Signaling Mechanisms of HIV-1 Tat-Induced Alterations of Claudin-5 Expression in Brain Endothelial Cells

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-κB (NF-κB) 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.

Keywords

blood—brain barrier brain endothelial cells HIV-1 dementia signal transduction tight junctions

Introduction

The cerebral microvascular endothelium constitutes the principal component of the blood–brain barrier (BBB). The morphologic basis of the BBB integrity is the formation of tight junctions, which are composed of specific junctional proteins, including claudin-5 (Huber et al, 2001; Pardridge, 2002). Considerable experimental evidence indicates BBB damage in human immunodeficiency virus-1 (HIV-1) infection (McArthur et al, 1992; Petito and Cash, 1992; Giovannoni et al, 1998). Such alterations were shown in pathologic studies (Rhodes and Ward, 1991; Krebs et al, 2000), CSF studies (Singer et al, 1994), and by dynamic magnetic resonance imaging (Avison et al, 2002). Disruption of the BBB appears to be critical for HIV-1 trafficking into the brain (Wu et al, 2000) and the development of HIV-1-associated central nervous system (CNS) complications. It has been shown that disruptions of the BBB are more frequent in AIDS patients with dementia as compared with nondemented AIDS patients or seronegative controls (Power et al, 1993). In addition, HIV-1 encephalitis was associated with the disruption of endothelial cell junctions and mononuclear cell infiltration (Dallasta et al, 1999; Boven et al, 2000).

Central nervous system manifestations of HIV-1 infection frequently do not correlate with the viral load. Therefore, it has been hypothesized that specific soluble factors released by HIV-infected cells can contribute to the pathogenesis of HIV-1 infection. One of such potential mediators is Tat protein, which has a crucial role in HIV-1 gene expression and replication (Nath and Geiger, 1998; Ansari et al, 1999). Tat can be released actively from HIV-1-infected cells (Chang et al, 1997), easily cross cell membranes (Vives et al, 2003), and interact with a variety of cell surface receptors, including vascular endothelial growth factor receptor type 2 (VEGFR-2). Elevated levels of Tat protein and mRNA were shown in brains of AIDS patients (Hudson et al, 2000; Valle et al, 2000).

In addition to its neurotoxic influence, Tat can evoke profound vascular effects (Acheampong et al, 2002; Kim et al, 2003; Khan et al, 2003). For example, Tat induced oxidative stress and disturbances in redox balance in brain microvascular endothelial cell (BMEC) and the brain tissue were associated with activation of redox-responsive transcription factors and inflammatory genes (Pu et al, 2003; Toborek et al, 2003; Lee et al, 2004). Furthermore, our studies indicate that alterations of expression and distribution of selected tight junction proteins, including claudin-5, may be one of the major cytotoxic effects of Tat (András et al, 2003). Such effects may affect the BBB permeability and can be critical for HIV-1 entry into the brain. Claudin-5 plays an important role in maintaining the integrity of brain endothelial cells because of its involvement in both paracellular sealing and membrane domain differentiation (Furuse et al, 1998; Morita et al, 1999; Tsukita and Furuse, 2000). Indeed, the size-selective loosening of tight junctions was observed in claudin-5 knockout mice (Nitta et al, 2003). It also has been proposed that the ratio of claudin-5 to other claudins can regulate the tightness of the BBB (Liebner et al, 2000a, b). Thus, because of the importance of claudin-5 in the regulation of the BBB integrity, the present study focused on the regulatory mechanisms of Tat-induced alterations of claudin-5 expression.

Materials and methods

Primary Cultures of Brain Microvascular Endothelial Cells

Brain microvascular endothelial cells were isolated from the forebrains of 2-week-old Wistar rats and cultured as previously described (Deli et al, 1997; András et al, 2003). Brain microvascular endothelial cells were cultured in Dulbecco's modified Eagle's medium with 20% plasma-derived bovine serum (PDBS, Animal Technologies Inc., Tyler, TX, USA), 40 μg/mL endothelial cell growth supplement (BD Biosciences, Bedford, MA, USA), 100 μg/mL heparin, 2 mmol/L glutamine, 5 μg/mL insulin, 5 μg/mL human transferrin, 5 ng/mL Na-selenite (Insulin-Transferrin-Sodium Selenite Media Supplement, Sigma, St Louis, MO, USA), and 25 μg/mL gentamicin. Cultures were incubated at 37°C in a humidified atmosphere of 5% CO₂. Brain microvascular endothelial cells can dedifferentiate very rapidly and lose their specific characteristics as early as after the first passage. Therefore, only primary cultures were used in the present study.

Tat Treatment and Inhibitors of Specific Signaling Pathways

Human immunodeficiency virus-1 Tat protein is encoded by a gene consisting of two exons. The first exon encodes the neurotoxic and membrane interactive domains. Therefore, the present study was performed on recombinant Tat protein derived from the first exon, which encodes for the first 72 amino acids (Tat_1–72). Tat was synthesized and purified as previously described by Ma and Nath (1997). The functional properties of Tat were confirmed by its ability to activate the β-galactosidase gene in an HIV long terminal repeat-β-galactosidase plasmid that had been transfected into HeLa cells (Ma and Nath, 1997).

To exclude the possibility that Tat-induced effects were secondary to endotoxin contaminants of the Tat preparation, Tat solutions were immunoabsorbed with Tat anti***sera conjugated to protein-A beads (Magnuson et al, 1995), and the supernatant was tested for proinflammatory activity.

Because Tat strongly binds to serum proteins, BMEC were treated with Tat in serum-free media. All experiments were performed on confluent endothelial cell cultures (normally 3 to 4 days after seeding). Similar experimental design was used in our previously published papers (András et al, 2003; Pu et al, 2003; Toborek et al, 2003; Lee et al, 2004). Tat concentrations used in the present study are consistent with the literature data (Bonavia et al, 2001; Prendergast et al, 2002; Speth et al, 2002). In addition, evidence indicates that pathologic concentrations of Tat in HIV-infected patients can reach the range of nanograms per milliliter of serum (Xiao et al, 2000).

