title: Mitochondrial E3 Ubiquitin Protein Ligase 1 Mediates Cigarette Smoke-Induced Endothelial Cell Death and Dysfunction.
abstract: By virtue of the critical roles of Akt in vascular endothelial cell (EC) survival and function, cigarette smoke-induced Akt reduction may contribute to EC death and dysfunction in smokers' lungs. One of the negative Akt regulatory mechanisms is K48-linked Akt ubiquitination and subsequent proteasomal degradation. Here, we assessed the involvement of mitochondrial E3 ubiquitin protein ligase 1 (MUL1), recently revealed as a novel Akt ubiquitin E3 ligase, in cigarette smoke-induced Akt ubiquitination and its contribution to pulmonary EC death and dysfunction. In human lung microvascular ECs (HLMVECs), cigarette smoke extract (CSE) noticeably elevated MUL1 expression and K48-linked Akt ubiquitination, whereas Akt, p-Akt, eNOS, and p-eNOS levels were decreased. MUL1 knockdown suppressed CSE-induced Akt ubiquitination/degradation and cytoplasmic reductions of Akt and p-Akt. Furthermore, MUL1 knockdown attenuated reductions of eNOS and p-eNOS and alleviated EC survival, migration, and tube formation in the presence of CSE exposure. In addition, overexpression of K284R Akt, a mutant for a MUL1-ubiquitination site, produced similar effects. In HLMVECs exposed to CSE, Akt-MUL1 interaction was increased in coimmunoprecipitation and in situ proximity ligation assays. Similarly, the proximity ligation assay signals were elevated in rat lungs exposed to cigarette smoke for 3 months, during which Mul1 levels were noticeably increased. Finally, we found that CSE-mediated MUL1 induction in HLMVECs is mediated by retinoic acid receptor-related orphan receptor α. Taken together, these data suggest that cigarette smoke-induced MUL1 elevation mediates Akt ubiquitination/degradation, potentially leading to pulmonary EC death and functional impairment.
authors.affiliation: 1 Department of Otolaryngology, Ajou University School of Medicine, Suwon.
authors.forename: Sun-Yong
authors.initials: SY
authors.lastname: Kim
authors.affiliation: 2 Department of Pharmacology, Sungkyunkwan University School of Medicine, Samsung Biomedical Research Institute, Suwon.
authors.forename: Hyo Jeong
authors.initials: HJ
authors.lastname: Kim
authors.affiliation: 2 Department of Pharmacology, Sungkyunkwan University School of Medicine, Samsung Biomedical Research Institute, Suwon.
authors.forename: Mi Kyeong
authors.initials: MK
authors.lastname: Park
authors.affiliation: 3 Department of Pulmonary and Critical Care Medicine, Asan Medical Center, University of Ulsan College of Medicine, Seoul; and.
authors.forename: Jin Won
authors.initials: JW
authors.lastname: Huh
authors.affiliation: 4 Division of Pulmonary and Critical Care Medicine, Department of Medicine and.
authors.forename: Hye Yun
authors.initials: HY
authors.lastname: Park
authors.affiliation: 5 Department of Pathology, Samsung Medical Center, Sungkyunkwan University School of Medicine, Seoul, Republic of Korea.
authors.forename: Sang Yun
authors.initials: SY
authors.lastname: Ha
authors.affiliation: 2 Department of Pharmacology, Sungkyunkwan University School of Medicine, Samsung Biomedical Research Institute, Suwon.
authors.forename: Joo-Ho
authors.initials: JH
authors.lastname: Shin
authors.affiliation: 2 Department of Pharmacology, Sungkyunkwan University School of Medicine, Samsung Biomedical Research Institute, Suwon.
authors.forename: Yun-Song
authors.identifier: http://orcid.org/0000-0003-1147-8993
authors.initials: YS
authors.lastname: Lee
mesh_terms: D000818:Animals; D016923:Cell Death; D002465:Cell Movement / Q000187:drug effects; D002478:Cells, Cultured; D004195:Disease Models, Animal; D004305:Dose-Response Relationship, Drug; D042783:Endothelial Cells / Q000187:drug effects* / Q000201:enzymology / Q000473:pathology; D006801:Humans; D018345:Mice, Knockout; D024101:Mitochondrial Proteins / Q000235:genetics / Q000378:metabolism*; D009154:Mutation; D052250:Nitric Oxide Synthase Type III / Q000378:metabolism; D057094:Nuclear Receptor Subfamily 1, Group F, Member 1 / Q000172:deficiency / Q000235:genetics / Q000378:metabolism; D010766:Phosphorylation; D059748:Proteolysis; D051057:Proto-Oncogene Proteins c-akt / Q000235:genetics / Q000378:metabolism; D011656:Pulmonary Emphysema / Q000139:chemically induced* / Q000201:enzymology / Q000235:genetics; D034622:RNA Interference; D051381:Rats; D012906:Smoke / Q000009:adverse effects*; D012907:Smoking / Q000009:adverse effects*; D013997:Time Factors; D014162:Transfection; D044767:Ubiquitin-Protein Ligases / Q000235:genetics / Q000378:metabolism*; D054875:Ubiquitination; D015854:Up-Regulation
chemical_list: D024101:Mitochondrial Proteins; D057094:Nuclear Receptor Subfamily 1, Group F, Member 1; C539738:RORA protein, human; C539739:Rora protein, mouse; D012906:Smoke; C496314:NOS3 protein, human; D052250:Nitric Oxide Synthase Type III; C496316:Nos3 protein, rat; C529067:MUL1 protein, human; C000608240:MUL1 protein, mouse; C000608241:Mul1 protein, rat; C108062:TTC3 protein, human; D044767:Ubiquitin-Protein Ligases; D051057:Proto-Oncogene Proteins c-akt
keywords: Akt ubiquitination; chronic obstructive pulmonary disease; endothelial cells; mitochondrial E3 ubiquitin protein ligase 1; retinoic acid receptor-related orphan receptor α