Recognition of apoptotic cells by macrophages activates the peroxisome proliferator-activated receptor-gamma and attenuates the oxidative burst

It is appreciated that phagocytosis of apoptotic cells (AC) is an immunological relevant process that shapes the pro- versus anti-inflammatory macrophage phenotype. It was our intention to study the respiratory burst, a prototype marker of macrophage activation, under the impact of AC. Following incubation of RAW264.7 macrophages with AC, we noticed attenuated production of reactive oxygen species (ROS) in response to PMA treatment, and observed a correlation between attenuated ROS formation and suppression of protein kinase Calpha (PKCalpha) activation. EMSA analysis demonstrated an immediate activation of peroxisome proliferator-activated receptor-gamma (PPARgamma) following supplementation of AC to macrophages. In macrophages carrying a dominant-negative PPARgamma mutant, recognition of AC no longer suppressed PKCalpha activation, and the initial phase of ROS formation was largely restored. Interference with actin polymerization and transwell experiments suggest that recognition of AC by macrophages suffices to attenuate the early phase of ROS formation that is attributed to PPARgamma activation.     

Delayed activation of PPARgamma by LPS and IFN-gamma attenuates the oxidative burst in macrophages.

Desensitization of macrophages is important during the development of sepsis. It was our intention to identify mechanisms that promote macrophage deactivation upon contact with endotoxin (LPS) and interferon-gamma (IFN-gamma) in vitro. Macrophage activation was achieved with 12-O-tetradecanoylphorbol 13-acetate (TPA), and the oxidative burst (i.e., oxygen radical formation) was followed by oxidation of the redox-sensitive dyes hydroethidine and dichlorodihydrofluorescein diacetate. Prestimulation of macrophages for 15 h with a combination of LPS/IFN-gamma attenuated oxygen radical formation in response to TPA. Taking the anti-inflammatory properties of the peroxisome proliferator-activating receptorgamma (PPARgamma) into consideration, we established activation of PPARgamma in response to LPS/IFN-gamma by an electrophoretic mobility shift, supershift, and a reporter gene assay. The reporter contains a triple PPAR-responsive element (PPRE) in front of a thymidine kinase minimal promoter driving the luciferase gene. We demonstrated that PPRE decoy oligonucleotides, supplied in front of LPS/IFN-gamma, allowed a full oxidative burst to recover upon TPA addition. Furthermore, we suppressed the oxidative burst by using the PPARgamma agonists 15-deoxy-Delta12,14-prostaglandin J2, BRL 49653, or ciglitazone. No effect was observed with WY 14643, a PPARalpha agonist. We conclude that activation of PPARs, most likely PPARgamma, promotes macrophage desensitization, thus attenuating the oxidative burst. This process appears important during development of sepsis.     

Activation of peroxisome proliferator-activated receptor gamma by nitric oxide in monocytes/macrophages down-regulates p47phox and attenuates the respiratory burst

NO appears as an important determinant in auto and paracrine macrophage function. We hypothesized that NO switches monocyte/macrophage function from a pro- to an anti-inflammatory phenotype by activating anti-inflammatory properties of the peroxisome proliferator-activated receptor (PPAR)gamma. NO-releasing compounds (100 micro M S-nitrosoglutathione or 50 micro M spermine-NONOate) as well as inducible NO synthase induction provoked activation of PPARgamma. This was proven by EMSAs, with the notion that supershift analysis pointed to the involvement of PPARgamma. PCR analysis ruled out induction of PPARgamma mRNA as a result of NO supplementation. Reporter assays, with a construct containing a triple PPAR response element in front of a thymidine kinase minimal promoter driving the luciferase gene, were positive in response to NO delivery. DNA binding capacity as well as the transactivating capability of PPARgamma were attenuated by addition of the antioxidant N-acetyl-cysteine or in the presence of the NO scavenger 2-phenyl-4,4,5,6-tetramethyl-imidazoline-1-oxyl 3-oxide. Having established that NO but not lipophilic cyclic GMP analogs activated PPARgamma, we verified potential anti-inflammatory consequences. The oxidative burst of macrophages, evoked by phorbol ester, was attenuated in association with NO-elicited PPARgamma activation. A cause-effect relationship was demonstrated when PPAR response element decoy oligonucleotides, supplied in front of NO delivery, allowed to regain an oxidative response. PPARgamma-mediated down-regulation of p47 phagocyte oxidase, a component of the NAD(P)H oxidase system, was identified as one molecular mechanism causing inhibition of superoxide radical formation. We conclude that NO participates in controlling the pro- vs anti-inflammatory phenotype of macrophages by modulating PPARgamma.     

