Airway smooth muscle cells produce extracellular matrix proteins, which in turn can promote smooth muscle survival, proliferation and migration. Currently available therapies have little effect on airway smooth muscle matrix production and migration. Peroxisome proliferator-activated receptor (PPAR) ligands are reported to decrease migration and matrix production in various cell lines. In this study, we examined the effect of PPAR ligands on human airway smooth muscle (HASM) matrix production and migration. PPAR expression was examined by RT-PCR and Western blotting. Endogenous PPAR activity was examined by transfecting cells with a PPAR response element-luciferase reporter plasmid. We observed that HASM cells express PPARα, β and γ. A six-fold induction of luciferase activity was observed by stimulating cells with a pan-agonist, indicating endogenous PPAR activity. The PPAR ligands ciglitazone, 15-deoxy-Δ12,14-prostaglandin J(2) and WY-14643 decreased migration towards platelet-derived growth factor receptor. This was not mediated by inhibiting Akt phosphorylation or promoting PTEN activity, but partly through cyclooxygenase-2 induction and prostaglandin E(2) production that increased cyclic AMP levels in the cells. All three ligands also caused an inhibition of collagen and fibronectin secretion by cultured smooth muscle cells. We conclude that PPAR ligands decrease HASM migration and matrix production and are, therefore, potentially useful for modulating airway remodelling.

Background Apoptosis caused by endoplasmic reticulum ( ER ) stress contributes to atherothrombosis, the underlying cause of cardiovascular disease ( CVD ). T ‐cell death‐associated gene 51 ( TDAG51 ), a member of the pleckstrin homology‐like domain gene family, is induced by ER stress, causes apoptosis when overexpressed, and is present in lesion‐resident macrophages and endothelial cells. Methods and Results To study the role of TDAG51 in atherosclerosis, male mice deficient in TDAG51 and apolipoprotein E ( TDAG51 −/− / ApoE −/− ) were generated and showed reduced atherosclerotic lesion growth (56±5% reduction at 40 weeks, relative to ApoE −/− controls, P <0.005) and necrosis (41±4% versus 63±8% lesion area in TDAG51 −/− /ApoE −/− and ApoE −/− , respectively; P <0.05) without changes in plasma levels of lipids, glucose, and inflammatory cytokines. TDAG51 deficiency caused several phenotypic changes in macrophages and endothelial cells that increase cytoprotection against oxidative and ER stress, enhance PPAR γ‐dependent reverse cholesterol transport, and upregulate peroxiredoxin‐1 (Prdx‐1), an antioxidant enzyme with antiatherogenic properties (1.8±0.1‐fold increase in Prdx‐1 protein expression, relative to control macrophages; P <0.005). Two independent case–control studies found that a genetic variant in the human TDAG51 gene region (rs2367446) is associated with CVD ( OR , 1.15; 95% CI , 1.07 to 1.24; P =0.0003). Conclusions These findings provide evidence that TDAG51 affects specific cellular pathways known to reduce atherogenesis, suggesting that modulation of TDAG51 expression or its activity may have therapeutic benefit for the treatment of CVD .

Liver X receptor alpha (LXRalpha), an oxysterol-activated nuclear hormone receptor, regulates the expression of genes involved in lipid and cholesterol homeostasis and inflammation. We show here that transactivation by LXRalpha in monkey kidney COS-1 (Cos-1) cells is decreased by activation of the protein kinase C (PKC) signaling pathway. In transient co-transfection assays, phorbol myristate acetate (PMA) suppressed LXR-dependent transactivation of LXR-responsive reporter genes or the natural promoter of the human ATP-binding cassette (ABC), ABCA1 gene. The decrease in LXR transactivation after PMA treatment was also observed in human embryonic kidney (HEK) 293 and human hepatocellular carcinoma (HepG2) cells. Moreover, endogenous LXR target genes, ABCA1 and sterol response element-binding protein-1c, were also decreased by PMA treatment in HEK293 cells as assessed by real-time PCR. The PMA-mediated decrease of LXR activity was blocked by the PKC inhibitor bisindolylmaleimide and mimicked by constitutively active PKCalpha. Nuclear extracts treated with PMA show no decrease in LXRalpha DNA binding as assessed by mobility shift and chromatin immunoprecipitation assays. Additionally, in vitro kinase assays demonstrate that PKCalpha can phosphorylate LXRalpha. Our findings reveal a mode of regulation of LXRalpha that may be relevant to disease conditions where aberrant PKC signaling is observed, such as diabetes.

