HIV-1 Tat C phosphorylates VE-cadherin complex and increases human brain microvascular endothelial cell permeability
© Mishra and Singh; licensee BioMed Central Ltd. 2014
Received: 29 January 2014
Accepted: 18 June 2014
Published: 26 June 2014
Human brain microvascular endothelial cells (hBMVECs) are integral part of the blood brain barrier. Post-translational modifications of adherens junction proteins regulate the permeability of human brain microvascular endothelial cells. Pro-inflammatory signals can induce tyrosine phosphorylation of adherens junction proteins. The primary objective of this work is to provide a molecular model; how the HIV-1 Tat protein can compromise the BBB integrity and eventually lead to neurological consequences. We exposed hBMVECs to recombinant HIV-1 clade C Tat protein to study the effect of HIV-1 Tat C on permeability of hBMVECs. Trans-endothelial electrical resistance and fluorescent dye migration assay have been used to check the permeability of hBMVECs. DCFDA staining has been used for intracellular reactive oxygen species (ROS) detection. Western blotting has been used to study the expression levels and co-immunoprecipitation has been used to study the interactions among adherens junction proteins.
HIV-1 Tat C protein induced NOX2 and NOX4 expression level and increased intracellular ROS level. Redox-sensitive kinase; PYK2 activation led to increased tyrosine phosphorylation of VE-cadherin and β-catenin, leading to disruption of junctional assembly. The dissociation of tyrosine phosphatases VE-PTP and SHP2 from cadherin complex resulted into increased tyrosine phosphorylation of VE-cadherin and β-catenin in HIV-1 Tat C treated hBMVECs.
Unrestricted phosphorylation of junctional proteins in hBMVECs, in response to HIV-1 Tat C protein; leads to the disruption of junctional complexes and increased endothelial permeability.
HIV enters the central nervous system (CNS) by crossing the blood brain barrier (BBB) during early stages of HIV infection through infected blood cells [1, 2]. HIV infected cells secrete the Tat protein extracellularly  and affect the functions of human brain microvascular endothelial cells (hBMVECs) . The hBMVECs form a selective barrier between peripheral circulation and the CNS. The barrier properties of hBMVECs are principally imparted by adherens junction proteins (AJPs) and tight junction proteins (TJPs) . VE-cadherin and catenin play major role in stabilization of adherens junctions (AJs) and barrier integrity . Cadherin complex is composed of vascular endothelial-cadherin (VE-cadherin), bound to β-catenin or plakoglobin, which in turn binds to α-catenin; vinculin, α-actinin, ZO-1 and actin . The tyrosine phosphorylation of VE-cadherin complex is critical for the integrity of the AJs and endothelial permeability [8, 9].
VE-PTP is a tyrosine phosphatase, transmembrane binding partner of VE-cadherin. It connects with VE-cadherin through an extracellular domain and maintains the unphosphorylated state of VE-cadherin . Another phosphatase; SHP2 is a ubiquitously expressed non-receptor PTP, consisting of two N-terminal tandem SH2 domains and a catalytic phosphatase (PTP) domain and a C-terminal tail with two tyrosine residues . Ukropec et al. ; reported SHP2 as a component of VE-cadherin complex, which regulates the tyrosine phosphorylation of both, β-catenin  and VE-cadherin .
Phosphorylated states of the VE-cadherin and β-catenin regulate their interactions, and control the adhesion forces . VE-cadherin has many putative phospho-tyrosine sites, but Y658, Y685, and Y731 phosphorylation are reported to be important for maintaining the barrier integrity . PYK2 is a redox-sensitive tyrosine kinase and gets activated by NADPH mediated ROS generation . Tyrosine phosphorylation of β-catenin is induced by PYK2 (proline rich kinase 2)  and dephosphorylated by SHP2 .
Pro-inflammatory factors affect the phosphorylated state of VE-cadherin complex and disrupt the barrier integrity [19, 20]. In this study, we have selected HIV-1 Tat C to study the effect on endothelial permeability, because HIV-1 clade C alone is responsible for more than 56% of global HIV infections. HIV-1 clade C infections are prevalent in South-east Asian countries including India . We have examined the effects of the HIV-1 Tat C protein on redox-sensitive kinase PYK2 and tyrosine phosphatases of VE-cadherin complex (VE-PTP and SHP2) and their impact on tyrosine phosphorylation of VE-cadherin, β-catenin and permeability of hBMVECs.