To study specific mechanisms of Tat-induced alterations of claudin-5 expression, the following inhibitors of signal transduction pathways were used (Figure 1): SU1498 (1 μmol/L, an inhibitor of VEGFR-2), ZM336372 (0.5 μmol/L, an inhibitor of c-Raf), U0126 (10 or 20 μmol/L, an inhibitor of mitogen-activated protein kinase kinase1/2 (MEK1/2)), LY294002 (100 nmol/L, an inhibitor of phosphatidylinositol-3 kinase (PI-3 K)), SN50 (2 μmol/L, an inhibitor of nuclear factor-κB (NF-κB) translocation) or BAPTA/AM (5 μmol/L, an intracellular calcium chelator). In addition, we used 1-(5-isoquinolinesulfonyl)-2-methyl-piperazine dichloride (H-7; an inhibitor of protein kinase C (PKC) and cAMP-dependent protein kinase (PKA), 10 μmol/L), and calphostin-C (CPC; an inhibitor of PKC, 10 nmol/L). SU1498, ZM336372, U0126, LY294002, SN50, and BAPTA/AM were purchased from Calbiochem (San Diego, CA, USA); CPC and H-7 were from Sigma (St. Louis, MO, USA).

Figure 1

The involvement of specific signaling pathways in Tat-mediated alterations of claudin-5 mRNA levels in brain microvascular endothelial cells. (A) Confluent cultures were treated with vehicle (control) or 100 nmol/L Tat for up to 24 h and claudin-5 mRNA levels were determined by real-time reverse transcriptase-polymerase chain reaction (RT-PCR). (B) The cells were preincubated with 1 μmol/L SU1498 for 15 mins, followed by a coexposure to 100 nmol/L Tat for 8 h. (C) The cells were preincubated with 10 μmol/L U0126 for 15 mins followed by a coexposure to 100 nmol/L Tat for 8 h. (D) The cells were preincubated with 5 mmol/L BAPTA/AM for 5 mins followed by media change and exposure to 100 nmol/L Tat for 8 h. Values are mean+s.e.m., n = 6. ^*Significantly different as compared with control. ^†Values in the group Tat plus U0126 are significantly different as compared with those in the Tat group.

Antibodies

Rabbit polyclonal anti-claudin-5 antibody was purchased from Zymed Laboratories (San Francisco, CA, USA) and mouse monoclonal anti-phosphotyrosine antibody was obtained from Upstate Group (Lake Placid, NY, USA). The following antibodies were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA): mouse monoclonal anti-pERK1/2 antibody, rabbit polyclonal anti-Akt antibody as well as HRP-conjugated anti-mouse and anti-rabbit secondary antibodies. Rabbit polyclonal anti-ERK1/2 and mouse monoclonal anti-phospho-Akt (Ser473) antibodies were obtained from Cell Signaling Technology (Beverly, MA, USA). FITC and Texas Red-conjugated anti-mouse as well as anti-rabbit secondary antibodies were purchased from Santa Cruz Biotechnology and Chemicon International (Temecula, CA, USA).

Real-Time Reverse-Transcription-Polymerase Chain Reaction

Quantitative real-time reverse transcription-polymerase chain reaction (RT-PCR) was used to study the claudin-5 mRNA level changes. Specific rat claudin-5 primers were designed using Primer Express 2.0 and the following sequences were used: claudin-5 forward, 5'-CAC GGG AGG AGC GCT TTA T-3’ and claudin-5 reverse, 5'-GGC ACC GTT GGA TCA TAG AAC T-3'. In addition, the following primers were used to detect β-actin as a housekeeping gene: β-actin forward, 5'-AGG CCA ACC GTG AAA AGA TG-3’ and β-actin reverse, 5'-ACC AGA GGC ATA CAG GGA CAA-3'.

Total RNA was isolated using RNeasy kit (Qiagen, Valencia, CA, USA), treated by DNase I to remove possible genomic DNA contamination, and then reverse transcribed into cDNA using the Reverse Transcription System (Promega, Madison, WI, USA). Real-time PCR was conducted on the ABI Prism 7000 system (Applied Biosystems, Foster City, CA, USA) using 100 ng cDNA (2 μL of RT product), 12.5 μL SYBR Green PCR Master Mix (2 x), 300 nmol/L of forward and reverse primers in a total volume of 25 μL. The following thermocycling conditions were used: 95°C for 10 mins, followed by 95°C for 15 secs and 60°C for 60 secs for 40 cycles. The standard curve was generated by plotting the threshold cycle (Ct) versus the log concentration of the serial dilutions of one specific cDNA sample served as a calibrator. All samples were prepared in duplicate, their concentrations were calculated based on the standard curve and normalized according to β-actin mRNA level.

Immunoblotting

Homogenates of cultured BMEC were prepared in lysis buffer (20 mmol/L Tris-HCl, pH 7.4, 150 mmol/L NaCl, 0.5% Triton X-100, 0.1 mg/mL phenylmethylsulfonyl fluoride, 0.5% Nonidet 40, 1 mmol/L EDTA, 2.5 μg/mL leupetin, 1 μg/mL pepstatin A, 1 μg/mL aprotinin). After centrifugation at 15000 g for 15 mins, the supernatants were collected and protein concentrations were determined using the Bradford protein assay. Samples (20 μg protein/lane) were separated on SDS-polyacrylamide gel, blotted onto nitrocellulose membrane (Bio-Rad Laboratories, Hercules, CA, USA) and incubated with the respective antibodies. For visualization of detected proteins, immunoblots were analyzed using an ECL Western blot detection kit (Amersham Biosciences, Piscataway, NJ, USA).

Immunofluorescence Microscopy

Brain microvascular endothelial cells cultured on 35 mm dishes were fixed with ethanol for 30 mins at 4°C. After washing with phosphate-buffered saline (PBS) and blocking with 3% bovine serum albumin (BSA)-PBS for 30 mins, samples were incubated overnight at 4°C with primary antibody. The excess of primary antibody was removed, slides were washed with PBS, and incubated with Texas Red or FITC-conjugated secondary antibody for 2 h at room temperature. After washing with PBS, slides were mounted in aqueous mounting media and covered with coverslips. Specimens were evaluated under an epifluorescence Nicon Eclipse E600 microscope and the images were captured using a Spot CCD camera system.

