Antiinflammatory effect of rosiglitazone via modulation of mRNA stability of interleukin 10 and cyclooxygenase 2 in astrocytes

Investigation of molecular mechanisms of proinflammatory stimuli signaling in astrocytes is important for understanding their role in pathogenesis of central nervous system diseases as well as in functioning of the innate immunity system in non-immune cells. Here we show that lipopolysaccharide (LPS) stimulation of primary rat astrocytes led to conventional inflammatory response: increase in both proinflammatory (tumor necrosis factor, TNFα; prostaglandin E₂, PGE₂) and antiinflammatory marker (interleukin 10, IL-10) levels. The protein level of cyclooxygenase 2 (COX-2) was also increased. Rosiglitazone strengthened LPS-induced mRNA expression of COX-2 and IL-10 but not TNFα. Rosiglitazone is an agonist of nuclear receptor PPARγ, but its impact on IL-10 expression was not influenced by a PPARγ antagonist, GW9662, suggesting PPARγ-independent effect of rosiglitazone. The degradation of mRNA is one of the steps of inflammation regulation and might be affected by small molecules. In experiments with actinomycin D, we found that mRNA half-lives of IL-10, COX-2, and TNFα in naive astrocytes were 70, 44, and 19 min, respectively. LPS stimulation caused 2-fold increase in IL-10 and COX-2 mRNA decay rates, whereas addition of rosiglitazone restored them to the initial level. TNFα decay rate was not changed by these stimulations. This suggests that mRNA decay rate could be regulated by small molecules. Moreover, rosiglitazone could be used as a substance stimulating the resolution of inflammation without influence on proinflammatory signals. These results open new perspectives in the search for inflammation resolution modulators.     

Expression of peroxisome proliferator-activated receptor isoforms in the rat uterus during early pregnancy

Peroxisome proliferator-activated receptors (PPARs) play an important role in different compartments of the female reproductive system in rodents and humans. However, expressional profiles and physiological functions of PPARs in the endometrium prior to the placentation are not well understood. In this study, we determined expressional profiles of the PPARs during early pregnancy. Immunocytochemistry revealed that both PPARα and PPARβ/δ were strongly detected in the endometrial stroma on days 4.5–6.5 of pregnancy, which is just a starting time of implantation. Delayed implantation animal model showed that the expressions of PPARα and PPARβ/δ occurred after the initiation of implantation in the endometrial stroma. Moreover, an in vitro decidualization model further revealed that the expression of PPARα increased in the cultured rat endometrial stromal cells at 24 h after the decidualization treatment, but the expression of PPARβ/δ was delayed and increased at 48 h after the treatment. PPARγ was expressed in the endometrial stroma and its expression decreased significantly at 2.5 days post-coitum and maintained a low level of expression during the period of implantation. These results indicate that PPARα is expressed and induced by the initiation of implantation, prior to the expression of PPARβ/δ in decidualized endometrium. Increasing expression of PPARγ during fertilization and its decline during the period of implantation further suggest that PPARs may play important roles during early pregnancy.     

Contrasting effects of peroxisome-proliferator-activated receptor (PPAR)γ agonists on membrane-associated prostaglandin E2 synthase-1 in IL-1β-stimulated rat chondrocytes: evidence for PPARγ-independent inhibition by 15-deoxy-Δ12,14prostaglandin J2