Fisetin, morin and myricetin attenuate CD36 expression and oxLDL uptake in U937-derived macrophages

Dietary flavonoid intake has been reported to be inversely associated with the incidence of coronary artery disease. To clarify the possible role of flavonoids in the prevention of atherosclerosis, we investigated the effects of some of these compounds, including fisetin, morin and myricetin, on the susceptibility of low-density lipoprotein (LDL) to oxidative modification and on oxLDL uptake in macrophages. The results demonstrated that fisetin had stronger inhibitory activity than the other two on inhibiting Cu(2+)-mediated LDL oxidation measured by thiobarbituric acid-reactive substances assay (TBARS), conjugated diene formation and electrophoretic mobility. The class B scavenger receptor, CD36, to which oxLDL binds, is present in atherosclerotic lesions. Treatment of U937-derived macrophages with myricetin (20 microM) significantly inhibited CD36 cell surface protein and mRNA expression (p<0.01). Fisetin, morin and myricetin (20 microM) also reduced the feed-forward induction of CD36 mRNA and surface protein expression by PPARgamma. The inhibition of CD36 by flavonols was mediated by interference with PPARgamma activation thus counteracting the deleterious autoamplification loop of CD36 expression stimulated by PPARgamma ligand. All three flavonols (10 and 20 microM) markedly decreased the uptake of 1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanide perchlorate (DiI)-labeled oxLDL uptake in U937-derived macrophages dose-dependently. Current evidences indicate that fisetin, morin and myricetin not only prevent LDL from oxidation but also block oxLDL uptake by macrophages at least in part through reducing CD36 gene expression on macrophages. In conclusion, flavonols may play a role in ameliorating atherosclerosis.     

Secreted frizzled‐related protein 5 protects against oxidative stress‐induced apoptosis in human aortic endothelial cells via downregulation of Bax

This study was undertaken to determine the role of secreted frizzled‐related protein 5 (SFRP5) in endothelial oxidative injury. Human aortic endothelial cells (HAECs) were exposed to different oxidative stimuli and examined for SFRP5 expression. The effects of SFRP5 overexpression and knockdown on cell viability, apoptosis, and reactive oxygen species production were measured. HAECs treated with angiotensin (Ang) II (1 μM) or oxidized low‐density lipoprotein (oxLDL) (150 μg/mL) showed a significant increase in SFRP5 expression. Overexpression of SFRP5 significantly attenuated the viability suppression and apoptosis induction by Ang II and oxLDL, whereas the knockdown of SFRP5 exerted opposite effects. Overexpression of SFRP5 prevented ROS formation and β‐catenin activation and reduced Bax expression. Co‐expression of Bax significantly reversed the anti‐apoptotic effect of SFRP5 overexpression, whereas knockdown of Bax restrained Ang II‐ and oxLDL‐induced apoptosis in HAECs. Taken together, SFRP5 confers protection against oxidative stress‐induced apoptosis through inhibition of β‐catenin activation and downregulation of Bax.     

Resveratrol attenuates oxLDL-stimulated NADPH oxidase activity and protects endothelial cells from oxidative functional damages.

trans-Resveratrol (RSV) has been shown to have cardioprotective effect during ischemia-reperfusion through reactive oxygen species (ROS)-scavenging activity. Elevated ROS has been implicated in the initiation and progression of atherosclerosis. The nicotinamide adenine dinucleotide phosphate oxidase (NOX) is a major source of vascular ROS formation. In the present study, we show that exposure of vascular endothelial cells (EC) to oxidized low-density lipoproteins (oxLDL) results in elevations of NOX activity and cellular ROS levels. The oxLDL effects are effectively suppressed by RSV or astringinin (AST), either before or after oxLDL exposure. In this study, we show that RSV or AST treatment appears to suppress NOX activity by reducing the membrane association of gp91(phox) and Rac1, two protein species required for the assembly of active NOX complex. Exposure to RSV or AST protects EC from oxidative functional damages, including antiplatelet activity and mononucleocyte adhesion. In addition, ANG II-induced NOX activation is also attenuated. These results suggest that RSV or AST protects EC from oxLDL-induced oxidative stress by both direct ROS scavenging and inhibition of NOX activity.     

