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.

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.

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.