John P. Vanden Heuvel

Hepatitis B Virus X-Associated Protein 2 Is a Subunit of the Unliganded Aryl Hydrocarbon Receptor Core Complex and Exhibits Transcriptional Enhancer Activity

ABSTRACT Prior to ligand activation, the unactivated aryl hydrocarbon receptor (AhR) exists in a heterotetrameric 9S core complex consisting of the AhR ligand-binding subunit, a dimer of hsp90, and an unknown subunit. Here we report the purification of an ∼38-kDa protein (p38) from COS-1 cell cytosol that is a member of this complex by coprecipitation with a FLAG-tagged AhR. Internal amino acid sequence information was obtained, and p38 was identified as the hepatitis B virus X-associated protein 2 (XAP2). The simian ortholog of XAP2 was cloned from a COS-1 cDNA library; it codes for a 330-amino-acid protein containing regions of homology to the immunophilins FKBP12 and FKBP52. A tetratricopeptide repeat (TPR) domain in the carboxy-terminal region of XAP2 was similar to the third and fourth TPR domains of human FKBP52 and the Saccharomyces cerevisiae transcriptional modulator SSN6, respectively. Polyclonal antibodies raised against XAP2 recognized p38 in the unliganded AhR complex in COS-1 and Hepa 1c1c7 cells. It was ubiquitously expressed in murine tissues at the protein and mRNA levels. It was not required for the assembly of an AhR-hsp90 complex in vitro. Additionally, XAP2 did not directly associate with hsp90 upon in vitro translation, but was present in a 9S form when cotranslated in vitro with murine AhR. XAP2 enhanced the ability of endogenous murine and human AhR complexes to activate a dioxin-responsive element–luciferase reporter twofold, following transient expression of XAP2 in Hepa 1c1c7 and HeLa cells.

Dose-response effects of omega-3 fatty acids on triglycerides, inflammation, and endothelial function in healthy persons with moderate hypertriglyceridemia

BACKGROUND Eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) have been shown to reduce cardiovascular mortality at a dose of ≈1 g/d. Studies using higher doses have shown evidence of reduced inflammation and improved endothelial function. Few studies have compared these doses. OBJECTIVE The objective of this study was to compare the effects of a nutritional dose of EPA+DHA (0.85 g/d) with those of a pharmaceutical dose (3.4 g/d) on serum triglycerides, inflammatory markers, and endothelial function in healthy subjects with moderately elevated triglycerides. DESIGN This was a placebo-controlled, double-blind, randomized, 3-period crossover trial (8 wk of treatment, 6 wk of washout) that compared the effects of 0.85 and 3.4 g EPA+DHA/d in 23 men and 3 postmenopausal women with moderate hypertriglyceridemia (150-500 mg/dL). RESULTS The higher dose of EPA+DHA lowered triglycerides by 27% compared with placebo (mean ± SEM: 173 ± 17.5 compared with 237 ± 17.5 mg/dL; P = 0.002), whereas no effect of the lower dose was observed on lipids. No effects on cholesterol (total, LDL, and HDL), endothelial function [as assessed by flow-mediated dilation, peripheral arterial tonometry/EndoPAT (Itamar Medical Ltd, Caesarea, Israel), or Doppler measures of hyperemia], inflammatory markers (interleukin-1β, interleukin-6, tumor necrosis factor-α, and high-sensitivity C-reactive protein), or the expression of inflammatory cytokine genes in isolated lymphocytes were observed. CONCLUSION The higher dose (3.4 g/d) of EPA+DHA significantly lowered triglycerides, but neither dose improved endothelial function or inflammatory status over 8 wk in healthy adults with moderate hypertriglyceridemia. The trial was registered at clinicaltrials.gov as NCT00504309.

Conjugated linoleic acid activates peroxisome proliferator-activated receptor α and β subtypes but does not induce hepatic peroxisome proliferation in Sprague–Dawley rats

Since conjugated linoleic acid (CLA) has structural and physiological characteristics similar to peroxisome proliferators, we hypothesized that CLA would activate peroxisome proliferator-activated receptor (PPAR). We compared the effects of dietary CLA (0.0, 0.5, 1.0 and 1.5% by weight) with a peroxisome proliferator (0.01% Wy-14,643) in female and male Sprague-Dawley (SD) rats. Dietary CLA had little effect on body weight, liver weight, and hepatic peroxisome proliferation, compared to male rats fed Wy-14,643 diet. Lipid content in livers from rats fed 1.5% CLA and Wy-14,643 diets was increased (P < 0.01) when compared to rats fed control diets regardless of gender. Hepatic acyl-CoA oxidase (ACO) mRNA levels were increased 3-fold in male rats fed 1.5% CLA diet compared to rats fed control diets while Wy-14,643 supported approximately 30-fold ACO mRNA accumulation. A similar response was observed for liver fatty acid-binding protein (L-FABP) mRNA. The effect of dietary treatments on hepatic PPAR-responsive genes in female rats was weaker than in male rats. The (9Z,11E)-CLA isomer activated PPAR alpha in transfected cells to a similar extent as Wy-14,643, whereas the furan-CLA metabolite was comparable to bezafibrate on activating PPAR beta. These data suggest that while CLA was able to activate PPARs it is not a peroxisome proliferator in SD rats.

