific target gene of PPARgamma through the binding of PPARgamma-retinoid X receptor complex to the PPRE region in its promoter.     

Cyclin D1 repression of peroxisome proliferator-activated receptor gamma expression and transactivation

The cyclin D1 gene is overexpressed in human breast cancers and is required for oncogene-induced tumorigenesis. Peroxisome proliferator-activated receptor gamma (PPAR gamma) is a nuclear receptor selectively activated by ligands of the thiazolidinedione class. PPAR gamma induces hepatic steatosis, and liganded PPAR gamma promotes adipocyte differentiation. Herein, cyclin D1 inhibited ligand-induced PPAR gamma function, transactivation, expression, and promoter activity. PPAR gamma transactivation induced by the ligand BRL49653 was inhibited by cyclin D1 through a pRB- and cdk-independent mechanism, requiring a region predicted to form an helix-loop-helix (HLH) structure. The cyclin D1 HLH region was also required for repression of the PPAR gamma ligand-binding domain linked to a heterologous DNA binding domain. Adipocyte differentiation by PPAR gamma-specific ligands (BRL49653, troglitazone) was enhanced in cyclin D1(-/-) fibroblasts and reversed by retroviral expression of cyclin D1. Homozygous deletion of the cyclin D1 gene, enhanced expression by PPAR gamma ligands of PPAR gamma and PPAR gamma-responsive genes, and cyclin D1(-/-) mice exhibit hepatic steatosis. Finally, reduction of cyclin D1 abundance in vivo using ponasterone-inducible cyclin D1 antisense transgenic mice, increased expression of PPAR gamma in vivo. The inhibition of PPAR gamma function by cyclin D1 is a new mechanism of signal transduction cross talk between PPAR gamma ligands and mitogenic signals that induce cyclin D1.     

Dendritic cell immunogenicity is regulated by peroxisome proliferator-activated receptor gamma

Dendritic cells (DC) are the most potent APCs known that play a key role for the initiation of immune responses. Ag presentation to T lymphocytes is likely a constitutive function of DC that continues during the steady state. This raises the question of which mechanism(s) determines whether the final outcome of Ag presentation will be induction of immunity or of tolerance. In this regard, the mechanisms controlling DC immunogenicity still remain largely uncharacterized. In this paper we report that the nuclear receptor peroxisome proliferator-activated receptor gamma (PPAR-gamma), which has anti-inflammatory properties, redirects DC toward a less stimulatory mode. We show that activation of PPAR-gamma during DC differentiation profoundly affects the expression of costimulatory molecules and of the DC hallmarker CD1a. PPAR-gamma activation in DC resulted in a reduced capacity to activate lymphocyte proliferation and to prime Ag-specific CTL responses. This effect might depend on the decreased expression of costimulatory molecules and on the impaired cytokine secretion, but not on increased IL-10 production, because this was reduced by PPAR-gamma activators. Moreover, activation of PPAR-gamma in DC inhibited the expression of EBI1 ligand chemokine and CCR7, both playing a pivotal role for DC migration to the lymph nodes. These effects were accompanied by down-regulation of LPS-induced nuclear localized RelB protein, which was shown to be important for DC differentiation and function. Our results suggest a novel regulatory pathway for DC function that could contribute to the regulated balance between immunity induction and self-tolerance maintenance.     

The peroxisome proliferator-activated receptor gamma regulates expression of the perilipin gene in adipocytes

Recent studies have shown that lipid droplets are covered with a proteinaceous coat, although the functions and identities of the component proteins have not yet been well elucidated. The first identified lipid droplet-specific proteins are the perilipins, a family of proteins coating the surfaces of lipid droplets of adipocytes. The generation of perilipin-null mice has revealed that although they consume more food than control mice, they have normal body weight and are resistant to diet-induced obesity. In one study (Martinez-Botas, J., Anderson, J. B., Tessier, D., Lapillonne, A., Chang, B. H. J., Quast, M. J., Gorenstein, D., Chen, K. H., and Chan, L. (2000) Nat. Genet. 26, 474-479) it was reported that in an animal model obesity was reversible by breeding perilipin -/- alleles into Lepr db/db obese mice, ostensibly by increasing the metabolic rate of the mice. To understand the exact mechanisms that drive the exclusive expression of the perilipin gene in adipocytes, we analyzed the 5'-flanking region of the mouse gene. Treatment of differentiating 3T3-L1 adipocytes with an agonist of proliferator-activated receptor (PPAR) gamma, the putative "master regulator" of adipocyte differentiation, significantly augmented perilipin gene expression. Reporter assays using the -2.0-kb promoter revealed that this region contains a functional PPARgamma-responsive element. Gel mobility shift and chromatin immunoprecipitation assays showed that endogenous PPARgamma protein binds to the perilipin promoter. PPARgamma2, an isoform exclusively expressed in adipocytes, was found to be the most potent regulator from among the PPAR family members including PPARalpha and PPARgamma1. These results make evident the fact that perilipin gene expression in differentiating adipocytes is crucially regulated by PPARgamma2, providing new insights into the adipogenic action of PPARgamma2 and adipose-specific gene expression, as well as potential anti-obesity pharmaceutical agents targeted to a reduction of the perilipin gene product.     

