Tat-promoted vascular cell locomotion and collagenase IV activation are mediated by the binding of Tat-RGD region to 5β1 and vβ3. (A) shows the results of the migration assays with KS cells (▪) and IC-HUVE cells (□). bFGF (20 ng/mL) and Tat (20 ng/mL) were used as the positive controls, whereas the peptide resuspension buffer (PBS-0.1% BSA) was the negative control. In the left panel, Tat peptides were used at concentrations equimolar to Tat. In the right panel, Tat peptides were serially diluted to test whether their effect was dose-dependent. The location of the peptides in Tat amino acid sequence is shown on the top of the figure. (B) shows KS (▪) and IC-HUVE cell (□), migration to Tat (20 ng/mL), [65-80] Tat (4 ng/mL), FN (30 μg/mL), or bFGF (20 ng/mL) after preincubation of the cells with MoAbs (2 μg/mL each) directed against the  and β chains of 5β1 (anti-5β1), vβ3 (anti-vβ3), or vβ5 (anti-vβ5). Immunohistochemical analyses indicated that KS and HUVE cells express all these integrin chains26 (data not shown). Antibodies directed against CD34 or factor VIII-related antigen (antigens that are expressed by KS and IC-HUVE cells, respectively)30,31 (data not shown) were used as controls (CR-Ab). Polyclonal antibodies neutralizing the activity of bFGF (anti-bFGF)22 were used to determine the role of this cytokine in Tat-induced cell migration. The antibody dilution buffer (PBS-0.1% BSA) was the negative control. For (A) and (B), results (from 4 experiments, each in duplicate) refer to the number of migrated cells/field (average of 5 fields/filter) and are expressed as the percentage increase of cell migration over the number of cells migrated toward buffer (0% increase), which was 20 (±2) cells/field for KS cells and 15 (±1) cells/field for IC-HUVE cells. (C) shows KS (▪) and IC-HUVE (□) cell invasion to Tat or bFGF (20 ng/mL/each) after preincubation of the cells with anti-5β1 and/or anti-vβ3 MoAbs (2 μg/mL) or with buffer (PBS-0.1% BSA), as described in Materials and Methods. The results shown are from 3 experiments, each in duplicate, and they are relative to the number of invaded cells per field (average of 5 fields/filter). Data are expressed as the percentage increase of cell invasion toward Tat or bFGF over the number of invaded cells in the presence of buffer (0% increase), which was 10 (±2) cells/field for KS cells and 9 (±1) cells/field for IC-HUVE cells. (D) shows the Northern blot analysis of collagenase IV 72-kD gene expression in HUVE cells incubated with Tat (10 ng/mL), [65-80] Tat (2 ng/mL), bFGF (1 μg/mL, positive control), or dilution buffer (PBS-0.1% BSA, negative control). The amount of RNA loaded in the gels was always monitored by ethidium bromide staining before Northern blotting. Repeated experiments (4 times) gave similar or identical results. The Tat concentration used is the most active in inducing collagenase IV 72-kD mRNA expression.15 At all the concentrations tested, Tat and [65-80] Tat were equally potent (data not shown). Preincubation of HUVE cells with [72-86] Tat reproduced the results obtained with [65-80] Tat (data not shown).