Reviews: current topicsrole of nuclear receptors in the regulation of gene expression by dietary fatty acids (review)

Long chain fatty acids, derived either from endogenous metabolism or by nutritional sources play significant roles in important biological processes of membrane structure, production of biologically active compounds, and participation in cellular signaling processes. Recently, the structure of dietary fatty acids has become an important issue in human health because ingestion of saturated fats (containing triglycerides composed of saturated fatty acids) is considered harmful, while unsaturated fats are viewed as beneficial. It is important to note that the molecular reason for this dichotomy still remains elusive. Since fatty acids are important players in development of pathology of cardiovascular and endocrine system, understanding the key molecular targets of fatty acids, in particular those that discriminate between saturated and unsaturated fats, is much needed. Recently, insights have been gained on several fatty acid-activated nuclear receptors involved in gene expression. In other words, we can now envision long chain fatty acids as regulators of signal transduction processes and gene regulation, which in turn will dictate their roles in health and disease. In this review, we will discuss fatty acid-mediated regulation of nuclear receptors. We will focus on peroxisome proliferators-activated receptors (PPARs), liver X receptors (LXR), retinoid X receptors (RXRs), and Hepatocyte Nuclear Factor alpha (HNF-4alpha), all of which play pivotal roles in dietary fatty acid-mediated effects. Also, the regulation of gene expression by Conjugated Linoleic Acids (CLA), a family of dienoic fatty acids with a variety of beneficial effects, will be discussed.

Dietary conjugated linoleic acid induces peroxisome-specific enzyme accumulation and ornithine decarboxylase activity in mouse liver☆

Abstract Previous studies have shown that the dietary fatty acids, conjugated linoleic acids (CLA), inhibit carcinogenesis in the colon, mammary gland, forestomach, and skin. Several properties of this chemoprotective polyunsaturated fatty acid suggest it will act as an hepatic peroxisome proliferator. This study evaluated the effect of dietary CLA on the accumulation of enzymes associated with peroxisome proliferation in rodent liver. Female SENCAR mice were fed one of four semipurified diets containing 5% corn oil without CLA (“control diet”) or supplemented with incremental levels of CLA (0.5%, 1.0% or 1.5% by weight of diets) for 6 weeks. Hepatic mRNA levels of several enzymes known to be induced during peroxisome proliferation [i.e., acyl-CoA oxidase (ACO), cytochrome P4504A1 (CYP4A1), and liver fatty acid binding protein (FABP)] were measured by quantitative reverse transcriptase-polymerase chain reaction (RT-PCR). Diets containing 0.5%, 1.0% or 1.5% CLA were associated with approximately 6-, 9-, and 9-fold increases in ACO mRNA, respectively, compared with mRNA levels in mice fed the control diet. The steady state levels of FABP and CYP4A1 mRNA accumulation were maximal in animals fed 1.0% CLA diets and less magnified in mice fed 1.5% CLA diets. Western blot analysis revealed that the relative abundance of ACO protein in livers of mice fed CLA-containing diet groups (0.5%, 1.0%, and 1.5% CLA) were 2.5, 3.0, and 3.0 times the level ACO protein of mice fed the control diet (0.0% CLA). Because most peroxisome proliferators are considered nongenotoxic hepatocarcinogens in rodents, the effect of dietary CLA on ornithine decarboxylase (ODC) activity, a measure of cell proliferation and tumor promotion, was quantified. Activity on hepatic ODC was increased by approximately 10-fold for mice fed 1.0% and 1.5% diets, respectively, compared with those fed the control or 0.5% CLA diets. These data suggest that CLA displays the typical peroxisome proliferation response, i.e., induction of ACO, CYP4A1 and FABP accumulation and cell proliferation in rodent liver.

