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

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

Research output: Contribution to journalArticle

51 Citations (Scopus)

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 p47phox 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.

Original languageEnglish (US)
Pages (from-to)779-789
Number of pages11
JournalJournal of Virology
Volume78
Issue number2
DOIs
StatePublished - Jan 2004

Fingerprint

p21-Activated Kinases
Human immunodeficiency virus 1
Glutathione Transferase
Oxidants
oxidants
endothelial cells
actin
HIV-1
Actins
glutathione transferase
phosphotransferases (kinases)
Endothelial Cells
NADPH Oxidase
Human Umbilical Vein Endothelial Cells
Actin Cytoskeleton
microfilaments
Phosphorylation
phosphorylation
tat Gene Products
Stress Fibers

ASJC Scopus subject areas

  • Immunology

Cite this

Human Immunodeficiency Virus Type 1 Tat Regulates Endothelial Cell Actin Cytoskeletal Dynamics through PAK1 Activation and Oxidant Production. / Wu, Ru Feng; Gu, Ying; Xu, You Cheng; Mitola, Stefania; Bussolino, Federico; Terada, Lance S.

In: Journal of Virology, Vol. 78, No. 2, 01.2004, p. 779-789.

Research output: Contribution to journalArticle

@article{daa2b32cec91430a9065c551a9d2b4e5,
title = "Human Immunodeficiency Virus Type 1 Tat Regulates Endothelial Cell Actin Cytoskeletal Dynamics through PAK1 Activation and Oxidant Production",
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 p47phox 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.",
author = "Wu, {Ru Feng} and Ying Gu and Xu, {You Cheng} and Stefania Mitola and Federico Bussolino and Terada, {Lance S.}",
year = "2004",
month = "1",
doi = "10.1128/JVI.78.2.779-789.2004",
language = "English (US)",
volume = "78",
pages = "779--789",
journal = "Journal of Virology",
issn = "0022-538X",
publisher = "American Society for Microbiology",
number = "2",

}

TY - JOUR

T1 - Human Immunodeficiency Virus Type 1 Tat Regulates Endothelial Cell Actin Cytoskeletal Dynamics through PAK1 Activation and Oxidant Production

AU - Wu, Ru Feng

AU - Gu, Ying

AU - Xu, You Cheng

AU - Mitola, Stefania

AU - Bussolino, Federico

AU - Terada, Lance S.

PY - 2004/1

Y1 - 2004/1

N2 - 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 p47phox 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.

AB - 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 p47phox 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.

UR - http://www.scopus.com/inward/record.url?scp=0347319110&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=0347319110&partnerID=8YFLogxK

U2 - 10.1128/JVI.78.2.779-789.2004

DO - 10.1128/JVI.78.2.779-789.2004

M3 - Article

VL - 78

SP - 779

EP - 789

JO - Journal of Virology

JF - Journal of Virology

SN - 0022-538X

IS - 2

ER -