αvβ3-integrin-dependent activation of focal adhesion kinase mediates NF-κB activation and motogenic activity by HIV-1 Tat in endothelial cells

Abstract

Once in the extracellular environment, the transactivator protein HIV-1 Tat exerts several pleiotropic effects by interacting with different cellular receptors, including integrin αvβ3. Real-time surface plasmon resonance analysis reveals that Tat/αVβ3 interaction occurs with rapid kinetics (association and dissociation rates equal to 1.16×107 M-1 s-1 and 3.78×10-1 s-1, respectively) and high affinity (dissociation constant = 32 nM). Through this interaction, substratum-immobilized Tat promotes adhesion and motogenic activity in endothelial cells. Also, αvβ3/Tat interaction triggers the activation of focal adhesion kinase, RhoA and pp60src. Overexpression of the dominant negative form of focal adhesion kinase, but not of an inactive Leu1034Ser substitution mutant isoform, impairs the activation of focal adhesion kinase and RhoA, but not that of pp60src, without affecting endothelial cell adhesion and spreading. αvβ3/Tat interaction triggers the activation of NF-κB in endothelial cells in a focal adhesion kinase-, RhoA- and pp60src-dependent manner, as shown in dominant negative focal adhesion kinase transfectants or using specific pharmacological inhibitors. Finally, the activation of focal adhesion kinase, RhoA, NF-κB and pp60src are required to mediate the motogenic activity of Tat in endothelial cells. Since Tat accumulates in an immobilized form in the extracellular matrix, these results provide new biochemical and biological insights about αvβ3/Tat interaction exploitable for the design of anti-Tat strategies.

BSA conjugates bearing multiple copies of the basic domain of HIV-1 Tat: Prototype for the development of multitarget inhibitors of extracellular Tat

“…* Peptides fused to BSA on the average of 4 copies/BSA monomer (Friedler et al, 1999). lial cells (ECs) (Rusnati and Presta, 2002a)], and inducing a variety of biological effects that contribute to the pathogenesis of AIDSassociated pathologies (Rusnati and Presta, 2002a). Tat exerts these effects by interacting with different cell-surface receptors including integrins (Urbinati et al, 2005a), the vascular endothelial growth factor receptor 2 (KDR) (kinase insert domain receptor) (Albini et al, 1996b) and the chemokine receptors CXCR4 and CCL5 (Albini et al, 1998). Interestingly, the interaction of Tat with integrins and KDR occurs, at least in part, through the basic domain/NLS of Tat (Rusnati et al, 2001b;Urbinati et al, 2005a), as already seen for HSPGs.…”

“…Tat exerts these effects by interacting with different cell-surface receptors including integrins (Urbinati et al, 2005a), the vascular endothelial growth factor receptor 2 (KDR) (kinase insert domain receptor) (Albini et al, 1996b) and the chemokine receptors CXCR4 and CCL5 (Albini et al, 1998). Interestingly, the interaction of Tat with integrins and KDR occurs, at least in part, through the basic domain/NLS of Tat (Rusnati et al, 2001b;Urbinati et al, 2005a), as already seen for HSPGs. At variance, the interaction of Tat with chemokine receptors occurs through its cystein-rich domain (amino acid sequence 24-51) (Albini et al, 1998).…”

“…For the study of BSA-Tat-NLS/␣ v ␤ 3 interaction, an experimental model already set up was used (Urbinati et al, 2005a). Briefly, 50 g/ml of BSA-Tat-NLS were allowed to react with a CM5 sensorchip activated as described above, leading to the immobilization of 10,200 RU (0.3 pmoles/mm 2 of BSA-Tat-NLS).…”

“…Photomicrographs were taken under an inverted microscope (Olympus 1×51 microscope with a Camedia C-4040 digital camera, ×10/0.25; Olympus Biosystem, Munich, Germany). The area of the scratch was evaluated by computerized image analysis using the Image Pro-Plus analysis system (Urbinati et al, 2005a).…”

“…As mentioned in the Introduction, the basic domain/NLS of Tat contributes to the interaction of extracellular Tat with integrins (Urbinati et al, 2005a) and KDR (Albini et al, 1996b) that, in turn, trigger a complex signal transduction pathway that leads to proangiogenic activation of ECs (Urbinati et al, 2005a,b). On these bases, we investigated the possibility that, besides HSPGs, BSA-Tat-NLS binds also integrin ␣ v ␤ 3 and KDR, masking them to native Tat.…”