The association of hypercholesterolemia and obesity with airway hyperresponsiveness has drawn increasing attention to the potential role of cholesterol and lipid homeostasis in lung physiology and in chronic pulmonary diseases such as asthma. We have recently shown that activation of the nuclear hormone receptor liver X receptor (LXR) stimulates cholesterol efflux in human airway smooth muscle (hASM) cells and induces expression of the ATP-binding cassette (ABC) transporters ABCA1 and ABCG1, members of a family of proteins that mediate reverse cholesterol and phospholipid transport. We show here that ABCA1 is responsible for all LXR-mediated cholesterol and phospholipid efflux to both apolipoprotein AI and high-density lipoprotein acceptors. In contrast, ABCG1 does not appear to be required for this process. Moreover, we show that hASM cells respond to increased levels of cholesterol by inducing expression of ABCA1 and ABCG1 transporters, a process that is dependent on LXR expression. These findings establish a critical role for ABCA1 in reverse cholesterol and phospholipid transport in airway smooth muscle cells and suggest that dysregulation of cholesterol homeostasis in these cells may be important in the pathogenesis of diseases such as asthma.

Human (h) airway smooth muscle (ASM) cells are important mediators of the inflammatory process observed in asthma and other respiratory diseases. We show here that primary hASM cells express liver X receptor (LXR; alpha and beta subtypes), an oxysterol-activated nuclear receptor that controls expression of genes involved in lipid and cholesterol homeostasis, and inflammation. LXR was functional as determined by transient assays using LXR-responsive reporter genes and by analysis of mRNA and protein expression of endogenous LXR target genes in cells exposed to LXR agonists. LXR activation induced expression of the ATP-binding cassette transporters ABCA1 and ABCG1 and increased efflux of cholesterol to apolipoprotein AI and high-density lipoprotein acceptors, pointing to a role for hASM cells in modulating cholesterol homeostasis in the airway. Under inflammatory conditions, hASM cells release a variety of chemokines and cytokines that contribute to inflammatory airway diseases. Activation of LXR inhibited the expression of multiple cytokines in response to proinflammatory mediators and blocked the release of both granulocyte macrophage colony-stimulating factor and granulocyte colony stimulating factor. LXR activation also inhibited proliferation of hASM cells and migration toward platelet-derived growth factor chemoattractant, two important processes that contribute to airway remodeling. Our findings reveal biological roles for LXR in ASM cells and suggest that modulation of LXR activity offers prospects for new therapeutic approaches in the treatment of asthma and other inflammatory respiratory diseases.

T-cell death associated gene 51 (TDAG51) is a pro-apoptotic gene that can be induced by endoplasmic reticulum (ER) stress agents, including homocysteine, tunicamycin, thapsigargin or dithiothreitol. Our previous studies have demonstrated that transient overexpression of TDAG51 elicited significant changes in cell morphology, decreased cell adhesion and promoted detachment-induced programmed cell death (PCD). In support of these in vitro findings, we have further shown that TDAG51 expression was increased and correlated with PCD in the atherosclerotic lesions from apolipoprotein E (apoE)-deficient mice fed hyperhomocysteinemic diets, compared to mice fed control diet. We designed the current study to investigate the effect of TDAG51 deficiency in the development and progression of atherosclerosis. To assess in vivo significance of TDAG51 on atherosclerosis, we have crossed TDAG51-deficient mice with apoE-deficient mice to obtain double knockout mice. Our findings have demonstrated that TDAG51/apoE-deficient mice have a significant decrease in atherosclerotic lesion area, compared to age- and sex-matched apoE-deficient mice. Total plasma cholesterol and triglycerides as well as lipoprotein profiles were similar in both groups. However, TDAG51/apoE-deficient mice presented with increased hepatic steatosis. Further, a significant upregulation of peroxisome proliferator-activated receptor γ (PPAR-γ), a transcription factor required for adipose tissue formation, was demonstrated in TDAG51-deficient mouse embryonic fibroblasts (MEFs), compared to control wildtype MEFs. Interestingly, earlier studies in mice have reported that overexpression of PPAR-γ decreases atherosclerotic lesion development and increases hepatic steatosis - a phenotype similar to that observed in the mouse deficient in both apoE and TDAG51. Collectively, these findings provide evidence that TDAG51 mediates atherosclerotic lesion development and hepatic steatosis through a mechanism involving PPAR-γ.

