Tat-Mediated p66shc Transduction Decreased Phosphorylation of Endothelial Nitric Oxide Synthase in Endothelial Cells

Abstract

We evaluated the role of Tat-mediated p66shc transduction on the activation of endothelial nitric oxide synthase in cultured mouse endothelial cells. To construct the Tat-p66shc fusion protein, human full length p66shc cDNA was fused with the Tat-protein transduction domain. Transduction of TAT-p66shc showed a concentration- and time-dependent manner in endothelial cells. Tat-mediated p66shc transduction showed increased hydrogen peroxide and superoxide production, compared with Tat-p66shc (S/A), serine 36 residue mutant of p66shc. Tat-mediated p66shc transduction decreased endothelial nitric oxide synthase phosphorylation in endothelial cells. Furthermore, Tat-mediated p66shc transduction augmented TNF-α-induced p38 MAPK phosphorylation in endothelial cells. These results suggest that Tat-mediated p66shc transduction efficiently inhibited endothelial nitric oxide synthase phosphorylation in endothelial cells.

Keywords: Endothelial cells; Endothelial nitric oxide synthase; Superoxide; Tat-mediated transduction; p66shc.

Fig. 1

Schematic diagrams, Commassie blue staining and purification of Tat-p66shc fusion proteins. (A) The human p66shc coding frame is presented with six histidines and the Tat PTD domain (RKKRRQRRR). Diagram of the p66shc fusion protein was represented on the vector. (B) Commassie blue staining of the Tat-p66shc protein. Lane 1 (L) : Escherichia coli BL21 (DE3) lysates after ITPG induction, Lane 2 (W): Eluate with 10 mM imidazole-containing buffer Z, Lanes 3, 4. The serial eluates with 250 mM imidazolecontaining buffer Z. (C) Purification steps for Tat-p66shc fusion proteins.

Fig. 2

Transduction of Tat-p66shc proteins in cultured MS-1 endothelial cells. (A) Time-dependent cellular uptake of Tat-p66shc. Purified Tat-p66shc (30 nM) was added to the culture medium for the indicated times, and then cell lysates were subjected to Western blot analysis with anti-SHC antibody. Densitometric data analysis is plotted at the bottom. Expression levels are shown as % expression to maximum. Each bar shows the mean±S.E. (n=3). (B) Dose-dependent transduction of Tat-p66shc. Purified Tat-p66shc proteins were incubated with the indicated concentration for 3 h. Densitometric analysis data is plotted at the bottom. Expression levels are shown as % of β-actin. Each bar shows the mean±S.E. (n=3). Arrows at 66, 52, and 46 indicate the endogenous SHC protein expression. Arrow at 70.9 indicates the Tat-p66shc protein.

Fig. 3

Tat-p66shc transduction increased hydrogen peroxide and superoxide production in culture MS-1 endothelial cells. (A) Effect of Tat-p66shc on hydrogen peroxide production. Intracellular hydrogen peroxide production was evaluated using the peroxide-sensitive fluorophore 2',7'-dichlorodihydrofluorescein diacetate (DCF-DA). Control: p66shc protein alone, Tat-GFP (30 nM), Tat-p66shc (30 nM), Tat-p66shc S/A (30 nM). (B) Effect of Tat-p66shc on superoxide production. Intracellular superoxide production was evaluated using lucigenin chemiluminescence. Superoxide production levels are expressed as relative luminescence units, RLU/10⁵ cells. Control: p66shc protein alone, Tat-GFP (30 nM), Tat-p66shc (30 nM), Tat-p66shc S/A (30 nM). Each bar shows the mean±S.E. (n=5). NS, not significant, ^*p<0.05, ^#p<0.05 versus p66shc.

Fig. 4

Tat-p66shc transduction decreased endothelial nitric oxide synthase (eNOS) phosphorylation in culture MS-1 endothelial cells. (A) Effect of Tat-p66shc on basal eNOS phosphorylation. Tat-p66shc and Tat-GFP were incubated for 3 h with endothelial cells at the indicated concentrations, and then cell lysates were subjected to Western blot analysis. Note: Cells were not serum starved to detect basal eNOS phosphorylation. (B) Effect of Tat-p66shc on eNOS phosphorylation in the tumor necrosis factor (TNF)-α-stimulated endothelial cells for 0~45 min. Tat-p66shc (30 nM) and Tat-GFP (30 nM) were incubated for 3 h, and then TNF-α was treated for the indicated times. The cells were serum starved for 18 h to reduce basal p38 MAPK and eNOS phosphorylation. This figure shows a representative experiment out of 3 made.