High glucose-induced NF-kappaB activation occurs via tyrosine phosphorylation of IkappaBalpha in human glomerular endothelial cells: involvement of Syk tyrosine kinase.
Activation of nuclear factor-kappaB (NF-kappaB) occurs by dissociation from IkappaB after serine or tyrosine phosphorylation of IkappaBalpha, but the way of NF-kappaB activation by high glucose has not been defined. High glucose is known to activate NF-kappaB via protein kinase C and reactive oxygen species (ROS). In this study, we investigated how high glucose activates NF-kappaB for CC chemokine ligand 2 production in cultured human glomerular endothelial cells. High glucose increased nuclear translocation of p65 and also increased NF-kappaB DNA binding activity. High glucose-induced NF-kappaB activation occurred without degradation of IkappaBalpha. In agreement with this, there was no increase in serine phosphorylation of IkappaBalpha, while tyrosine phosphorylation of IkappaBalpha was increased by high glucose. High glucose increased the generation of ROS, whereas both alpha-lipoic acid and N-acetylcysteine scavenged the ROS and decreased high glucose-induced tyrosine phosphorylation of IkappaBalpha, nuclear translocation of p65, and NF-kappaB DNA binding activity. Protein kinase C pseudosubstrate inhibited high glucose-induced ROS production, tyrosine phosphorylation of IkappaBalpha, and nuclear translocation of p65. Both BAY 61-3606, a specific inhibitor of Syk protein-tyrosine kinase, and small interfering RNA directed against Syk inhibited high glucose-induced tyrosine phosphorylation of IkappaBalpha as well as p65 nuclear translocation. High glucose increased tyrosine phosphorylation of Syk, while it was inhibited by alpha-lipoic acid and protein kinase C pseudosubstrate. In summary, high glucose-induced NF-kappaB activation occurred not by serine phosphorylation of IkappaBalpha. Our data suggest that ROS-mediated tyrosine phosphorylation of IkappaBalpha is the mechanism for high glucose-induced NF-kappaB activation, and Syk may play a role in tyrosine phosphorylation of IkappaBalpha.
Yang
Won Seok
WS
Department of Internal Medicine, Asan Medical Center, Univ. of Ulsan, Song-Pa, PO Box 145, Seoul 138-736, Korea.
Seo
Jang Won
JW
Han
Nam Jeong
NJ
Choi
Jung
J
Lee
Ki-Up
KU
Ahn
Hanjong
H
Lee
Sang Koo
SK
Park
Su-Kil
SK
D000975:Antioxidants / Q000494:pharmacology; D001711:Biotransformation / Q000187:drug effects; D015153:Blotting, Western; D002460:Cell Line; D018932:Chemokine CCL2 / Q000096:biosynthesis; D024202:Electrophoretic Mobility Shift Assay; D042783:Endothelial Cells / Q000187:drug effects / Q000378:metabolism; D004797:Enzyme-Linked Immunosorbent Assay; D005947:Glucose / Q000494:pharmacology*; D006801:Humans; D007150:Immunohistochemistry; D047468:Immunoprecipitation; D047908:Intracellular Signaling Peptides and Proteins / Q000378:metabolism*; D007678:Kidney Glomerulus / Q000166:cytology / Q000187:drug effects / Q000378:metabolism*; D016328:NF-kappa B / Q000378:metabolism*; D009841:Oligonucleotides / Q000138:chemical synthesis / Q000494:pharmacology; D010766:Phosphorylation; D011505:Protein-Tyrosine Kinases / Q000378:metabolism*; D034741:RNA, Small Interfering / Q000494:pharmacology; D017382:Reactive Oxygen Species / Q000378:metabolism; D000072377:Syk Kinase; D008063:Thioctic Acid / Q000494:pharmacology; D014162:Transfection; D014443:Tyrosine / Q000378:metabolism*
D000975:Antioxidants; D018932:Chemokine CCL2; D047908:Intracellular Signaling Peptides and Proteins; D016328:NF-kappa B; D009841:Oligonucleotides; D034741:RNA, Small Interfering; D017382:Reactive Oxygen Species; D014443:Tyrosine; D008063:Thioctic Acid; D011505:Protein-Tyrosine Kinases; C000606896:SYK protein, human; D000072377:Syk Kinase; D005947:Glucose