Host Cell Factor (HCF-1) is a conserved, essential protein initially identified as a co-regulator for the Herpes Simplex Virus transactivator VP16. HCF-1 is variously involved in regulating transcription, splicing, cell proliferation and cytokinesis; however, its mechanisms of action remain unknown. HCF-1 function is manifested through an increasing assortment of cellular factors that target different regions of the protein. Several HCF-1 partners target the amino-terminal kelch domain of HCF-1 (residues 1–380) via a consensus HCF-binding motif (HBM) comprising the tetrapeptide (D/E)HXY. Searches of sequence databases indicated that this motif is present in E2F1 and E2F4, two members of the E2F family of cell cycle regulators. We show here that E2F4 specifically and directly interacts with HCF-1. Mutational analysis showed E2F4 independently targets the kelch domain and the basic domain (residues 450–902) of HCF-1, both of which are required for normal cell-cycle progression via separate determinants. The HBM-containing domain of E2F4 was necessary for interaction with the kelch domain of HCF-1 but not for interaction with the basic domain. Mutations in the HCF-1 kelch domain known to block cell growth abrogated E2F4 binding to the kelch domain in the absence but not in the presence of the juxtaposed basic region. Functionally, HCF-1 co-activated E2F4/DP-1 in transient transfection assays, while E2F4 blocked HCF-1-dependent rescue of a cell line that harbors a temperature sensitive mutant of HCF-1 that causes growth arrest. Our findings show that HCF-1 and E2F4 interact via multiple determinants and suggest a linkage between E2F4 and HCF-1 cell growth pathways.

Peroxisome proliferator-activated nuclear hormone receptors (PPAR) are ligand-activated transcription factors that play pivotal roles in governing metabolic homeostasis and cell growth. PPARs are primarily in the nucleus but, under certain circumstances, can be found in the cytoplasm. We show here that PPARα interacts with the centrosome-associated protein CAP350. CAP350 also interacts with PPARδ, PPARγ and liver-X-receptor α, but not with the 9-cis retinoic acid receptor, RXRα. Immunofluorescence analysis indicated that PPARα is diffusely distributed in the nucleus and excluded from the cytoplasm. However, in the presence of coexpressed CAP350, PPARα colocalizes with CAP350 to discrete nuclear foci and to the centrosome, perinuclear region and intermediate filaments. In contrast, the subcellular distribution of RXRα or of thyroid hormone receptor α was not altered by coexpression of CAP350. An amino-terminal fragment of CAP350 was localized exclusively to nuclear foci and was sufficient to recruit PPARα to these sites. Mutation of the single putative nuclear hormone receptor interacting signature motif LXXLL present in this fragment had no effect on its subnuclear localization but abrogated recruitment of PPARα to nuclear foci. Surprisingly, mutation of the LXXLL motif in this CAP350 subfragment did not prevent its binding to PPARα in vitro, suggesting that this motif serves some function other than PPARα binding in recruiting PPARα to nuclear spots. CAP350 inhibited PPARα-mediated transactivation in an LXXLL-dependent manner, suggesting that CAP350 represses PPARα function. Our findings implicate CAP350 in a dynamic process that recruits PPARα to discrete nuclear and cytoplasmic compartments and suggest that altered intracellular compartmentalization represents a regulatory process that modulates PPAR function.

