title: Zinc regulates Nox1 expression through a NF-κB and mitochondrial ROS dependent mechanism to induce senescence of vascular smooth muscle cells.
abstract: The role of oxidative stress and inflammation in the development and progression of cardiovascular diseases (CVD) is well established. Increases in oxidative stress can further exacerbate the inflammatory response and lead to cellular senescence. We previously reported that angiotensin II (Ang II) and zinc increase reactive oxygen species (ROS) and cause senescence of vascular smooth muscle cells (VSMCs) and that senescence induced by Ang II is a zinc-dependent process. Zinc stimulated NADPH oxidase (Nox) activity; however, the role of Nox isoforms in zinc effects was not determined.
  
  
  
  Here, we show that downregulation of Nox1, but not Nox4, by siRNA prevented both Ang II- and zinc-induced senescence in VSMCs. On the other hand, overexpression of Nox1 induced senescence, which was associated with reduced proliferation, reduced expression of telomerase and increased DNA damage. Zinc increased Nox1 protein expression, which was inhibited by chelation of zinc with TPEN and by overexpression of the zinc exporters ZnT3 and ZnT10. These transporters work to reduce cytosolic zinc, suggesting that increased cytosolic zinc mediates Nox1 upregulation. Other metals including copper, iron, cobalt and manganese failed to upregulate Nox1, suggesting that this pathway is zinc specific. Nox1 upregulation was inhibited by actinomycin D (ACD), an inhibitor of transcription, by inhibition of NF-κB, a known Nox1 transcriptional regulator and by N-acetyl cysteine (NAC) and MitoTEMPO, suggesting that NF-κB and mitochondrial ROS mediate zinc effects. Supporting this idea, we found that zinc increased NF-κB activation in the cytosol, stimulated the translocation of the p65 subunit to the nucleus, and that zinc accumulated in mitochondria increasing mitochondrial ROS, measured using MitoSox. Further, zinc-induced senescence was reduced by inhibition of NF-κB or reduction of mitochondrial ROS with MitoTEMPO. NF-κB activity was also reduced by MitoTEMPO, suggesting that mitochondrial ROS is upstream of NF-κB.
  
  
  
  Our data demonstrate that altered zinc distribution leading to accumulation of zinc in the mitochondria increases mitochondrial ROS production causing NF-κB activation which in turn upregulates Nox1 expression inducing senescence of VSMCs.
authors.affiliation: Department of Nutrition, Food and Exercise Sciences, Florida State University, Tallahassee, FL 32306, USA; Center for Advancing Exercise and Nutrition Research on Aging (CAENRA), Florida State University, Tallahassee, FL 32306, USA. Electronic address: gsalazar@fsu.edu.
authors.forename: G
authors.initials: G
authors.lastname: Salazar
authors.affiliation: Department of Nutrition, Food and Exercise Sciences, Florida State University, Tallahassee, FL 32306, USA.
authors.forename: J
authors.initials: J
authors.lastname: Huang
authors.affiliation: Department of Dietetics and Nutrition, University of Arkansas for Medical Sciences, Little Rock, AR 72205, USA.
authors.forename: R G
authors.initials: RG
authors.lastname: Feresin
authors.affiliation: Department of Nutrition, Food and Exercise Sciences, Florida State University, Tallahassee, FL 32306, USA.
authors.forename: Y
authors.initials: Y
authors.lastname: Zhao
authors.affiliation: Department of Medicine, Division of Cardiology, Emory University, Atlanta, GA 30322, USA.
authors.forename: K K
authors.initials: KK
authors.lastname: Griendling
mesh_terms: D000804:Angiotensin II / Q000378:metabolism; D000818:Animals; D001013:Aorta, Thoracic / Q000473:pathology*; D002352:Carrier Proteins / Q000235:genetics / Q000378:metabolism*; D027682:Cation Transport Proteins; D002478:Cells, Cultured; D016922:Cellular Senescence; D003609:Dactinomycin / Q000494:pharmacology; D005029:Ethylenediamines / Q000494:pharmacology; D005786:Gene Expression Regulation; D008565:Membrane Proteins / Q000235:genetics / Q000378:metabolism*; D026901:Membrane Transport Proteins; D051379:Mice; D008810:Mice, Inbred C57BL; D018345:Mice, Knockout; D008928:Mitochondria / Q000378:metabolism*; D032389:Myocytes, Smooth Muscle / Q000502:physiology*; D000074624:NADPH Oxidase 1 / Q000235:genetics / Q000378:metabolism*; D000074663:NADPH Oxidase 4 / Q000235:genetics / Q000378:metabolism; D016328:NF-kappa B / Q000378:metabolism; D018384:Oxidative Stress; D034741:RNA, Small Interfering / Q000235:genetics; D051381:Rats; D017207:Rats, Sprague-Dawley; D017382:Reactive Oxygen Species / Q000378:metabolism; D015032:Zinc / Q000378:metabolism*
chemical_list: D002352:Carrier Proteins; D027682:Cation Transport Proteins; D005029:Ethylenediamines; D008565:Membrane Proteins; D026901:Membrane Transport Proteins; D016328:NF-kappa B; D034741:RNA, Small Interfering; D017382:Reactive Oxygen Species; C491177:Slc30a3 protein, mouse; D000804:Angiotensin II; D003609:Dactinomycin; D000074624:NADPH Oxidase 1; D000074663:NADPH Oxidase 4; D015032:Zinc; C044387:N,N,N',N'-tetrakis(2-pyridylmethyl)ethylenediamine
keywords: Mitochondria; NF-κB; Nox1; ROS; Senescence; VSMCs; Zinc; ZnT10; ZnT3