15-Lipoxygenase metabolism of 2-arachidonylglycerol. Generation of a peroxisome proliferator-activated receptor alpha agonist

The recent demonstrations that cyclooxygenase-2 and leukocyte-type 12-lipoxygenase (LOX) efficiently oxygenate 2-arachidonylglycerol (2-AG) prompted an investigation into related oxygenases capable of metabolizing this endogenous cannabinoid receptor ligand. We evaluated the ability of six LOXs to catalyze the hydroperoxidation of 2-AG. Soybean 15-LOX, rabbit reticulocyte 15-LOX, human 15-LOX-1, and human 15-LOX-2 oxygenate 2-AG, providing 15(S)-hydroperoxyeicosatetraenoic acid glyceryl ester. In contrast, potato and human 5-LOXs do not efficiently metabolize this endocannabinoid. Among a series of structurally related arachidonyl esters, arachidonylglycerols serve as the preferred substrates for 15-LOXs. Steady-state kinetic analysis demonstrates that both 15-LOX-1 and 15-LOX-2 oxygenate 2-AG comparably or preferably to arachidonic acid. Furthermore, 2-AG treatment of COS-7 cells transiently transfected with human 15-LOX expression vectors or normal human epidermal keratinocytes results in the production and extracellular release of 15-hydroxyeicosatetraenoic acid glyceryl ester (15-HETE-G), establishing that lipoxygenase metabolism of 2-AG occurs in an eukaryotic cellular environment. Investigations into the potential biological actions of 15-HETE-G indicate that this lipid, in contrast to its free-acid counterpart, acts as a peroxisome proliferator-activated receptor alpha agonist. The results demonstrate that 15-LOXs are capable of acting on 2-AG to provide 15-HETE-G and elucidate a potential role for endocannabinoid oxygenation in the generation of peroxisome proliferator-activated receptor alpha agonists.     

Opposing effects of 15-lipoxygenase-1 and -2 metabolites on MAPK signaling in prostate. Alteration in peroxisome proliferator-activated receptor gamma

Human prostate tumors have elevated levels of 15-lipoxygenase-1 (15-LOX-1) and data suggest that 15-LOX-1 may play a role in the development of prostate cancer. In contrast, 15-LOX-2 expression is higher in normal rather than in tumor prostate tissue and appears to suppress cancer development. We recently reported that 13-(S)-HODE, the 15-LOX-1 metabolite, up-regulates the MAP kinase signaling pathway and subsequently down-regulates PPARgamma in human colorectal carcinoma cells. To determine whether this mechanism is applicable to prostate cancer and what the effects of 15-LOX-2 are, we investigated the effect of 15-LOX-1, 15-LOX-2, and their metabolites on epidermal growth factor (EGF)- and insulin-like growth factor (IGF)-1 signaling in prostate carcinoma cells. In PC3 cells, 13-(S)-HODE, a 15-LOX-1 metabolite, up-regulated MAP kinase while in contrast 15-(S)-HETE, a 15-LOX-2 metabolite, down-regulated MAP kinase. As a result, 13-(S)-HODE increased PPARgamma phosphorylation while a subsequent decrease in PPARgamma phosphorylation was observed with 15-(S)-HETE. Thus, 15-LOX metabolites have opposing effects on the regulation of the MAP kinase signaling pathway and a downstream target of MAP kinase signaling like PPARgamma. In addition to the EGF signaling pathway, the IGF signaling pathway appears to be linked to prostate cancer. 13-(S)-HODE and 15-(S)-HETE up-regulate or down-regulate, respectively, both the MAPK and Akt pathways after activation with IGF-1. Thus, the effect of these lipid metabolites is not solely restricted to EGF signaling and not solely restricted to MAPK signaling. These results provide a plausible mechanism to explain the apparent opposing effects 15-LOX-1 and 15-LOX-2 play in prostate cancer.     

15-Deoxy-delta(12,14)-prostaglandin J(2) down-regulates CXCR4 on carcinoma cells through PPARgamma- and NFkappaB-mediated pathways

The chemokine receptor CXCR4 plays a key role in the metastasis of colorectal cancer and its growth at metastatic sites. Here, we have investigated the mechanisms by which CXCR4 on cancer cells might be regulated by eicosanoids present within the colorectal tumor microenvironment. We show that prostaglandins PGE(2), PGA(2), PGD(2), PGJ(2) and 15dPGJ(2) each down-regulates CXCR4 receptor expression on human colorectal carcinoma cells to differing degrees. The most potent of these were PGD(2) and its metabolites PGJ(2) and 15dPGJ(2). Down-regulation was most rapid with the end-product 15dPGJ(2) and was accompanied by a marked reduction in CXCR4 mRNA. 15dPGJ(2) is known to be a ligand for the nuclear receptor PPARgamma. Down-regulation of CXCR4 was also observed with the PPARgamma agonist rosiglitazone, while 15dPGJ(2)-induced CXCR4 down-regulation was substantially diminished by the PPARgamma antagonists GW9662 and T0070907. These data support the involvement of PPARgamma. However, the 15dPGJ(2) analogue CAY10410, which can act on PPARgamma but which lacks the intrinsic cyclopentenone structure found in 15dPGJ(2), down-regulated CXCR4 substantially less potently than 15dPGJ(2). The cyclopentenone grouping is known to inhibit the activity of NFkappaB. Consistent with an additional role for NFkappaB, we found that the cyclopentenone prostaglandin PGA(2) and cyclopentenone itself could also down-regulate CXCR4. Immunolocalization studies showed that the cellular context was sufficient to trigger a focal nuclear pattern of NFkappaB p50 and that 15dPGJ(2) interfered with this p50 nuclear localization. These data suggest that 15dPGJ(2) can down-regulate CXCR4 on cancer cells through both PPARgamma and NFkappaB. 15dPGJ(2), present within the tumor microenvironment, may act to down-regulate CXCR4 and impact upon the overall process of tumor expansion.     

