Peroxisome Proliferator-activated Receptor Gamma Ligands Research Articles

Atherosclerosis in LDLR-Knockout Mice Is Inhibited, but Not Reversed, by the PPARγ Ligand Pioglitazone

Thiazolidinediones, a class of drugs for the treatment of type-2 diabetes, are synthetic ligands for peroxisome proliferator-activated receptor-gamma. They have been demonstrated to possess cardioprotective effects in humans and anti-atherogenic properties in animal models. However, the question remains whether a peroxisome proliferator-activated receptor-gamma ligand can reverse the development of atherosclerosis. In this study, we tested the effects of pioglitazone on the development of established atherosclerosis in low-density lipoprotein receptor-null mice. We observed that atherosclerosis in low-density lipoprotein receptor-null mice progressed when mice were fed a high-fat diet. Pioglitazone treatment of atherogenic mice prevented this progression of atherosclerosis from its middle stages of disease, but was not able to reverse it. Withdrawal of the high-fat diet from mice with advanced atherosclerosis did not result in a reduction in lesion sizes. Pioglitazone treatment also had no effect on advanced atherosclerosis. Levels of high density lipoprotein cholesterol correlated inversely with lesion development when pioglitazone was given during lesion progression. However, pioglitazone had no effect on circulating high density lipoprotein levels in mice in which treatment was initiated following 14 weeks on the high-fat diet. These findings have implications for the analysis of therapeutic agents in murine models of atherosclerosis and the use of pioglitazone in patients with established atherosclerosis.

Activation of peroxisome proliferator-activated receptor-γ inhibits transforming growth factor-β1 induction of connective tissue growth factor and extracellular matrix in hypertrophic scar fibroblasts in vitro

Peroxisome proliferator-activated receptor-gamma (PPAR-gamma) ligands have been recently reported to have beneficial effects on organ fibrosis. However, their effects on extracellular matrix (ECM) turnover in hypertrophic scar fibroblasts (HSFs), and the related molecular mechanisms are unknown. HSFs were cultured and exposed to different concentration PPAR-gamma ligands in the presence of transforming growth factor-beta1 (TGF-beta1). In growth-arrested HSFs, a PPAR-gamma natural ligand (15-deoxy-D12,14-prostaglandin J2, 15d-PGJ2) and a synthetic ligand (GW7845) dose-dependently attenuated TGFbeta1-induced expression of Connective tissue growth factor (CTGF), collagens and fibronectin. Furthermore, the suppression of CTGF mRNA and protein expression are relieved by pretreatment with an antagonist of PPAR-gamma (GW9662). Moreover, GW7845 and 15d-PGJ2 partially inhibited the expression and phosphorylation of the TGF-beta1/Smad pathway. These results suggest that in TGFbeta1-stimulated HSFs, PPAR-gamma ligands caused an antiproliferative effect and reduced ECM production through mechanisms that included reducing CTGF expression, and a crosstalk between PPAR-gamma and Smad may be involved in the inhibitory effects of PPAR-gamma ligands.

Effect of a synthetic peroxisome proliferator-activated receptor-gamma (PPARγ) ligand on breast cancer cells

e14638 Background: Peroxisome proliferator-activated receptor-gamma (PPARγ) ligands have been identified as a potential source of therapy for human cancers. And, it is reported that PPAR-γ ligands could serve as negative regulators of breast cancer development and progression, but their mechanism is still unknown. The purpose of this study was to determine whether the PPAR- γ ligand induces cell cycle arrest and apoptosis of MDA-MB-231(ERα-negative) and MCF-7(ERα-positive) breast cancer cell. Methods: The effect of PPAR-γ ligands on the cell viability of breast cancer cells was determined using mitochondrial tetrazolium(MTT) assay. The cell cycle distribution and apoptosis induction were evaluated by using the flow cytometry. The expression of apoptosis-related proteins were measured with Western blot analysis. Results: The treatment of MDA-MB- 231 cell with PPAR-γ ligand, troglitazone was shown to induce cell cycle G1 arrest and induction of apoptosis. Moreover, troglitazone treatment, applied in a dose-dependent manner, caused a marked decrease in phosphorylated retinoblastoma(pRb), cyclin D1, D2, D3, cyclin dependent kinase(Cdk) 2, 4, and 6 expression as well as a significant increase in Cdk inhibitor, p21 and p27. Troglitazone showed antiproliferative effect on MCF-7 cell with tamoxifen, respectively and synergically. Troglitazone and tamoxifen could induce G1 arrest and apoptosis of MCF-7 cell, through upregulation of Bax and downregulation of Bcl-2 and cyclin D1. Conclusions: PPAR-γ ligand, troglitazone induces cell cycle arrest and apoptosis of MDA-MB-231 cell and increases the sensitivity of anti-hormonal therapy in MCF-7 cell. These results suggest that troglitazone has anticancer effect on both ERα-negative and positive breast cancer cells. No significant financial relationships to disclose.

