15-Deoxy-delta12,14-PGJ2, but not troglitazone, modulates IL-1beta effects in human chondrocytes by inhibiting NF-kappaB and AP-1 activation pathways.

The activation of peroxisome proliferator-activated receptor gamma (PPARgamma) has been shown to inhibit the production and the effects of proinflammatory cytokines. Since interleukin-1beta (IL-1beta) directly mediates cartilage degradation in osteoarthritis, we investigated the capability of PPARgamma ligands to modulate IL-1beta effects on human chondrocytes. RT-PCR and Western blot analysis revealed that PPARgamma expression was decreased by IL-1beta. 15-deoxy-Delta12,14-prostaglandin J2 (15d-PGJ2), in contrast to troglitazone, was highly potent to counteract IL-1beta-induced cyclooxygenase-2 and inductible nitric oxide synthase expression, NO production and the decrease in proteoglycan synthesis. Western blot and gel-shift analyses demonstrated that 15d-PGJ2 inhibited NF-kappaB activation, while troglitazone was ineffective. Although 15d-PGJ2 attenuated activator protein-1 binding on the DNA, it potentiated c-jun migration in the nucleus. The absence or the low effect of troglitazone suggests that 15d-PGJ2 action in human chondrocytes is mainly PPARgamma-independent.     

15-deoxy-delta 12,14-prostaglandin J(2) inhibits the synthesis of the acute phase protein SIP24 in cartilage: Involvement of COX-2 in resolution of inflammation

We previously demonstrated that, in the MC615 cartilage cell line, the p38/NF-kB pathway is activated both during differentiation and in response to an inflammatory stimulus. In both cases, the p38/NF-kB pathway activation leads to the expression of the lipocalin SIP24 and of COX-2. Given the fact that, in the same cells, the COX-2 expression is sustained during the inflammation resolution, at the same time that the SIP24 expression is suppressed, in the present study we tested the hypothesis that COX-2 products play a role in SIP24 repression. Taken together, our results suggest that, during the resolution of inflammation, COX-2 represses the acute phase protein SIP24 and restores physiological conditions, possibly through a pathway involving PPARgamma. Experimental evidences being the following: (1) 15-deoxy-delta 12,14-prostaglandin J(2), but not PGE(2): (i) inhibits the expression of SIP24 in the inflammatory phase and induces COX-2 synthesis; (ii) represses NF-kB activation induced by LPS; (iii) represses the synthesis of microsomal PGE Synthase-1 induced by LPS. (2) PPARgamma and PPARalpha are present in MC615 cells in both proliferating and hyperconfluent cultures. (3) PPARgamma ligand GW7845, but not PPARalpha ligand GW7647: (i) represses the expression of SIP24 induced by LPS; (ii) induces COX-2 expression. (4) p38 is involved in the PPARgamma mediated induction of COX-2. In fact 15-deoxy-delta 12,14-prostaglandin J(2) activates p38 and the cell pretreatment with the p38 specific inhibitor SB203580 represses the expression of COX-2 induced by both the 15-deoxy-delta12,14-prostaglandin J(2) and the PPARgamma ligand GW7845.     

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.     

Binding analyses between Human PPARgamma-LBD and ligands.

The binding characteristics of a series of PPARgamma ligands (GW9662, GI 262570, cis-parinaric acid, 15-deoxy-Delta(12,14)-prostaglandin J(2), LY171883, indomethacin, linoleic acid, palmitic acid and troglitazone) to human PPARgamma ligand binding domain have been investigated for the first time by using surface plasmon resonance biosensor technology, CD spectroscopy and molecular docking simulation. The surface plasmon resonance biosensor determined equilibrium dissociation constants (KD values) are in agreement with the results reported in the literature measured by other methods, indicating that the surface plasmon resonance biosensor can assume a direct assay method in screening new PPARgamma agonists or antagonists. Conformational changes of PPARgamma caused by the ligand binding were detected by CD determination. It is interesting that the thermal stability of the receptor, reflected by the increase of the transition temperature (T(m)), was enhanced by the binding of the ligands. The increment of the transition temperature (DeltaT(m)) of PPARgamma owing to ligand binding correlated well with the binding affinity. This finding implies that CD could possibly be a complementary technology with which to determine the binding affinities of ligands to PPARgamma. Molecular docking simulation provided reasonable and reliable binding models of the ligands to PPARgamma at the atomic level, which gave a good explanation of the structure-binding affinity relationship for the ligands interacting with PPARgamma. Moreover, the predicted binding free energies for the ligands correlated well with the binding constants measured by the surface plasmon resonance biosensor, indicating that the docking paradigm used in this study could possibly be employed in virtual screening to discover new PPARgamma ligands, although the docking program cannot accurately predict the absolute ligand-PPARgamma binding affinity.     