Effect of peroxisome proliferator activated receptor (PPAR)gamma agonists on prostaglandins cascade in joint cells

In response to inflammatory cytokines, chondrocytes and synovial fibroblasts produce high amounts of prostaglandins (PG) which self-perpetuate locally the inflammatory reaction. Prostaglandins act primarily through membrane receptors coupled to G proteins but also bind to nuclear Peroxisome Proliferator-Activated Receptors (PPARs). Amongst fatty acids, the cyclopentenone metabolite of PGD2, 15-deoxy-Delta12,14PGJ2 (15d-PGJ2), was shown to be a potent ligand of the PPARgamma isotype prone to inhibit the production of inflammatory mediators. As the stimulated synthesis of PGE2 originates from the preferential coupling of inducible enzymes, cyclooxygenase-2 (COX-2) and membrane PGE synthase-1 (mPGES-1), we investigated the potency of 15d-PGJ2 to regulate prostaglandins synthesis in rat chondrocytes stimulated with interleukin-1beta (IL-1beta). We demonstrated that 15d-PGJ2, but not the high-affinity PPARgamma ligand rosiglitazone, decreased almost completely PGE2 synthesis and mPGES-1 expression. The inhibitory potency of 15d-PGJ2 was unaffected by changes in PPARgamma expression and resulted from inhibition of NF-kappaB nuclear binding and IkappaBalpha sparing, secondary to reduced phosphorylation of IKKbeta. Consistently with 15d-PGJ2 being a putative endogenous regulator of the inflammatory reaction if synthesized in sufficient amounts, the present data confirm the variable PPARgamma-dependency of its effects in joint cells while underlining possible species and cell types specificities.     

Inhibition of Oxygen-Induced Ischemic Retinal Neovascularization with Adenoviral 15-Lipoxygenase-1 Gene Transfer via Up-Regulation of PPAR-γ and Down-Regulation of VEGFR-2 Expression

15-lipoxygenase-1 (15-LOX-1) plays an important role in angiogenesis, but how it works still remains a controversial subject. The aims of our study are focused on determining whether or not 15-LOX-1 inhibiting oxygen-induced ischemic retinal neovascularization (RNV) and the underlying regulatory mechanism involving of 15-LOX-1, peroxisome proliferator-activated receptor γ (PPAR-γ) and vascular endothelial growth factor receptor 2 (VEGFR-2) in oxygen-induced retinopathy (OIR). Recombinant adenoviral vectors that expressing the 15-LOX-1 gene (Ad-15-LOX-1-GFP) or the green fluorescence protein gene (Ad-GFP) were intravitreous injected into the OIR mice at postnatal day 12 (P12), the mice were sacrificed 5 days later (P17). Retinal 15-LOX-1 expression was significantly increased at both mRNA and protein levels after 15-LOX-1 gene transfer. Immunofluorescence staining of retinal sections revealed 15-LOX-1 expression was primarily in the outer plexiform layer (OPL), inner nuclear layer (INL) and ganglion cell layer (GCL) retina. Meanwhile, RNV was significantly inhibited indicated by fluorescein retinal angiography and quantification of the pre-retinal neovascular cells. The expression levels of PPAR-γ were significantly up-regulated while VEGFR-2 were significantly down-regulated both in mRNA and protein levels. Our results suggested 15-LOX-1 gene transfer inhibited RNV in OIR mouse model via up-regulation of PPAR-γ and further down-regulation of VEGFR-2 expression. This could be a potentially important regulatory mechanism involving 15-LOX-1, PPAR-γ and VEGFR-2 during RNV in OIR. In conclusion, 15-LOX-1 may be a new therapeutic target for treating neovascularization diseases.     

15-lipoxygenase-1 metabolites down-regulate peroxisome proliferator-activated receptor gamma via the MAPK signaling pathway.

Human colon tumors have elevated levels of 15-lipoxygenase-1 (15-LO-1), suggesting that 15-LO-1 may play a role in the development of colorectal cancer. Also, 15-LO-1 metabolites can up-regulate epidermal growth factor signaling pathways, which results in an increase in mitogenesis. However, metabolites of 15-LO-1 can serve as ligands for peroxisome proliferator-activated receptor gamma (PPARgamma), and activation of this receptor causes most colon cancer cell lines to undergo a differentiative response and reverse their malignant phenotype. Hence, the role 15-LO-1 plays in colon cancer is not clear. To clarify the role of 15-LO-1 in carcinogenesis, the effect of 15-LO-1 and its metabolites on epidermal growth factor signaling and PPARgamma was investigated. In HCT-116 cells, exogenously added 15-LO-1 metabolites, 13-(S)-hydroxyoctadecadienoic acid, 13-(R)-hydroxyoctadecadienoic acid, and 13-(S)-hydroperoxyoctadecadienoic acid, up-regulated the MAPK signaling pathway, and an increase in PPARgamma phosphorylation was observed. Furthermore, in stable overexpressing 15-LO-1 HCT-116 cells, which produce endogenous 15-LO-1 metabolites, an up-regulation in mitogen-activated protein kinase and PPARgamma phosphorylation was observed. Incubation with a MAPK inhibitor ablated MAPK and PPARgamma phosphorylation. The 15-LO-1 up-regulates MAPK activity and increases PPARgamma phosphorylation, resulting in a down-regulation of PPARgamma activity. Thus, 15-LO-1 metabolites may not only serve as ligands for PPARgamma but can down-regulate PPARgamma activity via the MAPK signaling pathway.