Mildly oxidised LDL induces more macrophage death than moderately oxidised LDL: roles of peroxidation, lipoprotein-associated phospholipase A2 and PPARgamma

Death of macrophages and smooth muscle cells (SMC) can lead to progression of atherosclerosis. Mildly oxidised low-density lipoprotein (mildly-oxLDL) induced more overall death and apoptosis than moderately oxidised LDL, in human monocyte-macrophages (HMM). Mildly-oxLDL also induced more overall death in human SMC than did moderately-oxLDL. Mildly-oxLDL contained more hydroperoxides, but less oxysterol, malondialdehyde and negative charge than moderately-oxLDL. Specific inhibition of lipoprotein-associated phospholipase A(2) (by SB222657) diminished death induction in HMM by both oxLDL types. Peroxisome proliferator-activated receptor gamma (PPARgamma) antagonist (GW9662) and agonist (ciglitazone) experiments suggested that non-hydrolysed, oxidised phospholipids in oxLDL activate PPARgamma as a cellular defence mechanism. These results may be relevant to LDL oxidation within atherosclerotic plaques and may suggest strategies for combating atherosclerosis progression.     

Enhanced VDUP-1 gene expression by PPARgamma agonist induces apoptosis in human macrophage

The fate and phenotype of lesion macrophages is regulated by cellular oxidative stress. Thioredoxin-1 (Trx-1) plays a major role in the regulation of cellular redox balance, with resultant effects on gene expression and cellular responses including cell growth and death. Trx-1 activity is inhibited by interaction with vitamin D-upregulated protein-1 (VDUP-1). Peroxisome proliferator-activated receptor gamma (PPARgamma) is expressed by human monocyte-derived macrophages (HMDM) and PPARgamma agonism has been reported to decrease expression of inflammatory genes and to promote apoptosis of these cells. To determine whether VDUP-1 may be involved in regulating the effects of PPARgamma agonists in macrophages, we investigated the effect of a synthetic PPARgamma agonist (GW929) on the expression of VDUP-1 in HMDM. GW929 concentration-dependently increased HMDM expression of VDUP-1 (mRNA and protein). Transfection of different fragments of the VDUP-1 promoter as well as gel shift analysis revealed the presence of functional PPARgamma response elements (PPRE) in the promoter. Under conditions in which PPAR agonism altered levels of VDUP-1, caspase-3 activity, and macrophage apoptosis were also elevated. The results suggest that PPARgamma activation stimulates apoptosis in human macrophages by altering the cellular redox balance via regulation of VDUP-1.     

Antagonistic effects of oxidized low density lipoprotein and alpha-tocopherol on CD36 scavenger receptor expression in monocytes: involvement of protein kinase B and peroxisome proliferator-activated receptor-gamma

Vitamin E deficiency increases expression of the CD36 scavenger receptor, suggesting specific molecular mechanisms and signaling pathways modulated by alpha-tocopherol. We show here that alpha-tocopherol down-regulated CD36 expression (mRNA and protein) in oxidized low density lipoprotein (oxLDL)-stimulated THP-1 monocytes, but not in unstimulated cells. Furthermore, alpha-tocopherol treatment of monocytes led to reduction of fluorescent oxLDL-3,3'-dioctadecyloxacarbocyanine perchlorate binding and uptake. Protein kinase C (PKC) appears not to be involved because neither activation of PKC by phorbol 12-myristate 13-acetate nor inhibition by PKC412 was affected by alpha-tocopherol. However, alpha-tocopherol could partially prevent CD36 induction after stimulation with a specific agonist of peroxisome proliferator-activated receptor-gamma (PPARgamma; troglitazone), indicating that this pathway is susceptible to alpha-tocopherol action. Phosphorylation of protein kinase B (PKB) at Ser473 was increased by oxLDL, and alpha-tocopherol could prevent this event. Expression of PKB stimulated the CD36 promoter as well as a PPARgamma element-driven reporter gene, whereas an inactive PKB mutant had no effect. Moreover, coexpression of PPARgamma and PKB led to additive induction of CD36 expression. Altogether, our results support the existence of PKB/PPARgamma signaling pathways that mediate CD36 expression in response to oxLDL. The activation of CD36 expression by PKB suggests that both lipid biosynthesis and fatty acid uptake are stimulated by PKB.     