ABSTRACT In addition to its well-established role in the activation of herpes simplex virus immediate-early gene transcription, VP16 interacts with and downregulates the function of the virion host shutoff protein (vhs), thereby attenuating vhs-mediated destruction of viral mRNAs and translational arrest at late times of infection. We have carried out two-hybrid analysis in vivo and protein-protein interaction assays in vitro to identify determinants in VP16 necessary for interaction with vhs. The minimal amino-terminal subfragment of VP16 capable of binding to vhs encompassed residues 1 to 345. Alteration of a single leucine at position 344 to alanine (L344A) in the context of the amino-terminal fragment of VP16 containing residues 1 to 404 was sufficient to abolish interaction with vhs in vitro and in vivo. Leu344 could be replaced with hydrophobic amino acids (Ile, Phe, Met, or Val) but not by Asn, Lys, or Pro, indicating that hydrophobicity is an important property of binding to vhs. VP16 harboring a loss-of-function mutation at L344 was not compromised in its ability to interact with host cell factor (HCF-1) or to activate transcription of viral immediate-early genes in transient-transfection assays. Virus complementation assays using the VP16-null virus 8MA and the VP16/vhs double-mutant virus 8MAΔSma showed that VP16(L344A) was able to complement the growth of 8MAΔSma but not 8MA. Thus, a single point mutation in VP16 uncouples binding to vhs from other functions of VP16 required for virus growth and indicates that direct physical association between VP16 and vhs is necessary to sustain a productive infection.

Human host cell factor-1 (HCF-1) is essential for cell cycle progression and is required, in conjunction with the herpes simplex virus transactivator VP16, for induction of viral immediate-early gene expression. We show here that HCF-1 directly binds to the Myc-interacting protein Miz-1, a transcription factor that induces cell cycle arrest at G(1), in part by directly stimulating expression of the cyclin-dependent kinase inhibitor p15(INK4b). A domain encompassing amino acids 750-836, contained within a subregion of HCF-1 required for cell cycle progression, was sufficient to bind Miz-1. Conversely, HCF-1 interacted with two separate regions in Miz-1: the N-terminal POZ domain and a C-terminal domain (residues 637-803) previously shown to harbor determinants for interaction with c-Myc and the coactivator p300. The latter functioned as a potent transactivation domain when tethered to DNA, indicating that HCF-1 targets a transactivation function in Miz-1. HCF-1 or a Miz-1-binding fragment of HCF-1 repressed transactivation by Gal4-Miz-1 in transfection assays. Moreover, HCF-1 repressed Miz-1-mediated transactivation of a reporter gene linked to the p15(INK4b) promoter. Protein/protein interaction studies and transient transfection assays demonstrated that HCF-1 interferes with recruitment of p300 to Miz-1, similar to what has been reported with c-Myc. Our findings identify Miz-1 as a novel HCF-1-interacting partner and illustrate cross-talk between these two proteins that may be of consequence to their respective functions in gene regulation and their opposing effects on the cell cycle.

The promoter regions of the genes encoding the first two enzymes of the peroxisomal beta-oxidation pathway, acyl-CoA oxidase (AOx) and enoyl-CoA hydratase/3-hydroxyacyl-CoA dehydrogenase (HD), contain transcriptional regulatory sequences termed peroxisome proliferator-response elements (PPRE) that are bound by the peroxisome proliferator-activated receptor alpha (PPARalpha) and 9-cis-retinoic acid receptor (RXRalpha) heterodimeric complex. In this study, the role of the short heterodimer partner (SHP) receptor in modulating PPARalpha-mediated gene transcription from the PPREs of the genes encoding AOx and HD was investigated both in vitro and in vivo. In vitro binding assays using glutathione-S-transferase-tagged chimeric receptors for PPARalpha and SHP were used to verify the interaction between PPARalpha and SHP. This interaction was unaffected by the presence of the peroxisome proliferator, Wy-14,643. SHP has been proposed to act as a negative regulator of nuclear hormone receptor activity, and SHP inhibited transcription by PPARalpha/RXRalpha heterodimers from the AOx-PPRE. Surprisingly, SHP potentiated transcription by PPARalpha/RXRalpha heterodimers from the HD-PPRE. This is the first demonstration of positive transcriptional activity attributable to SHP. Together, these results suggest that SHP can modulate PPARalpha/RXRalpha-mediated transcription in a response element-specific manner.

