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. 2004 Jan;78(2):779-89.
doi: 10.1128/jvi.78.2.779-789.2004.

Human immunodeficiency virus type 1 Tat regulates endothelial cell actin cytoskeletal dynamics through PAK1 activation and oxidant production

Ru Feng Wu  1 , Ying Gu, You Cheng Xu, Stefania Mitola, Federico Bussolino, Lance S Terada
Affiliations

Human immunodeficiency virus type 1 Tat regulates endothelial cell actin cytoskeletal dynamics through PAK1 activation and oxidant production

Ru Feng Wu et al. J Virol. 2004 Jan.

Abstract

Human immunodeficiency virus type 1 Tat exerts prominent angiogenic effects which may lead to a variety of vasculopathic conditions in AIDS patients. Because endothelial cells undergo prominent cytoskeletal rearrangement during angiogenesis, we investigated the specific effects of Tat on the endothelial cell actin cytoskeleton. Glutathione S-transferase (GST)-Tat, at a level of 200 ng/ml (equivalent to 52 ng of Tat/ml), caused stress fiber disassembly, peripheral retraction, and ruffle formation in human umbilical vein endothelial cells (HUVEC) and human lung microvascular endothelial cells. At 600 ng of GST-Tat/ml (157 ng of Tat/ml), actin structures were lost, and severe cytoskeletal collapse occurred. In contrast, GST-Tat harboring mutations within either the cysteine-rich or basic domains exerted minimal effects on the endothelial cytoskeleton. HUVEC expressing a DsRed-Tat fusion protein displayed similar actin rearrangements, followed by actin collapse, whereas neighboring nontransfected cells retained normal actin structures. Because active mutants of p21-activated kinase 1 (PAK1) induce identical changes in actin dynamics, we hypothesized that Tat exerts its cytoskeletal effects through PAK1. GST-Tat activated PAK1 within 5 min, and adenovirus delivery of a kinase-dead PAK1 [PAK1(K298A)] completely prevented cytoskeletal collapse induced by GST-Tat or DsRed-Tat and also blocked downstream activation of c-Jun N-terminal kinase. Further, GST-Tat increased phosphorylation of the NADPH oxidase subunit p47(phox) and caused its rapid redistribution to membrane ruffles. PAK1(K298A) blocked p47(phox) phosphorylation, and interference with NADPH oxidase function through superoxide scavenging or through expression of a transdominant inhibitor, p67(V204A), prevented GST-Tat-induced alterations in the actin cytoskeleton. We conclude that Tat induces actin cytoskeletal rearrangements through PAK1 and downstream activation of the endothelial NADPH oxidase.