Primary hBMVECs were obtained from Dr. Joan Berman, department of pathology, Albert Einstein College of Medicine, New York as a kind gift. hBMVECs were cultured as described previously . For all hBMVECs cultures, the culture flasks were coated with 0.2% bovine gelatin solution. CEM-GFP cells (NIH-AIDS Reagent Programme) were used for transactivation assay to check the biological activity of recombinant HIV-1 Tat C protein. CEM-GFP cells were grown in RPMI 1640 (Gibco) supplemented with 10% fetal bovine serum (Gibco), 2 mM glutamine, 100 units of penicillin/streptomycin/ml. Cell cultures were maintained at 37°C and constant supply of 5% CO2 in a humidified incubator.
Expression and purification of HIV-1 Tat C protein
HIV-1 Tat C protein has been expressed and purified as per our standardized protocol, described elsewhere in detail . Immunoconfirmation of recombinant HIV-1 Tat C protein was done by western blot analysis using anti-Tat antibody (NIH AIDS Research and Reference Reagent Program). Endotoxin level of purified recombinant HIV-1 Tat C protein was measured by Limulus Amebocyte Lysate (LAL) assay (Lonza) as per manufacturer’s protocol. The level of endotoxin was found in range of 0.04 EU/μg of purified protein; which was much below the acceptable limit set by international standards. Transcriptional activity of recombinant HIV-1 Tat C protein was checked by transactivation assay as described previously . CEM-GFP cells are T-cell lines, carrying a stably integrated GFP gene under the control of HIV-1 subtype-B LTR. Purified HIV-1 Tat C protein (5 μg) was transfected in CEM-GFP cells, with proteo-juice protein transfection reagent (Novagen). GFP expressions were visualized with fluorescence microscope (Axio Observer-A1, Carl Zeiss, Germany). All protocols were approved by the Centre for Cellular and Molecular Biology (CCMB), Hyderabad Institutional Biosafety Committee.
HIV-1 Tat C treatment on human brain microvascular endothelial cells
hBMVECs were grown as confluent monolayer and exposed with HIV-1 Tat C protein at various doses (100 ng/ml, 200 ng/ml, 500 ng/ml, and 1 μg/ml) in serum-free M199 media. Control cells were treated with Tat buffer (30 mM phosphate buffer, pH 6.8 supplemented with 1 mM DTT). BMVECs have been exposed to Tat C at different concentration ranges due to its closeness to reported serum levels and hBMVECs would normally encounter such concentrations of Tat in NeuroAIDS patients [3, 24, 25]. hBMVECs were harvested for RNA and protein sample preparation after 12 hours of HIV-1 Tat C protein treatments.
Western blot analysis
Cell lysis were done in RIPA buffer (150 mM NaCl, 50 mM Tris.HCL pH 7.5, 1% NP-40, 0.5% sodium doxycholate, 0.1% SDS and 1× protease inhibitor cocktail). Estimation of protein concentrations was done by Bradford assay (Bio-Rad). Proteins samples were boiled in SDS-loading buffer and run on 12% SDS-gel, transferred on PVDF membrane (Millipore) at 100 V for 2 hrs. Primary antibody incubations were given overnight at 4°C with dilutions (1:1000). HRP conjugated secondary antibody were applied for 1 hour at room temperature and developed by using Super Signal developing reagent (Pierce, Thermo Scientific). Antibodies against p-Y-731-VE-cadherin (Invitrogen), VE-cadherin, anti-β-tubulin, NOX2 and NOX4 (Abcam), β-catenin (Santacruz biotechnology), PYK2 (Cell signaling technology), SHP2 (Sigma) have been used in the study. VE-PTP antibody was provided as a kind gift by Astrid F. Nottebaum, Max Planck Institute of Molecular Medicine, Muenster, Germany.
Transendothelial Electrical Resistance (TEER) Assay
EVOM2 (World precision Instruments) was used to measure electrical resistance across the endothelial monolayer. PTFE membrane inserts (12 well plate) (Corning Life sciences) consisting of 3.0 μm pore sizes were used in the permeability assays. 0.2% bovine gelatin coating was done on insert membrane before cell seeding for better adherence of hBMVECs. Cell seeding density was 2 × 104 in 500 μl of complete M199 media. To check the development of junctional integrity, TEER values were checked on regular intervals until reached at consistent resistance in endothelial cells. After attaining the optimal confluency, the hBMVECs were treated with different doses of HIV-1 Tat C protein. TEER of hBMVECs was measured after 12 hrs of HIV-1 Tat C protein exposure. The resistance of empty insert membranes, without cell seeding was deducted from recorded TEER values.
Fluorescein migration assay
The fluorescein migration assay was performed as described previously elsewhere . hBMVECs were seeded on membrane inserts in similar way as for TEER assay. After treatment with different doses of Tat C proteins, cells were washed with HBSS buffer and incubated with 0.005% sodium fluorescein dye (SRL # 064738, mol weight- 376.28) on upper chamber of the insert membrane. hBMVECs were incubated with sodium fluorescein dye for 30 minutes at 37°C, followed by collection of 100 μl of the flowthrough from lower chambers. Fluorescence was measured at 480/530 nm wavelength by using fluorimeter (Infinite M200, Tecon).