Microsomal prostaglandin E synthase (mPGES)-1 is a newly identified inducible enzyme of the arachidonic acid cascade with a key function in prostaglandin (PG)E₂ synthesis. We investigated the kinetics of inducible cyclo-oxygenase (COX)-2 and mPGES-1 expression with respect to the production of 6-keto-PGF_1α and PGE₂ in rat chondrocytes stimulated with 10 ng/ml IL-1β, and compared their modulation by peroxisome-proliferator-activated receptor (PPAR)γ agonists. Real-time PCR analysis showed that IL-1β induced COX-2 expression maximally (37-fold) at 12 hours and mPGES-1 expression maximally (68-fold) at 24 hours. Levels of 6-keto-PGF_1α and PGE₂ peaked 24 hours after stimulation with IL-1β; the induction of PGE₂ was greater (11-fold versus 70-fold, respectively). The cyclopentenone 15-deoxy-Δ^12,14prostaglandin J₂ (15d-PGJ₂) decreased prostaglandin synthesis in a dose-dependent manner (0.1 to 10 μM), with more potency on PGE₂ level than on 6-keto-PGF_1α level (-90% versus -66% at 10 μM). A high dose of 15d-PGJ₂ partly decreased COX-2 expression but decreased mPGES-1 expression almost completely at both the mRNA and protein levels. Rosiglitazone was poorly effective on these parameters even at 10 μM. Inhibitory effects of 10 μM 15d-PGJ₂ were neither reduced by PPARγ blockade with GW-9662 nor enhanced by PPARγ overexpression, supporting a PPARγ-independent mechanism. EMSA and TransAM^? analyses demonstrated that mutated IκBα almost completely suppressed the stimulating effect of IL-1β on mPGES-1 expression and PGE₂ production, whereas 15d-PGJ₂ inhibited NF-κB transactivation. These data demonstrate the following in IL-1-stimulated rat chondrocytes: first, mPGES-1 is rate limiting for PGE₂ synthesis; second, activation of the prostaglandin cascade requires NF-κB activation; third, 15d-PGJ₂ strongly inhibits the synthesis of prostaglandins, in contrast with rosiglitazone; fourth, inhibition by 15d-PGJ₂ occurs independently of PPARγ through inhibition of the NF-κB pathway; fifth, mPGES-1 is the main target of 15d-PGJ₂.     

Pioglitazone, a synthetic ligand for PPARγ, induces apoptosis in RB-deficient human colorectal cancer cells

No published data are available about the expression of peroxisome proliferator-activated receptor γ (PPARγ) and the role of PPARγ in retinoblastoma protein (RB)-deficient human colorectal cancer (CRC) cells (SNU-C4 and SNU-C2A). Our aim was to investigate whether PPARγ is expressed in SNU-C4 and SNU-C2A cells and to elucidate possible molecular mechanisms underlying the effect of pioglitazone, a synthetic ligand for PPARγ, on cell growth in these cell lines. RT-PCR and Western blot analysis showed that both human CRC cell lines expressed PPARγ mRNA and protein. Pioglitazone inhibited the cell growth of both cell lines through G2/M phase block and apoptosis. In addition, pioglitazone caused a down-regulation of the X chromosome-linked inhibitor of apoptosis (XIAP), Bcl-2, and cyclooxygenase-2 (COX-2) under conditions leading to PPARγ down-regulation. These results suggest that pioglitazone may have therapeutic relevance or significance in the treatment of human CRC, and the down-regulation of XIAP, Bcl-2, and COX-2 may contribute to pioglitazone-induced apoptosis in these and other RB-deficient cell lines and tumors.     

Expression of cytochrome P-450 4 enzymes in the kidney and liver: Regulation by PPAR and species-difference between rat and human

Members of the cytochrome P-450 4 (CYP4) family catalyze the ω-hydroxylation of fatty acids, and some of them have the PPAR response element in the promoter area of the genes. The localization of CYP4A and PPAR isoforms and the effect of PPAR agonists on CYP4A protein level and activity were determined in rat kidney and liver. Immunoblot analysis showed that CYP4A was expressed in the liver and proximal tubule, with lower expression in the preglomerular microvessel, glomerulus and thick ascending limb (TAL), but the expression was not detected in the collecting duct. PPARα was expressed in the liver, proximal tubule and TAL. PPARγ was expressed in the collecting duct, with lower expression in the TAL, but no expression in the proximal tubule and liver. The PPARα agonist clofibrate induced CYP4A protein levels and activity in the renal cortex and liver. The PPARγ agonist pioglitazone did not modulate them in these tissues. The localization of CYP4A and CYP4F were further determined in human kidney and liver by immunohistochemical technique. Immunostainings for CYP4A and CYP4F were observed in the hepatocytes of the liver lobule and the proximal tubules, with lower stainings in the TALs and collecting ducts, but no staining in the glomeruli or renal vasculatures. These results indicate that the inducibility of CYP4A by PPAR agonists in the rat tissues correlates with the expression of the respective PPAR isoforms, and that the localization of CYP4 in the kidney has a species-difference between rat and human.     