Interleukin-2 Suppression by 2-Arachidonyl Glycerol Is Mediated through Peroxisome Proliferator-Activated Receptor γ Independently of Cannabinoid Receptors 1 and 2

2-Arachidonyl glycerol (2-AG) is an endogenous arachidonic acid derivative that binds cannabinoid receptors CB1 and CB2 and is hence termed an endocannabinoid. 2-AG also modulates a variety of immunological responses, including expression of the autocrine/paracrine T cell growth factor interleukin (IL)-2. The objective of the present studies was to determine the mechanism responsible for IL-2 suppression by 2-AG. Because of the labile properties of 2-AG, 2-AG ether, a nonhydrolyzable analog of 2-AG, was also used. Both 2-AG and 2-AG ether suppressed IL-2 expression independently of CB1 and CB2, as demonstrated in leukocytes derived from CB1/CB2-null mice. Moreover, we demonstrated that both 2-AG and 2-AG ether treatment activated peroxisome proliferator-activated receptor γ (PPARγ), as evidenced by forced differentiation of 3T3-L1 cells into adipocytes, induction of aP2 mRNA levels, and activation of a PPARγ-specific luciferase reporter in transiently transfected 3T3-L1 cells. Consequently, the putative role of PPARγ in IL-2 suppression by 2-AG and 2-AG ether was examined in Jurkat T cells. Concordant with PPARγ involvement, the PPARγ-specific antagonist 2-chloro-5-nitro-N-(4-pyridyl)-benzamide (T0070907) blocked 2-AG- and 2-AG ether-mediated IL-2 suppression. Likewise, 2-AG suppressed the transcriptional activity of two transcription factors crucial for IL-2 expression, nuclear factor of activated T cells and nuclear factor κB, in the absence but not in the presence of T0070907. 2-AG treatment also induced PPARγ binding to a PPAR response element in activated Jurkat T cells. Together, the aforementioned studies identify PPARγ as a novel intracellular target of 2-AG, which mediates the suppression of IL-2 by 2-AG in a manner that is independent of CB1 and/or CB2.

Evidence That Peroxisome Proliferator-activated Receptor α Is Complexed with the 90-kDa Heat Shock Protein and the Hepatitis Virus B X-associated Protein 2

The peroxisome proliferator-activated receptor α (PPARα) is a ligand-inducible transcription factor, which belongs to the nuclear receptor superfamily. PPARα mediates the carcinogenic effects of peroxisome proliferators in rodents. In humans, PPARα plays a fundamental role in regulating energy homeostasis via control of lipid metabolism. To study the possible role of chaperone proteins in the regulation of PPARα activity, a monoclonal antibody (mAb) was made against PPARα and designated as 3B6/PPAR. The specificity of mAb 3B6/PPAR in recognizing PPARα was tested in immunoprecipitations using in vitrotranslated PPAR subtypes. The mAb 3B6/PPAR recognized PPARα, failed to bind to PPARβ or PPARγ, and is efficient in both immunoprecipitating and visualizing the receptor on protein blots. The immunoprecipitation of PPARα in mouse liver cytosol using mAb 3B6/PPAR has resulted in the detection of two co-immunoprecipitated proteins, which are heat shock protein 90 (hsp90) and the hepatitis B virus X-associated protein 2 (XAP2). The concomitant depletion of PPARα in hsp90-depleted mouse liver cytosol was also detected. Complex formation between XAP2 and PPARα/FLAG was also demonstrated in an in vitro translation binding assay. hsp90 interacts with PPARα in a mammalian two-hybrid assay and binds to the E/F domain. Transient expression of XAP2 co-expressed with PPARα resulted in down-regulation of a peroxisome proliferator response element-driven reporter gene activity. Taken together, these results indicate that PPARα is in a complex with hsp90 and XAP2, and XAP2 appears to function as a repressor. This is the first demonstration that PPARα is stably associated with other proteins in tissue extracts and the first nuclear receptor shown to functionally interact with XAP2.

Selenium attenuates pro-inflammatory gene expression in macrophages.

Selenium (Se) is an important element required for the optimal functioning of the immune system. Particularly in macrophages, which play a pivotal role in immune regulation, Se acts as a major antioxidant in the form of selenoproteins to mitigate the cytotoxic effects of reactive oxygen species. Here we describe the role of Se as an anti-inflammatory agent and its effect on the macrophage signal transduction pathways elicited by bacterial endotoxin, LPS. Our studies demonstrate that supplementation of Se to macrophages (Se-deficient) leads to a significant decrease in the LPS-induced expression of two important pro-inflammatory genes, cyclooxygenase-2 (COX-2) and tumor necrosis factor-alpha (TNF-alpha) via the inhibition of MAP kinase pathways. Furthermore, Se-deficiency in mice exacerbated the LPS-mediated infiltration of macrophages into the lungs suggesting that Se status is a crucial host factor that regulates inflammation. In summary, our results indicate that Se plays an important role as an anti-inflammatory agent by tightly regulating the expression of pro-inflammatory genes in immune cells.