Analysis of peroxisome proliferator-activated receptor polymorphisms in inflammatory bowel disease

Peroxisome proliferator-activated receptor gamma (PPARg), is a ligand-activated transcription factor which regulates gene expression. PPARg is highly expressed in the colon, inhibiting the NF-kB activated pathway, an anti-inflammatory effect of PPARg is observed. 5-aminosalicylic acid (5-ASA) is a PPARg ligand which can induce inflammatory genes repression.

Caspase-mediated Cleavage of β-Catenin Precedes Drug-induced Apoptosis in Resistant Cancer Cells

A delicate balance between cell death and survival pathways maintains normal physiology, which is altered in many cancers, shifting the balance toward increased survival. Several studies have established a close connection between the Wnt/beta-catenin pathway and tumorigenesis, aberrant activation of which might contribute toward increased cancer cell growth and survival. Extensive research is underway to identify therapeutic agents that can induce apoptosis specifically in cancer cells with minimal collateral damage to normal cells. Although tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) can induce apoptosis specifically in tumor cells, many cancer cells develop resistance, which can be overcome by combinatorial treatment with other agents: for example, peroxisome proliferator-activated receptor gamma (PPARgamma) ligands. To identify the molecular target mediating combinatorial drug-induced apoptosis, we focused on beta-catenin, a protein implicated in oncogenesis. Our results show that co-treatment of TRAIL-resistant cancer cells with TRAIL and the PPARgamma ligand troglitazone leads to a reduction of beta-catenin expression, coinciding with maximal apoptosis. Modulation of beta-catenin levels via ectopic overexpression or small interference RNA-mediated gene silencing modulates drug-induced apoptosis, indicating involvement of beta-catenin in regulating this pathway. More in-depth studies indicated a post-translational mechanism, independent of glycogen synthase kinase-3beta activity regulating beta-catenin expression following combinatorial drug treatment. Furthermore, TRAIL- and troglitazone-induced apoptosis was preceded by a cleavage of beta-catenin, which was complete in a fully apoptotic population, and was mediated by caspases-3 and -8. These results demonstrate beta-catenin as a promising new target of drug-induced apoptosis, which can be targeted to sensitize apoptosis-resistant cancer cells.

Peroxisome proliferator‐activated receptor‐γ abrogates Smad‐dependent collagen stimulation by targeting the p300 transcriptional coactivator

Ligands of peroxisome proliferator-activated receptor-gamma (PPAR-gamma) abrogate the stimulation of collagen gene transcription induced by transforming growth factor-beta (TGF-beta). Here, we delineate the mechanisms underlying this important novel physiological function for PPAR-gamma in connective tissue homeostasis. First, we demonstrated that antagonistic regulation of TGF-beta activity by PPAR-gamma ligands involves cellular PPAR-gamma, since 15-deoxy-Delta12,14-prostaglandin J(2) (15d-PGJ(2)) failed to block TGF-beta-induced responses in either primary cultures of PPAR-gamma-null murine embryonic fibroblasts, or in normal human skin fibroblasts with RNAi-mediated knockdown of PPAR-gamma. Next, we examined the molecular basis underlying the abrogation of TGF-beta signaling by PPAR-gamma in normal human fibroblasts in culture. The results demonstrated that Smad-dependent transcriptional responses were blocked by PPAR-gamma without preventing Smad2/3 activation. In contrast, the interaction between activated Smad2/3 and the transcriptional coactivator and histone acetyltransferase p300 induced by TGF-beta, and the accumulation of p300 on consensus Smad-binding DNA sequences and histone H4 hyperacetylation at the COL1A2 locus, were all prevented by PPAR-gamma. Wild-type p300, but not a mutant form of p300 lacking functional histone acetyltransferase, was able to restore TGF-beta-induced stimulation of COL1A2 in the presence of PPAR-gamma ligands. Collectively, these results indicate that PPAR-gamma blocked Smad-mediated transcriptional responses by preventing p300 recruitment and histone H4 hyperacetylation, resulting in the inhibition of TGF-beta-induced collagen gene expression. Pharmacological activation of PPAR-gamma thus may represent a novel therapeutic approach to target p300-dependent TGF-beta profibrotic responses such as stimulation of collagen gene expression.

