Peroxisome proliferator-activated receptor gamma ligands, 15-deoxy-Delta12,14-prostaglandin J2, and ciglitazone, induce growth inhibition and cell cycle arrest in hepatic oval cells

There is growing evidence to show that hepatic oval cells contribute to liver regeneration, dysplastic nodule formation, and hepato-carcinogenesis. Peroxisome proliferator-activated receptors (PPARs) and their ligands play an important role in cell growth, inflammatory responses, and liver pathogenesis including fibrosis and cancer. However, little is known about the role of PPARgamma/its ligands in the growth and differentiation of hepatic oval cells. In this study, we found that OC15-5, a rat hepatic oval cell line, expressed PPARgamma at mRNA and protein levels, and a natural ligand for PPARgamma, 15-deoxy-Delta12,14-prostaglandin J2 (15d-PGJ2), and a synthetic ligand, ciglitazone, inhibited growth of OC15-5 cells by arresting at G1-S in a dose-dependent manner. Apoptosis was also induced in OC15-5 cells by 15d-PGJ2 treatment. In OC15-5 cells treated with 15d-PGJ2, the expression of CDK inhibitor, p27(Kip1), was up-regulated, while that of p21(WAF1/Cip1), p18(INK4C) CDK2, CDK4, and cyclin E was unchanged. In addition, delayed up-regulation of AFP expression was observed in OC15-5 cells after 15d-PGJ2 or ciglitazone treatment. This is the first report to show that the PPARgamma ligand was involved in the growth, cell cycle, and differentiation of hepatic oval cells, raising the possibility that the PPARgamma ligands may regulate liver regeneration and hepato-carcinogenesis.     

Inhibition of cellular proliferation through IkappaB kinase-independent and peroxisome proliferator-activated receptor gamma-dependent repression of cyclin D1

The nuclear receptor peroxisome proliferator-activated receptor gamma (PPARgamma) is a ligand-regulated nuclear receptor superfamily member. Liganded PPARgamma exerts diverse biological effects, promoting adipocyte differentiation, inhibiting tumor cellular proliferation, and regulating monocyte/macrophage and anti-inflammatory activities in vitro. In vivo studies with PPARgamma ligands showed enhancement of tumor growth, raising the possibility that reduced immune function and tumor surveillance may outweigh the direct inhibitory effects of PPARgamma ligands on cellular proliferation. Recent findings that PPARgamma ligands convey PPARgamma-independent activities through IkappaB kinase (IKK) raises important questions about the specific mechanisms through which PPARgamma ligands inhibit cellular proliferation. We investigated the mechanisms regulating the antiproliferative effect of PPARgamma. Herein PPARgamma, liganded by either natural (15d-PGJ(2) and PGD(2)) or synthetic ligands (BRL49653 and troglitazone), selectively inhibited expression of the cyclin D1 gene. The inhibition of S-phase entry and activity of the cyclin D1-dependent serine-threonine kinase (Cdk) by 15d-PGJ(2) was not observed in PPARgamma-deficient cells. Cyclin D1 overexpression reversed the S-phase inhibition by 15d-PGJ(2). Cyclin D1 repression was independent of IKK, as prostaglandins (PGs) which bound PPARgamma but lacked the IKK interactive cyclopentone ring carbonyl group repressed cyclin D1. Cyclin D1 repression by PPARgamma involved competition for limiting abundance of p300, directed through a c-Fos binding site of the cyclin D1 promoter. 15d-PGJ(2) enhanced recruitment of p300 to PPARgamma but reduced binding to c-Fos. The identification of distinct pathways through which eicosanoids regulate anti-inflammatory and antiproliferative effects may improve the utility of COX2 inhibitors.     

4-Hydroxynonenal and PPARgamma ligands affect proliferation, differentiation, and apoptosis in colon cancer cells

PPARgamma ligands inhibit growth and induce apoptosis of various cancer cells. 4-Hydroxynonenal (HNE), a product of lipid peroxidation, inhibits proliferation and induces differentiation or apoptosis in neoplastic cells. The aim of this work was to investigate the effects of PPARgamma ligands (rosiglitazone and 15-deoxy-prostaglandin J2 (15d-PGJ2)) and HNE, alone or in association, on proliferation, apoptosis, differentiation, and growth-related and apoptosis-related gene expression in colon cancer cells (CaCo-2 cells). PPARgamma ligands inhibited cell proliferation (IC50 was 37.47+/-6.6 microM, for 15d-PGJ2, and 170.34+/-20 microM for rosiglitazone). HNE (1 microM) inhibited cell growth by 70%. Apoptosis was induced by 15d-PGJ2 and HNE and, to a minor extent, rosiglitazone. Differentiation was induced by rosiglitazone and by 15d-PGJ2, but not by HNE. PPARgamma ligands inhibited c-myc expression. HNE induced a transitory increase in c-myc expression and a subsequent down-regulation. HNE induced p21 expression, whereas PPARgamma ligands did not. Expression of the bax gene was increased by HNE and 15d-PGJ2, but not by rosiglitazone. No synergism or antagonism was found between HNE and PPARgamma ligands. Both apoptosis and differentiation induction may be responsible for the inhibition of proliferation by PPARgamma ligands; apoptosis and c-myc and p21 expression seem to be involved in the inhibition of proliferation by HNE.     

