title: Activating transcription factor 6 mediates oxidized LDL-induced cholesterol accumulation and apoptosis in macrophages by up-regulating CHOP expression.
abstract: OBJECTIVE
  This study was to explore whether activating transcription factor 6 (ATF6), an important sensor to endoplasmic reticulum (ER) stress, would mediate oxidized low-density lipoprotein (ox-LDL)- induced cholesterol accumulation and apoptosis in cultured macrophages and the underlying molecular mechanisms.
  
  
  METHODS
  Intracellular lipid droplets and total cholesterol levels were assayed by oil red O staining and enzymatic colorimetry, respectively. Cell viability and apoptosis were determined using MTT assay and AnnexinV-FITC apoptosis detection kit, respectively. The nuclear translocation of ATF6 in cells was detected by immunofluorescence analysis. Protein and mRNA levels were examined by Western blot analysis and real time-PCR, respectively. ATF6 siRNA was transfected to RAW264.7 cells by lipofectamin.
  
  
  RESULTS
  Exposure of cells to ox-LDL induced glucose-regulated protein 78 (GRP78). C/EBP homologous protein (CHOP), a key-signaling component of ER stress-induced apoptosis, was up-regulated in ox-LDL-treated cells. ATF6, a factor that positively regulates CHOP expression, was activated by ox-LDL in a concentration- and time- dependent manner. The role of the ATF6-mediated ER stress pathway was further confirmed through the siRNA-mediated knockdown of ATF6, which attenuated ox-LDL-induced upregulation of CHOP, cholesterol accumulation and apoptosis in macrophages. In addition, the phosphorylation of double-stranded RNA-activated protein kinase-like endoplasmic reticulum kinase (PERK), another factor that positively regulates CHOP expression, was induced in the presence of ox-LDL, and PERK-specific siRNA also inhibited the ox-LDL-induced upregulation of CHOP and apoptosis in RAW264.7 cells.
  
  
  CONCLUSIONS
  These results demonstrate that ER stress-related proteins, particularly ATF6 and its downstream molecule CHOP, are involved in ox-LDL-induced cholesterol accumulation and apoptosis in macrophages.
authors.affiliation: Institute of Atherosclerosis, Key Laboratory of Atherosclerosis in Universities of Shandong, Taishan Medical University, Taian, China.
authors.forename: Shutong
authors.initials: S
authors.lastname: Yao
authors.forename: Chuanlong
authors.initials: C
authors.lastname: Zong
authors.forename: Ying
authors.initials: Y
authors.lastname: Zhang
authors.forename: Hui
authors.initials: H
authors.lastname: Sang
authors.forename: Mingfeng
authors.initials: M
authors.lastname: Yang
authors.forename: Peng
authors.initials: P
authors.lastname: Jiao
authors.forename: Yongqi
authors.initials: Y
authors.lastname: Fang
authors.forename: Nana
authors.initials: N
authors.lastname: Yang
authors.forename: Guohua
authors.initials: G
authors.lastname: Song
authors.forename: Shucun
authors.initials: S
authors.lastname: Qin
mesh_terms: D051702:Activating Transcription Factor 6 / Q000235:genetics / Q000502:physiology*; D000818:Animals; D017209:Apoptosis / Q000502:physiology*; D001483:Base Sequence; D015153:Blotting, Western; D002460:Cell Line; D002784:Cholesterol / Q000378:metabolism*; D017931:DNA Primers; D000091342:Endoplasmic Reticulum Chaperone BiP; D005434:Flow Cytometry; D005455:Fluorescent Antibody Technique; D006360:Heat-Shock Proteins / Q000096:biosynthesis; D008077:Lipoproteins, LDL / Q000502:physiology*; D008264:Macrophages / Q000166:cytology / Q000378:metabolism*; D051379:Mice; D016133:Polymerase Chain Reaction; D012333:RNA, Messenger / Q000235:genetics; D060888:Real-Time Polymerase Chain Reaction; D051742:Transcription Factor CHOP / Q000378:metabolism*; D015854:Up-Regulation / Q000502:physiology*
chemical_list: D051702:Activating Transcription Factor 6; D017931:DNA Primers; C497250:Ddit3 protein, mouse; D000091342:Endoplasmic Reticulum Chaperone BiP; D006360:Heat-Shock Proteins; C000717248:Hspa5 protein, mouse; D008077:Lipoproteins, LDL; D012333:RNA, Messenger; C099252:oxidized low density lipoprotein; D051742:Transcription Factor CHOP; D002784:Cholesterol