Electrophilic peroxisome proliferator-activated receptor-gamma ligands have potent antifibrotic effects in human lung fibroblasts.

Pulmonary fibrosis is a progressive scarring disease with no effective treatment. Transforming growth factor (TGF)-beta is up-regulated in fibrotic diseases, where it stimulates differentiation of fibroblasts to myofibroblasts and production of excess extracellular matrix. Peroxisome proliferator-activated receptor (PPAR) gamma is a transcription factor that regulates adipogenesis, insulin sensitization, and inflammation. We report here that a novel PPARgamma ligand, 2-cyano-3,12-dioxoolean-1,9-dien-28-oic acid (CDDO), is a potent inhibitor of TGF-beta-stimulated differentiation of human lung fibroblasts to myofibroblasts, and suppresses up-regulation of alpha-smooth muscle actin, fibronectin, collagen, and the novel myofibroblast marker, calponin. The inhibitory concentration causing a 50% decrease in aSMA for CDDO was 20-fold lower than the endogenous PPARgamma ligand, 15-deoxy-Delta(12,14)-prostaglandin J(2) (15 d-PGJ(2)), and 400-fold lower than the synthetic ligand, rosiglitazone. Pharmacologic and genetic approaches were used to demonstrate that CDDO mediates its activity via a PPARgamma-independent pathway. CDDO and 15 d-PGJ(2) contain an alpha/beta unsaturated ketone, which acts as an electrophilic center that can form covalent bonds with cellular proteins. Prostaglandin A(1) and diphenyl diselenide, both strong electrophiles, also inhibit myofibroblast differentiation, but a structural analog of 15 d-PGJ(2) lacking the electrophilic center is much less potent. CDDO does not alter TGF-beta-induced Smad or AP-1 signaling, but does inhibit acetylation of CREB binding protein/p300, a critical coactivator in the transcriptional regulation of TGF-beta-responsive genes. Overall, these data indicate that certain PPARgamma ligands, and other small molecules with electrophilic centers, are potent inhibitors of critical TGF-beta-mediated profibrogenic activities through pathways independent of PPARgamma. As the inhibitory concentration causing a 50% decrease in aSMA for CDDO is 400-fold lower than that in rosiglitazone, the translational potential of CDDO for treatment of fibrotic diseases is high.

15-Deoxy-Δ12,14-prostaglandin J2 inhibits fibrogenic response in human hepatoma cells

Liver fibrosis can be induced by environmental chemicals or toxicants, and finally stimulates fibrogenic cytokines expression, such as transforming growth factor-beta (TGF-beta) and its downstream mediator connective tissue growth factor (CTGF). 15-deoxy-Delta(12,14)-prostaglandin J(2) (15d-PGJ(2)) is a metabolite of arachidonic acid, can act as a peroxisome proliferator-activated receptor gamma (PPARgamma) ligand, and function as either anti-inflammatory or inflammatory agents in different cell types. In this study, CTGF was detected in three human hepatoma cell lines, Hep3B, HepG2, and Huh-7, and it was up-regulated by TGF-beta. 15d-PGJ(2) significantly inhibited TGF-beta-induced CTGF protein and mRNA expressions, and promoter activity in hepatoma cells. 15d-PGJ(2) suppressed TGF-beta-induced Smad2 phosphorylation, however enhancing the phosphorylation of ERK, c-Jun N-terminal kinase (JNK), and p38 in TGF-beta-treated Hep3B cells. Other PPAR ligands like the PPARgamma agonist, troglitazone; the PPARalpha agonist, Wy-14643, and bezafibrate were also able to inhibit TGF-beta-induced CTGF. The results suggest that 15d-PGJ(2) inhibits TGF-beta-induced CTGF expression by inhibiting the phosphorylation of Smad2, which is independent of PPAR, and 15d-PGJ(2) might also act through a PPAR-dependent mechanism in human hepatoma cells. 15d-PGJ(2) might have a beneficent effect on prevention of liver fibrosis induced by environmental toxicants.

