Subcellular localization of Nox4 and regulation in diabetes.
Oxidative stress is implicated in human diseases. Some of the oxidative pathways are harbored in the mitochondria. NAD(P)H oxidases have been identified not only in phagocytic but also in somatic cells. Nox4 is the most ubiquitous of these oxidases and is a major source of reactive oxygen species (ROS) in many cell types and in kidney tissue of diabetic animals. We generated specific Nox4 antibodies, and found that Nox4 localizes to mitochondria. (i) Immunoblot analysis in cultured mesangial cells and kidney cortex revealed that Nox4 is present in crude mitochondria, in mitochondria-enriched heavy fractions, and in purified mitochondria; (ii) immunofluorescence confocal microscopy also revealed that Nox4 localizes with the mitochondrial marker Mitotracker; and (iii) the mitochondrial localization prediction program MitoProt indicated that the probability score for Nox4 is identical to mitochondrial protein cytochrome c oxidase subunit IV. We also show that in purified mitochondria, siRNA-mediated knockdown of Nox4 significantly reduces NADPH oxidase activity in pure mitochondria and blocks glucose-induced mitochondrial superoxide generation. In a rat model of diabetes, mitochondrial Nox4 expression is increased in kidney cortex. Our data provide evidence that a functional Nox4 is present and regulated in mitochondria, indicating the existence of a previously undescribed source of ROS in this organelle.
Block
Karen
K
Department of Medicine, University of Texas Health Science Center, San Antonio, TX 78229, USA.
Gorin
Yves
Y
Abboud
Hanna E
HE
D000818:Animals; D015153:Blotting, Western; D002478:Cells, Cultured; D003921:Diabetes Mellitus, Experimental / Q000235:genetics / Q000378:metabolism*; D003922:Diabetes Mellitus, Type 1 / Q000235:genetics / Q000378:metabolism*; D005786:Gene Expression Regulation; D047468:Immunoprecipitation; D007672:Kidney Cortex / Q000166:cytology / Q000378:metabolism; D008297:Male; D050527:Mesangial Cells / Q000166:cytology / Q000378:metabolism; D018613:Microscopy, Confocal; D008928:Mitochondria / Q000378:metabolism*; D000074663:NADPH Oxidase 4; D019255:NADPH Oxidases / Q000235:genetics / Q000378:metabolism*; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D034741:RNA, Small Interfering / Q000235:genetics; D051381:Rats; D017207:Rats, Sprague-Dawley; D017382:Reactive Oxygen Species / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D014162:Transfection
D012333:RNA, Messenger; D034741:RNA, Small Interfering; D017382:Reactive Oxygen Species; D000074663:NADPH Oxidase 4; D019255:NADPH Oxidases; C498911:Nox4 protein, rat
FASEB J. 2005 Sep;19(11):1504-6
15972297
J Cell Biol. 2008 Jun 30;181(7):1129-39
18573911
Sci STKE. 2006 Aug 22;2006(349):re8
16926363
J Biol Chem. 2005 Nov 25;280(47):39616-26
16135519
Am J Physiol Renal Physiol. 2006 Feb;290(2):F345-56
16131649
Arterioscler Thromb Vasc Biol. 2004 Apr;24(4):677-83
14670934
Free Radic Biol Med. 2005 Aug 1;39(3):381-91
15993337
Physiol Rev. 2007 Jan;87(1):245-313
17237347
Am J Physiol Renal Physiol. 2003 Aug;285(2):F219-29
12842860
Diabetes. 2003 Oct;52(10):2570-7
14514642
Proc Natl Acad Sci U S A. 2000 Jul 5;97(14):8010-4
10869423
Am J Physiol Renal Physiol. 2005 Oct;289(4):F816-25
16159901
J Biol Chem. 2008 Dec 12;283(50):35273-82
18849343
Biochim Biophys Acta. 2009 Jan;1793(1):154-70
18694785
Nature. 2000 Apr 13;404(6779):787-90
10783895
Curr Opin Pharmacol. 2002 Apr;2(2):121-5
11950621
Genes Dev. 2003 Jun 15;17(12):1475-86
12783855
Genes Cells. 2005 Dec;10(12):1139-51
16324151
J Biol Chem. 1997 Oct 24;272(43):27338-44
9341184
Physiol Rev. 2002 Jan;82(1):47-95
11773609
Biochem Biophys Res Commun. 2005 Apr 1;329(1):32-9
15721269
J Leukoc Biol. 2004 Oct;76(4):760-81
15240752
J Biol Chem. 2004 Oct 29;279(44):45935-41
15322091
Cardiovasc Res. 2006 Jul 15;71(2):289-99
16765921
Free Radic Biol Med. 2008 Nov 1;45(9):1340-51
18760347
J Biol Chem. 2005 Apr 29;280(17):17149-53
15749715
Biochim Biophys Acta. 2002 Sep 2;1592(1):63-77
12191769
J Cell Biol. 2007 Nov 19;179(4):585-91
17998403
Cell Signal. 2006 Jan;18(1):69-82
15927447
Diabetes. 2004 Feb;53 Suppl 1:S96-102
14749273
Diabetes. 2005 Jun;54(6):1615-25
15919781
Kidney Int. 2005 May;67(5):1890-8
15840036