The effects of distinct classes of peroxisome proliferator-activated receptor γ (PPARγ) ligands on myogenesis and MyoD gene expression were examined in mouse skeletal muscle C2C12 myoblasts. Treatment of C2C12 cells with the PPARγ ligand, 15-deoxy-Δ12,14-prostaglandin J2 (15d-PGJ2), repressed morphologically defined myogenesis and reduced endogenous mRNA levels of the myogenic differentiation markers MyoD, myogenin, and α-actin. In contrast, two synthetic PPARγ ligands, L-805645 and ciglitazone, exhibited no effects. In transient transfection assays, 15d-PGJ2 specifically inhibited the expression of a MyoD promoter-luciferase reporter gene (MyoDLuc) in a cell type- and promoter-specific manner, indicating that 15d-PGJ2 functions in part by repressing MyoD gene transcription. The inhibition of MyoD gene expression by 15d-PGJ2 is mediated by the distal region of the MyoD gene promoter. PPARγ on its own also inhibited MyoDLuc expression and further augmented the 15d-PGJ2 response. In contrast, L-805645 and ciglitazone did not inhibit MyoDLuc expression on their own but did so in the presence of ectopically expressed PPARγ. Interestingly, a transdominant inhibitor of PPARγ (hPPARγ2Δ500) had no effect on the 15d-PGJ2-dependent repression of MyoDLucexpression but overcame L-805645/PPARγ-dependent repression. Finally, saturating concentrations of L-805645, which did not affect myogenesis, failed to ablate 15d-PGJ2-mediated repression of the myogenic program. Thus, distinct PPARγ ligands may repress MyoD gene expression through PPARγ-dependent and -independent pathways, and 15d-PGJ2 can inhibit the myogenic program independent of its cognate receptor, PPARγ.

Liver X receptor alpha (LXRalpha), is a nuclear hormone receptor that is activated by oxysterols and plays a crucial role in regulating cholesterol and lipid metabolism in liver and cholesterol efflux from lipid-loaded macrophages. Here we show that treatment of human peripheral blood monocytes or monocytic THP-1 cells with the LXR ligand 22(R)-hydroxycholesterol (22(R)-HC), in combination with 9-cis-retinoic acid (9cRA), a ligand for the LXR heterodimerization partner retinoid X receptor (RXR), results in the specific induction of the potent pro-apoptotic and pro-inflammatory cytokine tumor necrosis factor-alpha (TNF-alpha). Promoter analysis, inhibitor studies, and order-of-addition experiments demonstrated that TNF-alpha induction by 22(R)-HC and 9cRA occurs by a novel two-step process. The initial step involves 22(R)-HC-dependent induction of TNF-alpha mRNA, and intracellular accumulation of TNF-alpha protein, mediated by binding of LXRalpha/RXRalpha to an LXR response element at position -879 of the TNF-alpha promoter. Subsequent cell release of TNF-alpha protein occurs via a separable 9cRA-dependent, LXRalpha-independent step that requires de novo transcription and protein synthesis. Our findings reveal a potentially new dimension of the physiological role of LXRalpha and identify a unique multistep pathway of TNF-alpha production that may be of consequence to the normal function of LXR in monocyte/macrophages and in disease conditions such as atherosclerosis.

The peroxisomal proliferator-activated receptors (PPARs) are members of the nuclear receptor superfamily that act as ligand-activated transcription factors. PPARgamma plays a critical role in regulating adipocyte differentiation and lipid metabolism. Recently, thiazolidinedione (TZD) and select non-TZD antidiabetic agents have been identified as PPARgamma agonists. To further characterize this receptor subclass, a mutant hPPARgamma lacking five carboxyl-terminal amino acids was produced (hPPARgamma2Delta500). In COS-1 cells transfected with PPAR-responsive reporter constructs, the mutant receptor could not be activated by a potent PPARgamma agonist. When cotransfected with hPPARgamma2 or hPPARalpha, hPPARgamma2Delta500 abrogated wild-type receptor activity in a dose-responsive manner. hPPARgamma2Delta500 was also impaired with respect to binding of a high-affinity radioligand. In addition, its conformation was unaffected by normally saturating concentrations of PPARgamma agonist as determined by protease protection experiments. Electrophoretic mobility shift assays demonstrated that hPPARgamma2Delta500 and hPPARgamma2 both formed heterodimeric complexes with human retinoidxreceptor alpha (hRXRalpha) and could bind a peroxisome proliferator-responsive element (PPRE) with similar affinity. Therefore, hPPARgamma2Delta500 appears to repress PPAR activity by competing with wild type receptor to dimerize with RXR and bind the PPRE. In addition, the mutant receptor may titrate out factors required for PPAR-regulated transcriptional activation. Both hPPARgamma2 and hPPARgamma2Delta500 localized to the nucleus of transiently transfected COS-1 cells as determined by immunofluorescence using a PPARgamma-specific antibody. Thus, nuclear localization of PPARgamma occurs independently of its activation state. The dominant negative mutant, hPPARgamma2Delta500, may prove useful in further studies to characterize PPAR functions both in vitro and in vivo