Induction of cell cycle arrest and apoptosis in breast cancer cells by troglitazone, a peroxisome proliferator-activated receptor-gamma (PPAR-γ) ligand.

Abstract Abstract #5034 Background: Peroxisome proliferator-activated receptor-gamma(PPAR-γ) ligands inhibit cell proliferation and induce apoptosis in cancer cells. And, it is reported that PPAR-γ ligands could serve as negative regulators of breast cancer development and progression, but their mechanism is still unknown. Here we wished to determine whether the PPAR-γ ligand induces cell cycle arrest and apoptosis of MDA-MB-231(ERα-negative) and MCF-7(ERα-positive) breast cancer cell.
 Methods: The effect of PPAR-γ ligands on the cell viability of breast cancer cells was determined using mitochondrial tetrazolium(MTT) assay. The cell cycle distribution and apoptosis induction were evaluated by using the flow cytometry. The expression of apoptosis-related proteins were measured with Western blot analysis. Statistical analysis was performed using Student's t test, and p<0.05 was considered significant.
 Results: The treatment of MDA-MB-231 cell with PPAR-γ ligand, troglitazone was shown to induce cell cycle G1 arrest and induction of apoptosis. Moreover, troglitazone treatment, applied in a dose-dependent manner, caused a marked decrease in phosphorylated retinoblastoma(pRb), cyclin D1, D2, D3, cyclin dependent kinase(Cdk) 2, 4, and 6 expression as well as a significant increase in Cdk inhibitor, p21 and p27. Troglitazone showed antiproliferative effect on MCF-7 cell with tamoxifen, respectively and synergically. Troglitazone and tamoxifen could induce G1 arrest and apoptosis of MCF-7 cell, through upregulation of Bax and downregulation of Bcl-2 and cyclin D1.
 Conclusion: PPAR-γ ligand, troglitazone induces cell cycle arrest and apoptosis of MDA-MB-231 cell and increases the sensitivity of anti-hormonal therapy in MCF-7 cell. These results suggest that troglitazone has anticancer effect on both ERα-negative and positive breast cancer cells. Citation Information: Cancer Res 2009;69(2 Suppl):Abstract nr 5034.

Telmisartan induces proliferation of human endothelial progenitor cells via PPARγ-dependent PI3K/Akt pathway

Objective Although recent clinical trials have suggested that angiotensin II type 1 receptor blockers (ARBs) reduced cardiovascular events, the precise mechanisms involved are still unknown. Telmisartan, an ARB, has recently been identified as a ligand of peroxisome proliferator-activated receptor-gamma (PPARγ). On the other hand, since endothelial progenitor cells (EPCs) are thought to play a critical role in ischemic diseases, we investigated effects of telmisartan on proliferation of EPCs. Methods and results Human peripheral blood mononuclear cells were isolated from healthy volunteers, and cultured on fibronectin-coated dishes in the presence or absence of telmisartan. Four days after starting culture, adherent cells were collected, and equal numbers of cells were reseeded into methylcellulose medium with or without telmisartan. In the presence of telmisartan, numbers of colonies increased in a dose-dependent manner. DiI-AcLDL uptake and lectin and CD31, CD34 staining revealed that these colonies were EPCs. Increase in colony number by treatment with telmisartan was absolutely inhibited when cultured with a specific inhibitor of PPARγ. In addition, we observed that specific inhibitors of phosphoinositide-3 kinase (PI3K) abolished telmisartan-stimulated increase of monocytic EPC-like cells and telmisartan induced phosphorylation of Akt. Furthermore, mRNA expression of p21 was downregulated in a dose dependent manner, suggesting that growth inductive effects of telmisartan might be regulated by the PI3K/Akt and p21 signaling pathway. Conclusions These findings suggest that telmisartan might contribute to endothelial integrity and vasculogenesis in ischemic regions by increasing numbers of EPCs.

