The Tat Protein of Human Immunodeficiency Virus Type-1 Promotes Vascular Cell Growth and Locomotion by Engaging the 5β1 and vβ3 Integrins and by Mobilizing Sequestered Basic Fibroblast Growth Factor

DURING ACUTE INFECTION of T cells by human immunodeficiency virus type-1 (HIV-1), Tat, a transactivator of viral gene expression,1 is released extracellularly.2-4 In this form, Tat exerts activities that have linked the protein to the pathogenesis of Kaposi’s sarcoma (KS), a disease of vascular origin that is very common and aggressive in HIV-1–infected individuals (acquired immunodeficiency syndrome-KS [AIDS-KS]) but mild and indolent in the absence of HIV infection.5 In particular, Tat promotes the locomotion and growth of spindle cells of endothelial origin derived from AIDS-KS lesions (KS cells) and of normal endothelial cells,2,3,6-10which are considered to be the precursors of KS cells.11However, endothelial cells become responsive to the effects of Tat only after activation with inflammatory cytokines (IC), such as interleukin-1β (IL-1β), tumor necrosis factor-α (TNF-α), and interferon-γ (IFN-γ).6-10 These IC are the same found to be increased in the lesions and blood of KS patients.12-14 

The requirement of other factors for Tat angiogenic effects is also observed in vivo, because inoculation of Tat protein alone in nude mice has little or no effect.15 In contrast, when Tat is injected with IC or with suboptimal (non–lesion-forming) amounts of basic fibroblast growth factor (bFGF), it promotes the development of angioproliferative KS-like lesions in the inoculated animals.9,15 It is of interest that bFGF production is enhanced in KS cells or induced in endothelial cells by the same IC that are required to promote endothelial cell responsiveness to Tat.16-18 

Tat has also been shown to bind F1K-1/KDR, one of the receptors for the vascular endothelial growth factor (VEGF),19 suggesting an additional mechanism for Tat to exert angiogenic activity. Both bFGF and VEGF, two potent angiogenic factors,20,21 are highly expressed in AIDS-KS lesions,15,22-25 where they synergize in promoting neoangiogenesis and edema.18,25 

Tat can also promote KS and endothelial cell adhesion through the binding of its arginine-glycine-aspartic acid (RGD) region to the α5β1 and αvβ3 integrins.26 These receptors, which bind the RGD sequence of extracellular matrix (ECM) proteins, such as fibronectin (FN) and vitronectin (VN),27 are constitutively expressed by KS cells both in vitro and in primary lesions,15,26 and their levels are increased in normal endothelial cells by the same IC that induce bFGF expression and cellular responsiveness to Tat.10,26 Other data indicated that the basic region of Tat binds αvβ5, an integrin that recognizes similar sequences in VN.28 

Altogether, these results indicated that Tat has properties similar to both angiogenic factors and ECM proteins and that it requires the cooperation of inflammatory or angiogenic cytokines to exert its effects. However, they did not explain the mechanism(s) by which Tat can promote angiogenesis and KS progression.

We report here that the angiogenic effects of Tat are mediated by two domains of the protein. Specifically, the RGD region of Tat induces the migration and invasion of KS and endothelial cells by binding to the α5β1 and αvβ3 integrins. Additionally, the Tat basic sequence, because of its affinity for heparin, releases preformed extracellular-bound bFGF into a soluble form that mediates Tat-promoted vascular cell growth.