Thrombospondin-1 inhibits nitric oxide signaling via CD36 by inhibiting myristic acid uptake

Although CD36 is generally recognized to be an inhibitory signaling receptor for thrombospondin-1 (TSP1), the molecular mechanism for transduction of this signal remains unclear. Based on evidence that myristic acid and TSP1 each modulate endothelial cell nitric oxide signaling in a CD36-dependent manner, we examined the ability of TSP1 to modulate the fatty acid translocase activity of CD36. TSP1 and a CD36 antibody that mimics the activity of TSP1 inhibited myristate uptake. Recombinant TSP1 type 1 repeats were weakly inhibitory, but an anti-angiogenic peptide derived from this domain potently inhibited myristate uptake. This peptide also inhibited membrane translocation of the myristoylated CD36 signaling target Fyn and activation of Src family kinases. Myristate uptake stimulated cGMP synthesis via endothelial nitric-oxide synthase and soluble guanylyl cyclase. CD36 ligands blocked myristate-stimulated cGMP accumulation in proportion to their ability to inhibit myristate uptake. TSP1 also inhibited myristate-stimulated cGMP synthesis by engaging its receptor CD47. Myristate stimulated endothelial and vascular smooth muscle cell adhesion on type I collagen via the NO/cGMP pathway, and CD36 ligands that inhibit myristate uptake blocked this response. Therefore, the fatty acid translocase activity of CD36 elicits proangiogenic signaling in vascular cells, and TSP1 inhibits this response by simultaneously inhibiting fatty acid uptake via CD36 and downstream cGMP signaling via CD47.     

Evaluation of eicosanoids and NSAIDs as PPARgamma ligands in colorectal carcinoma cells

The activation of peroxisome proliferator activated receptor gamma (PPARgamma) may play a role in the control of colorectal carcinogenesis. The expression of PPARgamma was examined by Western blotting in human colorectal tumors and matched normal adjacent tissues, as well as in various colorectal carcinoma cell lines. In the tissues, the expression of PPARgamma was elevated in tumors relative to the adjacent normal tissues. Each colorectal carcinoma cell line expressed PPARgamma. The ability of various eicosanoids to bind PPARgamma in colorectal carcinoma cells was investigated using luciferase reporter assays. The well-known PPARgamma ligands, troglitazone and 15-deoxy-Delta(12,14)-prostaglandin J(2) strongly induced PPARgamma binding activity. Products of lipoxygenases displayed moderate binding activity, while other prostaglandins and fatty acids displayed little or no reporter activation. The activation of PPARgamma by 13(S)-HODE, the major metabolite of 15-lipoxygenase-1 from linoleic acid, was concentration dependent reaching maximum at 10 micro M (35-fold activation). The endogenous production of 13(S)-HODE by expression of 15-LO-1 did not activate PPARgamma. The ability of various nonsteroidal anti-inflammatory drugs (NSAIDs) to induce PPARgamma activation was also evaluated. The conventional NSAIDs that inhibit both cyclooxygenases (COX-1 and COX-2) also induced PPARgamma binding activity. In general, however, neither COX-1- nor COX-2-specific inhibitors induced the activation of PPARgamma. Taken together, the metabolites of 15-lipoxygenase and the conventional NSAIDs were confirmed as exogenous ligands for PPARgamma in colorectal carcinoma cells.     

In vivo upregulation of CD95 and CD95L causes synergistic inhibition of angiogenesis by TSP1 peptide and metronomic doxorubicin treatment

Antiangiogenic thrombospondin-1 (TSP1) induces endothelial cell death via a CD95-mediated cascade. We used this signaling pathway, where CD95/Fas is a rate-limiting intermediate, as a target to optimize the efficacy of TSP1 active peptide, DI-TSP. Like TSP1, DI-TSP upregulated endothelial CD95L in vivo. To modulate CD95 levels, we chose chemotherapy agent doxorubicin (DXR). DXR caused sustained upregulation of CD95 in the activated endothelium at 1/100 of the maximal tolerated dose. DI-TSP and DXR synergistically induced endothelial apoptosis in vitro, and in vivo, in developing murine vessels. Fas decoy, TSP1 receptor antibody and Pifithrin, a p53 inhibitor, severely decreased apoptosis and restored angiogenesis by DXR-DI-TSP combination, evidencing critical roles of CD95 and TSP1. Combined therapy synergistically blocked neovascularization and progression of the bladder and prostate carcinoma. Such informed design of a complex antiangiogenic therapy based on the rate-limiting molecular targets is a novel concept, which may yield new approaches to cancer treatment.     