Protective effects of honokiol against oxidized LDL-induced cytotoxicity and adhesion molecule expression in endothelial cells

Honokiol, a compound extracted from Chinese medicinal herb Magnolia officinalis, has several biological effects. However, its protective effects against endothelial injury remain unclarified. In this study, we examined whether honokiol prevented oxidized low-density lipoprotein (oxLDL)-induced vascular endothelial dysfunction. Incubation of oxLDL with honokiol (2.5-20 microM) inhibited copper-induced oxidative modification as demonstrated by diene formation, thiobarbituric acid reactive substances (TBARS) assay and electrophoretic mobility assay. Expression of adhesion molecules (ICAM, VCAM and E-selectin) and endothelial NO synthase (eNOS) affected by oxLDL was investigated by flow cytometry and Western blot. We also measured the production of reactive oxygen species (ROS) using the fluorescent probe 2',7'-dichlorofluorescein acetoxymethyl ester (DCF-AM). Furthermore, several apoptotic phenomena including increased cytosolic calcium, alteration of mitochondrial membrane potential, cytochrome c release and activation of caspase-3 were also investigated. Apoptotic cell death was characterized by terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling (TUNEL) stain. The results showed that honokiol prevented the copper-induced oxidative modification of LDL. Honokiol also ameliorated the oxLDL-diminished eNOS protein expression and reduced the oxLDL-induced adhesion molecules and the adherence of THP-1 cells to HUVECs. Furthermore, honokiol attenuated the oxLDL-induced cytotoxicity, apoptotic features, ROS generation, intracellular calcium accumulation and the subsequent mitochondrial membrane potential collapse, cytochrome c release and activation of caspase-3. Our results suggest that honokiol may have clinical implications in the prevention of atherosclerotic vascular disease.     

Dual role of peroxiredoxin I in macrophage-derived foam cells

We and others have shown that foam cell formation initiated by exposing macrophages to oxidized low density lipoprotein (oxLDL) triggers the differential expression of a number of proteins. Specifically, our experiments have identified peroxiredoxin I (Prx I) as one of these up-regulated proteins. The peroxiredoxins, a family of peroxidases initially described for their antioxidant capability, have generated recent interest for their potential to regulate signaling pathways. Those studies, however, have not examined peroxiredoxin for a potential dual functionality as both cytoprotective antioxidant and signal modulator in a single, oxidant-stressed system. In this report, we examine the up-regulation of Prx I in macrophages in response to oxLDL exposure and its ability to function as both antioxidant enzyme and regulator of p38 MAPK activation. As an antioxidant, induction of Prx I expression led to improved cell survival following treatment with oxLDL or tert-butyl hydroperoxide. The improved survival coincided with a decrease in measurable reactive oxygen species (ROS), and both the increased survival and reduced ROS were reversed by Prx I small interfering RNA transfection. Additionally, our data show that activation of p38 MAPK in oxLDL-treated macrophages was dependent on the up-regulation of Prx I. Reduction of Prx I expression by small interfering RNA transfection resulted in a significant decrease in p38 MAPK activation, whereas the up-regulation of Prx I expression with either oxLDL or ethoxyquin led to increased p38 MAPK activation. These results are consistent with multiple roles for Prx I in macrophage-derived foam cells that include functionality as both an antioxidant and a regulator of oxidant-sensitive signal transduction.     

Regulation of LPS stimulated ROS production in peritoneal macrophages from alloxan-induced diabetic rats: involvement of high glucose and PPARgamma

An increased occurrence of long term bacterial infections is common in diabetic patients. Bacterial cell wall components are described as the main antigenic agents from these microorganisms and high blood glucose levels are suggested to be involved in altered immune response. Hyperglycemia is reported to alter macrophages response to lipopolysaccharide (LPS) and peroxisome proliferators activated receptor gamma (PPARgamma) expression. Additionally, glucose is the main metabolic fuel for reduced nicotinamide adenine dinucleotide phosphate (NADPH) production by pentose phosphate shunt. In this work, lipopolysaccharide (LPS) stimulated reactive oxygen species (ROS) and nitrite production were evaluated in peritoneal macrophages from alloxan-induced diabetic rats. Cytosolic dehydrogenases and PPARgamma expression were also investigated. LPS was ineffective to stimulate ROS and nitrite production in peritoneal macrophages from diabetic rats, which presented increased glucose-6-phosphate dehydrogenase and malate dehydrogenase activity. In RAW 264.7 macrophages, acute high glucose treatment abolished LPS stimulated ROS production, with no effect on nitrite and dehydrogenase activities. Peritoneal macrophages from alloxan-treated rats presented reduced PPARgamma expression. Treating RAW 264.7 macrophages with a PPARgamma antagonist resulted in defective ROS production in response to LPS, however, stimulated nitrite production was unaltered. In conclusion, in the present study we have reported reduced nitric oxide and reactive oxygen species production in LPS-treated peritoneal macrophages from alloxan-induced diabetic rats. The reduced production of reactive oxygen species seems to be dependent on elevated glucose levels and reduced PPARgamma expression.     