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Figures

FIG. 1.
FIG. 1.
Effect of GST-Tat on endothelial cell actin dynamics. (a) HUVEC were transfected with actin-GFP to visualize actin microfilament structures. Cells were subsequently treated with GST-Tat or GST alone for the indicated times and observed live and unfixed. At a concentration of 200 ng/ml, loss of stress fibers and peripheral retraction were seen within 5 min (b and c), and ruffling was observed within 5 min and peaking at approximately 20 min (b to d, arrows). (e) GST alone had no effect on actin structures. (g) At a concentration of 600 ng/ml, ruffling occurred early and was followed by the appearance of actin retraction fibers. (h) By 60 min, all cells showed marked collapse of the actin skeleton, with most cells displaying a reticular or a stellate cytosolic remnant. (i) Treatment with GST alone (600 ng/ml) had no effect on actin dynamics. Collapse of the actin cytoskeleton induced by cytochalasin D (10 μM) is shown in panel j for comparison.
FIG.2.
FIG.2.
Effect of GST-Tat on HLMVEC. (a and c) Unstimulated HLMVEC or GST-treated HLMVEC transfected with actin-GFP displayed pronounced flattening and prominent stress fibers. (b) After treatment with 200 ng of GST-Tat/ml, stress fibers resorbed and edge ruffles appeared within 20 to 30 min, similar in character to changes observed in HUVEC. (d) At 600 ng of GST-Tat/ml, actin collapse and focal edge accumulation were seen.
FIG. 3.
FIG. 3.
Effect of Tat basic and cysteine-rich domains. HUVEC were transfected with actin-GFP and exposed to 600 ng of GST/ml (a), wild-type GST-Tat (b), GST-Tat R(49,52,53,55,56,57)A (c), or GST-Tat C(22,25,27)A (d). Whereas all cells exposed to wild-type GST-Tat displayed marked cytoskeletal collapse, cells exposed to GST-Tat containing mutations within the basic domain remained identical to GST-treated cells. Rare cells treated with the cysteine-rich domain mutant GST-Tat developed retraction fibers.
FIG. 4.
FIG. 4.
Effect of DsRed-Tat on endothelial cell actin dynamics. HUVEC were cotransfected with actin-GFP and DsRed-Tat and observed at the indicated times. Actin-GFP was imaged in the green channel (a, d, g, and j), and DsRed-Tat or DsRed was imaged in the red channel (b, e, h, and k). At 15 h, frequent ruffle formation was observed (a, arrows), and DsRed-Tat was seen accumulating in nucleoli, with occasional visualization in cytosol (b). Peripheral retraction started within 24 h (d), seen in DsRed-Tat-expressing cells (d to f). (g to i) By 48 h, DsRed-Tat-expressing cells displayed actin collapse identical to that after GST-Tat treatment. Actin changes were visualized only in DsRed-Tat-expressing cells and not in neighboring cells (i, arrow). Cells transfected with DsRed alone did not exhibit alterations in actin dynamics at 24 h (j to l) or 48 h (not shown).
FIG. 5.
FIG. 5.
Activation of PAK1 by GST-Tat. HUVEC were treated with GST-Tat or GST (600 ng/ml) for the indicated times. PAK1 activation was assessed by IP kinase and phosphorylation of myelin basic protein (MBP). TNF (100 ng/ml) was added for 15 min as a positive control. The lower panel shows an immunoblot of immunoprecipitated PAK1 with anti-PAK1. HC, immunoglobulin heavy chain.
FIG. 6.
FIG. 6.
Effect of PAK1(K298A) on actin rearrangement. HUVEC were cotransfected with actin-GFP and DsRed-Tat and then infected with either Ad-lacZ or Ad-PAK1(K298A) (MOI = 100:1, 1 h) the following day. Cells were examined after 48 h. The upper panel shows immunoblot with anti-Myc, demonstrating expression of Myc-tagged PAK1(K298A) (DN-PAK1). Actin-GFP was imaged in the green channel (a and d), and DsRed-Tat was imaged in the red channel (b and e). DsRed-Tat-expressing cells infected with Ad-lacZ displayed prominent actin collapse (a to c), whereas DsRed-Tat-expressing cells infected with Ad-PAK1(K298A) retained normal morphology (d to f). (g and h) HUVEC were transfected with actin-GFP, infected with the corresponding adenoviruses, and treated with GST-Tat (600 ng/ml for 60 min). GFP-actin is shown. Cytoskeletal collapse seen in Ad-lacZ-infected cells was absent in Ad-PAK1(K298A)-infected cells.
FIG. 7.
FIG. 7.
Effect of PAK1(K298A) on JNK activation. HUVEC were cotransfected with either empty vector or PAK1(K298A) (DN-PAK1), and with HA-JNK2. After 24 h, cells were treated with GST-Tat (600 ng/ml, 15 min), and HA-JNK2 activation was assessed by IP kinase. Capture of HA-JNK2 was assessed by immunoblotting for JNK (lower panel). PAK1(K298A) blocked JNK activation by GST-Tat.
FIG. 8.
FIG. 8.
DsRed-p47 translocates to Tat-induced membrane ruffles. HUVEC were cotransfected with actin-GFP and DsRed-p47. After stimulation of cells with GST (a to c) or GST-Tat (d to g) (200 ng/ml, 15 to 20 min), DsRed-p47 was imaged in the red channel (a and d), and actin-GFP was imaged in the green channel (b and e). DsRed-p47 translocated avidly to actin ruffles upon stimulation with GST-Tat (d to f). The green/red fluorescence ratio image of inset from panel f shows cancellation of actin-GFP signal by DsRed-p47 within ruffles (g), indicating colocalization of the two proteins. DsRed itself, when cotransfected with actin-GFP, did not translocate to ruffles (h to j). The green/red ratio image of inset from panel j shows relative enhancement of actin-GFP within ruffle relative to DsRed (k).
FIG. 9.
FIG. 9.
Effect of PAK1(K298A) on p47phox phosphorylation. HUVEC were cotransfected with either empty vector or PAK1(K298A) (DN-PAK1) and with Flag-p47 and then stimulated with GST or GST-Tat (600 ng/ml). Flag-p47 was immunoprecipitated, and phosphorylation (upper panel) and protein capture (anti-Flag, lower panel) were assessed. GST-Tat increased phosphorylation of p47phox, whereas PAK1(K298A) completely blocked basal and Tat-induced p47phox phosphorylation.
FIG. 10.
FIG. 10.
Effect of p67(V204A) on actin rearrangement. HUVEC were transfected with actin-GFP and then infected with Ad-lacZ (a and b) or Ad-p67(V204A) (c and d) (MOI = 100:1, 1 h). The upper panel shows immunoblot with anti-p67phox, demonstrating expression of p67(V204A) (DN-p67). (b) Cells infected with Ad-lacZ displayed prominent retraction and actin collapse after treatment with GST-Tat (600 ng/ml). (c and d) Expression of p67(V204A) completely suppressed actin cytoskeletal rearrangements. (e and f) HUVEC were transfected with actin-GFP and then pretreated with the superoxide dismutase mimetic MnTMPyP (100 μM, 1 h) prior to treatment with GST-Tat (600 ng/ml). MnTMPyP prevented the actin cytoskeletal changes induced by GST-Tat (f).
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