Pathways of Proliferation

See related article, pages 1155–1163 Excessive proliferation of vascular smooth muscle cells (VSMCs) contributes to the pathogenesis of many cardiovascular diseases, including atherosclerosis and pulmonary arterial hypertension (PAH). VSMC proliferation also underlies the failure of many therapies, notable examples being restenosis following coronary angioplasty, vein graft failure in patients with coronary artery bypass grafts, and transplant vasculopathy. Few therapies directly target excess VSMC proliferation, in part, because the underlying pathways have been unknown. Recently, several pathways of VSMC proliferation have been defined, and new therapeutic targets have emerged. An example of the power of preventing VSMC proliferation in reducing human cardiovascular disease is the rapamycin (sirolimus)-coated coronary stent. After dozens of agents failed to prevent the 30% restenosis rate postangioplasty, this VSMC proliferation inhibitor reduced the number to ≈6%.1 However, rapamycin has toxicities, limiting its systemic use. Moreover, the mechanisms of accelerated VSMC proliferation may vary by disease, and, thus, the efficacy of an antiproliferative drug will likely be contextual (ie, disease dependent). Understanding the pathways controlling proliferation offers hope for identifying drugs that selectively target proliferating cells. The contribution by Gizard et al in this issue of Circulation Research identifies such a pathway and suggests new therapeutic targets.2 Members of this group expand on their previous work showing that peroxisome proliferator-activated receptor (PPAR)α activation suppresses G1→S cell cycle progression by increasing the expression of the cyclin dependent kinase (CDK) inhibitor p16INK4a.3 The present study is built on a solid body of knowledge of the cell cycle. Although growth factors are necessary to initiate proliferation in normal cells, cells become independent of these external stimuli during the G1 phase. This “point of no return” is highly regulated by the interaction among cyclins, CDKs, and CDK inhibitors.4 In nonproliferating cells, the …

Mechanisms of pancreatic fibrosis and applications to the treatment of chronic pancreatitis

Pancreatic stellate cells (PSCs) play a crucial role in pancreatic fibrogenesis in chronic pancreatitis and in the desmoplastic reaction of pancreatic cancer. When PSCs are stimulated by oxidative stress, ethanol and its metabolite acetaldehyde, and cytokines, the phenotype of quiescent fat-storing cells converts to myofibroblastlike activated PSCs, which then produce extracellular matrix, adhesion molecules, and various chemokines in response to cytokines and growth factors. Recent data suggest that PSCs have a phagocytic function. Plateletderived growth factor is a potent stimulator of PSC proliferation. Transforming growth factor beta, activin A, and connective tissue growth factor also play a role in PSC-mediated pancreatic fibrogenesis through autocrine and paracrine loops. Following pancreatic damage, pathophysiological processes that occur in the pancreas, including pancreas tissue pressure, hyperglycemia, intracellular reactive oxygen species production, activation of protease-activated receptor 2, induction of cyclooxygenase 2, and bacterial infection play a role in sustaining pancreatic fibrosis through increased PSC proliferation and collagen production by PSCs. Targeting PSCs might be an effective therapeutic approach in chronic pancreatitis. Various substances including vitamin A, vitamin E, polyphenols, peroxisome proliferator-activated receptor gamma ligands, and inhibitors of the renin-angiotensin system show great promise of being useful in the treatment of chronic pancreatitis.

15-deoxy-Delta12,14 prostaglandin J2-induced heme oxygenase-1 in megakaryocytes regulates thrombopoiesis.