The genes encoding the first two enzymes of the peroxisomal β-oxidation pathway, acyl-CoA oxidase (AOx) and enoyl-CoA hydratase/3-hydroxyacyl-CoA dehydrogenase (HD), contain upstreamcis-acting regulatory regions termed peroxisome proliferator response elements (PPRE). Transcription of these genes is mediated through the binding of peroxisome proliferator-activated receptor α (PPARα), which binds to a PPRE as a heterodimer with the 9-cis-retinoic acid receptor (RXRα). Here we demonstrate that the HD-PPRE is also a target for the constitutive androstane receptor β (CARβ). In vitro binding analysis showed that CARβ bound the HD-PPRE, but not the AOx-PPRE, as a heterodimer with RXRα. Binding of CARβ/RXRα to the HD-PPRE occurred via determinants that overlap partially with those required for PPARα/RXRα binding. In vivo, CARβ/RXRα activated transcription from an HD-PPRE luciferase reporter construct. Interestingly, CARβ was shown to also modulate PPARα/RXRα-mediated transactivation in a response element-specific manner. In the presence of the peroxisome proliferator, Wy-14,643, CARβ had no effect on PPARα/RXRα-mediated transactivation from the HD-PPRE but antagonized transactivation from the AOx-PPRE in both the presence and the absence of proliferator. Our results illustrate that transcription of the AOx and HD genes is differentially regulated by CARβ and that the HD gene is a specific target for regulation by CARβ. Overall, this study proposes a novel role for CARβ in the regulation of peroxisomal β-oxidation.

Peroxisome proliferator-activated receptor alpha (PPAR alpha) heterodimerizes with the 9-cis-retinoic acid receptor (RXR alpha) to bind to peroxisome proliferator-response elements (PPRE) present in the upstream regions of a number of genes involved in metabolic homeostasis. Among these genes are those encoding fatty acyl-CoA oxidase (AOx) and enoyl-CoA hydratase/3-hydroxyacyl-CoA dehydrogenase (HD), the first two enzymes of the peroxisomal beta-oxidation pathway. Here we demonstrate that the orphan nuclear hormone receptor, RevErb alpha, modulates PPAR alpha/RXR alpha-dependent transactivation in a response element specific manner. In vitro binding analysis showed that RevErb alpha bound the HD-PPRE but not the AOx-PPRE. Determinants within the HD-PPRE required for RevErb alpha binding were distinct from those required for PPAR alpha/RXR alpha binding. In transient transfections, RevErb alpha antagonized transactivation by PPAR alpha/RXR alpha from an HD-PPRE luciferase reporter construct, whereas no effects were observed with an AOx-PPRE reporter construct. These data identify the HD gene as a target for RevErb alpha and illustrate cross-talk between the RevErb alpha and PPAR alpha signaling pathways on the HD-PPRE. Our results suggest a novel role for RevErb alpha in regulating peroxisomal beta-oxidation.

p62 is a recently identified ubiquitin-binding, cytosolic phosphoprotein that interacts with several signal transduction molecules including the tyrosine kinase p56(lck) and the protein kinase C-zeta. p62 is therefore suggested to serve an important role in signal transduction in the cell, although the physiological function of p62 remains undefined. Here we demonstrate by transient transfection assays that p62 stimulates the transcription of reporter genes linked to the simian virus 40 (SV40) enhancer. A putative p62-responsive element was localized to the B domain of the distal 72-base pair repeat of the SV40 enhancer. p62 was unable to bind this element in vitro, nor was it able to activate transcription when directly tethered to a promoter, suggesting that p62 stimulates transcription via an indirect mechanism. Stimulation of transcription mediated by p62 was dependent on its amino-terminal region, which is also necessary for interaction with cell surface signaling molecules. These findings indicate that p62 may link extracellular signals directly to transcriptional responses, and identify the SV40 enhancer as a downstream target for signal transduction pathways in which p62 participates.