Statistical Analysis

Routine statistical analysis of data was completed using SigmaStat 2.0 (SPSS Inc., Chicago, IL, USA). One-way ANOVA was used to compare responses among treatments. Treatment means were compared using Bonferroni's least significant difference procedure. P<0.05 was considered significant.

Results

We hypothesize that disturbances of cellular redox status may be the underlying mechanism of Tat-induced vascular toxicity. Therefore, to study the mechanisms of Tat-mediated alterations of claudin-5 expression, we concentrated on redox-responsive signal transduction pathways, such as the VEGFR-2/Ras/ERK1/2 pathway, the PI-3K/Akt/NF-κB pathway, and calcium-dependent signaling. The dose and time-dependent effects of Tat on claudin-5 expression were presented previously (András et al, 2003).

Tat Exposure Diminishes Claudin-5 mRNA Levels

To investigate whether Tat-induced alterations of claudin-5 expression is regulated at the transcriptional level, claudin-5 mRNA levels were determined using real-time RT-PCR in BMEC exposed to 100 nmol/L Tat for up to 24 h. As indicated in Figure 1A, a 4-h exposure to Tat resulted in a significant decrease in claudin-5 mRNA levels. These levels reached the minimal levels in cells treated with Tat for 8 h and returned to the normal values after a 24-h Tat exposure.

Tat-induced decrease in claudin-5 mRNA level was prevented by pretreatment with U0126, a specific inhibitor of MEK1/2 (Figure 1C). However, pretreatment with the VEGFR-2 inhibitor SU1498 or calcium chelator BAPTA/AM did not affect Tat-induced alterations of claudin-5 mRNA levels (Figures 1B and 1D, respectively). In all experiments presented in Figures 1B-1D, BMEC were exposed to Tat for 8 h.

VEGFR-2 and the Ras/ERK1/2 Pathway are Involved in Tat-Induced Downregulation of Claudin-5 Protein Expression

Evidence suggests that Tat-induced cellular effects can be mediated, at least in part, by VEGFR-2. Therefore, tyrosine phosphorylation of VEGFR-2 was assessed to evaluate if’ Tat can interact with this receptor in BMEC. In addition, to better visualize Tat-induced changes, the nuclei of control and Tat-treated cells were stained with Hoechst dye. As illustrated in Figure 2A, exposure to Tat resulted in marked alterations in VEGFR-2 tyrosine phosphorylation pattern, indicating the involvement of this receptor.

Figure 2

(A) Tat-mediated alterations in the vascular endothelial growth factor receptor type 2 (VEGFR-2) phosphorylation pattern in brain microvascular endothelial cells (BMEC). Confluent cultures were treated with vehicle (control) or 100 nmol/L Tat for 5 mins, followed by the detection of VEGFR-2 immunoreactivity (left panel), tyrosine phosphorylation (second panel), or Hoechst staining to visualize the nuclei (third panel). The merged images are shown on the right panel. Exposure to Tat resulted in marked alterations in VEGFR-2 tyrosine phosphorylation pattern. The microphotographs are representative images of three independent experiments. Magnification x 400. (B) The involvement of VEGFR-2 in Tat-induced activation of ERK1/2 in BMEC. Confluent cultures were treated with vehicle (control) or 100 nmol/L Tat for 15 or 30 mins and phosphorylated ERK1/2 (pERK1/2) as well as total ERK1/2 were detected by Western blot. In addition, selected cultures were pretreated with 1 μmol/L SU1498 for 15 mins, followed by a coexposure to 100 nmol/L Tat for 30 min (right bands). The blot reflects representative data of three independent experiments.

We hypothesized that activation of VEGFR-2 may lead to stimulation of the Ras/c-Raf/ERK1/2 pathway in BMEC. Therefore, activation of extracellular signal regulated kinase 1/2 (ERK1/2) was determined in control and Tat-treated BMEC by immunoblotting with antibodies for the active, dually phosphorylated ERK1/2. Figure 2B depicts that exposure to Tat resulted in a time-dependent increase in phosphorylated ERK1/2. In addition, inhibition of the VEGFR-2 by SU1498 attenuated Tat-induced phosphorylation of ERK1/2, confirming that this effect is mediated via activation of VEGFR-2 (Figure 2B).

A possible involvement of VEGFR-2 and the Ras/c-Raf/ERK1/2 pathway in Tat-mediated alterations of claudin-5 expression was studied in a series of Western blots and immunofluorescence analyses. As shown in Figures 3A and 7C, a functional inhibition of VEGFR-2 by SU1498 partially blocked Tat-induced alterations of claudin-5 protein expression. Similarly, a selective c-Raf inhibitor ZM336372 markedly attenuated a decrease in claudin-5 protein in Tat-treated BMEC as determined by Western blot (Figure 3B) and immunostaining (Figure 7D). Finally, a blockage of ERK1/2 activation by the MAPK kinase 1/2 (MEK1/2) inhibitor U0126 effectively prevented Tat-induced downregulation of claudin-5 protein expression, indicating the important role of the ERK1/2 signaling in this effect (Figures 3C and 7E).

Figure 3

The involvement of the VEGFR-2/Ras/ERK1/2 signaling in Tat-induced alterations of claudin-5 expression in brain microvascular endothelial cells. Confluent cultures were treated with vehicle (control) or 100 nmol/L Tat for 24 h and claudin-5 protein levels were detected by Western blot. In addition, selected cultures were pretreated for 15 mins with: (A) SU1498 (1 μmol/L), (B) ZM336372 (0.5 μmol/L), or (C) U0126 (10 or 20 μmol/L), followed by a coexposure to 100 nmol/L Tat for 24 h. The bar graphs are the combined densitometry data from four independent experiments and the blots are the representative images from these experiments. ^*Significantly different as compared with control. ^†Values in the group Tat plus specific inhibitor are significantly different as compared with those in the Tat group.

