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Comparative Study
. 2008 Jul 30;28(31):7788-96.
doi: 10.1523/JNEUROSCI.0061-08.2008.

Caveolin-1 regulates human immunodeficiency virus-1 Tat-induced alterations of tight junction protein expression via modulation of the Ras signaling

Yu Zhong  1 , Eric J Smart, Babette Weksler, Pierre-Olivier Couraud, Bernhard Hennig, Michal Toborek
Affiliations
Comparative Study

Caveolin-1 regulates human immunodeficiency virus-1 Tat-induced alterations of tight junction protein expression via modulation of the Ras signaling

Yu Zhong et al. J Neurosci. .

Abstract

The blood-brain barrier (BBB) is the critical structure for preventing human immunodeficiency virus (HIV) trafficking into the brain. Specific HIV proteins, such as Tat protein, can contribute to the dysfunction of tight junctions at the BBB and HIV entry into the brain. Tat is released by HIV-1-infected cells and can interact with a variety of cell surface receptors activating several signal transduction pathways, including those localized in caveolae. The present study focused on the mechanisms of Tat-induced caveolae-associated Ras signaling at the level of the BBB. Treatment with Tat activated the Ras pathway in human brain microvascular endothelial cells (HBMECs). However, caveolin-1 silencing markedly attenuated these effects. Because the integrity of the brain endothelium is regulated by intercellular tight junctions, these structural elements of the BBB were also evaluated in the present study. Exposure to Tat diminished the expression of several tight junction proteins, namely, occludin, zonula occludens (ZO)-1, and ZO-2 in the caveolar fraction of HBMECs. These effects were effectively protected by pharmacological inhibition of the Ras signaling and by silencing of caveolin-1. The present data indicate the importance of caveolae-associated signaling in the disruption of tight junctions on Tat exposure. They also demonstrate that caveolin-1 may constitute an early and critical modulator that controls signaling pathways leading to the disruption of tight junction proteins. Thus, caveolin-1 may provide an effective target to protect against Tat-induced HBMEC dysfunction and the disruption of the BBB in HIV-1-infected patients.

