Peroxisome proliferator-activated receptor alpha plays a vital role in inducing a detoxification system against plant compounds with crosstalk with other xenobiotic nuclear receptors

Peroxisome proliferator-activated receptor alpha (PPARalpha) is thought to play an important role in lipid metabolism in the liver. To clarify the extra-hepatic and/or unknown function of PPARalpha, we previously performed a proteome analysis of the intestinal proteins and identified 17beta-hydroxysteroid dehydrogenase type 11 as a mostly induced protein by a PPARalpha ligand [Motojima, K. (2004) Eur. J. Biochem. 271, 4141-4146]. Because of its supposed wide substrate specificity, we examined the possibility that PPARalpha plays an important role in inducing detoxification systems for some natural foods by feeding mice with various plant seeds and grains. Feeding with sesame but not others often killed PPARalpha knockout mice but not wild-type mice. A microarray analysis of the sesame-induced mRNAs in the intestine revealed that PPARalpha plays a vital role in inducing various xenobiotic metabolizing enzymes in the mouse intestine and liver. A PPARalpha ligand alone could not induce most of these enzymes, suggesting that there is an essential crosstalk among PPARalpha and other xenobiotic nuclear receptors to induce a detoxification system for plant compounds.     

First-generation monoclonal antibodies identifying the human leukotriene B(4) receptor-1

The leukotriene B(4) receptor (BLTR) is a seven-transmembrane chemoattractant receptor that is important in pro-inflammatory responses. We have produced the first widely applicable monoclonal antibodies against the human BLTR and confirmed the antibody specificity using flow cytometric analysis of three different cell lines stably expressing the recombinant receptor. The antibodies did not cross-react with the recently cloned second LTB(4) receptor, BLTR2, or the Cys LT1 and Cys LT2 receptors. Functional analysis in combination with two-color flow cytometry showed that the BLTR antibodies bind to cells that are activated by LTB(4). The antibodies were shown to recognize BLTR in cell ELISA and immunocytochemistry. Endogenous expression of BLTR in CD15-positive blood leukocytes and in differentiated HL-60 cells was also demonstrated with the antibodies.     

A rapid, homogeneous, fluorescence polarization binding assay for peroxisome proliferator-activated receptors alpha and gamma using a fluorescein-tagged dual PPARalpha/gamma activator

Peroxisome proliferator-activated receptors (PPARs) and other members of the nuclear hormone receptor family are important drug targets for the treatment of metabolic diseases. PPARalpha and PPARgamma play crucial roles in lipid and glucose metabolism, respectively. Therefore, screening methods that help to rapidly identify activators of these receptors should be of considerable value. A homogeneous fluorescence polarization (FP) ligand binding assay capable of rapidly identifying ligands that bind to both PPARalpha and PPARgamma has been developed using purified PPARalpha or PPARgamma ligand binding domains and a fluorescein-labeled analog (FLA) of a potent dual PPARalpha/gamma activator. FLA activator showed good binding affinity toward both PPARalpha (K(i)=0.7microM) and PPARgamma (K(i)=0.4microM). The binding of FLA activator was rapid and reached a plateau within 10 min. The resulting FP signal was stable for at least 18h. The FP binding assay performed robustly in a 384-well format, and the average Z' value was 0.77. There was a good correlation between the binding potency (IC(50) values) and rank order of binding potency for a panel of standard PPAR ligands obtained in FP binding assay and scintillation proximity assay or gel filtration binding assays using (3)H-labeled PPARalpha (r(2)=0.99) and PPARgamma (r(2)=0.99) ligands. There was also a good correlation of IC(50) values obtained by FP binding assay and scintillation proximity assay for the clinically used PPAR activators. Thus, the FP binding assay with a single fluorescein-labeled PPARalpha/gamma dual activator offers a homogeneous nonradioactive, sensitive, robust, and less expensive high-throughput assay for detecting compounds that bind to both PPARgamma and PPARalpha. Using this FP binding assay, we have identified a large number of PPARalpha/gamma dual activators. A similar assay platform may be easily adapted to other members of the nuclear hormone receptor family.     

Hydroxyeicosanoids bind to and activate the low affinity leukotriene B4 receptor, BLT2

Leukotriene B(4), an arachidonate metabolite, is a potent chemoattractant of leukocytes involved in various inflammatory diseases. Two G-protein-coupled receptors for leukotriene B(4) have been cloned and characterized. BLT1 (Yokomizo, T., Izumi, T., Chang, K., Takuwa, Y., and Shimizu, T. (1997) Nature 387, 620-624) is a high affinity receptor exclusively expressed in leukocytes, and BLT2 (Yokomizo, T., Kato, K., Terawaki, K., Izumi, T., and Shimizu, T. (2000) J. Exp. Med. 192, 421-432) is a low affinity receptor expressed more ubiquitously. Here we report the binding profiles of various BLT antagonists and eicosanoids to either BLT1 or BLT2 using the membrane fractions of Chinese hamster ovary cells stably expressing the receptor. BLT antagonists are grouped into three classes: BLT1-specific U-75302, BLT2-specific LY255283, and BLT1/BLT2 dual-specific ZK 158252 and CP 195543. We also show that 12(S)-hydroxyeicosatetraenoic acid, 12(S)-hydroperxyeicosatetraenoic acid, and 15(S)-hydroxyeicosatetraenoic acid competed with [(3)H]LTB(4) binding to BLT2, but not BLT1, dose dependently. These eicosanoids also cause calcium mobilization and chemotaxis through BLT2, again in contrast to BLT1. These findings suggest that BLT2 functions as a low affinity receptor, with broader ligand specificity for various eicosanoids, and mediates distinct biological and pathophysiological roles from BLT1.     