Platelet production is an intricate process that is poorly understood. Recently, we demonstrated that the natural peroxisome proliferator-activated receptor gamma (PPARgamma) ligand, 15-deoxy-Delta(12,14) prostaglandin J(2) (15d-PGJ(2)), augments platelet numbers by increasing platelet release from megakaryocytes through the induction of reactive oxygen species (ROS). 15d-PGJ(2) can exert effects independent of PPARgamma, such as increasing oxidative stress. Heme oxygenase-1 (HO-1) is a potent antioxidant and may influence platelet production. To further investigate the influence of 15d-PGJ(2) on megakaryocytes and to understand whether HO-1 plays a role in platelet production. Meg-01 cells (a primary megakaryoblastic cell line) and primary human megakaryocytes derived from cord blood were used to examine the effects of 15d-PGJ(2) on HO-1 expression in megakaryocytes and their daughter platelets. The role of HO-1 activity in thrombopoiesis was studied using established in vitro models of platelet production. 15d-PGJ(2) potently induced HO-1 protein expression in Meg-01 cells and primary human megakaryocytes. The platelets produced from these megakaryocytes also expressed elevated levels of HO-1. 15d-PGJ(2)-induced HO-1 was independent of PPARgamma, but could be replicated using other electrophilic prostaglandins, suggesting that the electrophilic properties of 15d-PGJ(2) were important for HO-1 induction. Interestingly, inhibiting HO-1 activity enhanced ROS generation and augmented 15d-PGJ(2)-induced platelet production, which could be attenuated by antioxidants. These new data reveal that HO-1 negatively regulates thrombopoiesis by inhibiting ROS.

Ciglitazone ameliorates lung inflammation by modulating the inhibitor κB protein kinase/nuclear factor-κB pathway after hemorrhagic shock

Peroxisome proliferator-activated receptor-gamma is a ligand-activated transcription factor. Ciglitazone, a peroxisome proliferator-activated receptor-gamma ligand, has been shown to provide beneficial effects in experimental models of sepsis and ischemia/reperfusion injury. We investigated the effects of ciglitazone on lung inflammation after severe hemorrhage. Prospective, laboratory study, rodent model of hemorrhagic shock. University hospital laboratory. Male rats. Hemorrhagic shock was induced by withdrawing blood to a mean arterial pressure of 50 mm Hg. At 3 hrs after hemorrhage, rats were rapidly resuscitated by returning their shed blood. At the time of resuscitation and every hour thereafter, animals received ciglitazone (10 mg/kg) or vehicle intraperitoneally. Heart rate and mean arterial pressure were measured throughout the experiment. Plasma and lung tissue were collected for analysis up to 3 hrs after resuscitation. Ciglitazone treatment ameliorated mean arterial pressure, reduced lung injury, significantly blunted lung neutrophil infiltration, and lowered plasma interleukin-6, interleukin-10, and monocyte chemoattractant protein-1 levels. In a time course analysis, vehicle-treated rats had a significant increase in nuclear factor-kappaB DNA binding, which was preceded by increased inhibitor kappaB protein kinase activity and inhibitor kappaB alpha degradation in the lung. Treatment with ciglitazone significantly reduced inhibitor kappaB protein kinase activity and inhibitor kappaB alpha degradation and completely inhibited nuclear factor-kappaB DNA binding. This reduction of inhibitor kappaB protein kinase activity afforded by ciglitazone appeared to be a consequence of a physical interaction between peroxisome proliferator-activated receptor-gamma and increased inhibitor kappaB protein kinase. Ciglitazone ameliorates the inflammatory response and may reduce lung injury after hemorrhagic shock. These protective effects appear to be mediated through inhibition of the inhibitor kappaB protein kinase/nuclear factor-kappaB pathway.

Systemic PPARγ Ligation Inhibits Allergic Immune Response in the Skin

We have shown previously that specific ligands of the peroxisome proliferator-activated receptor-gamma (PPARgamma) inhibit the systemic allergic immune response. The objective of this study was to investigate the impact of PPARgamma-ligand treatment on the local allergic immune response. We established a murine model exhibiting clinical and histological features of AD-like skin lesions with high reproducibility. In this model, the PPARgamma ligand was applied in an either preventive or therapeutic manner via systemic and local routes. The affected skin areas were assessed by standardized skin score, histological analyses, and immunohistochemical examinations. Our data show that systemic application of PPARgamma ligand by a preventive protocol led to significantly reduced onset of eczematous skin lesions. This was confirmed by histology, showing decreased skin thickness accompanied by significantly reduced infiltrations of CD4+ and CD8+ lymphocytes but also mast cells. Additionally, early allergen-specific IgE and IgG1 responses were reduced (day 21/35), whereas IgG2a levels remained unchanged. In conclusion, our results demonstrate that PPARgamma-ligand treatment inhibits not only systemic allergic immune response, but also local allergen-mediated dermatitis. Our findings point to therapeutic strategies, including a PPARgamma-ligand-based treatment.

