Purchase this article with an account.
J. M. Tarr, N. Ding, L. A. Peréz-Jurado, A. Antonell, T. L. Leto, E. M. Kohner, R. Chibber; TNF--Induced Activity of Core 2 β-1, 6-N Acetylglucosamineyltransferase in Diabetic Leukocytes Requires a NADPH Oxidase Signalling Pathway. Invest. Ophthalmol. Vis. Sci. 2010;51(13):5084.
Download citation file:
© ARVO (1962-2015); The Authors (2016-present)
To explore the NADPH oxidase signalling pathway in the tumour necrosis factor-alpha (TNF-α)-induced activity of core 2 β-1, 6-N-acetylglucosamineyltransferase (C2GNT) (Ben-Mahmud et al., 2006) in diabetic leukocytes.
Human leukocytes (U937 cells) and a lymphoblastoid cell line (FI0007) from a patient with p47phox-deficient chronic granulomatous disease were cultured in RPMI medium containing normal (5.6mM) glucose. Cells were exposed to TNF-α (8pg/ml) for 24h in the presence and absence of (i) NADPH oxidase inhibitors (30µM apocynin and 1µM scrambled and unscrambled gp91ds-tat), (ii) LY379196 (specific PKCβ1/2 inhibitor, 50nM) and (iii) antioxidants N-acetyl cysteine (NAC, 15mM) and Tiron (5mM). NADPH oxidase activity was measured using cytochrome c reduction assay. The FI0007 cell line was transfected with p47phox DNA using Roche FuGENE 6 transfection reagent.
Compared to control medium, exposure to TNF-α raised C2GNT [927.5±199.4 vs. 166.5±54.16 (n=12), P<0.001] and NADPH oxidase [100±0 vs. 142.0±16.89 (n=5), P<0.005] activity in U937 cells that was significantly reversed with apocynin and LY379196. These findings were supported using antioxidants (NAC and Tiron) and gp91ds-tat (scrambled and unscrambled), a specific NADPH oxidase inhibitor. Treatment of FI0007 cells with TNF-α failed to increase C2GNT and NADPH oxidase activity. The C2GNT and NADPH oxidase response to TNF-α was restored [400.1±164.4 vs. 990.4±294.9 (n=10), P<0.02] by transfection with an expression plasmid containing a p47phox cDNA inserted in the sense direction.
Our results demonstrate a novel signalling crosstalk between TNF-α, C2GNT, NADPH oxidase and PKCβ1/2 in diabetic leukocytes.
This PDF is available to Subscribers Only