Retinoid X receptor and peroxisome proliferator-activated receptor-gamma agonists cooperate to inhibit matrix metalloproteinase gene expression

Introduction  

We recently described the ability of retinoid X receptor (RXR) ligand LG100268 (LG268) to inhibit interleukin-1-beta (IL-1-β)-driven matrix metalloproteinase-1 (MMP-1) and MMP-13 gene expression in SW-1353 chondrosarcoma cells. Other investigators have demonstrated similar effects in chondrocytes treated with rosiglitazone, a ligand for peroxisome proliferator-activated receptor-gamma (PPARγ), for which RXR is an obligate dimerization partner. The goals of this study were to evaluate the inhibition of IL-1-β-induced expression of MMP-1 and MMP-13 by combinatorial treatment with RXR and PPARγ ligands and to investigate the molecular mechanisms of this inhibition.     

All-<Emphasis Type="Italic">trans</Emphasis>- and 9-<Emphasis Type="Italic">cis</Emphasis>-retinoic acids activate the human cyclooxynase-2 gene: a role for DR1 as RARE or RXRE

The human COX-2 promoter contains a direct repeat 1 (DR1) which was shown to confer responsiveness to PPARs. We found that in AN₃CA and F9 cells, this hCOX-2 DR1 mediates responsiveness to all-trans-retinoic acid (tRA) or 9-cis-retinoic acid (9cRA), but this effect was suppressed by PPARδ. Truncated PPARδ lacking the activation domain AF2 cannot suppress RA-induced activation of the hCOX-2 gene via DR1, suggesting that cofactor recruitment by AF2 is required for the suppression by PPARδ. Gel shift assay showed that PPAR/RXR, RARβ/RXR, and RXR/RXR, bind to hCOX-2 DR1, revealing the promiscuity of this DR1. Particularly, RXR homodimer was able to bind to this DR1 only in the presence of 9cRA. Our results established that tRA and 9cRA are potent inducers of hCOX-2 and that the hCOX-2 DR1 could either serve as RARE or RXRE depending on cellular contexts.     

KDT501, a Derivative from Hops,Normalizes Glucose Metabolism and Body Weight in Rodent Models of Diabetes

Aims/Hypothesis

We developed KDT501, a novel substituted 1,3-cyclopentadione chemically derived from hop extracts, and evaluated it in various in vitro and in vivo models of diabetes and insulin sensitivity.

Methods

KDT501 was evaluated for anti-inflammatory effects in monocyte/macrophage cells; agonistic activity for peroxisome proliferator-activated receptors (PPAR); lipogenesis and gene expression profile in human subcutaneous adipocytes. Body composition, glucose, insulin sensitivity, and lipids were assessed in diet-induced obesity (DIO) mice and Zucker Diabetic Fatty (ZDF) rats after oral administration.

Results

KDT501 mediated lipogenesis in 3T3L1 and human subcutaneous adipocytes; however, the gene expression profile of KDT501 differed from that of the full PPARγ agonist rosiglitazone, suggesting that KDT501 has pleiotropic biological activities. In addition, KDT501 showed only modest, partial PPARγ agonist activity and exhibited anti-inflammatory effects in monocytes/macrophages that were not observed with rosiglitazone. In a DIO mouse model, oral administration of KDT501 significantly reduced fed blood glucose, glucose/insulin AUC following an oral glucose bolus, and body fat. In ZDF rats, oral administration of KDT501 significantly reduced fed glucose, fasting plasma glucose, and glucose AUC after an oral glucose bolus. Significant, dose-dependent reductions of plasma hemoglobin A1c, weight gain, total cholesterol, and triglycerides were also observed in animals receiving KDT501.

Conclusion

These results indicate that KDT501 produces a unique anti-diabetic profile that is distinct in its spectrum of pharmacological effects and biological mechanism from both metformin and pioglitazone. KDT501 may thus constitute a novel therapeutic agent for the treatment of Type 2 diabetes and associated conditions.     

