Mouse MS-1 endothelial cells were purchased from the American Type Culture Collection (Manassas, VA, USA). Cells were grown in Dulbecco's-modified Eagle medium (DMEM) with 10% fetal bovine serum, 10 U/ml penicillin, and 10 µg/ml streptomycin. Antibodies to Shc (SC-1695, Santa Cruz Biotechnology, Santa Cruz, CA, USA), phospho-ser36-p66shc (CN566807, Calbiochem, La Jolla, CA, USA), eNOS (SC-654, Santa Cruz Biotechnology, Santa Cruz, CA, USA), phospho-ser1199-eNOS (#9571, Cell Signaling Technology, Danvers, MA, USA), and β-actin (SC-1616, Santa Cruz Biotechnology, Santa Cruz, CA, USA) were used. Horseradish peroxidase (HRP)-labeled anti-rabbit antibody was obtained from Pierce biotechnology (Rockford, IL USA). Ni-nitrilotriacetic acid Sepharose was purchased from Qiagen (Valencia, CA, USA). Isopropyl-β-thiogalactoside (IPTG) was purchased from USB (Cleveland, OH, USA).

Tat-p66shc was generated by inserting p66shc cDNA into pTAT-2.1. The human p66shc was isolated from Xpress-tagged p66shc cDNA in pcDNA3/1/His A (Invitrogen, Carlsbad, CA, USA) [10] by PCR using the following two primers; the sense primer was 5'-CGG GAT CCC GGA ATT CGG CTT ATG GAT CTC C-3' (containing an EcoRI restriction site), and the antisense primer was 5'-CGA AGC TTT CAC AGT TTC CGC TCC ACA GG-3' (containing a HindIII restriction site). After digesting with EcoRI and Hind III, the full length p66shc constructs were cloned into the pTAT bacterial expression vector (pTAT-2.1, kindly donated by Steven Dowdy), which contains a six-histidine tag, for easy purification. p66shc (S36A) cDNA was kindly donated by Kaikobad Irani (Pittsburgh University). pTAT-p66shc plasmids were then transformed into the BL21 (DE3) strain of E. coli. Following 4 hours of induction with IPTG, the cells were sonicated in buffer Z (8 M urea, 100 mM NaCl, 20 mM HEPES) and recombinant proteins were purified on a Ni-NTA agarose column (Qiagen). After washing, TAT-p66shc was eluted using 250 mM imidazole containing Buffer Z followed by desalting on a PD-10 column (Amersham Pharmacia Biotech, Piscataway, NJ, USA) in PBS. The eluate was frozen in 10% glycerol at -80℃. p66shc protein as control protein was purified from pET28b/p66shc expression vector with same protocol for Tat-p66shc.

MS-1 cells were grown in DMEM at 37℃ in humidified 95% air/5% CO₂. The cells were grown to confluence on a 6-well plate and then treated with various concentrations of Tat-p66shc for the indicated times. Cell extracts were prepared for Western blot analysis.

MS-1 cells were harvested with 100 µl of lysis buffer containing 20 mM Tris-Cl, pH 7.5, 100 mM NaCl, 2 mM EDTA, 2 mM EGTA, 1 mM Na₃VO₃, 1 mM beta-glycerophosphate, 4 mM Na pyrophosphate, 5 mM NaF, 1% Triton X-100, and a protease inhibitor cocktail. The lysate was centrifuged at 12,000 rpm for 20 min, and the supernatant was collected. Protein (30 µg) was separated by 10% SDS-PAGE and was electrotransfered onto nitrocellulose membranes. After blocking with 5% skim milk for 2 h at room temperature, blots were incubated overnight at 4℃ with specific primary antibody (1:1,000) and subsequently with horseradish peroxidase (HRP)-conjugated secondary antibody. Blots were developed for visualization using an enhanced chemiluminescence detection kit (Pierce). Cells were serum starved for 18 h in some experiments to reduce basal phosphorylation of specific proteins.