Figure 4

(A) Vascular endothelial growth factor receptor type 2(VEGFR-2) is not involved in Tat-mediated activation of Akt in brain microvascular endothelial cells (BMEC). Confluent cultures were treated with vehicle (control) or 100 nmol/L Tat for 15 or 30 mins and phosphorylated Akt (pAkt) was detected by Western blot. In addition, selected cultures were pretreated with 1 μmol/L SU1498 for 15 mins, followed by a coexposure to 100 nmol/L Tat (right bands). Tat treatment markedly activated Akt; however, inhibition of VEGFR-2 did not influence this effect. The blot reflects representative data of three independent experiments. (B) The involvement of the PI-3K/Akt signaling in Tat-induced alterations of claudin-5 expression in RMEC. Confluent cultures were treated with vehicle (control) or 100 nmol/L Tat for 24 h and claudin-5 protein levels were detected by Western blot. In addition, selected cultures were pretreated for 15 mins with 100 nmol/L LY294002, followed by a coexposure to 100 nmol/L Tat for 24 h. The bar graph is the combined densitometry data from four independent experiments and the blot is the representative image from these experiments. ^*Significantly different as compared with control.

Tat-Induced Stimulation of the PI-3K/Akt/NF-κB Pathway Contributes to Decreased Expression of Claudin-5 Protein

The PI-3K/Akt/NF-κB pathway is a classical redox-regulated signaling mechanism; therefore, its involvement in Tat-induced alterations of claudin-5 expression also was investigated in the present study. To elucidate the regulatory effects of Tat on the PI-3K/Akt/NF-κB signaling, BMEC were incubated with 100 nmol/L Tat, and phosphorylation of Akt, a target of PI-3 K, was determined by Western blot. As illustrated in Figure 4A, Akt phosphorylation was apparent after 15 mins of Tat exposure with a further increase after a 30-min treatment. However, Tat-mediated activation of the PI-3K/Akt signaling appeared to be independent on stimulation of VEGFR-2. Indeed, VEGFR-2 inhibitor SU1498 had no effect on Tat-mediated Akt activation (Figure 4A). In parallel experiments, inhibition of PI-3 K by LY294002 also did not affect Tat-simulated activation of ERK1/2 (data not shown). Thus, no evident crosstalk occurs between Tat-activated VEGFR-2/Ras/ERK1/2 and the PI-3K/Akt signaling pathways in BMEC.

As illustrated in Figures 4B (Western blot) and 7F (immunoreactivity), Tat-induced stimulation of the PI-3K/Akt pathway is involved in alterations of claudin-5 expression. Specifically, pretreatment with the PI-3 K inhibitor LY294002 protected against decreased claudin-5 protein levels in Tat-treated BMEC.

Stimulation of the PI-3K/Akt pathway can lead to the activation of NF-κB DNA-binding activity. Indeed, using double immunofluorescence studies, we detected that treatment with Tat can induce translocation of NF-κB from the cytoplasm into the nuclei of BMEC (Figure 5A). Activation of NF-κB also appeared to be involved in Tat-mediated downregulation of claudin-5 expression. As shown in Figures 5B and 7G, inhibition of NF-κB translocation into the nuclei by treatment with SN50 markedly attenuated Tat-induced alterations of claudin-5 protein expression.

Figure 5

(A) Tat-mediated activation of nuclear factor-κB (NF-κB) in brain microvascular endothelial cells (BMEC). Confluent cultures were treated with vehicle (control) or 100 nmol/LTat for 4 h, followed by detection of p65 immunoreactivity (an NF-κB subunit, left panel) or Hoechst staining to visualize the nuclei (second panel). The merged images are shown on the right panel. Exposure to Tat resulted in a marked increase in p65 translocation into the nuclei. The microphotographs are representative images of three independent experiments. Magnification x 200. (B) The involvement of NF-κB in Tat-induced alterations of claudin-5 expression in BMEC. Confluent cultures were treated with vehicle (control) or 100 nmol/L Tat for 24 h and claudin-5 protein levels were detected by Western blot. In addition, selected cultures were pretreated for 15 mins with 2 μmol/L SN50, followed by a coexposure to 100 nmol/L Tat for 24 h. The bar graph is the combined densitometry data from four independent experiments and the blot is the representative image from these experiments. ^*Significantly different as compared with control. ^†Values in the group Tat plus SN50 are significantly different as compared with those in the Tat group.

Chelation of Intracellular Calcium Inhibits HIV-1 Tat-Evoked Alterations of Claudin-5 Protein Expression

Treatment with Tat can increase intracellular free calcium levels (Haughey and Mattson, 2002). Therefore, we tested the hypothesis that intracellular calcium also may be involved in the Tat-induced downregulation of claudin-5 expression. As illustrated in Figures 6 (Western blot) and 7 H (immunoreactivity), pretreatment with an intracellular calcium chelator BAPTA/AM efficiently blocked Tat-mediated decrease in claudin-5 protein expression. It should be pointed out that these protective effects were at the posttranscriptional level because exposure to BAPTA/AM did not affect Tat-induced downregulation of claudin-5 mRNA levels (Figure 1D).

Figure 6

The involvement of protein kinase C (PKC) and intracellular calcium in Tat-induced alterations of claudin-5 expression in brain microvascular endothelial cells. Confluent cultures were treated with vehicle (control) or 100 nmol/L Tat for 24 h and claudin-5 protein levels were detected by Western blot. In addition, selected cultures were pretreated with H-7 (10 μmol/L, 15 mins preexposure), calphostin C (CPC; 10 μmol/L, 15 min preexposure), or BAPTA/AM (5 μmol/L, 5 mins preexposure), followed by treatment with 100 nmol/L Tat for 24 h. The bar graph is the combined densitometry data from four independent experiments and the blot is the representative image from these experiments. ^*Significantly different as compared with control. ^†Values in the group Tat plus BAPTA/AM are significantly different as compared with those in the Tat group.