A novel dual peroxisome proliferator-activated receptors α and γ agonist with beneficial effects on insulin resistance and lipid metabolism

Peroxisome proliferator-activated receptors (PPARs) α and γ are key regulators of lipid homeostasis and insulin resistance. In this study␣we show that a novel compound, 3-{4-[2-(5-methyl-2-phenyl-oxazol-4-yl)-ethoxy]-phenyl}- 2-[2-(2-nitro-phenoxy)-acetyl amino]-propionic acid (O325H), is an agonist with dual effect on PPARα/γ by using dual-luciferase reporter gene assay. By activating PPARα and PPARγ simultaneously, O325H promotes pre-adipocyte differentiation and up-regulates the expression of glucose and lipid metabolic target genes. In diabetic mice, administration of O325H at 10 mg/kg decreases the blood lipid and glucose levels. Therefore, O325H has dual action on PPARα and PPARγ and is a promising agent for the amelioration of lipid metabolic disorders and diabetes associated with insulin resistance.     

The contrasting roles of PPARδ and PPARγ in regulating the metabolic switch between oxidation and storage of fats in white adipose tissue

Background  

The nuclear receptors peroxisome proliferator-activated receptor γ (PPARγ) and peroxisome proliferator-activated receptor δ (PPARδ) play central roles in regulating metabolism in adipose tissue, as well as being targets for the treatment of insulin resistance. While the role of PPARγ in regulating insulin sensitivity has been well defined, research into PPARδ has been limited until recently due to a scarcity of selective PPARδ agonists.     

Reduction of Nuclear Peroxisome Proliferator-Activated Receptor γ Expression in Methamphetamine-Induced Neurotoxicity and Neuroprotective Effects of Ibuprofen

We examined changes in nuclear peroxisome proliferator-activated receptor γ (PPARγ) in the striatum in methamphetamine (METH)-induced dopaminergic neurotoxicity, and also examined effects of treatment with drugs possessing PPARγ agonistic properties. The marked reduction of nuclear PPARγ-expressed cells was seen in the striatum 3 days after METH injections (4 mg/kg × 4, i.p. with 2-h interval). The reduction of dopamine transporter (DAT)-positive signals and PPARγ expression, and accumulation of activated microglial cells were significantly and dose-dependently attenuated by four injections of a nonsteroidal anti-inflammatory drug and a PPARγ ligand, ibuprofen (10 or 20 mg/kg × 4, s.c.) given 30 min prior to each METH injection, but not by either a low or high dose of aspirin. Either treatment of ibuprofen or aspirin, that showed no effects on METH-induced hyperthermia, significantly blocked the METH-induced striatal cyclooxygenase (COX) expression. Furthermore, the treatment of an intrinsic PPARγ ligand 15d-PG J2 also attenuated METH injections-induced reduction of striatal DAT. Therefore, the present study suggests the involvement of reduction of PPARγ expression in METH-induced neurotoxicity. Taken together with the previous report showing protective effects of other PPARγ ligand, these results imply that the protective effects of ibuprofen against METH-induced neurotoxicity may be based, in part, on its anti-inflammatory PPARγ agonistic properties, but not on its COX-inhibiting property or hypothermic effect. Special issue article in honor of Dr. Akitane Mori.     

PPAR Agonist-Induced Reduction of Mcp1 in Atherosclerotic Plaques of Obese,Insulin-Resistant Mice Depends on Adiponectin-Induced Irak3 Expression