Bile acids induce the expression of the human peroxisome proliferator-activated receptor alpha gene via activation of the farnesoid X receptor

Peroxisome proliferator-activated receptor alpha (PPARalpha) is a nuclear receptor that controls lipid and glucose metabolism and exerts antiinflammatory activities. PPARalpha is also reported to influence bile acid formation and bile composition. Farnesoid X receptor (FXR) is a bile acid-activated nuclear receptor that mediates the effects of bile acids on gene expression and plays a major role in bile acid and possibly also in lipid metabolism. Thus, both PPARalpha and FXR appear to act on common metabolic pathways. To determine the existence of a molecular cross-talk between these two nuclear receptors, the regulation of PPARalpha expression by bile acids was investigated. Incubation of human hepatoma HepG2 cells with the natural FXR ligand chenodeoxycholic acid (CDCA) as well as with the nonsteroidal FXR agonist GW4064 resulted in a significant induction of PPARalpha mRNA levels. In addition, hPPARalpha gene expression was up-regulated by taurocholic acid in human primary hepatocytes. Cotransfection of FXR/retinoid X receptor in the presence of CDCA led to up to a 3-fold induction of human PPARalpha promoter activity in HepG2 cells. Mutation analysis identified a FXR response element in the human PPARalpha promoter (alpha-FXR response element (alphaFXRE)] that mediates bile acid regulation of this promoter. FXR bound the alphaFXRE site as demonstrated by gel shift analysis, and CDCA specifically increased the activity of a heterologous promoter driven by four copies of the alphaFXRE. In contrast, neither the murine PPARalpha promoter, in which the alphaFXRE is not conserved, nor a mouse alphaFXRE-driven heterologous reporter, were responsive to CDCA treatment. Moreover, PPARalpha expression was not regulated in taurocholic acid-fed mice. Finally, induction of hPPARalpha mRNA levels by CDCA resulted in an enhanced induction of the expression of the PPARalpha target gene carnitine palmitoyltransferase I by PPARalpha ligands. In concert, these results demonstrate that bile acids stimulate PPARalpha expression in a species-specific manner via a FXRE located within the human PPARalpha promoter. These results provide molecular evidence for a cross-talk between the FXR and PPARalpha pathways in humans.     

Regulation of peroxisome proliferator-activated receptor alpha by protein kinase C

Peroxisome proliferator-activated receptor alpha (PPARalpha) is a nuclear receptor activated by fatty acids, hypolipidemic drugs, and peroxisome proliferators (PPs). Like other nuclear receptors, PPARalpha is a phosphoprotein whose activity is affected by a variety of growth factor signaling cascades. In this study, the effects of protein kinase C (PKC) on PPARalpha activity were explored. In vivo phosphorylation studies in COS-1 cells transfected with murine PPARalpha showed that the level of phosphorylated PPARalpha is increased by treatment with the PP Wy-14,643 as well as the PKC activator phorbol myristol acetate (PMA). In addition, inhibitors of PKC decreased Wy-14,643-induced PPARalpha activity in a variety of reporter assays. Overexpressing PKCalpha, -beta, -delta, and -zeta affected both basal and Wy-14,643-induced PPARalpha activity. Four consensus PKC phosphorylation sites are contained within the DNA binding (C-domain) and hinge (D-domain) regions of rat PPARalpha (S110, T129, S142, and S179), and their contribution to receptor function was examined. Mutation of T129 or S179 to alanine prevented heterodimerization of PPARalpha with RXRalpha, lowered the level of phosphorylation by PKCalpha and PKCdelta in vitro, and lowered the level of phosphorylation of transfected PPARalpha in transfected cells. In addition, the T129A mutation prevented PPARalpha from binding DNA in an electromobility shift assay. Together, these studies demonstrate a direct role for PKC in the regulation of PPARalpha, and suggest several PKCs can regulate PPARalpha activity through multiple phosphorylation sites.     

Comparison of full-length versus ligand binding domain constructs in cell-free and cell-based peroxisome proliferator-activated receptor alpha assays

Peroxisome proliferator-activated receptor alpha (PPARalpha) is the nuclear receptor responsible for regulating genes that control lipid homeostasis. Because of this role, PPARalpha has become a target of interest for the development of drugs to treat diseases such as dyslipidemia, obesity, and atherosclerosis. Assays currently employed to determine potency and efficacy of potential drug candidates typically utilize a truncated form of the native receptor, one which lacks the entire N-terminal region of the protein. The amino terminus, containing the regions that encode the ligand-independent activation function AF-1 and DNA binding domains, is highly structured and contributes significantly to the overall tertiary structure of the native protein. We report that differences in PPARalpha full-length and ligand binding domain constructs result in differences in binding affinity for coactivator peptides but have little effect on potency of agonists in both cell-free and cell-based nuclear receptor assays.