Cytochrome P450 (CYP) 2J2 gene transfection attenuates MMP-9 via inhibition of NF-kappabeta in hyperhomocysteinemia.
Hyperhomocysteinemia (HHcy) is associated with atherosclerotic events involving the modulation of arachidonic acid (AA) metabolism and the activation of matrix metalloproteinase-9 (MMP-9). Cytochrome P450 (CYP) epoxygenase-2J2 (CYP2J2) is abundant in the heart endothelium, and its AA metabolites epoxyeicosatrienoic acids (EETs) mitigates inflammation through NF-kappabeta. However, the underlying molecular mechanisms for MMP-9 regulation by CYP2J2 in HHcy remain obscure. We sought to determine the molecular mechanisms by which P450 epoxygenase gene transfection or EETs supplementation attenuate homocysteine (Hcy)-induced MMP-9 activation. CYP2J2 was over-expressed in mouse aortic endothelial cells (MAECs) by transfection with the pcDNA3.1/CYP2J2 vector. The effects of P450 epoxygenase transfection or exogenous supplementation of EETs on NF-kappabeta-mediated MMP-9 regulation were evaluated using Western blot, in-gel gelatin zymography, electromobility shift assay, immunocytochemistry. The result suggested that Hcy downregulated CYP2J2 protein expression and dephosphorylated PI3K-dependent AKT signal. Hcy induced the nuclear translocation of NF-kappabeta via downregulation of IKbetaalpha (endogenous cytoplasmic inhibitor of NF-kappabeta). Hcy induced MMP-9 activation by increasing NF-kappabeta-DNA binding. Moreover, P450 epoxygenase transfection or exogenous addition of 8,9-EET phosphorylated the AKT and attenuated Hcy-induced MMP-9 activation. This occurred, in part, by the inhibition of NF-kappabeta nuclear translocation, NF-kappabeta-DNA binding and activation of IKbetaalpha. The study unequivocally suggested the pivotal role of EETs in the modulation of Hcy/MMP-9 signal.
Moshal
Karni S
KS
Department of Physiology and Biophysics, School of Medicine University of Louisville, Louisville, Kentucky 40202, USA.
Zeldin
Darryl C
DC
Sithu
Srinivas D
SD
Sen
Utpal
U
Tyagi
Neetu
N
Kumar
Munish
M
Hughes
William M
WM
Metreveli
Naira
N
Rosenberger
Dorothea S E
DS
Singh
Mahavir
M
Vacek
Thomas P
TP
Rodriguez
Walter E
WE
Ayotunde
Adeagbo
A
Tyagi
Suresh C
SC
D015126:8,11,14-Eicosatrienoic Acid / Q000031:analogs & derivatives / Q000494:pharmacology; D000818:Animals; D002478:Cells, Cultured; D000090762:Cytochrome P-450 CYP2J2; D003577:Cytochrome P-450 Enzyme System / Q000235:genetics* / Q000378:metabolism*; D004789:Enzyme Activation / Q000187:drug effects; D004790:Enzyme Induction / Q000187:drug effects; D006710:Homocysteine / Q000494:pharmacology; D020138:Hyperhomocysteinemia / Q000201:enzymology / Q000378:metabolism*; D020672:I-kappa B Proteins / Q000378:metabolism; D020780:Matrix Metalloproteinase 9 / Q000096:biosynthesis / Q000378:metabolism*; D051379:Mice; D000072000:NF-KappaB Inhibitor alpha; D010105:Oxygenases / Q000235:genetics* / Q000378:metabolism*; D019869:Phosphatidylinositol 3-Kinases / Q000378:metabolism; D010766:Phosphorylation / Q000187:drug effects; D051057:Proto-Oncogene Proteins c-akt / Q000378:metabolism; D051996:Transcription Factor RelA / Q000037:antagonists & inhibitors* / Q000378:metabolism; D014162:Transfection
D015126:8,11,14-Eicosatrienoic Acid; D000090762:Cytochrome P-450 CYP2J2; D003577:Cytochrome P-450 Enzyme System; D006710:Homocysteine; D020672:I-kappa B Proteins; D020780:Matrix Metalloproteinase 9; D000072000:NF-KappaB Inhibitor alpha; D010105:Oxygenases; D019869:Phosphatidylinositol 3-Kinases; D051057:Proto-Oncogene Proteins c-akt; D051996:Transcription Factor RelA; C000606205:Nfkbia protein, mouse; C050715:8,9-epoxyeicosatrienoic acid