Figure 7

The involvement of specific signaling pathways in Tat-mediated alterations of claudin-5 immunoreactivity in brain microvascular endothelial cells. Confluent cultures were treated with (A) vehicle (control) or (B) 100 nmol/L Tat for 24 h and claudin-5 protein expression was determined by immunostaining. In addition, selected cultures were pretreated for 15 mins with: (C) SU1498 (1 μmol/L), (D) ZM336372 (0.5 μmol/L), (E) U0126 (20 μmol/L), (F) LY294002 (100 nmol/L), (G) SN50 (2 μmol/L) or (H) BAPTA/AM (5 μmol/L), followed by exposure to 100 nmol/L Tat for 24 h. Magnification x 200.

An increase in intracellular calcium can regulate PKC activity. In addition, PKC was suggested to be involved in PMA-mediated decrease in claudin-5 immunoreactivity in choroid plexus (Lippoldt et al, 2000). However, pretreatment of BMEC with two different PKC inhibitors, H-7 and calphostin-C, did not affect Tat-mediated alterations of claudin-5 expression in BMEC (Figure 6).

Discussion

Evidence indicates that exposure to Tat can increase BMEC permeability in vitro and in vivo (Oshima et al, 2000; Arese et al, 2001; Kim et al, 2003; Pu et al, 2003), suggesting the disruption of the BBB integrity. However, signaling pathways induced by this protein in BMEC are poorly understood. In particular, the mechanisms involved in Tat-induced changes of tight junction protein expression are not known. The results of the present study show that Tat can stimulate several redox-regulated signal transduction pathways including the Ras/ERK1/2, PI-3K/Akt/NF-κB, and calcium signaling. In addition, activation of these signaling pathways provides the regulatory mechanisms of Tat-mediated alterations of claudin-5 expression (Figure 8). Exposure to Tat induces a transient decrease in claudin-5 mRNA levels, reaching minimal levels after an 8-h treatment (Figure 1). The recovery of claudin-5 mRNA levels after a 24-h exposure to Tat suggests a self-defense mechanism of BMEC in an attempt to restore the BBB integrity. In contrast, a decrease in claudin-5 protein expression is persistent and the levels of this tight junction protein almost completely disappear in BMEC treated with Tat for 24 h (András et al, 2003).

Figure 8

Redox-responsive signaling pathways that can participate in Tat-mediated alterations of claudin-5 expression in brain microvascular endothelial cells.

In the present study, we indicated that Tat could induce the Ras/ERK1/2 signaling through activation of VEGFR-2 (Figure 2B). These results are supported by the literature data, which show the importance of VEGFR-2 in Tat-mediated cellular effects. Specifically, VEGFR-2 was proposed to serve as a high-affinity receptor for Tat in endothelial cells (Albini et al, 1996) and regulate Tat-mediated effects on cell growth and angiogenic activity. In fact, Tat-stimulated angiogenesis was attenuated by a cotreatment with anti-VEGFR-2 antibody (Albini et al 1996; Barillari et al, 1999). It was shown that Tat can interact with VEGFR-2 through the cysteine-rich and basic domains encoded by the first exon of tat gene (Mitola et al, 2000). In contrast, the C-terminal domain of Tat encoded by the second exon is not required for VEGFR-2 activation (Mitola et al, 2000). This is an important fact, because the form of Tat peptide encoded by the first exon (amino acids 1–72) was used in the present study.

Tat-mediated activation of the Ras/ERK1/2 pathway via VEGFR-2 appears to be specific, because inhibition of VEGFR-2 had no effect on Tat-induced activation of Akt (Figure 4A). However, it should be pointed out that blocking VEGFR-2 by SU1498 offered only a partial inhibition of ERK1/2 phosphorylation (Figure 2B), indicating that other Tat-mediated signaling pathways also may be involved in this process. The form of Tat (1–72) used in the present study can interact with other cell surface receptors, such as chemokine receptors CCR2, CCR3 (Albini et al, 1998), CXCR4 (Xiao et al, 2000; Poggi et al, 2004), and VEGFR-1 (Mitola et al, 1997). In addition, Tat can be internalized by cells through the interaction of its basic domain with cell surface heparan-sulfate proteoglycans (Rusnati et al, 1999). Thus, Tat may affect signaling mechanisms and cellular metabolism without interacting with cell surface receptors.

The ERK1/2 pathway is known to be involved in multiple cascades of serine/threonine and tyrosine phosphorylation reactions that mediate diverse cellular responses to growth factors, physical stress, and cytokines (Kyriakis and Avruch, 2001). In agreement with the results of the present study, it was reported that Tat can induce ERK1/2 activation in vascular endothelial cells (Rusnati et al, 2001). However, the novelty of the current study is to show that the VEGFR-2/Ras/ERK1/2 signaling can participate in Tat-mediated alterations of claudin-5 expression. This involvement appears to be complex, for example, inhibition of VEGFR-2 by SU1498 had no effect on Tat-induced alterations of claudin-5 mRNA levels (Figure 1B) but partially protected against changes in claudin-5 protein expression (Figure 3A). In contrast, inhibition of ERK1/2 phosphorylation by U0126 protected against both Tat-induced diminished claudin-5 mRNA and protein levels (Figures 1C and 3C, respectively). In strong support of the importance of the ERK1/2 signaling in Tat-induced cell toxicity, it was shown that Tat-evoked permeability changes can be mediated through a MAPK and tyrosine kinase-dependent pathway (Oshima et al, 2000). Evidence also indicates that activation of the ERK1/2 can stimulate HIV-1 replication by phosphorylation of Tat and other HIV-1 proteins and enhancing the infectivity of HIV-1 virions in vitro (Yang and Gabuzda, 1999). Thus, Tat-mediated activation of ERK1/2 may not only participate in alterations of claudin-5 expression and endothelial cell permeability but also contribute to increased viral infectivity, generating a self-intensifying mechanism of cell toxicity.

lts of the present study show that Tat can activate the PI-3K/Akt signaling in BMEC (Figure 4A), a pathway which is well known to lead to activation of NF-κB (Thomas et al, 2002). Indeed, exposure of BMEC to Tat resulted in a marked activation of NF-κB as illustrated by nuclear translocation of this transcription factor (Figure 5A). These findings are confirmed by the literature data that documented Tat-mediated activation of the PI-3K signaling in PC12 cells (Milani et al, 1996). In addition, Tat-induced activation of NF-κB was shown in a variety of cell types, including brain endothelial cells (Toborek et al, 2003).

