Differential recruitment of the coactivator proteins CREB-binding protein and steroid receptor coactivator-1 to peroxisome proliferator-activated receptor gamma/9-cis-retinoic acid receptor heterodimers by ligands present in oxidized low-density lipoprotein.

Peroxisome proliferator-activated receptor gamma (PPARgamma) colocalizes with oxidized low-density lipoprotein (LDL) in foam cells in atherosclerotic lesions. We have explored a potential role of oxidized fatty acids in LDL as PPARgamma activators. LDL from patients suffering from intermittent claudication due to atherosclerosis was analyzed using HPLC and gas chromatography/mass spectrophotometry and found to contain 9-hydroxy and 13-hydroxyoctadecadienoic acid (9- and 13-HODE), as well as 5-hydroxy-, 12-hydroxy- and 15-hydroxyeicosatetraenoic acid (5-, 12- and 15-HETE respectively). PPARgamma was potently activated by 13(S)-HODE and 15(S)-HETE, as judged by transient transfection assays in macrophages or CV-1 cells. 5(S)- and 12(S)-HETE as well as 15-deoxy-Delta(12,14)-prostaglandin J(2) also activated PPARgamma but were less potent. Interestingly, the effect of the lipoxygenase products 13(S)-HODE and 15(S)-HETE as well as of the drug rosiglitazone were preferentially enhanced by the coactivator CREB-binding protein, whereas the effect of the cyclooxygenase product 15-deoxy-Delta(12,14)-prostaglandin J(2) was preferentially enhanced by steroid receptor coactivator-1. We interpret these results, which may have relevance to the pathogenesis of atherosclerosis, to indicate that the lipoxygenase products on the one hand and the cyclooxygenase product on the other exert specific effects on the transcription of target genes through differential coactivator recruitment by PPARgamma/9-cis retinoic acid receptor heterodimer complexes.

Identification of subunits of the 650 kDa 12(S)-HETE binding complex in carcinoma cells.

Cytosol and nuclei of Lewis lung carcinoma (LLC) cells contain high affinity binding sites specific for the arachidonic acid metabolite 12(S)-hydroxy-5,8,10,14-eicosatetraenoic acid (12(S)-HETE). In this report we present evidence that the cytosolic 12(S)-HETE binding complex also occurs in human erythroleukemia (HEL) and promonocytic leukemia (U937) cells as well as in murine 3T3-L1 preadipocytes but not in intestinal epithelial cells (Int407). The cytosolic 650 kDa 12(S)-HETE-binding complex was found to consist of subunits; raising the ATP concentration in cytosol led to conversion of the 650 kDa complex to a 50 kDa binding component, presumably the actual 12(S)-HETE binding polypeptide. Lowering of the cytosolic concentration of ATP had the opposite effect, i.e., the amount of the 650 kDa complex increased. Another subunit of the 650 kDa complex was identified as heat shock protein 70 (hsp70) by Western blot analyses and coimmunoprecipitation. Hsp70 was present in substoichiometric amounts, in an approximate 1:6 ratio. The multimeric nature of the binding complex and the identification of hsp70 as a subunit suggest that there are similarities between the 12(S)-HETE binding protein and receptors of the steroid/thyroid hormone superfamily.

Synergistic induction of acyl-CoA oxidase activity, an indicator of peroxisome proliferation, by arachidonic acid and retinoic acid in Morris hepatoma 7800C1 cells.

Morris hepatoma 7800C1 cells (a Wistar rat cell line) were exposed to 100 microM arachidonic acid in the medium for seven days. This treatment resulted in 150% and 60% increases (above control activities) in acyl-CoA oxidase (which catalyzes the first step in peroxisomal beta-oxidation) and catalase activities, respectively. Arachidonic acid (C20:4) can be metabolized to 20- and 19-hydroxy-arachidonic acid by cytochrome P-450IVA and it was shown that our cells are capable of forming 20-hydroxyarachidonic acid. However, 20-hydroxyarachidonic acid (0.1-0.8 microM, 4 days) had no effects on lauroyl-CoA oxidase and catalase activities in Morris hepatoma cells. Treatment of 7800C1 cells with 100 microM all-trans-retinoic acid resulted in inductions of catalase (160% above the control activity) and carnitine acetyltransferase (140% above the control activity) activities. The activity of lauroyl-CoA oxidase was often, but not always, slightly induced by treatment with all-trans-retinoic acid. When all-trans-retinoic acid was administered together with arachidonic acid, these two compounds had a synergistic effect on the induction of acyl-CoA oxidase activity (almost 700% above the control activity). However, treatment of Morris hepatoma cells with the man-made peroxisome proliferator, perfluorooctanoic acid, together with all-trans-retinoic acid did not result in any synergistic effect on this same enzyme activity. In summary, this study (1) corroborates findings from transfection experiments indicating that the heterodimer PPAR-RXR alpha activates transcription of the acyl-CoA oxidase gene using the Morris hepatoma cell line; (2) shows that arachidonic acid induces the activity of lauroyl-CoA oxidase; (3) suggests that transcription of the catalase gene is not regulated by a PPAR-RXR alpha heterodimer in this system; and (4) demonstrates that peroxisome proliferation in Morris hepatoma cells by perfluorooctanoic acid is not as dependent on the level of retinoic acid as is the same process caused by arachidonic acid.

Metabolism of 12(S)-hydroxy-5,8,10,14-eicosatetraenoic acid by kidney and liver peroxisomes.

12(S)-Hydroperoxy-5,8,10,14-eicosatetraenoic acid (12(S)-HPETE) is one of the main products formed from arachidonic acid by animal lipoxygenases. It is usually reduced to the corresponding hydroxy acid, 12(S)-hydroxy-5,8,10,14-eicosatetraenoic acid (12(S)-HETE) or it can serve as an intermediate in the biosynthesis of hepoxilins. The functions of 12(S)-HETE are incompletely understood but it has been proposed that it plays a role in the metastatic process of certain cancer cells, including Lewis lung carcinoma cells. 12(S)-HETE is metabolically degraded by beta- or omega-oxidation. Indirect evidence has suggested involvement of peroxisomes in the beta-oxidative degradation. The purpose of the present investigation was to study 12(S)-HETE metabolism by peroxisomes isolated from rat liver or kidney by sucrose density gradient centrifugation. The purity of subcellular fractions was determined by marker enzyme analyses and electron microscopy. 12(S)-HETE was quantitatively converted by liver or kidney peroxisomes into a single, diethyl ether-extractable metabolite. This was identified by gas-liquid chromatography-mass spectrometry as 8-hydroxyhexadecatrienoic acid. Lewis lung carcinoma cells also metabolized 12(S)-HETE to 8-hydroxyhexadecatrienoic acid.