Recombinant HIV-1 Tat protein (from the IIIB isolate) was obtained and handled as previously described.2-4,15 The peptides [11-24] Tat, [36-50] Tat, [46-60] Tat, [56-70] Tat, [65-80] Tat, and [72-86] Tat were purchased from American Biotechnologies Inc (Cambridge, MA). The peptide [48-53] Tat, the FN cyclic peptide GRGDSP, and the mutated cyclic peptide GKGESP were purchased from Research Genetics (Huntsville, AL). Human recombinant IL-1α and IL-1β, IL-2, IL-6, oncostatin M, TNF-α and TNF-β, IFN-γ, bFGF, and FN (from human plasma) were purchased from Boehringer Mannheim (Indianapolis, IN). VN (from human plasma), heparin (sodium salt, from porcine intestinal mucosa), human collagen IV, and bovine serum albumine (BSA) fraction V were from Sigma (St Louis, MO). Human recombinant acidic FGF (aFGF) or endothelial cell growth supplement (ECGS), an extract from bovine hypothalamus containing aFGF,29 were purchased from Collaborative Research Inc (Bedford, MA). Human recombinant VEGF, anti-bFGF rabbit polyclonal antibodies, and bFGF enzyme-linked immunosorbent assay (ELISA) kit were from R&D Systems (Minneapolis, MN). The affinity-purified monoclonal antibodies (MoAbs) directed against CDw49e (α5 chain of one of the FN receptors), CD29 (β1 chain of one of the FN receptors), CD51 (αv chain of one of the VN receptors), CD61 (β3 chain of one of the VN receptors; for review, see Hynes27), and the MoAb QBEND/10 directed against CD34, a marker expressed by both KS and endothelial cells,30 were purchased from Amac Inc (Westbrook, ME). MoAbs raised against the whole α5β1 or αvβ3 integrins were purchased from Chemicon (Temecula, CA). MoAbs raised against αvβ5 were a gift from Dr E. Ruoslahti (La Jolla Cancer Research Foundation, La Jolla, CA) or were purchased from Telios, Inc (La Jolla, CA). The MoAb directed against factor VIII-related antigen31 was from Dakopatt (Carpinteria, CA). The sequence, synthesis, and purification of antisense bFGF or sense bFGF phosphorothioate oligodeoxynucleotides (24 mers) directed against the splice donor-acceptor site 1 of bFGF RNA have been previously described.24 Cell culture media and media supplements were purchased from GIBCO-BRL Life Technologies, Inc (Gaithersburg, MD).

Different strains of KS cells (AIDS-KS3, KS4, KS6, KS7, and KS8; passage 6-12) were established and cultured as described previously.32 Three different strains of endothelial cell derived from human umbilical vein (HUVE cells; passage 4-10) were activated with combined IC (IC-HUVE cells), as reported elsewhere.6-10,17 

The migration assays were performed in the Boyden chamber, as previously described.8 Polycarbonate filters (12-μm pore; Nucleoprobe Inc, Cabin John, MD) were coated with type IV collagen or recombinant Tat protein. The invasion assays were performed as the migration assays, with the difference that the filters were coated first with collagen IV and then with matrigel (Collaborative Research), a reconstituted basement membrane derived from a tumor cell line,33 to prevent the migration of noninvasive cells.8 Growth assays were performed by both the cell counting and the thymidine incorporation methods, as described previously,2,3,6,7 with cells seeded onto plates precoated with 1.5% gelatin or with recombinant HIV-1 Tat protein.

In the blocking experiments with antibodies or competitor peptides, cells were seeded onto culture plates (for the growth assays) or resuspended by trypsinization (for the migration and invasion assays) and then preincubated on rotation in RPMI-0.01% BSA containing the competitor peptides or antibodies at the indicated concentrations for either 2 hours at 4°C or for 30 minutes at room temperature. Growth, migration, and invasion assays were then performed as described above.

HUVE cells were incubated for 12 to 14 hours with Tat, [65-80] Tat, bFGF, or their dilution buffer (phosphate-buffered saline [PBS]-0.1% BSA). Total RNA was then extracted from the cells and subjected to electrophoresis (10 μg for each lane) and Northern blot analysis. A³²[P]-labeled oligodeoxynucleotide corresponding to the sequence +59 to +99 of collagenase IV cDNA (encoding the 72-kD form), which detects a 3.4-kb transcript, was used as a probe, as previously described.15 The amount of RNA loaded in each lane was the same as detected by ethidium bromide staining of the gels.

