Deficiency of TDAG51 protects against atherosclerosis by modulating apoptosis, cholesterol efflux, and peroxiredoxin-1 expression.

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 PPARγ-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.

PPAR ligands decrease human airway smooth muscle cell migration and extracellular matrix synthesis.

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, ß 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.

LXR-induced reverse cholesterol transport in human airway smooth muscle is mediated exclusively by ABCA1.

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.

Protein kinase C alpha modulates liver X receptor alpha transactivation.

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.

Liver X receptor stimulates cholesterol efflux and inhibits expression of proinflammatory mediators in human airway smooth muscle cells.

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.

Host cell factor-1 and E2F4 interact via multiple determinants in each protein.

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.

Activity and subcellular compartmentalization of peroxisome proliferator-activated receptor alpha are altered by the centrosome-associated protein CAP350.

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 PPAR(alpha) interacts with the centrosome-associated protein CAP350. CAP350 also interacts with PPAR(delta), PPAR(gamma) and liver-X-receptor alpha, but not with the 9-cis retinoic acid receptor, RXR(alpha). Immunofluorescence analysis indicated that PPAR(alpha) is diffusely distributed in the nucleus and excluded from the cytoplasm. However, in the presence of coexpressed CAP350, PPAR(alpha) colocalizes with CAP350 to discrete nuclear foci and to the centrosome, perinuclear region and intermediate filaments. In contrast, the subcellular distribution of RXR(alpha) or of thyroid hormone receptor alpha was not altered by coexpression of CAP350. An amino-terminal fragment of CAP350 was localized exclusively to nuclear foci and was sufficient to recruit PPAR(alpha) 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 PPAR(alpha) to nuclear foci. Surprisingly, mutation of the LXXLL motif in this CAP350 subfragment did not prevent its binding to PPAR(alpha) in vitro, suggesting that this motif serves some function other than PPAR(alpha) binding in recruiting PPAR(alpha) to nuclear spots. CAP350 inhibited PPAR(alpha)-mediated transactivation in an LXXLL-dependent manner, suggesting that CAP350 represses PPAR(alpha) function. Our findings implicate CAP350 in a dynamic process that recruits PPAR(alpha) to discrete nuclear and cytoplasmic compartments and suggest that altered intracellular compartmentalization represents a regulatory process that modulates PPAR function.

Host cell factor-1 interacts with and antagonizes transactivation by the cell cycle regulatory factor Miz-1.

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.

Oxysterol activators of liver X receptor and 9-cis-retinoic acid promote sequential steps in the synthesis and secretion of tumor necrosis factor-alpha from human monocytes.

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.