Co-immunoprecipitation of VE-cadherin/VE-PTP/β-catenin and SHP2
Cells were pelleted after 12 hours of HIV-1 Tat C treatment (500 ng/ml), washed with cold PBS and lysed in NP-40 mild lysis buffer (NP-40 1%, Tris–HCl 50 mM, NaCL 150 mM, NaF 1 mM, PMSF 2 mM, Sodium orthovanadate 1 mM, Sodium pyrophosphate 10 mM, Protease inhibiter cocktail 1×). Equal amounts of protein samples were incubated with VE-cadherin antibody overnight at 4°C. These samples were incubated with protein-G-sepharose beads for 3–4 hours followed by centrifugation. The immunoprecipitated samples along with beads were washed three times with lysis buffer and finally eluted by boiling in SDS-loading buffer. The samples were run on 12/% SDS-Page gel and probed with respective antibodies.
Intracellular Reactive Oxygen Species (ROS) detection by Microscopy
hBMVECs were grown in 6 well culture dishes till confluency. HIV-1 Tat treatment was given for 12 hours at the different doses as described earlier. Cells were washed with HBSS and treated with final concentration of 10 μM of cell permeant fluorogenic dye 2’ 7’ dichlorofluorescein diacetate (DCFDA) for 15 minutes at 37°C in incubator chamber. After 15 minutes of incubation, cells were washed and fluorescence was visualized under fluorescence microscope (Axio Observer-A1, Carl Zeiss, and Germany).
Chemical inhibiter against PYK2 (PF-431396) (Tocris Bioscience) has been used to inhibit the kinase activity. hBMVECs were cultured till confluency and treated with 22 nM PF-431396. Cells treated with equal volume of DMSO have been taken as control. Diphenyleneiodonium chloride (DPI) (Sigma Aldrich) dissolved in DMSO has been used as ROS scavenger, at 100 nM concentration. hBMVECs were harvested for lysate preparation after 12 hours of treatment.
Results have been expressed as the mean and standard error of the mean. Level of significance in treated group versus untreated group has been measured by using Student’s t test. P-values < 0.05 have been taken as significant in one tailed array.
HIV-1 Tat mediated phosphorylation of adherens junction protein (VE-cadherin and β-catenin)
Downregulation and dissociation of VE-PTP and SHP2 from VE-cadherin complex in HIV-1 Tat treated human BMVECs
HIV-1 Tat dissociates β-catenin from the VE-cadherin/β-catenin complex and induces endothelial permeability
HIV-1 Tat upregulates NADPH oxidase (NOX2 and NOX4) expression and increases ROS production in human BMVECs
HIV-1 Tat activates PYK-2 in dose dependent manner
ROS scavenger (DPI) mediated rescue of SHP2 expression and downregulation in phosphorylation of β-catenin
PYK2 inhibiter abrogates the phosphorylation of β-catenin and rescues endothelial permeability
Neurological consequences still persist in the era of HAART therapy, where viral load has been effectively controlled in infected patients. However HIV-1 protein such as Tat has been detected in the CSF of patients kept on anti-retroviral therapy and show effectively controlled viremia . Therefore, we have focused on the bystander effect of HIV-1 Tat protein on human brain microvascular endothelial cells. In brain microvascular endothelial cells, the levels of VE-cadherin regulate the permeability of endothelial cells . Vascular endothelial cells are connected by homophilic adhesion through AJPs and TJPs . We previously reported the post-transcriptional regulation of VE-cadherin by miR-101 and permeability in hBMVECs exposed to HIV-1 Tat C protein . Increased tyrosine phosphorylation of VE-cadherin and associated catenins are known to compromises the junctional architecture of endothelial cells, which disrupts their physical interactions at cell-cell junctions [8, 19, 20, 31]. Inflammatory conditions change the magnitude of tyrosine phosphorylation of AJPs [19, 32–34]. Previously, phosphorylation of Y731 amino acid position in VE-cadherin has been reported to be responsible for impairing the association of VE-cadherin with p120-catenin and β-catenin  during leukocyte extravasation .
Different mechanisms and pathways have been reported for the regulation of endothelial permeability. Involvement of PPAR/lipid raft-dependent MMP-2 and MMP-3 activation lead to the degradation of occludin and tricellulin, which has been reported in C12-HSL-induced perturbations of epithelial barrier functions . Role of activation of ERK1/2 and Akt signaling has been described for increased BBB permeability through decreased tight junction (TJ) protein expressions in brain microvascular endothelial cells. Tat mediated effects can be attenuated by application of PPAR antagonist . Another in vitro BBB model study showed that HIV-1 infection of pericytes can also result into compromised BBB integrity . Another recent study highlighted the role of Rho signaling and CREB in triggering the nuclear localization of ZO-1 and perturbing the endothelial permeability after HIV-1 Tat exposure .