KS cells were incubated for 2 days with conditioned media from activated T cells or with combined IC, which increase bFGF production and release,16 or were cultured in the presence of exogenous bFGF. Cells were lifted nonenzymatically with a dissociation buffer (PBS-based chelating solution; GIBCO-BRL), washed with PBS, and resuspended in RPMI. The plates were also rinsed with PBS. Cells or plates were then treated for 20 minutes with the control buffer (PBS-0.1% BSA), Tat, equimolar concentrations of Tat peptides, or heparin. A limited trypsin digest of cells or plates was used to retrieve the total bFGF bound to cells or ECM.16Supernatants were centrifuged and tested for bFGF content by ELISA. To avoid the loss of bFGF, all samples were handled in plastic ware precoated with PBS-0.1% BSA.

To elucidate the mechanism(s) of Tat activity, initial studies were focused on identifying the domains required for the effect of Tat on KS and endothelial cell migration and invasion. This was performed by using overlapping Tat peptides. These mapping studies indicated that only the peptides containing the RGD region, namely [65-80] Tat and [72-86] Tat, can induce the migration of KS and IC-activated HUVE cells that was observed at concentrations equimolar to Tat (Fig1A, left panel). The migration induced by Tat-RGD region was dose-dependent (Fig1A, right panel).

Therefore, blocking experiments were performed with antibodies directed against RGD-binding integrins expressed by KS and IC-HUVE cells, such as α5β1, αvβ3, and αvβ5.26 As shown in Fig 1B, KS and IC-HUVE cell migration to Tat or to the [65-80] Tat RGD peptide was inhibited by either anti-α5 and anti-β1 antibodies or anti-αv and anti-β3 antibodies but not by anti-αvβ5 antibodies. In addition, inhibition was complete when both anti-α5β1 and anti-αvβ3 antibodies were added together to the cells. To the contrary, antibodies directed against other antigens (CD34 and factor VIII-related antigen) expressed by KS and IC-HUVE cells, respectively26,30,31 (data not shown), had no effects on Tat-promoted cell migration.

Additional experiments performed with MoAbs directed against the whole α5β1 or αvβ3 yielded similar results. Specifically, 70%, 75%, and 90% inhibition of Tat-induced IC-HUVE cell migration was observed with anti-α5β1, anti-αvβ3, or combined anti-α5β1 and anti-αvβ3 antibodies, respectively. Moreover, the anti-α5β1 or anti-αvβ3 antibodies inhibited FN-induced, but not bFGF-induced, cell migration (Fig 1B), indicating that the antibodies were specific34 and nontoxic to the cells. Lastly, migration to Tat was not affected by anti-bFGF antibodies (Fig 1B), indicating that the chemiotactic effect of Tat is not due to the bFGF that is produced by the cells.

Experiments were then performed to investigate the role of the interaction between the Tat-RGD region and integrins in Tat-promoted cellular invasion. As for Tat-induced migration, anti-α5β1 or anti-αvβ3 antibodies inhibited KS and IC-HUVE cell invasion induced by Tat. Complete inhibition was again observed by the simultaneous addition of antibodies directed against both integrins (Fig 1C). The effect of the antibodies was specific, because they did not inhibit cellular invasion promoted by bFGF (Fig 1C). Consistent with these data, the Tat RGD peptide also promoted collagenase IV 72-kD gene expression at levels comparable with those induced by full-length Tat (Fig 1D), which is known to activate collagenase expression during cell invasion.15 Thus, Tat-promoted migration and invasion are mediated by the binding of the RGD region of the protein to the α5β1 and αvβ3 integrins. In contrast, αvβ5, which is known to bind the basic region of Tat,28 is not involved in these effects.

Soluble Tat protein promotes KS and IC-HUVE cell growth.2,3,6,7,10 To clarify the mechanism(s) of this Tat effect, cell growth experiments were performed with the same Tat peptides employed in the previous experiments. As shown in Fig2A, the peptides containing the Tat basic region, [46-60] and [48-53] Tat, induced the growth of both KS and IC-HUVE cells, whereas the RGD-containing peptides, [65-80] and [72-86] Tat, promoted only KS cell growth. In addition, both [46-60] Tat and [65-80] Tat promoted KS cell growth in a dose-dependent fashion (Fig 2A, right panel). Consistent with these results, when the RGD and the basic Tat peptides were added together, KS cell growth increased and reached levels similar to those observed with the Tat protein. Differently from what was observed with KS cells, the combination of [46-60] Tat and [65-80] Tat did not augment HUVE cell proliferation induced by [46-60] Tat alone. Moreover, FN, an RGD-containing molecule,27 promoted KS cell growth at levels similar to those observed with RGD peptides but had no effect on endothelial cells.