Gene expression profile in colon cancer cells with respect to XIAP expression status

We observed a marked synergism between peroxisome proliferator-activated receptor gamma (PPARgamma) ligands and X-linked inhibitor of apoptosis (XIAP) down-regulation in colon cancer. In the current study, we detected the gene expression profile in HCT116 cells treated with or without PPARgamma ligand troglitazone. HCT116-XIAP(+/+) and HCT116-XIAP(-/-) cells were treated with or without 50 microM troglitazone for 48 h. Gene expressions were detected by microarray, and selected genes were validated by reverse-transcriptase polymerase chain reaction (PCR), real-time PCR, and Western blot. Relative to HCT116-XIAP(+/+) cells, 58 genes were up-regulated and 33 genes down-regulated in HCT116-XIAP(-/-) cells, all by > or =4-fold. These genes could be classified into a wide variety of functional classes, but we focused on those related to angiogenesis, apoptosis, and proliferation. Thus, two pro-apoptotic genes and one pro-proliferation gene were up-regulated in HCT116-XIAP(-/-) cells. Two pro-proliferation genes, one pro-angiogenesis gene, one anti-angiogenesis gene, and one anti-apoptosis gene were down-regulated in HCT116-XIAP(-/-) cells. Relative to HCT116-XIAP(+/+) cells treated with troglitazone, 137 genes were up-regulated, and 31 genes were down-regulated in troglitazone-treated HCT116-XIAP(-/-) cells, all by > or =4-fold. Among the up-regulated genes were two anti-angiogenesis genes, seven pro-apoptosis genes, and six anti-proliferation genes. Among the down-regulated genes were one anti-angiogenesis gene, one pro-angiogenesis gene, one anti-apoptosis gene, one anti-proliferation gene, and two pro-proliferation genes. Down-regulation of XIAP in HCT116 cells with or without troglitazone treatment was associated with changes of gene expression that favor increased tendency of apoptosis, decreased cell proliferation, and angiogenesis potential.

Activation of peroxisome proliferator‐activated receptor gamma suppresses mast cell maturation involved in allergic diseases

Mast cells play a central role in allergic and inflammatory diseases. Several reports indicated role of peroxisome proliferator-activated receptor gamma (PPARgamma) on mast cell function. However, there is no report about the role of PPARgamma on differentiation of mast cells from the progenitors. In this study, we investigated the role of PPARgamma in regulating bone marrow-derived mast cell maturation and the therapeutic implications for mast cell-related diseases such as atopic or contact dermatitis. We used in vitro cell culture system for mast cell differentiation from bone marrow-progenitors using specific ligands and lentiviral-mediated short hairpin RNA of PPARgamma, and in vivo murine dermatitis models. Activation of PPARgamma inhibited the maturation of bone marrow progenitors into connective tissue-type mast cells (CTMCs) through up-regulation of GATA-4 and GATA-6 resulting in a decrease in expression of histidine decarboxylase and mast cell histamine content. In comparison, the differentiation of bone marrow progenitors into CTMCs was significantly accelerated by the knockdown of PPARgamma expression by lentiviral-mediated short hairpin RNA. Peroxisome proliferator-activated receptor gamma ligand administration to mice inhibited the maturation of mast cells resulting in attenuation of atopic and contact dermatitis via diminishment of the number of mature mast cells. Our results indicate that PPARgamma is one of master regulators on mast cell maturation and potentially useful for the therapy in various disorders involving mast cell activation.

