PMC

Time-course analysis of ERK1/2 activation induced by Tat in HUVEC and EA-hy 926 cells. HUVEC (A) and EA-hy 926 (B) cells starved for 24 and 36 h in serum-free medium, respectively, were stimulated with Tat conjugated to polystyrene beads for different time points. The bottom panels of each set of immunoblots represent total ERK present in the cell lysates.

Tat induces Shc phosphorylation and recruitment of its adaptor Grb2. Equal amounts of total proteins from both type of cells treated with 1 μg of Tat (middle panels) were immunoprecipitated with anti-Shc antibody followed by immunoblotting with either anti-phosphotyrosine or anti-Grb2 mAbs. Tat induces the Shc phosphorylation on tyrosine residues. (A and B) Shc associates with Grb2 within 5 min in HUVEC and EA-hy 926 cells. Intensities of the phosphorylated bands of Shc (arbitrary units, AU) were measured, and values are given on bottom of the top panels.

Activation of Ras and Rac upon stimulation of endothelial cells with Tat. Active GTP-bound Ras was pulled down from lysates of EA-hy 926 cells with GST-Raf-RBD coupled to glutathione agarose, and Ras was determined by immunoblotting with a Ras antibody. Tat rapidly activates Ras within 1 min; the activation is still persistent at 3 min and starts to decrease at 5 min. A pulldown assay with a GST-fusion protein corresponding to the p21-binding domain of PAK-1 was performed on EA-hy 926 cells stimulated for different times with Tat. Activation of Rac by Tat, FN, or BSA is shown in the top panel. The bottom panels of each set of immunoblots represent either total Ras or total Rac present in the cells lysates.

ERK phosphorylation by Tat is induced via activation of Ras. (A) EA-hy 926 cells were transiently transfected with a dominant-negative of Ras (S17N) that prevented the phosporylation of ERK1/2 induced by Tat. To confirm these data and to clarify the role of Rac, EA-hy 926 cells were transiently transfected with siRNAs previously identified for their efficiency to target key sites of the mRNA encoding for Ras or Rac. (B) The silencing of Ras decreased ERK1/2 phosphorylation and no induction was observed after stimulation with Tat. (C) In contrast, the silencing of Rac caused a reduction of its expression, but had only a small effect on phosphorylation of ERK1/2 stimulated by Tat. The blots were stripped and reprobed with anti-ERK antibody to confirm equal loading (middle panels). Intensities of both 42 and 44 phosphorylated bands of ERK1/2 (arbitrary units, AU) were measured and values are given on bottom of the top panels.

Tat promotes endothelial cell cycle progression through ERK1/2 activation. The EA-hy 926 cells were seeded on type I collagen and preincubated with 25 μM PD 98059 or 10 μM SB 203580 for 20 min. Cells were then stimulated with 10 μg/ml Tat for 24 h and pulsed with BrdU. (A) A reduced percentage of cells incubated with PD 98059 and Tat (61%, b) entered into the S phase compared with the percentage of cells incubated with Tat and DMSO (80%, a) or SB 203580 and Tat (82%, c). The role of ERK1/2 was further analyzed in EA-hy 926 cells transfected with the kinase-dead MEK-K97M and then incubated for 24 h in a medium containing bFGF, and Tat or FN. (B) Entry into the S phase was monitored by BrdU incorporation, as described above. Only 8% of the cells transfected and treated with Tat incorporated BrdU compared with 70% of the cells transfected with the control vector (a and c). Similar results have been obtained in cells treated with FN (b and d).

Tat promotes endothelial cell cycle progression through the induction of Cyclin D1 expression. (A) HUVEC and EA-hy 926 cells were plated on type I collagen, synchronized in G0, and incubated for 24 h in a medium containing 25 ng/ml bFGF in the presence of 10 μg/ml Tat, or 25 μg/ml FN or BSA as control. Entry into the S phase was examined by 5′-bromo-2′-deoxy-uridine (BrdU) incorporation and anti-BrdU staining. A consistent amount of HUVECs incubated with Tat (18%) or FN (19%), compared with BSA (2%), entered into the S phase during the 24 h of the assay. Similarly, stimulation of EA-hy 926 cells with Tat and FN induced 40 and 67% of the cells to incorporate BrdU, whereas only 13% of the cells incubated in the presence of BSA entered the S phase. (B) HUVEC and EA-hy 926 cells synchronized in G0 were treated as described above. Immunoblotting with anti-cyclin D1 antibody was then performed on equal amounts of total proteins obtained from the cell lysates.

Tat promotes, through its RGD region, ERK1/2 activation on endothelial cells. (A) HUVECs were stimulated with the [46-60] and [66-80]Tat peptides mapping in the basic and RGD region, respectively. [1-20]Tat and [57-70]Tat peptides were used as controls. As a positive control, the cells were stimulated with FN, whereas BSA was used as negative control. Immunoblotting with anti-phosphoERK antibody was then performed on equal amounts of total proteins obtained from the cell lysates, as shown in the bottom panel. (B) The cells were pretreated with two different doses (1 or 2 μg/10⁶ cells) of the blocking mAbs against integrins α5β1, αvβ3, α4β1, or a mAb anti-MHC class I as control and were then stimulated with 1 μg of Tat. Immunoblotting with anti-phosphoERK antibody on cell lysates indicates that ERK1/2 activation by Tat is decreased by treatment with 2 μg of the antibodies against α5β1 and αvβ3. Combination of the antibodies against α5β1 and αvβ3 (1 μg each) synergistically reduces ERK phosphorylation induced by Tat. In contrast, both doses of mAb anti-α4β1 do not significantly influence the activation of ERK promoted by Tat. Intensities of both 42 and 44 phosphorylated bands of ERK1/2 (arbitrary units, AU) were measured and values are given on bottom of the top panels. An equal amount of ERK was used in this analysis (bottom panel).