The genes encoding the peroxisomal beta-oxidation enzymes enoyl-CoA hydratase/3-hydroxyacyl-CoA dehydrogenase (HD) and fatty acyl-CoA oxidase (AOx) are coordinately regulated by peroxisome proliferator-activated receptor alpha (PPAR alpha)/9-cis-retinoic acid receptor (RXR alpha) heterodimers that transactivate these genes in a ligand-dependent manner via upstream peroxisome proliferator response elements (PPRE). Here we demonstrate that the monomeric orphan nuclear hormone receptor, RZR alpha, modulates PPAR alpha/RXR alpha-dependent transactivation in a response-element dependent manner. Electrophoretic mobility shift analysis showed that RZR alpha bound specifically as a monomer to the HD-PPRE but not the AOx-PPRE. Determinants in the HD-PPRE for binding of RZRa were distinct from those required for interaction with PPAR alpha/RXR alpha heterodimers. In transient transfections, RZR alpha stimulated ligand-mediated transactivation by PPAR alpha from an HD-PPRE luciferase reporter in the absence of exogenously added RXR alpha, but did not affect PPAR alpha-dependent transactivation of an AOx-PPRE reporter gene. These data illustrate cross-talk between the RZR alpha and PPAR alpha signaling pathways at the level of the HD-PPRE in the regulation of the HD gene and characterize additional factors governing the regulation of peroxisomal beta-oxidation.

Receptor interacting protein 140 (RIP140), a previously identified putative ligand-dependent coactivator of nuclear hormone receptors, was isolated by yeast two-hybrid cloning as a factor that interacts with peroxisome proliferator-activated receptor α (PPARα). This interaction in yeast required the integrity of the carboxyl-terminal, ligand-dependent activation domain of PPARα. However, protein binding studies carried out in vitro showed that full-length RIP140 bound efficiently to PPARα in the absence of exogenously added ligand. RIP140 also bound strongly to the liver-X-receptor (LXRα) in the absence of an activator for this receptor. In contrast, a strong interaction of RIP140 with the PPARα and LXRα heterodimerization partner retinoid-X-receptor α (RXRα) required the presence of its cognate ligand, 9-cis retinoic acid. Transfection analysis in mammalian cells demonstrated that RIP140 antagonized PPARα/RXRα- and LXRα/RXRα-mediated signaling. Our findings identify RIP140 as a novel modulator of transcriptional activation mediated by PPARα and LXRα and indicate that RIP140 can also bind to nuclear hormone receptors in a ligand-independent manner and repress their activity.

Mitochondrial 3‐hydroxy‐3‐methylglutaryl‐CoA synthase (mHMG‐CoAS) is a key enzyme in ketogenesis, catalyzing the condensation of acetyl‐CoA and acetoacetyl‐CoA to generate HMG‐CoA, which is eventually converted to ketone bodies. Transcription of the nuclear‐encoded gene for mHMG‐CoAS is stimulated by peroxisome proliferator‐activated receptor (PPAR) α, a fatty acid‐activated nuclear hormone receptor. Here we show that the mHMG‐CoAS protein physically interacts with PPARα in vitro, and potentiates PPARα‐dependent transcriptional activation via the cognate PPAR response element of the mHMG‐CoAS gene in vivo. Immunofluorescence of transiently transfected cells demonstrated that in the presence of PPARα, mHMG‐CoAS is translocated into the nucleus. Binding to PPARα, stimulation of PPARα activity and nuclear penetration require the integrity of the sequence LXXLL in mHMG‐CoAS, a motif known to mediate the interaction between nuclear hormone receptors and coactivators. These findings reveal a novel mechanism of gene regulation whereby the product of a PPARα‐responsive gene, normally resident in the mitochondria, directly interacts with this nuclear hormone receptor to autoregulate its own nuclear transcription.