Peptide competition experiments were then performed to clarify these data. As shown in Fig 2B, Tat-promoted KS and endothelial cell growth were inhibited only partially when the cells were preincubated with an excess of [46-60] Tat or [65-80] Tat, and complete inhibition of KS and HUVE cell growth was observed only when cells were preincubated with an excess of the two peptides added together to the cells. Thus, both the RGD and the basic region of Tat appear to participate in Tat-promoted cell proliferation, suggesting that more than one pathway is involved in this process. Therefore, the role of these two regions of Tat on cell growth was further analyzed.

To investigate the role of the RGD region and of the Tat-integrin interaction in cellular growth, proliferative assays were performed with Tat in the presence of integrin competitors, such as RGD peptides,27 or with anti-α5β1, anti-αvβ3, or anti-αvβ5 antibodies. RGD peptides, but not the KGE-mutated peptides that were used as controls, and anti-α5β1 or anti-αvβ3 antibodies, but not anti-αvβ5 or control antibodies, blocked Tat-induced KS and IC-HUVE cell growth (Fig3, upper left panel). These results were obtained at concentrations of antibodies or peptides that did not cause cell detachment or affect basal cell growth (Fig 3, upper right panel). Similarly, anti-α5β1 or anti-αvβ3 antibodies inhibited KS cell growth induced by TNF-α, a mitogen for these cells,6 and HUVE cell proliferation induced by ECGS (Fig 3, lower left panel). Thus, as with other growth factors, Tat-induced cell growth requires integrin engagement.

This suggested that, by binding to α5β1 and αvβ3, Tat may provide endothelial cells with the same adhesion signal that is normally provided by ECM molecules and is required by the cells to proliferate in response to mitogens.35,36 In fact, anti-α5β1 and anti-αvβ3, but not anti-αvβ5, antibodies inhibited the proliferation of HUVE cells seeded on Tat-coated plates and stimulated to grow with ECGS (Fig 3, lower right panel). Thus, the interaction of Tat-RGD region with α5β1 and αvβ3 provides the adhesion signal required for cell growth in response to mitogens.

Further studies focused to elucidate the mechanism(s) by which Tat basic region induces the growth of KS and endothelial cells. Basic residues similar to those in Tat are also present in many growth factors, and they bind HSPG associated with the cell membrane and the ECM.37,38 For this reason, basic sequences can displace extracellular-bound angiogenic factors through a competitive effect for heparin-binding sites.38,39 After their release from the cells, both Tat and bFGF bind to cell surface- and ECM-associated HSPG through their basic region, which has a strong binding affinity for heparin.4,37,40 The finding that the binding of Tat to heparin is competed out by bFGF4 suggested that Tat and bFGF could compete for the same heparin-binding sites.