The 15-deoxy-delta12,14-prostaglandin J2 inhibits the inflammatory response in primary rat astrocytes via down-regulating multiple steps in phosphatidylinositol 3-kinase-Akt-NF-kappaB-p300 pathway independent of peroxisome proliferator-activated receptor gamma

Ligands for peroxisome proliferator-activated receptor gamma (PPARgamma), such as 15-deoxy-12,14-PGJ2 (15d-PGJ2), have been proposed as a new class of anti-inflammatory compounds because 15d-PGJ2 was able to inhibit the induction of inflammatory response genes such as inducible NO synthase (iNOS) and TNF (TNF-alpha) in a PPAR-dependent manner in various cell types. In primary astrocytes, the anti-inflammatory effects (inhibition of TNF-alpha, IL-1beta, IL-6, and iNOS gene expression) of 15d-PGJ2 are observed to be independent of PPARgamma. Overexpression (wild-type and dominant-negative forms) of PPARgamma and its antagonist (GW9662) did not alter the 15d-PGJ2-induced inhibition of LPS/IFN-gamma-mediated iNOS and NF-kappaB activation. The 15d-PGJ2 inhibited the inflammatory response by inhibiting IkappaB kinase activity, which leads to the inhibition of degradation of IkappaB and nuclear translocation of p65, thereby regulating the NF-kappaB pathway. Moreover, 15d-PGJ2 also inhibited the LPS/IFN-gamma-induced PI3K-Akt pathway. The 15d-PGJ2 inhibited the recruitment of p300 by NF-kappaB (p65) and down-regulated the p300-mediated induction of iNOS and NF-kappaB luciferase reporter activity. Coexpression of constitutive active Akt and PI3K (p110) reversed the 15d-PGJ2-mediated inhibition of p300-induced iNOS and NF-kappaB luciferase activity. This study demonstrates that 15d-PGJ2 suppresses inflammatory response by inhibiting NF-kappaB signaling at multiple steps as well as by inhibiting the PI3K/Akt pathway independent of PPARgamma in primary astrocytes.     

Peroxisome proliferator-activated receptor gamma ligands attenuate immunological symptoms of experimental allergic asthma

Asthma is characterized by a predominant T(H)2 type immune response to airborne allergens. Controlling T(H)2 cell function has been proposed as therapy for this disease. We show here that ligands for the nuclear receptor peroxisome proliferator activated receptor (PPAR)gamma significantly reduced the immunological symptoms of allergic asthma in a murine model of this disease. A PPARgamma ligand, 15-deoxy-delta(12,14)-prostaglandin J(2), significantly inhibited production of the T(H)2 type cytokine IL-5 from T cells activated in vitro. More importantly, in a murine model of allergic asthma, mice treated orally with ciglitazone, a potent synthetic PPARgamma ligand, had significantly reduced lung inflammation and mucous production following induction of allergic asthma. T cells from these ciglitazone treated mice also produced less IFNgamma, IL-4, and IL-2 upon rechallenge in vitro with the model allergen. Our results suggest that ligands for PPARgamma may be effective treatments for asthmatic patients.     

Suppression of prostaglandin E2 receptor subtype EP2 by PPARgamma ligands inhibits human lung carcinoma cell growth

Prostaglandin E(2) (PGE(2)), a major cyclooxygenase (COX-2) metabolite, plays important roles in tumor biology and its functions are mediated through one or more of its receptors EP1, EP2, EP3, and EP4. We have shown that the matrix glycoprotein fibronectin stimulates lung carcinoma cell proliferation via induction of COX-2 expression with subsequent PGE(2) protein biosynthesis. Ligands of peroxisome proliferator-activated receptor gamma (PPARgamma) inhibited this effect and induced cellular apoptosis. Here, we explore the role of the PGE(2) receptor EP2 in this process and whether the inhibition observed with PPARgamma ligands is related to effects on this receptor. We found that human non-small cell lung carcinoma cell lines (H1838 and H2106) express EP2 receptors, and that the inhibition of cell growth by PPARgamma ligands (GW1929, PGJ2, ciglitazone, troglitazone, and rosiglitazone [also known as BRL49653]) was associated with a significant decrease in EP2 mRNA and protein levels. The inhibitory effects of BRL49653 and ciglitazone, but not PGJ2, were reversed by a specific PPARgamma antagonist GW9662, suggesting the involvement of PPARgamma-dependent and -independent mechanisms. PPARgamma ligand treatment was associated with phosphorylation of extracellular regulated kinase (Erk), and inhibition of EP2 receptor expression by PPARgamma ligands was prevented by PD98095, an inhibitor of the MEK-1/Erk pathway. Butaprost, an EP2 agonist, like exogenous PGE(2) (dmPGE(2)), increased lung carcinoma cell growth, however, GW1929 and troglitazone blocked their effects. Our studies reveal a novel role for EP2 in mediating the proliferative effects of PGE(2) on lung carcinoma cells. PPARgamma ligands inhibit human lung carcinoma cell growth by decreasing the expression of EP2 receptors through Erk signaling and PPARgamma-dependent and -independent pathways.     