Induction of G1 phase arrest and apoptosis in MDA‐MB‐231 breast cancer cells by troglitazone, a synthetic peroxisome proliferator‐activated receptor γ (PPARγ) ligand

Peroxisome proliferator-activated receptor gamma (PPARgamma) ligands inhibit cell proliferation and induce apoptosis in cancer cells. Here we wished to determine whether the PPARgamma ligand induces apoptosis and cell cycle arrest of the MDA-MB-231 cell, an estrogen receptor alpha negative breast cancer cell line. The treatment of MDA-MB-231 cell with PPARgamma ligands was shown to induce inhibition of cell growth in a dose-dependent manner as determined by MTT assay. Cell cycle analysis showed a G1 arrest in MDA-MB-231 cells exposed to troglitazone. An apoptotic effect by troglitazone demonstrated that apoptotic cells elevated by 2.5-fold from the control level at 10 microM, to 3.1-fold at 50 microM and to 3.5-fold at 75 microM. Moreover, troglitazone treatment, applied in a dose-dependent manner, caused a marked decrease in pRb, cyclin D1, cyclin D2, cyclin D3, Cdk2, Cdk4 and Cdk6 expression as well as a significant increase in p21 and p27 expression. These results indicate that troglitazone causes growth inhibition, G1 arrest and apoptotic death of MDA-MB-231 cells.

Cytoprotective effect of tauroursodeoxycholate on hepatocyte apoptosis induced by peroxisome proliferator‐activated receptor gamma ligand

Peroxisome proliferator-activated receptor gamma (PPARgamma) ligands inhibit cell growth and induce apoptosis in various cancer cells. Bile acids are also known to cause hepatocyte apoptosis through nuclear receptor-mediated mechanisms. The aim of this study was to examine the effect of two different bile acid species on the inhibitory action of PPARgamma in cell growth with paying attention to the role of the mitogen-activated protein kinase pathway as an underlying mechanism. Immortalized human hepatocytes (OUMS-29) and hepatoma cells (HepG2 and Huh7) were incubated with troglitazone (TGZ), a PPARgamma ligand with or without pre-incubation of either hydrophobic glycochenodeoxycholate (GCDC) or hydrophilic tauroursodeoxycholate (TUDC). TGZ induced cell apoptosis in all cell types, resulting in the reduction of cell viability. While pre-incubation with GCDC enhanced the apoptotic effects of TGZ, TUDC significantly attenuated it. Both bile acids enhanced p38 and c-Jun N-terminal kinase (JNK) phosphorylation in a similar way, whereas there was more drastic enhancement of extracellular signal-regulated kinase (ERK) 1/2 phosphorylation in the presence of TUDC compared to GCDC. In addition, ERK inhibitors suppressed the action of TUDC against apoptotic effect of TGZ. This study demonstrates that TUDC exhibits anti-apoptotic and cytoprotective effects against TGZ-induced cell apoptosis, presumably through the ERK signaling pathway. We speculate that the administration of TUDC might be one of the potential strategies for the hepatotoxicity caused by TGZ.

Eicosapentaenoic acid inhibits interleukin-6 production in interleukin-1 β-stimulated C6 glioma cells through peroxisome proliferator-activated receptor-gamma

Epidemiological studies suggest that intake of omega-3 polyunsaturated fatty acids improves neurological disorders such as Alzheimer's disease which exhibit inflammatory pathology. We therefore investigated the anti-inflammatory effects of eicosapentaenoic acid (EPA) on interleukin (IL)-1 β-stimulated C6 glioma cells. In the present study, EPA inhibited pro-inflammatory cytokine IL-6 production, a characteristic of certain neurodegenerative disorders, in IL-1 β-stimulated C6 glioma cells in dose-dependent fashion. EPA down-regulated the expression of IL-6 at mRNA level, indicating that the effect of EPA occurs at the transcriptional level. In addition, peroxisome proliferator-activated receptor (PPAR) γ antagonists abolished the inhibitory effect of EPA on IL-1 β-induced IL-6 production, whereas PPAR α antagonist did not block the inhibitory effect of EPA. EPA might thus contribute to the regulation of pro-inflammatory cytokine production in astrocytes through interaction with PPAR γ. Among the PPAR γ ligands tested in this study, ciglitazone, a synthetic agonist of PPAR γ, effectively inhibited IL-6 production, but while neither rosiglitazone nor 15-deoxy-Δ 12,14-prostaglandin J 2 did. These findings indicate that the coordination of PPAR gamma ligands is important in inhibiting the production of IL-6 in C6 glioma cells.