Intracellular hydrogen peroxide production was detected using the peroxide-sensitive fluorophore 2',7'-dichlorodihydrofluorescein diacetate (DCF-DA), as described previously [6,16]. The cells were cultured in chamber slides (2×10⁵ cells/well) (Nalgen Nunc International, Rochester, NY, USA) to detect DCF-DA fluorescence. The cells were rinsed three times and incubated for 30 min with 10 µM DCF-DA at 37℃ in Krebs-HEPES buffer and Hanks buffered salts solution, respectively. The absolute fluorescence of 20~25 random cells was quantified using MetaMorph software (Molecular Devices, Sunnyvale, CA, USA). Superoxide production was assayed in the cultured endothelial cells using lucigenin chemiluminescence. In briefly, darkadapted lucigenin solution (5 µM) was prepared in aerated Krebs-Hepes buffer. Cells were immersed in lucigenin solution and chemiluminescence detected with a Monolight luminometer. The chemiluminescence signal was integrated over 2 minutes.

Values are expressed as means±SEM. The statistical evaluation was conducted a one-way analysis of variance followed by a Tukey's post-hoc test, and p<0.05 was considered statistically significant.

First, we constructed the Tat-p66shc fusion protein using recombinant DNA technology. The Tat-p66shc expression vector contained a consecutive cDNA sequence encoding human p66shc, the Tat-PTD (RKKRRQRRR), and six histidine residues at the amino-terminus (Fig. 1). Tat-p66shc proteins were serially eluted with the treatment of 250 mM imidazole containing buffer Z. The purified Tat-p66shc proteins were confirmed by Commassie blue staining, which showed a molecular weight of approximately 70.5 kDa (Fig. 1).

Purified Tat-p66shc (30 nM) was added to cultured endothelial cells for various incubation times to evaluate the transduction ability in cultured endothelial cells. After the incubation, the cells were harvested and the change in transduced p66shc was analyzed with Western blotting using the anti-Shc antibody. As shown in Fig. 2A, the Tat-p66shc fusion protein was detected in the cell lysates within 15 min and its transduction reached a maximum at 3 h. A significant level of transduced Tat-p66shc was present in the cells at 24 h (Fig. 2A). Moreover, Tat-p66shc fusion proteins were transduced into the cells in a dose-dependent manner in the range of 1~100 nM (Fig. 2B).

We next studied whether transduced Tat-p66shc affected hydrogen peroxide and superoxide production in cultured endothelial cells. Endothelial cells were incubated with Tat-p66shc, Tat-p66shc (S/A), p66shc, and Tat-GFP as a control for 3 h. After the incubation, hydrogen peroxide and superoxide production was measured in the endothelial cells using DCF-DA and lucigenin chemiluminescence. As shown in Fig. 3, Tat-p66shc (30 nM) for 3 h significantly elevated intracellular hydrogen peroxide compared with Tat-GFP and p66shc as control in endothelial cells. Also, the Tat-p66shc incubation (30 nM) increased superoxide production, as accessed by lucigenin chemiluminescence. However, Tat-p66shc-mediated ROS productions were greater than that of Tat-p66shc (S/A), suggesting important role of serine 36 residue of p66shc in ROS productions in endothelial cells.

To understand the acute effect of p66shc on eNOS activity, we examined the effect of Tat-p66shc transduction on eNOS phosphorylation in cultured endothelial cells. In comparison to Tat-GFP treated cells, Tat-p66shc transduction did not affect eNOS protein expression; however, it decreased eNOS phosphorylation at the serine 1177 residue (Fig. 4A).

Finally, we investigated the role of Tat-p66shc protein transduction on eNOS phosphorylation in tumor necrosis factor (TNF)-α-stimulated endothelial cells. As shown in Fig. 4B, TNF-α increased p38 MAPK and eNOS phosphorylation. In contrast, Tat-p66shc protein transduction (30 nM for 3 h) augmented TNF-α-induced p38 MAPK phosphorylation and abrogated TNF-α-induced eNOS phosphorylation in endothelial cells.