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,
* Department of Medicine,
Department of Microbiology, Immunology and Molecular Genetics, and
Department of Human Genetics, University of California at Los Angeles, CA 90095
To understand pathways mediating the inflammatory responses of human aortic endothelial cells to oxidized phospholipids, we previously used a combination of genetics and genomics to model a coexpression network encompassing >1000 genes. CHAC1 (cation transport regulator-like protein 1), a novel gene regulated by ox-PAPC (oxidized 1-palmitoyl-2-arachidonyl-sn-3-glycero-phosphorylcholine), was identified in a co-regulated group of genes enriched for components of the ATF4 (activating transcription factor 4) arm of the unfolded protein response pathway. Herein, we characterize the role of CHAC1 and validate the network model. We first define the activation of CHAC1 mRNA by chemical unfolded protein response-inducers, but not other cell stressors. We then define activation of CHAC1 by the ATF4-ATF3-CHOP (C/EBP homologous protein), and not parallel XBP1 (X box-binding protein 1) or ATF6 pathways, using siRNA and/or overexpression plasmids. To examine the subset of genes downstream of CHAC1, we used expression microarray analysis to identify a list of 227 differentially regulated genes. We validated the activation of TNFRSF6B (tumor necrosis factor receptor superfamily, member 6b), a FASL decoy receptor, in cells treated with CHAC1 small interfering RNA. Finally, we showed that CHAC1 overexpression enhanced apoptosis, while CHAC1 small interfering RNA suppressed apoptosis, as determined by TUNEL, PARP (poly(ADP-ribose) polymerase) cleavage, and AIF (apoptosis-inducing factor) nuclear translocation.
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
1 I.N.M. was supported by the Heart and Stroke Foundation of Canada and by Canadian Institutes of Health Research postdoctoral fellowships. This work was supported by National Institutes of Health Grant HL30568.
2 Address correspondence and reprint requests to Dr. Imran N. Mungrue, Division of Cardiology, University of California at Los Angeles, 675 Charles E. Young Drive South, MRL 3210, Los Angeles, CA 90095. E-mail address: imungrue{at}yahoo.com
3 Abbreviations used in this paper: ox-PAPC, oxidized 1-palmitoyl-2-arachidonyl-sn-3-glycero-phosphorylcholine; AIF, apoptosis-inducing factor; ATF, activating transcription factor; CHAC1, cation transport regulator-like protein 1; CHOP, C/EBP homologous protein; cPARP, cleaved PARP; DAPI, 4',6-diamidino-2-phenylindole; eGFP, enhanced GFP; ER, endoplasmic reticulum; HAEC, human aortic endothelial cells; KLF, Kruppel-like factor; PARP, poly(ADP-ribose) polymerase; siRNA, small interfering RNA; TNFRSF6B, tumor necrosis factor receptor superfamily, member 6b, a FASL decoy receptor; UPR, unfolded protein response; XBP1, X box-binding protein 1.
4 The online version of this article contains supplemental material.
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