Several lines of evidence indicate that the PI-3K/Akt/NF-κB pathway can be involved in Tat-mediated cell toxicity. For example, it was shown that blocking PI-3 K resulted in inhibition of Tat-induced apoptosis of brain endothelial cells (Kim et al, 2003). In addition, the role of NF-κB activation in Tat-mediated inflammatory responses is well documented (Toborek et al, 2003; Lee et al, 2004). However, the novelty of the present study is to show a connection between the PI-3K/Akt/NF-κB signaling pathway and Tat-induced alterations in claudin-5 expression. Several different mechanisms can be responsible for these effects. Activation of PI-3 K was shown to be involved in induction of Snail expression (Peinado et al, 2003), a transcriptional repressor for occludin and claudin-3, 4, and 7 (Ikenouchi et al, 2003). Snail can bind directly to the E-boxes of the promoter regions of claudin/occludin genes, repressing their activity. However, the specific effects of Snail on claudin-5 expression are not known.

In this study, inhibition of NF-κB translocation into the nuclei also resulted in attenuation of Tat-mediated alterations of claudin-5 protein levels (Figures 5B and 7G). Therefore, we suggest that activation of this transcription factor may serve as a negative regulator of claudin-5 expression. Such a potential mechanism is supported by the literature data, which showed that NF-κB can inhibit expression of occludin, a tight junction protein associated with claudin-5 (Wachtel et al, 2001). Thus, activation of NF-κB can not only induce inflammatory responses in the brain endothelium but also may directly affect the BBB integrity by interference with tight junction protein expression.

Tat-induced cell toxicity has been linked to an increase in intracellular calcium level through its release from the inositol triphoshate (IP₃) sensitive pool (Hegg et al, 2000; Haughey and Mattson, 2002). Increased calcium ions can trigger several signaling cascades, including the ERK1/2 and PKC pathways. Therefore, a possible involvement of intracellular calcium in Tat-mediated changes in claudin-5 expression was also examined in this study. A pretreatment with intracellular calcium chelator BAPTA/AM markedly prevented Tat-induced alterations of claudin-5 expression (Figures 6 and 7(H). However, it should be pointed out that these effects were observed only at the protein level and BAPTA/AM did not affect Tat-mediated changes in claudin-5 mRNA expression (Figure 1D). In addition, blockage of PKC activity with two different inhibitors, H-7 and calphostin-C, did not influence Tat-mediated alterations of claudin-5 expression (Figure 6). These were unexpected results, because PKC signaling was suggested to be involved in diminished claudin-5 immunoreactivity in choroid plexus in response to phorbolester treatment (Lippoldt et al, 2000). However, different experimental models and treatment factors can be responsible for the discrepancy of these results.

In summary, the present study shows that Tat can decrease claudin-5 expression both at the transcriptional and posttranscriptional levels. In addition, several signal transduction mechanisms, including the VEGFR-2/Ras/ERK1/2, PI-3K/Akt/NF-κB, and calcium signaling pathways, are involved in Tat-mediated alterations of claudin-5 levels. The effects appear to be specific, because inhibition of PKC did not affect Tat-mediated downregulation of claudin-5 expression. Tat-induced alterations of claudin-5 levels may constitute a critical mechanism of the BBB disturbances in the course of HIV-1 infection.

References

Acheampong

Mukhtar

Parveen

Ngoubilly

Ahmad

Patel

Pomerantz

(2002) Ethanol strongly potentiates apoptosis induced by HIV-1 proteins in primary human brain microvascular endothelial cells. Virology 304:222–34.

Albini

Ferrini

Benelli

Sforzini

Giunciuglio

Aluigi

Proudfoot

Alouani

Wells

Mariani

Rabin

Farber

Noonan

(1998) HIV-1 Tat protein mimicry of chemokines. Proc Natl Acad Sci USA 95:13153–8.

Albini

Soldi

Giunciuglio

Giraudo

Benelli

Primo

Noonan

Salio

Camussi

Rockl

Bussolino

(1996) The angiogenesis induced by HIV-1 tat protein is mediated by the Flk-1/KDR receptor on vascular endothelial cells. Nat Med 2:1371–5.

András

Deli

Nath

Hennig

Toborek

(2003) HIV-1 Tat protein alters tight junction protein expression and distribution in cultured brain endothelial cells. J Neurosci Res 74:255–65.

Ansari

Safak

Gallia

Sawaya

Amini

Khalili

(1999) Interaction of YB-1 with human immunodeficiency virus type 1 Tat and TAR RNA modulates viral promoter activity. J Gen Virol 80: 2629–38.

Arese

Ferrandi

Primo

Camussi

Bussolino

(2001) HIV-1 Tat protein stimulates in vivo vascular permeability and lymphomononuclear cell recruitment. J Immunol 166:1380–8.

Avison

Nath

Berger

(2002) Understanding pathogenesis and treatment of HIV dementia: a role for magnetic resonance? Trends Neurosci 25:468–73.

Barillari

Sgadari

Fiorelli

Samaniego

Colombini

Manzari

Modesti

Nair

Cafaro

Sturzl

Ensoli

(1999) The Tat protein of human immunodeficiency virus type-1 promotes vascular cell growth and locomotion by engaging the alpha5beta1 and alphavbeta3 integrins and by mobilizing sequestered basic fibroblast growth factor. Blood 94:663–72.

Bonavia

Bajetto

Barbero

Albini

Noonan

Schettini

(2001) HIV-1 Tat causes apoptotic death and calcium homeostasis alterations in rat neurons. Biochem Biophys Res Commun 288:301–8.

