1. ¹Department of Surgery, University of Kentucky, Lexington, Kentucky, USA
2. ²Biological Research Center, Szeged, Hungary
3. ³Department of Neurology, Johns Hopkins University, Baltimore, Maryland, USA
4. ⁴College of Agriculture, University of Kentucky, Lexington, Kentucky, USA

Correspondence: Dr M Toborek, Molecular Neuroscience and Vascular Biology Laboratory, Department of Surgery/Neurosurgery, University of Kentucky Medical Center, 593 Wethington Bldg., 900 S. Limestone, Lexington, KY 40536, USA. E-mail: mjtobo00@uky.edu

Received 13 October 2004; Revised 16 December 2004; Accepted 21 December 2004; Published online 30 March 2005.

Abstract

Exposure of brain microvascular endothelial cells (BMEC) to human immunodeficiency virus-1 (HIV-1) Tat protein can decrease expression and change distribution of tight junction proteins, including claudin-5. Owing to the importance of claudin-5 in maintaining the blood–brain barrier (BBB) integrity, the present study focused on the regulatory mechanisms of Tat-induced alterations of claudin-5 mRNA and protein levels. Real-time reverse-transcription-polymerase chain reaction revealed that claudin-5 mRNA was markedly diminished in BMEC exposed to Tat. However, U0126 (an inhibitor of mitogen-activated protein kinase kinase1/2, MEK1/2) protected against this effect. In addition, inhibition of the vascular endothelial growth factor receptor type 2 (VEGFR-2) by SU1498, phosphatidylinositol-3 kinase (PI-3 K) by LY294002, nuclear factor-B (NF-B) by peptide SN50, and intracellular calcium by BAPTA/AM partially prevented Tat-mediated alterations in claudin-5 protein levels and immunoreactivity patterns. In contrast, inhibition of protein kinase C did not affect claudin-5 expression in Tat-treated cells. The present findings indicate that activation of VEGFR-2 and multiple redox-regulated signal transduction pathways are involved in Tat-induced alterations of claudin-5 expression. Because claudins constitute the major backbone of tight junctions, the present data are relevant to the disturbances of the BBB in the course of HIV-1 infection.