Synergism between platelet-activating factor-like phospholipids and peroxisome proliferator-activated receptor gamma agonists generated during low density lipoprotein oxidation that induces lipid body formation in leukocytes

Oxidized low density lipoprotein (LDL) has an important proinflammatory role in atherogenesis. In this study, we investigated the ability of oxidized LDL (oxLDL) and its phospholipid components to induce lipid body formation in leukocytes. Incubation of mouse peritoneal macrophages with oxidized, but not with native LDL led to lipid body formation within 1 h. This was blocked by platelet-activating factor (PAF) receptor antagonists or by preincubation of oxLDL with rPAF acetylhydrolase. HPLC fractions of phospholipids purified from oxLDL induced calcium flux in neutrophils as well as lipid body formation in macrophages. Injection of the bioactive phospholipid fractions or butanoyl and butenoyl PAF, a phospholipid previously shown to be present in oxLDL, into the pleural cavity of mice induced lipid body formation in leukocytes recovered after 3 h. The 5-lipoxygenase and cyclooxygenase-2 colocalized within lipid bodies formed after stimulation with oxLDL, bioactive phospholipid fractions, or butanoyl and butenoyl PAF. Lipid body formation was inhibited by 5-lipoxygenase antagonists, but not by cyclooxygenase-2 inhibitors. Azelaoyl-phosphatidylcholine, a peroxisome proliferator-activated receptor-gamma agonist in oxLDL phospholipid fractions, induced formation of lipid bodies at late time points (6 h) and synergized with suboptimal concentrations of oxLDL. We conclude that lipid body formation is an important proinflammatory effect of oxLDL and that PAF-like phospholipids and peroxisome proliferator-activated receptor-gamma agonists generated during LDL oxidation are important mediators in this phenomenon.     

Phorbol ester potentiates the growth inhibitory effects of troglitazone via up-regulation of PPARgamma in A549 cells

The activation of peroxisome proliferator-activated receptor gamma (PPARgamma) has been shown to induce growth arrest and differentiation of various cancer cells. In the current study, we investigated the effect of 12-O-tetradecanoylphorbol-13-acetate (TPA) on the expression of PPARgamma and proliferation of A549 cells. TPA elicited a dose- and time-dependent increase in PPARgamma mRNA and protein levels. PPARgamma expression in response to TPA was attenuated by pretreatment with bisindolylmaleimide I, N-acetyl-L-cysteine (NAC) and PD98059. TPA-induced protein kinase C (PKC) activation was linked to the generation of reactive oxygen species (ROS), both of which were indispensable for PPARgamma expression in A549 cells. Pretreatment with bisindolylmaleimide I or NAC blocked TPA-induced phosphorylation of extracellular signal-regulated kinase (ERK), suggesting that ERK-mediated signaling is also involved in the induction of PPARgamma. Furthermore, the growth inhibitory effect of troglitazone was significantly potentiated by prolonged incubation with TPA and was attenuated in the presence of GW9662, a specific inhibitor of PPARgamma. These effects were associated with an induction of cell cycle arrest at G0/G1 phase, which was accompanied by the induction of p21Waf1/Cip1 expression and decreased cyclin D1 expression. Taken together, these observations indicate that TPA synergizes with PPARgamma ligand to inhibit cell growth through up-regulation of PPARgamma expression.     

Pro-MMP-2 activation by the PPARgamma agonist, ciglitazone, induces cell invasion through the generation of ROS and the activation of ERK