10.

Boven

Middel

Verhoef

De Groot

Nottet

(2000) Monocyte infiltration is highly associated with loss of the tight junction protein zonula occludens in HIV-1 associated dementia. Neuropathol Appl Neurobiol 26:356–60.

11.

Chang

Samaniego

Nair

Buonaguro

Ensoli

(1997) HIV-1 Tat protein exits from cells via a leaderless secretory pathway and binds to extracellular matrix-associated heparan sulfate proteoglycans through its basic region. AIDS 11:1421–31.

12.

Dallasta

Pisarov

Esplen

Werley

Moses

Nelson

Achim

(1999) Blood–brain barrier tight junction disruption in human immunodeficiency virus-1 encephalitis. Am J Pathol 155:1915–27.

13.

Deli

Szabó

Dung

NTK

Joó

(1997) Immunohistochemical and electron microscopy detections on primary cultures of rat cerebral endothelial cells. In: Drug transport across the blood–brain barrier: in vivo and in vitro techniques ( De Boer

Sutanto

, eds), Amsterdam: Harwood Academic Publishers, 23–8.

14.

Furuse

Fujita

Hiiragi

Fujimoto

Tsukita

(1998) Claudin-1 and −2: novel integral membrane proteins localizing at tight junctions with no sequence similarity to occludin. J Cell Biol 141:1539–50.

15.

Giovannoni

Miller

Heales

Land

Harrison

Thompson

(1998) Elevated cerebrospinal fluid and serum nitrate and nitrite levels in patients with central nervous system complications of HIV-1 infection: a correlation with blood–brain-barrier dysfunction. J Neurol Sci 156:53–8.

16.

Haughey

Mattson

(2002) Calcium dysregulation and neuronal apoptosis by the HIV-1 proteins Tat and gp120. J Acquir Immune Defic Syndr 31(Suppl 2): S55–61.

17.

Hegg

Peterson

Thayer

(2000) Betachemokines and human immunodeficiency virus type-1 proteins evoke intracellular calcium increases in human microglia. Neuroscience 98:191–9.

18.

Huber

Egleton

Davis

(2001) Molecular physiology and pathophysiology of tight junctions in the blood–brain barrier. Trends Neurosci 24:719–25.

19.

Hudson

Liu

Nath

Jones

Raghavan

Narayan

Everall

I (2000) Detection of the human immunodeficiency virus regulatory protein tat in CNS tissues. J Neurovirol 6:145–55.

20.

Ikenouchi

Matsuda

Furuse

Tsukita

(2003) Regulation of tight junctions during the epithelium—mesenchyme transition: direct repression of the gene expression of claudins/occludin by Snail. J Cell Sci 116:1959–67.

21.

Khan

Di Cello

Nath

Kim

(2003) Human immunodeficiency virus type 1 tat-mediated cytotoxicity of human brain microvascular endothelial cells. J Neurovirol 9:584–93.

22.

Kim

Avraham

Koh

Jiang

Park

Avraham

(2003) HIV-1 Tat-mediated apoptosis in human brain microvascular endothelial cells. J Immunol 170: 2629–2637.

23.

Krebs

Ross

McAllister

Wigdahl

(2000) HIV-1-associated central nervous system dysfunction. Adv Pharmacol 49:315–85.

24.

Kyriakis

Avruch

(2001) Mammalian mitogen-activated protein kinase signal transduction pathways activated by stress and inflammation. Physiol Rev 81: 807–69.

25.

Lee

Eum

Nath

Toborek

(2004) Estrogen-mediated protection against HIV Tat protein-induced inflammatory pathways in human vascular endothelial cells. Cardiovasc Res 63:139–48.

26.

Liebner

Fischmann

Rascher

Duffner

Grote

Kalbacher

Wolburg

(2000a) Claudin-1 and claudin-5 expression and tight junction morphology are altered in blood vessels of human glioblastoma multi∗forme. Acta Neuropathol (Berlin) 100:323–31.

27.

Liebner

Kniesel

Kalbacher

Wolburg

(2000b) Correlation of tight junction morphology with the expression of tight junction proteins in blood–brain barrier endothelial cells. Eur J Cell Biol 79:707–17.

28.

Lippoldt

Liebner

Andbjer

Kalbacher

Wolburg

Haller

Fuxe

(2000) Organization of choroid plexus epithelial and endothelial cell tight junctions and regulation of claudin-1, −2 and −5 expression by protein kinase C. Neuroreport 11:1427–31.

29.

Nath

(1997) Molecular determinants for cellular uptake of Tat protein of human immunodeficiency virus type 1 in brain cells. J Virol 71:2495–9.

30.

Magnuson

Knudsen

Geiger

Brownstone

RM.

Nath

(1995) Human immunodeficiency virus type 1 tat activates non-N-methyl-d-aspartate excitatory amino acid receptors and causes neurotoxicity. Ann Neurol 37:373–80.

31.

McArthur

Nance-Sproson

Griffin

Hoover

Selnes

Miller

Margolick

Cohen

Farzadegan

Saah

(1992) The diagnostic utility of elevation in cerebrospinal fluid beta 2-microglobulin in HIV-1 dementia. Multicenter AIDS Cohort Study. Neurology 42:1707–12.

32.

Milani

Mazzoni

Borgatti

Zauli

Cantley

Capitani

(1996) Extracellular human immunodeficiency virus type-1 Tat protein activates phosphatidylinositol 3-kinase in PC12 neuronal cells. J Biol Chem 271:22961–4.

33.

Mitola

Soldi

Zanon

Barra

Gutierrez

Berkhout

Giacca

Bussolino

(2000) Identification of specific molecular structures of human immunodeficiency virus type 1 Tat relevant for its biological effects on vascular endothelial cells. J Virol 74:344–53.

34.

Mitola

Sozzani

Luini

Primo

Borsatti

Weich

Bussolino

(1997) Tat-human immunodeficiency virus-1 induces human monocyte chemotaxis by activation of vascular endothelial growth factor receptor-1. Blood 90:1365–72.