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.     

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.     

Structure-activity relationships of rosiglitazone for peroxisome proliferator-activated receptor gamma transrepression

Anti-inflammatory effects of peroxisome proliferator-activated receptor gamma (PPRAγ) ligands are thought to be largely due to PPARγ-mediated transrepression. Thus, transrepression-selective PPARγ ligands without agonistic activity or with only partial agonistic activity should exhibit anti-inflammatory properties with reduced side effects. Here, we investigated the structure-activity relationships (SARs) of PPARγ agonist rosiglitazone, focusing on transrepression activity. Alkenic analogs showed slightly more potent transrepression with reduced efficacy of transactivating agonistic activity. Removal of the alkyl group on the nitrogen atom improved selectivity for transrepression over transactivation. Among the synthesized compounds, 3l exhibited stronger transrepressional activity (IC₅₀: 14 μM) and weaker agonistic efficacy (11%) than rosiglitazone or pioglitazone.     

Differentiation-dependent expression of the brown adipocyte uncoupling protein gene: regulation by peroxisome proliferator-activated receptor gamma.

Uncoupling protein (UCP) is expressed only in brown adipocytes and is responsible for the unique thermogenic properties of this cell type. The novel brown preadipocyte cell line, HIB-1B, expresses UCP in a strictly differentiation-dependent manner. Transgenic mice studies have shown that a region from kb -2.8 to -1.0 of the marine UCP gene is required for brown adipocyte-specific expression. Subsequent analysis identified a potent 220-bp enhancer from kb -2.5 to -2.3. We show that this enhancer is active only in differentiated HIB-1B adipocytes, and we identify a peroxisome proliferator-activated receptor gamma (PPARgamma) response element, referred to as UCP regulatory element 1 (URE1), within the enhancer. URE1 has differentiation-dependent enhancing activity in HIB-1B cells and is required for enhancer action, since mutations of URE1 that block protein binding abolish enhancer activity. We also show that PPAR gamma antibodies block binding to URE1 of nuclear extracts from cultured brown adipocytes and from the brown adipose tissue of cold-exposed mice. Protein binding to URE1 increases substantially during differentiation of HIB-1B preadipocytes, and PPAR-gamma mRNA levels increase correspondingly. Although forced expression of PPAR gamma and retinoid X receptor alpha activates the enhancer in HIB-1B preadipocytes, these receptors are not capable of activating the enhancer in NIH 3T3 fibroblasts. Our results show that PPAR gamma is a regulator of the differentiation-dependent expression of UCP and suggest that there are additional factors in HIB-1B cells required for brown adipocyte-specific UCP expression.     

Activated liver X receptors stimulate adipocyte differentiation through induction of peroxisome proliferator-activated receptor gamma expression

Liver X receptors (LXRs) are nuclear hormone receptors that regulate cholesterol and fatty acid metabolism in liver tissue and in macrophages. Although LXR activation enhances lipogenesis, it is not well understood whether LXRs are involved in adipocyte differentiation. Here, we show that LXR activation stimulated the execution of adipogenesis, as determined by lipid droplet accumulation and adipocyte-specific gene expression in vivo and in vitro. In adipocytes, LXR activation with T0901317 primarily enhanced the expression of lipogenic genes such as the ADD1/SREBP1c and FAS genes and substantially increased the expression of the adipocyte-specific genes encoding PPARγ (peroxisome proliferator-activated receptor γ) and aP2. Administration of the LXR agonist T0901317 to lean mice promoted the expression of most lipogenic and adipogenic genes in fat and liver tissues. It is of interest that the PPARγ gene is a novel target gene of LXR, since the PPARγ promoter contains the conserved binding site of LXR and was transactivated by the expression of LXRα. Moreover, activated LXRα exhibited an increase of DNA binding to its target gene promoters, such as ADD1/SREBP1c and PPARγ, which appeared to be closely associated with hyperacetylation of histone H3 in the promoter regions of those genes. Furthermore, the suppression of LXRα by small interfering RNA attenuated adipocyte differentiation. Taken together, these results suggest that LXR plays a role in the execution of adipocyte differentiation by regulation of lipogenesis and adipocyte-specific gene expression.     

Adipose tissue resistin expression is severely suppressed in obesity and stimulated by peroxisome proliferator-activated receptor gamma agonists

Elevated levels of the hormone resistin, which is secreted by fat cells, are proposed to cause insulin resistance and to serve as a link between obesity and type 2 diabetes. In this report we show that resistin expression is significantly decreased in the white adipose tissue of several different models of obesity including the ob/ob, db/db, tub/tub, and KKA(y) mice compared with their lean counterparts. Furthermore, in response to several different classes of antidiabetic peroxisome proliferator-activated receptor gamma agonists, adipose tissue resistin expression is increased in both ob/ob mice and Zucker diabetic fatty rats. These data demonstrate that experimental obesity in rodents is associated with severely defective resistin expression, and decreases in resistin expression are not required for the antidiabetic actions of peroxisome proliferator-activated receptor gamma agonists.