Asynchronous Shear Stress and Circumferential Strain Reduces Endothelial NO Synthase and Cyclooxygenase-2 but Induces Endothelin-1 Gene Expression in Endothelial Cells

Objective—Endothelium-derived vasoactive agents NO, endothelin-1 (ET-1), and prostacyclin (PGI2) not only regulate vascular tone but also influence atherogenic processes, including smooth muscle migration and proliferation, as well as monocyte and platelet adhesion. Complex hemodynamics characterized by the temporal phase angle between mechanical factors circumferential strain and wall shear stress (stress phase angle [SPA]) have been implicated in regions prone to pathologic development, such as atherosclerosis and intimal hyperplasia, in coronary and peripheral arteries where the mechanical forces are highly asynchronous (SPA=−180°). We determined the gene expression of endothelial NO synthase (eNOS), ET-1, and cyclooxygenase-2 (COX-2) affected by asynchronous hemodynamics (SPA=−180°) relative to normal hemodynamics (SPA=0°) in bovine aortic endothelial cells. Methods and Results—Quantitative competitive RT-PCR analysis showed that eNOS production (at 5 and 12 hours) and COX-2 production (at 5 hours) were reduced at the gene expression level by asynchronous hemodynamics (SPA=−180°) compared with synchronous hemodynamics (SPA=0°), whereas ET-1 exhibited an opposite trend (at 5 and 12 hours). NO, ET-1, and PGI2 secretion followed their respective gene expression profiles after 5 and 12 hours. Conclusion—Together, these data suggest that highly asynchronous mechanical force patterns (SPA=−180°) can elicit proatherogenic vasoactive responses in endothelial cells at the gene expression level, indicating a novel mechanism that induces cardiovascular pathology.

Beef in an Optimal Lean Diet study: effects on lipids, lipoproteins, and apolipoproteins

Background: A Step I diet with lean beef compared with lean white meat both decrease LDL cholesterol. To our knowledge, no studies have evaluated a low–saturated fatty acid (SFA) (<7% calories) diet that contains lean beef. Objective: We studied the effect on LDL cholesterol of cholesterol-lowering diets with varying amounts of lean beef [ie, Dietary Approaches to Stop Hypertension (DASH): 28 g beef/d; Beef in an Optimal Lean Diet (BOLD): 113 g beef/d; and Beef in an Optimal Lean Diet plus additional protein (BOLD+): 153 g beef/d] compared with that of a healthy American diet (HAD). Design: Thirty-six hypercholesterolemic participants (with LDL-cholesterol concentrations >2.8 mmol/L) were randomly assigned to consume each of the 4 diets (HAD: 33% total fat, 12% SFA, 17% protein, and 20 g beef/d), DASH (27% total fat, 6% SFA, 18% protein, and 28 g beef/d), BOLD (28% total fat, 6% SFA, 19% protein, and 113 g beef/d), and BOLD+ (28% total fat, 6% SFA, 27% protein, and 153 g beef/d) for 5 wk. Results: There was a decrease in total cholesterol (TC) and LDL-cholesterol concentrations (P < 0.05) after consumption of the DASH (−0.49 ± 0.11 and −0.37 ± 0.09 mmol/L, respectively), BOLD (−0.48 ± 0.10 and −0.35 ± 0.9 mmol/L, respectively), and BOLD+ (−0.50 ± 0.10 and −0.345 ± 0.09 mmol/L, respectively) diets compared with after consumption of the HAD (−0.22 ± 0.10 and −0.14 ± 0.10 mmol/L, respectively). Apolipoprotein A-I, C-III, and C-III bound to apolipoprotein A1 particles decreased after BOLD and BOLD+ diets compared with after the HAD, and there was a greater decrease in apolipoprotein B after consumption of the BOLD+ diet than after consumption of the HAD (P < 0.05 for both). LDL cholesterol and TC decreased after consumption of the DASH, BOLD, and BOLD+ diets when the baseline C-reactive protein (CRP) concentration was <1 mg/L; LDL cholesterol and TC decreased when baseline CRP concentration was >1 mg/L with the BOLD and BOLD+ diets. Conclusions: Low-SFA, heart-healthy dietary patterns that contain lean beef elicit favorable effects on cardiovascular disease (CVD) lipid and lipoprotein risk factors that are comparable to those elicited by a DASH dietary pattern. These results, in conjunction with the beneficial effects on apolipoprotein CVD risk factors after consumption of the BOLD and BOLD+ diets, which were greater with the BOLD+ diet, provide support for including lean beef in a heart-healthy dietary pattern. This trial was registered at clinicaltrials.gov as NCT00937898.