35.

Morita

Sasaki

Furuse

Tsukita

(1999) Endothelial claudin: claudin-5/TMVCF constitutes tight junction strands in endothelial cells. J Cell Biol 147:185–94.

36.

Nath

Geiger

(1998) Neurobiological aspects of human immunodeficiency virus infection: neurotoxic mechanisms. Prog Neurobiol 54:19–33.

37.

Nitta

Hata

Gotoh

Seo

Sasaki

Hashimoto

Furuse

Tsukita

(2003) Size-selective loosening of the blood–brain barrier in claudin-5-deficient mice. J Cell Biol 161:653–60.

38.

Oshima

Flores

Vaitaitis

Coe

Joh

Park

Zhu

Alexander

(2000) HIV-1 Tat increases endothelial solute permeability through tyrosine kinase and mitogen-activated protein kinase-dependent pathways. AIDS 14:475–82.

39.

Pardridge

(2002) Drug and gene delivery to the brain: the vascular route. Neuron 36:555–8.

40.

Peinado

Quintanilla

Cano

(2003) Transforming growth factor beta-1 induces snail transcription factor in epithelial cell lines: mechanisms for epithelial mesenchymal transitions. J Biol Chem 278:21113–23.

41.

Petito

Cash

(1992) Blood–brain barrier abnormalities in the acquired immunodeficiency syndrome: immunohistochemical localization of serum proteins in postmortem brain. Ann Neurol 32:658–66.

42.

Poggi

Carosio

Fenoglio

Brenci

Murdaca

Setti

Indiveri

Scabini

Ferrero

Zocchi

(2004) Migration of V delta 1 and V delta 2 T cells in response to CXCR3 and CXCR4 ligands in healthy donors and HIV-1-infected patients: competition by HIV-1 Tat. Blood 103:2205–13.

43.

Power

Kong

Crawford

Wesselingh

Glass

McArthur

Trapp

(1993) Cerebral white matter changes in acquired immunodeficiency syndrome dementia: alterations of the blood-brain barrier. Ann Neurol 34:339–50.

44.

Prendergast

Rogers

Mulholland

Littleton

Wilkins

Jr Self

Nath

(2002) Neurotoxic effects of the human immunodeficiency virus type-1 transcription factor Tat require function of a polyamine sensitive-site on the N-methyl-D-aspartate receptor. Brain Res 954:300–7.

45.

Tian

Flora

Lee

Nath

Hennig

Toborek

(2003) HIV-1 Tat protein upregulates inflammatory mediators and induces monocyte invasion into the brain. Mol Cell Neurosci 24:224–37.

46.

Rhodes

Ward

(1991) AIDS meningoencephalomyelitis. Pathogenesis and changing neuropathologic findings. Pathol Annu 26:Pt 2:247–276.

47.

Rusnati

Tulipano

Spillmann

Tanghetti

Oreste

Zoppetti

Giacca

Presta

(1999) Multiple interactions of HIV-I Tat protein with size-defined heparin oligosaccharides. J Biol Chem 274:28198–205.

48.

Rusnati

Urbinati

Musulin

Ribatti

Albini

Noonan

Marchisone

Waltenberger

Presta

(2001) Activation of endothelial cell mitogen activated protein kinase ERK(1/2) by extracellular HIV-1 Tat protein. Endothelium 8:65–74.

49.

Singer

Syndulko

Fahy-Chandon

Schmid

Conrad

Tourtellotte

(1994) Intrathecal IgG synthesis and albumin leakage are increased in subjects with HIV-1 neurologic disease. J Acquir Immune Defic Syndr 7:265–71.

50.

Speth

Schabetsberger

Mohsenipour

Stockl

Wurzner

Stoiber

Lass-Florl

Dierich

(2002) Mechanism of human immunodeficiency virus-induced complement expression in astrocytes and neurons. J Virol 76:3179–88.

51.

Thomas

Monick

Staber

Yarovinsky

Carter

Hunninghake

(2002) Respiratory syncytial virus inhibits apoptosis and induces NF-kappa B activity through a phosphatidylinositol 3-kinase-dependent pathway. J Biol Chem 277:492–501.

52.

Toborek

Lee

Malecki

Flora

Garrido

Hennig

Bauer

Nath

(2003) HIV-Tat protein induces oxidative and inflammatory pathways in brain endothelium. J Neurochem 84:169–79.

53.

Tsukita

Furuse

(2000) The structure and function of claudins, cell adhesion molecules at tight junctions. Ann NY Acad Sci 915:129–35.

54.

Valle

Croul

Morgello

Amini

Rappaport

Khalili

(2000) Detection of HIV-1 Tat and JCV capsid protein, VP1, in AIDS brain with progressive multifocal leukoencephalopathy. J Neurovirol 6:221–8.

55.

Vives

Richard

Rispal

Lebleu

(2003) TAT peptide internalization: seeking the mechanism of entry. Curr Protein Pept Sci 4:125–32.

56.

Wachtel

Bolliger

Ishihara

Frei

Bluethmann

Gloor

(2001) Down-regulation of occludin expression in astrocytes by tumour necrosis factor (TNF) is mediated via TNF type-1 receptor and nuclear factor-kappaB activation. J Neurochem 78: 155–162.

57.

Woodman

Weiss

McManus

D'Aversa

Hesselgesser

Major

Nath

Berman

(2000) Mechanisms of leukocyte trafficking into the CNS. J Neurovirol 6(Suppl 1):S82–5.

58.

Xiao

Neuveut

Tiffany

Benkirane

Rich

Murphy

Jeang

(2000) Selective CXCR4 antagonism by Tat: implications for in vivo expansion of coreceptor use by HIV-1. Proc Natl Acad Sci USA 97:11466–71.

59.

Yang

Gabuzda

(1999) Regulation of human immunodeficiency virus type 1 infectivity by the ERK mitogen-activated protein kinase signaling pathway. J Virol 73: 3460–6.