Peroxisome proliferator-activated receptor gamma (PPARgamma) is a nuclear receptor modulating a variety of biological functions including cancer cell proliferation and differentiation. However, the role of PPARgamma and its ligands in tumor invasion is unclear. To evaluate a possible role for PPARgamma ligands in tumor invasion, we examined whether PPARgamma agonists including pioglitazone, troglitazone, rosiglitazone, and ciglitazone could affect the activity of matrix metalloproteinases (MMPs) in the HT1080 cell line, a well-studied and well-characterized cell line for MMP research. The gelatin zymography assay showed that ciglitazone activated pro-MMP-2 significantly. In addition, ciglitazone increased the expression of MMP-2, which was accompanied by an increase of membrane type 1-MMP (MT1-MMP) expression. The PPARgamma antagonist, GW9662 attenuated the ciglitazone-induced PPARgamma activation but it did not affect the pro-MMP2 activation by ciglitazone, suggesting that the action of ciglitazone on the pro-MMP-2 activation bypassed the PPARgamma pathway. Antioxidants and various inhibitors of signal transduction were used to investigate the mechanism of ciglitazone-induced pro-MMP-2 activation. We found that the sustained production of reactive oxygen species (ROS) was required for pro-MMP-2 activation by ciglitazone. We also found that PB98059, an inhibitor of MEK-ERK, significantly blocked ciglitazone-induced pro-MMP-2 activation and that extracellular signal-regulated kinase (ERK) was hyperphosphorylated by ciglitazone. Moreover, cell invasion was significantly increased by ciglitazone in the HT1080 cell lines, whereas cell motility was not affected. This study suggests that ciglitazone-induced pro-MMP-2 activation increases PPARgamma-independent tumor cell invasion through ROS production and ERK activation in some types of cancer cells.     

IL-13 attenuates gastrointestinal candidiasis in normal and immunodeficient RAG-2(-/-) mice via peroxisome proliferator-activated receptor-gamma activation

We recently demonstrated that in vitro peroxisome proliferator-activated receptor-gamma (PPARgamma) activation of mouse peritoneal macrophages by IL-13 or PPARgamma ligands promotes uptake and killing of Candida albicans through mannose receptor overexpression. In this study, we demonstrate that i.p. treatment of immunocompetent and immunodeficient (RAG-2(-/-)) mice with natural and synthetic PPARgamma-specific ligands or with IL-13 decreases C. albicans colonization of the gastrointestinal (GI) tract 8 days following oral infection with the yeast. We also showed that Candida GI infection triggers macrophage recruitment in cecum mucosa. These mucosal macrophages, as well as peritoneal macrophages, overexpress the mannose receptor after IL-13 and rosiglitazone treatments. The treatments promote macrophage activation against C. albicans as suggested by the increased ability of peritoneal macrophages to phagocyte C. albicans and to produce reactive oxygen intermediates after yeast challenge. These effects on C. albicans GI infection and on macrophage activation are suppressed by treatment of mice with GW9662, a selective PPARgamma antagonist, and are reduced in PPARgamma(+/-) mice. Overall, these data demonstrate that IL-13 or PPARgamma ligands attenuate C. albicans infection of the GI tract through PPARgamma activation and hence suggest that PPARgamma ligands may be of therapeutic value in esophageal and GI candidiasis in immunocompromised patients.     

Oxidized alkyl phospholipids are specific, high affinity peroxisome proliferator-activated receptor gamma ligands and agonists

Synthetic high affinity peroxisome proliferator-activated receptor (PPAR) agonists are known, but biologic ligands are of low affinity. Oxidized low density lipoprotein (oxLDL) is inflammatory and signals through PPARs. We showed, by phospholipase A(1) digestion, that PPARgamma agonists in oxLDL arise from the small pool of alkyl phosphatidylcholines in LDL. We identified an abundant oxidatively fragmented alkyl phospholipid in oxLDL, hexadecyl azelaoyl phosphatidylcholine (azPC), as a high affinity ligand and agonist for PPARgamma. [(3)H]azPC bound recombinant PPARgamma with an affinity (K(d)((app)) approximately 40 nm) that was equivalent to rosiglitazone (BRL49653), and competition with rosiglitazone showed that binding occurred in the ligand-binding pocket. azPC induced PPRE reporter gene expression, as did rosiglitazone, with a half-maximal effect at 100 nm. Overexpression of PPARalpha or PPARgamma revealed that azPC was a specific PPARgamma agonist. The scavenger receptor CD36 is encoded by a PPRE-responsive gene, and azPC enhanced expression of CD36 in primary human monocytes. We found that anti-CD36 inhibited azPC uptake, and it inhibited PPRE reporter induction. Results with a small molecule phospholipid flippase mimetic suggest azPC acts intracellularly and that cellular azPC accumulation was efficient. Thus, certain alkyl phospholipid oxidation products in oxLDL are specific, high affinity extracellular ligands and agonists for PPARgamma that induce PPAR-responsive genes.