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Figures

Figure 1.
Figure 1.
Tat-mediated activation of Ras in HBMECs. A, B, HBMECs were exposed to Tat for the indicated time points (A) or concentration (B). Active GTP-Ras was pulled down from the cell lysates with Raf-1 RBD (Ras binding domain) coupled to glutathione agarose, and Ras protein was determined by immunoblotting with monoclonal anti-Ras antibody. The bottom blots represent total Ras expression in HBMECs. C, The membrane fraction was separated as described in Materials and Methods, and Ras expression was detected using monoclonal anti-Ras antibody. The whole-cell lysate was used to determine the total Ras protein. In A–C, treatment with AA-Tat and/or BSA was used as negative controls. Cells were exposed to AA-Tat or BSA for 3 min. The blots reflect representative data from three different experiments and the bar graphs represent quantified results (mean ± SD) from these experiments as analyzed by densitometry. *p < 0.05, **p < 0.01 compared with control cells.
Figure 2.
Figure 2.
Tat-mediated activation of MEK1/2 in HBMECs is Ras dependent. A, Time course analysis of MEK1/2 activation induced by Tat. HBMECs were treated with 100 nm Tat for the indicated time periods. Activated (phosphorylated) MEK1/2 (p-MEK1/2) was analyzed by immunoblotting with a specific phospho-MEK1/2 (Ser217/221) antibody. In addition, total MEK1/2 was determined using a specific anti-MEK1/2 antibody. BSA was used as a negative control. B, HBMECs were pretreated for 2 h with Ras inhibitor FTS (20 μm) and exposed to 100 nm Tat for 30 min. Phosphorylated and total MEK1/2 were determined as described in A. In A and B, the blots reflect representative data from three different experiments, and the bar graphs represent quantified results (mean ± SD) from these experiments as analyzed by densitometry. **p < 0.01 compared with controls. †††Values in the group Tat+FTS are statistically different from those in the Tat group at p < 0.001.
Figure 3.
Figure 3.
Tat-mediated activation of ERK1/2 in HBMECs is downstream from Ras and MEK1/2. A, Time course analysis of ERK1/2 activation induced by Tat. HBMECs were treated with 100 nm Tat for the indicated time periods. Phosphorylated (p-ERK1/2) and total ERK1/2 were analyzed by immunoblotting with specific antibodies. BSA was used as a negative control. B, HBMECs were pretreated for 2 h with Ras inhibitor FTS (20 μm) and exposed to 100 nm Tat for 30 min. Phosphorylated and total ERK1/2 were determined as described in A. C, HBMECs were pretreated with the indicated concentrations of MEK1/2 inhibitor U0126, followed by exposure to 100 nm Tat for 30 min. Phosphorylated and total ERK1/2 were analyzed as in A. In A–C, the blots reflect representative data from three different experiments, and the bar graphs represent quantified results (mean ± SD) from these experiments as analyzed by densitometry. *p < 0.05, **p < 0.01, ***p < 0.001 compared with controls; values in the group Tat+FTS or Tat+U0126 are statistically different from those in the Tat group at ††p < 0.01 or †††p < 0.001.
Figure 4.
Figure 4.
Exposure to Tat upregulates membrane caveolin-1 levels in HBMECs. A, HBMECs were treated with indicated concentrations of Tat for 3 min. Caveolin-1 protein was analyzed in the membrane fractions and total cell extracts. Treatment with AA-Tat (100 nm) for 3 min was used as negative control. B, HBMECs were exposed to 100 nm Tat for the indicated time periods and membrane and total caveolin-1 were detected as in A. The blots reflect representative data from three different experiments, and the bar graphs represent quantified results (mean ± SD) from these experiments as analyzed by densitometry. *p < 0.05; **p < 0.01.
Figure 5.
Figure 5.
Tat-mediated activation of Ras is regulated by caveolin-1. A, Ras coimmunoprecipitates with caveolin-1. Lysates of control and Tat-treated HBMEC cultures were immunoprecipitated using monoclonal pan-Ras antibody, followed by Western blotting with anti-caveolin-1 antibody or anti-pan-Ras antibody (control). IP, Immunoprecipitation; WB, Western blotting. B, HBMECs were transfected with specific caveolin-1 siRNA or with control siRNA and caveolin-1 levels were analyzed by immunoblotting in total cell extracts. The silencing procedure resulted in ∼70% decrease in cellular caveolin-1 levels. C, Caveolin-1 was silenced in HBMECs as in A, followed by treatment with Tat (100 nm) for 3 min. Active GTP-Ras and total Ras were determined as in Figure 1. The blots reflect representative data from four different experiments, and the bar graphs represent quantified results (mean ± SD) from these experiments as analyzed, by densitometry. *p < 0.05, ***p < 0.001 compared with controls or mock transfection. ††Data in the Tat+cav-1 siRNA are significantly different from those in the Tat group at p < 0.01.
Figure 6.
Figure 6.
Exposure to Tat alters expression of tight junction proteins in caveolae fraction of HBMECs via the Ras-regulated pathway. A, Confluent HBMECs were exposed to 100 nm Tat for the indicated time points. Tight junction proteins (occludin, ZO-1, and ZO-2) were detected by immunoblotting in total cell extracts. Treatment with AA-Tat (100 nm for 15 h) was used as a negative control. B, Confluent HBMECs were treated with 100 nm Tat for 15 or 24 h, and cells were fractionated using a detergent-free method. Expression level of tight junction proteins was detected by immunoblotting in postnuclear soup (PNS), cytosol (CYTO), intracellular membranes (IM), plasma membrane (PM), and caveolae membranes (CM). C, Confluent HBMECs were pretreated with 5 μm FTS for 3 h, and then incubated with 100 nm Tat for 15 h. FTS was retained in cell culture medium for the duration of Tat treatment. Tight junction proteins were determined in caveolae membrane fraction as in B. Actin was determined as housekeeping protein and loading control. The blots reflect representative data from four different experiments, and the bar graphs represent quantified results (mean ± SD) from these experiments as analyzed by densitometry. *p < 0.05 compared with control (mock transfection). †Data in the Tat+FTS are significantly different from those in the Tat group at p < 0.05.
Figure 7.
Figure 7.
Caveolin-1 modulates Tat-induced alterations of tight junction protein expression. A, Caveolin-1 was silenced in HBMECs as in Figure 5, followed by exposure to 100 nm Tat for 15 h. Expression of tight junction proteins (occludin, ZO-1, and ZO-2) was analyzed by immunoblotting in total cell extracts. Control experiments include mock transfection and transfection with control siRNA. The blots reflect representative data from three different experiments. *p < 0.05 compared with control (mock transfection). †Data in the cultures exposed to Tat and caveolin-1 siRNA are significantly different from those in the cultures exposed to Tat alone or Tat plus control siRNA at p < 0.05. B, Brain microvascular endothelial cells were isolated from cavoelin-1-deficient (cav-1−/−) mice and the respective controls. Cells were cultured until confluence and treated with 100 nm Tat for 15 h. ZO-1 immunoreactivity was determined by immunofluorescence (red staining). In addition, DAPI staining was performed to visualize the nuclei (blue staining). Tat treatment resulted in diminished and fragmented expression of ZO-1 immunoreactivity at the cell–cell borders (longer arrows) and redistribution of ZO-1 from the cell borders into the cytoplasm (shorter and open arrows) in cells from control mice. These effects were markedly attenuated in cells from cav-1−/− mice. The images were taken using a 60× objective and a 10× ocular lens.
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