Human Immunodeficiency Virus Type 1 Tat Regulates Endothelial Cell Actin Cytoskeletal Dynamics through PAK1 Activation and Oxidant Production

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.