title: Homocysteine increases the expression of vascular endothelial growth factor by a mechanism involving endoplasmic reticulum stress and transcription factor ATF4.
abstract: Vascular endothelial growth factor (VEGF) plays a key role in the development and progression of diabetic retinopathy. We previously demonstrated that amino acid deprivation and other inducers of endoplasmic reticulum-stress (ER stress) up-regulate the expression of VEGF in the retinal-pigmented epithelial cell line ARPE-19. Because homocysteine causes ER stress, we hypothesized that VEGF expression is increased by ambient homocysteine. dl-Homocysteine-induced VEGF expression was investigated in confluent ARPE-19 cultures. Northern analysis showed that homocysteine increased steady state VEGF mRNA levels 4.4-fold. Other thiol-containing compounds, including l-homocysteine thiolactone and DTT, induced VEGF expression 7.9- and 8.8-fold. Transcriptional run-on assays and mRNA decay studies demonstrated that the increase in VEGF mRNA levels was caused by increased transcription rather than mRNA stabilization. VEGF mRNA induction paralleled that of the ER-stress gene GRP78. Homocysteine treatment caused transient phosphorylation of eIF2alpha and an increase in ATF4 protein level. Overexpression of a dominant-negative ATF4 abolished the VEGF response to homocysteine treatment and to amino acid deprivation. VEGF mRNA expression by ATF4-/- MEF did not respond to homocysteine treatment and the response was restored with expression of wild-type ATF4. These studies indicate that expression of the pro-angiogenic factor VEGF is increased by homocysteine and other thiol-containing reductive compounds via ATF4-dependent activation of VEGF transcription.
authors.affiliation: Department of Biochemistry and Molecular Biology, University of New Mexico School of Medicine, Albuquerque, New Mexico 87131-0001, USA.
authors.forename: C Nathaniel
authors.initials: CN
authors.lastname: Roybal
authors.forename: Shujie
authors.initials: S
authors.lastname: Yang
authors.forename: Chiao-Wang
authors.initials: CW
authors.lastname: Sun
authors.forename: Diego
authors.initials: D
authors.lastname: Hurtado
authors.forename: David L
authors.initials: DL
authors.lastname: Vander Jagt
authors.forename: Tim M
authors.initials: TM
authors.lastname: Townes
authors.forename: Steve F
authors.initials: SF
authors.lastname: Abcouwer
mesh_terms: D051701:Activating Transcription Factor 4; D015152:Blotting, Northern; D015153:Blotting, Western; D022762:CCAAT-Enhancer-Binding Proteins / Q000378:metabolism; D002352:Carrier Proteins / Q000378:metabolism; D002460:Cell Line; D002467:Cell Nucleus / Q000378:metabolism; D018076:DNA, Complementary / Q000378:metabolism; D003609:Dactinomycin / Q000494:pharmacology; D004305:Dose-Response Relationship, Drug; D004721:Endoplasmic Reticulum / Q000378:metabolism*; D000091342:Endoplasmic Reticulum Chaperone BiP; D004797:Enzyme-Linked Immunosorbent Assay; D004847:Epithelial Cells / Q000166:cytology; D005799:Genes, Dominant; D006360:Heat-Shock Proteins; D006710:Homocysteine / Q000737:chemistry / Q000502:physiology*; D006801:Humans; D018832:Molecular Chaperones; D009154:Mutation; D009389:Neovascularization, Pathologic; D019384:Nucleic Acid Synthesis Inhibitors / Q000494:pharmacology; D010766:Phosphorylation; D011401:Promoter Regions, Genetic; D011485:Protein Binding; D012333:RNA, Messenger / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D013312:Stress, Physiological; D013997:Time Factors; D051742:Transcription Factor CHOP; D014157:Transcription Factors / Q000378:metabolism*; D014158:Transcription, Genetic; D015854:Up-Regulation; D042461:Vascular Endothelial Growth Factor A / Q000096:biosynthesis* / Q000378:metabolism
chemical_list: C497109:ATF4 protein, human; D022762:CCAAT-Enhancer-Binding Proteins; D002352:Carrier Proteins; C497249:DDIT3 protein, human; D018076:DNA, Complementary; D000091342:Endoplasmic Reticulum Chaperone BiP; C000717247:HSPA5 protein, human; D006360:Heat-Shock Proteins; D018832:Molecular Chaperones; D019384:Nucleic Acid Synthesis Inhibitors; D012333:RNA, Messenger; D014157:Transcription Factors; D042461:Vascular Endothelial Growth Factor A; D006710:Homocysteine; D051701:Activating Transcription Factor 4; D051742:Transcription Factor CHOP; D003609:Dactinomycin