Synthetic peroxisome proliferator-activated receptor (PPAR) agonists are used to treat dyslipidemia and insulin resistance. In this study, we examined molecular mechanisms that explain differential effects of a PPARα agonist (fenofibrate) and a PPARγ agonist (rosiglitazone) on macrophages during obesity-induced atherogenesis. Twelve-week-old mice with combined leptin and LDL-receptor deficiency (DKO) were treated with fenofibrate, rosiglitazone or placebo for 12 weeks. Only rosiglitazone improved adipocyte function, restored insulin sensitivity, and inhibited atherosclerosis by decreasing lipid-loaded macrophages. In addition, it increased interleukin-1 receptor-associated kinase-3 (Irak3) and decreased monocyte chemoattractant protein-1 (Mcp1) expressions, indicative of a switch from M1 to M2 macrophages. The differences between fenofibrate and rosiglitazone were independent of Pparγ expression. In bone marrow-derived macrophages (BMDM), we identified the rosiglitazone-associated increase in adiponectin as cause of the increase in Irak3. Interestingly, the deletion of Irak3 in BMDM (IRAK3^−/− BMDM) resulted in activation of the canonical NFκB signaling pathway and increased Mcp1 protein secretion. Rosiglitazone could not decrease the elevated Mcp1 secretion in IRAK3^−/− BMDM directly and fenofibrate even increased the secretion, possibly due to increased mitochondrial reactive oxygen species production. Furthermore, aortic extracts of high-fat insulin-resistant LDL-receptor deficient mice, with lower adiponectin and Irak3 and higher Mcp1, showed accelerated atherosclerosis. In aggregate, our results emphasize an interaction between PPAR agonist-mediated increase in adiponectin and macrophage-associated Irak3 in the protection against atherosclerosis by PPAR agonists.     

Using yeast two-hybrid system to detect interactions of ATP synthase subunits from Spinacia oleracea

Subunit interactions among the chloroplast ATP synthase subunits were studied using the yeast two-hybrid system. Various pairwise combinations of genes encoding α, β, γ, δ and ε subunits ofSpinach ATP synthase fused to the binding domain or activation domain of GAL4 DNA were introduced into yeast and then expression of a reporter gene encoding β-galactosidase was detected. Of all the combinations, that of γ and ε subunit genes showed the highest level of reporter gene expression, while those of α and β, a and ε, β and ε and β and δ induced stable and significant reporter gene expression. The combination of δ and ε as well as that of δ and γ induced weak and unstable reporter gene expression. However, combinations of α and γ, β and γ and α and δ did not induce reporter gene expression. These results suggested that specific and strong interactions between γ and ε, α and β, α and ε, β and ε and β and δ subunits, and weak and transient interactions between δ and ε and δ and γ subunits occurred in the yeast cell in the two-hybrid system. These results give a new look into the structural change of ATP synthase during catalysis.     

Interleukin-15 effectively potentiates the in vitro tumor-specific activity and proliferation of peripheral blood γδT cells isolated from glioblastoma patients

γδT cells play a regulatory role in both primary and metastatic tumor growth in humans. The mechanisms responsible for the activation and proliferation of circulating γδT cells should be fully understood prior to their adoptive transfer to cancer patients. We have examined in vitro functional effects of interleukin-15 (IL-15) on highly purified γδT cells isolated from glioblastoma patients. γδT cells constitutively express the heterotrimeric IL-2 receptor (IL-2R) αβγ, but the levels of IL-2Rβ or γ expression were not increased by incubation with saturating amounts of IL-15. IL-15 was shown to induce a maximal γδT cell proliferation, although at much higher concentrations (at least 2000 U/ml) than IL-2 (100 U/ml). Submaximal concentrations of IL-15 plus low concentrations of IL-2 produced an additive proliferative response. In contrast to the IL-2-induced response, this activity was completely or partially abrogated by anti-IL-2Rβ, or anti-IL-2Rγ antibodies, but not by anti-IL-2Rα antibodies. Incubation of γδT cells in the presence of IL-15 resulted not only in the appearance of NK and LAK activity, but also in specific autologous tumor cell killing activity, an additive effect being seen with IL-15 and IL-2. This IL-15-induced tumor-specific activity could be significantly blocked by anti-IL-2Rγ and anti-IL-2R-β mAb, but not by anti-IL-2Rα mAb. Thus, in contrast to IL-2, IL-15 activates tumor-specific γδT cells through the components of IL-2Rβ and IL-2Rγ, but not IL-2Rα. These enhanced in vitro tumor-specific and proliferative responses of γδT cells seen with IL-15 suggest a rational adjuvant imunotherapeutic use of γδT cells in cancer patients. Received: 23 January 1998 / Accepted: 20 May 1998