To determine this, KS cells were grown with IC that further increase production and release of bFGF16 and then treated with scalar concentrations (from 0.1 ng/mL to 10 μg/mL) of Tat or Tat basic peptide. A gentle trypsin digest or heparin was used as a positive control, because they are known to release HSPG-bound bFGF into a soluble form.16,37 Soluble bFGF was then measured by ELISA. As compared with trypsin treatment, angiogenic concentrations of Tat or equimolar concentrations of [46-60] Tat basic peptide released about 26% of cell-bound bFGF and 41% of ECM-bound bFGF produced by KS cells (Fig4A). These levels of bFGF were similar to those retrieved by heparin (used to evaluate the total retrievable bFGF).16,37 In contrast, [56-70] Tat peptide (used as a control) was not capable of releasing bFGF. The increase of soluble bFGF was detected 20 minutes after the addition of Tat or heparin to the cells, and the levels remained elevated for about 24 hours and returned to baseline after 48 hours (data not shown). As observed for heparin, Tat was also able to retrieve and maintain into a soluble form exogenous bFGF added to the cells (Fig 4B). Because the addition of Tat did not increase bFGF mRNA or intracellular bFGF content, as determined by Northern blot analysis and ELISA, respectively (data not shown), the increase of extracellular soluble bFGF by Tat is caused by the release of HSPG-bound extracellular protein. Thus, the growth effect of Tat basic region could be due to its capability of retrieving HSPG-bound bFGF produced by KS and IC-activated endothelial cells. In fact, antisense oligomers directed against bFGF mRNA, previously shown to specifically inhibit bFGF expression24 but not control sense bFGF oligomers, blocked Tat-promoted growth of KS cells that constitutively produce bFGF (Fig 4C). This was associated with a reduction of intracellular- and extracellular-bound bFGF content as determined by ELISA after normalization to total protein content. In fact, intracellular bFGF content was reduced upon antisense treatment from 9,606 μg/100 μg of total protein to 3,840 μg/100 μg. Similarly, extracellular bound bFGF was reduced from 1,162 to 770 pg/mL.

Consistent with this result, neutralizing antibodies directed against bFGF22 blocked KS cell growth induced by Tat or by the Tat basic peptide (Fig 4C). Anti-bFGF antibodies also blocked Tat-induced growth of IC-HUVE cells that produce bFGF (Fig 4D). In contrast, anti-bFGF antibodies did not block endothelial cell growth induced by aFGF, which was used to control the antibody specificity (Fig 4D). In agreement with its capability of maintaining exogenously added bFGF in a soluble form, Tat enhanced bFGF-promoted proliferation of nonactivated endothelial cells that do not produce bFGF (Fig 4E). In contrast, Tat slightly inhibited the mitogenic effect of VEGF on endothelial cells (Fig 4E). These results indicated that bFGF specifically triggers Tat-induced vascular cell growth.

In this study, we have analyzed the mechanisms responsible for the angiogenic, KS-promoting effect of Tat. The results indicate that KS and endothelial cell migration and invasion induced by Tat are mediated by the binding of its RGD region to the α5β1 and αvβ3 integrins (Fig 1). The RGD sequence of Tat is also sufficient to activate the expression of collagenase IV 72-kD, which plays a key role in cell invasion and angiogenesis.15,41,42 These data are consistent with results obtained by others with RGD-containing molecules, such as FN or VN,34,41-43 and demonstrate that Tat induces KS and endothelial cell locomotion by a molecular mimicry of ECM molecules. They are also in agreement with the fact that Tat induces the synthesis and release of collagenase IV 92 kD in monocytes44 and with the recent finding that Tat activates members of the focal adhesion kinase family that are induced by integrin triggering and that play a major role in cellular migration.45,46 

Differently from Tat-induced cell locomotion, the basic and RGD region of Tat are both required for Tat-induced cell growth (Fig 2). It appears that the interaction of Tat with α5β1 or αvβ3 provides KS and endothelial cells with the adhesion signal that is required for their growth in response to mitogens. Consistent with this, endothelial cells adhere to immobilized Tat in a fashion similar to FN or VN,26 and under these conditions, the addition of exogenous bFGF dramatically increases cell growth,15 as previously described for ECM molecules.35,36,47 The involvement of the RGD region in Tat-induced cell growth is also consistent with previous data indicating that α5β1, which recognizes the RGD region of both FN and Tat, mediates cellular growth promoted by FN in other systems,48,49 and that the RGD region of Tat promotes in KS cells the expression of the same mitogen-activated protein kinases that are induced by integrin triggering.45 

However, the adhesion signal is not sufficient to induce growth of normal endothelial cells that also need the presence of an angiogenic factor that triggers cell proliferation.50 However, Tat RGD peptide or FN are mitogenic for KS cells (Fig 2). Because this does not occur with normal cells (for review, see Levesque et al51), these data suggest that KS cells may possess a difference in intracellular signaling through integrins, as indicated by previous studies with tumor cells.52 

Differently from the Tat RGD peptide, full-length Tat protein or Tat basic peptide are capable of promoting the growth of both KS and normal endothelial cells, suggesting that Tat provides cells additionally with a cell growth triggering signal.