Peroxisome proliferator-activated receptor gamma ligands inhibit retinoblastoma phosphorylation and G1--> S transition in vascular smooth muscle cells

Peroxisome proliferator-activated receptor gamma (PPARgamma) is a member of the nuclear receptor superfamily that is activated by binding certain fatty acids, eicosanoids, and insulin-sensitizing thiazolidinediones (TZD). The TZD troglitazone (TRO) inhibits vascular smooth muscle cell proliferation and migration both in vitro and in vivo. The precise mechanism of its antiproliferative activity, however, has not been elucidated. We report here that PPARgamma ligands inhibit rat aortic vascular smooth muscle cell proliferation by blocking the events critical for G(1) --> S progression. Flow cytometry demonstrated that both TRO and another TZD, rosiglitazone, prevented G(1) --> S progression induced by platelet-derived growth factor and insulin. Movement of cells from G(1) --> S was also inhibited by the non-TZD, natural PPARgamma ligand 15-deoxy-(12,14)Delta prostaglandin J(2) (15d-PGJ(2)), and the mitogen-activated protein kinase pathway inhibitor PD98059. Inhibition of G(1) --> S exit by these compounds was accompanied by a substantial blockade of retinoblastoma protein phosphorylation. TRO and rosiglitazone attenuated both the mitogen-induced degradation of p27(kip1) and the mitogenic induction of p21(cip1). 15d-PGJ(2) and PD98059 inhibited both the degradation of p27(kip1) and the induction of cyclin D1 in response to mitogens. These effects resulted in the inhibition of mitogenic stimulation of cyclin-dependent kinases activated by cyclins D1 and E. These data demonstrate that PPARgamma ligands are antiproliferative drugs that act by modulating cyclin-dependent kinase inhibitors; they may provide a new therapeutic approach for proliferative vascular diseases.     

Activation of peroxisome proliferator-activated receptor gamma does not inhibit IL-6 or TNF-alpha responses of macrophages to lipopolysaccharide in vitro or in vivo

We have investigated the potential use of peroxisome proliferator-activated receptor gamma (PPARgamma) agonists as anti-inflammatory agents in cell-based assays and in a mouse model of endotoxemia. Human peripheral blood monocytes were treated with LPS or PMA and a variety of PPARgamma agonists. Although 15-deoxy-Delta12,14-prostaglandin J2 (15d-PGJ2) at micromolar concentrations significantly inhibited the production of TNF-alpha and IL-6, four other high affinity PPARgamma ligands failed to affect cytokine production. Similar results were obtained when the monocytes were allowed to differentiate in culture into macrophages that expressed significantly higher levels of PPARgamma or when the murine macrophage cell line RAW 264.7 was used. Furthermore, saturating concentrations of a potent PPARgamma ligand not only failed to block cytokine production, but also were unable to block the inhibitory activity of 15d-PGJ2. Thus, activation of PPARgamma does not appear to inhibit the production of cytokines by either monocytes or macrophages, and the inhibitory effect observed with 15d-PGJ2 is most likely mediated by a PPARgamma-independent mechanism. To examine the anti-inflammatory activity of PPARgamma agonists in vivo, db/db mice were treated with a potent thiazolidinedione that lowered their elevated blood glucose and triglyceride levels as expected. When thiazolidinedione-treated mice were challenged with LPS, they displayed no suppression of cytokine production. Rather, their blood levels of TNF-alpha and IL-6 were elevated beyond the levels observed in control db/db mice challenged with LPS. Comparable results were obtained with the corresponding lean mice. Our data suggest that compounds capable of activating PPARgamma in leukocytes will not be useful for the treatment of acute inflammation.     

Roles of peroxisome proliferator-activated receptors delta and gamma in myoblast transdifferentiation

Dietary long chain fatty acids and thiazolidinediones act as potent activators of adipogenesis in established preadipose cell lines. High concentrations of thiazolidinediones have also been shown to induce terminal differentiation of non-preadipose cells, such as fibroblasts and myoblasts, into adipose-like cells. This transdifferentiation was observed in both rodent and human myoblasts. In this report, we show that PPARdelta mediates some of the effects exerted by long chain fatty acids on myogenesis and adipogenesis. Activation of PPARdelta by long chain fatty acids impairs the expression of the determination factor MyoD1 and alpha-actin, abolishes the development of multinucleated myotubes, and in parallel induces the expression of PPARgamma gene, a master regulator of adipogenesis. Ectopic expression of PPARdelta in C2C12 myoblasts potentiated the fatty acid-induced expression of adipogenic markers, while expression of a dominant negative PPARdelta mutant exerted opposite effects. Furthermore, a sequential activation of first PPARdelta with long chain fatty acids and then PPARgamma with thiazolidinediones is required for adipogenesis in C2C12 myoblasts. This study demonstrates that PPARdelta, at least in part, is responsible for the dual effects of long chain fatty acids as inhibitors of myogenesis and inducers of transdifferentiation into preadipose-like cells.