A novel method for analysis of nuclear receptor function at natural promoters: peroxisome proliferator-activated receptor gamma agonist actions on aP2 gene expression detected using branched DNA messenger RNA quantitation

Peroxisome proliferator-activated receptor-gamma (PPARgamma), a member of the nuclear hormone receptor superfamily, plays an essential role in the mediation of the actions of antidiabetic drugs known as thiazolidinediones (TZDs). PPARgamma activates many target genes involved in lipid anabolism including the adipocyte fatty acid binding protein (aP2). In this study, induction of aP2 gene expression by PPARgamma agonists was examined in both cultured cells and diabetic mice using branched DNA (bDNA)-mediated mRNA quantitation. bDNA technology allows for the direct measurement of a particular mRNA directly within cellular lysate using a 96-well plate format in a time frame comparable to a reporter gene assay. In cultured human subcutaneous preadipocytes, the TZDs, troglitazone and BRL-49653, both rapidly induced aP2 mRNA as detected with the bDNA method. In these cells, the effect of BRL-49653 on aP2 mRNA levels was detectable as early as 30 min after treatment (47% increase) and was maximal after 24 h of treatment (12-fold increase). The effects of troglitazone on aP2 mRNA induction were similar to those of BRL-49653 except that the maximal level of induction was consistently lower (e.g. 24 h treatment = 4-fold increase). Dose-response relationships for both of the TZDs were also determined using the 24-h treatment time point. EC50s for both BRL-49653 and troglitazone were estimated to be 80 nM and 690 nM, respectively. A natural PPARgamma ligand, 15-deoxy-delta12,14-PGJ2, was also active in this assay with a maximal induction of aP2 mRNA of approximately 5-fold when tested at 1 microM. Since the PPARgamma:retinoid X receptor (RXR) heterodimer has been characterized as a permissive heterodimer with respect to RXR ligands, the ability of 9-cis-retinoic acid (9-cis-RA) to induce aP2 mRNA was examined. Although 9-cis-RA had very low efficacy (2-fold induction), the maximal effect was reached at 100 nM. No synergism or additivity in aP2 mRNA induction was detected when 9-cis-RA was included with either of the TZDs used in this study. Significant induction of aP2 mRNA in bone marrow of db/db mice treated with either troglitazone or BRL-49653 was also detected, indicating that the bDNA assay may be a simple method to monitor nuclear receptor target gene induction in vivo.     

Expression and function of PPARgamma in rat placental development

Peroxisome proliferator-activated receptor (PPAR) gamma is a nuclear receptor known to regulate adipogenesis. Deletion of the PPARgamma gene in the mouse results in death by embryonic day 10.0 (E10.0) due to the failure of establishment of a labyrinth layer in the placenta, which suggests that PPARgamma is involved in trophoblast differentiation. To define PPARgamma function further in placental development, the expression and localization of the PPARgamma gene in the rat placenta was investigated. RT-PCR analysis shows the presence of PPARgamma mRNA in the placenta of day 11 of pregnancy (d11). The expression level is higher at d13 and then later decreased. Immunohistochemistry detects both PPARgamma and its putative intrinsic ligand, 15-deoxy-Delta(12,14)-prostaglandin J(2), in the trophoblast of layer I which lined the maternal sinus. Oral administration of troglitazone, an agonist of PPARgamma, to pregnant rats between d9 and d11 increases the expression level of PPARgamma in the placenta and reduces the mortality of the fetuses by half. These results suggest that PPARgamma is required not only for trophoblast differentiation but also trophoblast maturation to establish maternal-fetal transport.     

PPARgamma ligands suppress proliferation of human urothelial basal cells in vitro

Expression of peroxisome proliferator-activated receptor (PPAR) gamma in the human urinary tract through embryonic development suggests its possible roles in the development, proliferation, and differentiation of uroepithelium. Little is known, however, about physiological roles of PPARgamma in the urinary tract. We investigated effects of PPARgamma ligands on the proliferation of normal human urothelial cells and stromal cells cultivated from surgical specimens. Active proliferation in vitro as well as high molecular weight cytokeratin expression indicated that cultured urothelial cells possess basal cell phenotype. PPARgamma protein, expressed predominantly in the epithelial layer of the normal human urinary tract in vivo, was abundantly expressed in urothelial cells but barely detectable in stromal cells in vitro. Natural ligand for PPARgamma, 15-deoxy-Delta(12,14) prostaglandin J(2) (15d-PGJ(2)), as well as synthetic ones, troglitazone and pioglitazone, suppressed proliferation of the urothelial cells dose-dependently. These effects were PPARgamma specific because clofibrate or PGF(2alpha) did not affect proliferation of urothelial cells. Neither 9-cis retinoic acid or all-trans retinoic acid (ATRA) at 1 microM showed any synergism on the antiproliferative effects of PPARgamma ligands. Urothelial cells treated with PPARgamma ligands showed drastic morphologic changes and cell cycle arrest at G0/G1 phase accompanied with increased mRNA level of a cyclin-dependent kinase inhibitor p21(WAF1/CIP1). Since 15d-PGJ(2) is present in vivo during the resolution phase of inflammation, these results indicated that PPARgamma might be involved in the terminal phase of urothelial re-epithelialization processes.