Previous studies suggested that bFGF, which is a KS and endothelial cell growth factor,20,22,24 may mediate Tat-induced vascular cell proliferation. Basic FGF, in fact, is present in all systems in which Tat has a growth effect. Specifically, exogenous bFGF is required to observe the angiogenic effect of Tat in vivo.15 Moreover, bFGF is produced and released extracellularly by KS cells that spontaneously proliferate in response to Tat2,3,16,22 and by IC-activated endothelial cells that, after this activation, proliferate with Tat.6,7,10,17 After its release, bFGF binds the cell surface and the ECM-associated HSPG remaining soluble only in a small fraction.16,37 The bound bFGF fraction represents a localized storage of the growth factor that is protected from proteolytic degradation and can be retrieved by treatment of the cells with heparin, heparinase, or trypsin.16,37 

Several growth factors can bind HSPG through their basic residues (for review, see Raines and Ross38). In contrast, other angiogenic molecules, such as the majority of VEGF isoforms, lack basic residues and, therefore, do not bind the HSPG associated to the surface of producer cells or to ECM, remaining soluble and diffusible.21 Similarly to bFGF, Tat binds heparin through its basic sequence and can compete with bFGF for binding to heparin.4 Heparin, in fact, can inhibit the mitogenic effect of Tat (data not shown), as previously found for cellular adhesion to immobilized Tat28 and transactivation of HIV-1 gene expression by extracellular Tat.4 Our data indicate that the basic residues of Tat can displace preformed HSPG-bound bFGF by competing for cell surface- and ECM-associated heparin binding sites (Fig 4). This leads to an increase of the soluble fraction of bFGF at levels promoting KS and endothelial cell growth, as shown by the inhibition of Tat-induced cell growth by antisense bFGF oligomers (reducing both the intracellular and extracellular content of bFGF) or by neutralizing anti-bFGF antibodies (Fig 4). Consistent with its capability of maintaining exogenously added bFGF in a soluble and highly diffusible form, Tat enhances endothelial cell proliferation promoted by bFGF. In contrast, Tat does not augment the growth effect of VEGF on endothelial cells (Fig 4). Thus, although Tat binds the VEGF receptor Flk-1/KDR,19,45 this does not lead to cell growth. This is consistent with the finding that, differently from Tat or bFGF, VEGF does not promote KS cell proliferation.18 Moreover, Tat promotes the growth of IC-activated endothelial cells that produce bFGF but not VEGF.18 Furthermore, in primary KS lesions, the VEGF amounts are much higher than that of Tat,15,25making unlikely an action (either activatory or inhibitory) of Tat on the VEGF receptor. Thus, although both VEGF and bFGF are highly expressed in AIDS-KS lesions, Tat synergizes with bFGF, and not with VEGF, in promoting neoangiogenesis and, therefore, KS development and progression.

In conclusion, the results described herein demonstrate that the Tat protein of HIV-1 is not directly angiogenic, but it enhances angiogenesis by mimicking the effects of ECM proteins on cell migration, invasion, and adhesion, and by mobilizing bFGF, a true angiogenic factor, which acts as the final mediator of Tat-induced KS and endothelial cell growth. This explains why Tat needs exogenous bFGF or factors promoting bFGF expression, such as IC, to exert its angiogenic effect.

IC and bFGF are highly expressed in AIDS-KS lesions, where extracellular Tat costains with the α5β1 and αvβ3 integrins on both spindle cells and vessels.15 This suggests that the mechanisms of Tat action described here are operative in vivo and that integrin, bFGF, and Tat competitors should be considered as a therapeutic strategy for AIDS-KS.

The authors thank Dr E. Ruoslahti for some of the anti-αvβ5 antibodies and A. Lippa and F.M. Regini for editorial assistance.