In our study, we observed a dose-dependent increase in tyrosine phosphorylation of VE-cadherin at p-Y731 position and β-catenin at p-Y654 position (Figure 1A-E). The Tyr-731 site is considered unique to VE-cadherin in reference to endothelial permeability . VE-cadherin and β-catenin have shown induced phosphorylation at their tyrosine positions accompanied by reduced expression of respective phosphatases VE-PTP and SHP2. Tyrosine phosphorylation of VE-cadherin and β-catenin which disrupts their physical interactions explains increased endothelial permeability and leakage .
We studied the role of kinases and phosphatases in HIV-1 Tat induced tyrosine phosphorylation of AJPs. We focused on PYK2 kinase because PYK2 is highly redox sensitive kinase and its signaling gets affected by intracellular ROS levels . PYK2 has been described as a critical regulator of endothelial inflammation via phosphorylating IKK-RelA/p65 pathway, therefore facilitating the nuclear translocation of the RelA/p65 . In our study, increased generation of ROS was observed with the increasing doses of HIV-1 Tat C exposure (Figure 4D). Role of NOX2 in HIV-1 Tat induced generation of ROS has been reported in human astrocytes, where enhanced monocyte adhesion was regulated by ROS mediated increase in ICAM/VCAM expression . Secretion of TNF-α has also been reported to induce NADPH oxidase-derived ROS generation and PYK2 activation in H9c2 cells . In addition, PYK2-dependent tyrosine phosphorylation has been reported to disrupt the association of zonula occludens-2 with the endothelial tight junctions . Our study showed increased intracellular ROS generation, followed by increase in phosphorylation and activation of PYK2, in HIV-1 Tat exposed hBMVECs (Figure 5A, B). This explains the higher levels of tyrosine phosphorylation in β-catenin and VE-cadherin which will lead to increased endothelial cell permeability (Figure 3C). As expected, ROS scavenging has significantly reduced the activation of PYK2 (Figure 5C, D) even in presence of HIV-1 Tat in hBMVECs. PTPs act together with cellular protein tyrosine kinases and control the phosphorylation and dephosphorylation of substrate proteins and associated signaling .
VE-PTP enhances VE-cadherin mediated cell–cell adhesion and plays a major role in maintaining endothelial barrier permeability . The significant downregulation of VE-PTP (p ≤0.05) (Figure 2A, E) and significant decrease in association of VE-PTP with VE-cadherin were observed in HIV-1 Tat C exposed hBMVECs (Figure 2B, F). In a recent report, Vockel et. al. (2013) reported the generation of ROS through activation of NADPH oxidases and activation of redox-sensitive tyrosine kinase PYK2 and their effect on cell-cell junctions. They suggested that activation of PYK2 could be essential for VE-cadherin/VE-PTP dissociation . In our study, the downregulation and dissociation of VE-PTP from VE-cadherin complex explains the high level of tyrosine phosphorylation of VE-cadherin in HIV-1 Tat treated hBMVECs. Another, non-receptor cytosolic tyrosine phosphatase SHP2 has been reported to be associated with β-catenin  and dephosphorylates β-catenin and VE-cadherin in quiscent cells. We observed the significant downregulation of SHP2 in HIV-1 Tat treated hBMVECs in dose dependent manner (Figure 2C) and a significant dissociation of SHP2 from VE-cadherin complex.
Pro-inflammatory insults affect the binding of VE-cadherin and β-catenin . We observed the separation of β-catenin from VE-cadherin complex in pull down experiment in HIV-1 Tat exposed hBMVECs (Figure 3A). The disaggregation of VE-cadherin and β-catenin is most likely due to the increased level of their tyrosine phosphorylation. The disassembly of these proteins led to the enhanced endothelial permeability in HIV-1 Tat treated hBMVECs.ROS generation activated the tyrosine kinases and elevated phosphorylation of AJPs and finally disruption of endothelial integrity. We scavenged the ROS by using DPI followed by HIV-1 Tat treatment. ROS scavenging significantly abolished the PYK2 activation (Figure 5C), sustained SHP2 expression level (Figure 6A), and reduced tyrosine phosphorylation of β-catenin (Figure 6C) in the hBMVECs. These changes resulted into improved permeability in DPI treated hBMVECs (Figure 6E).
We checked the tyrosine phosphorylation of β-catenin in hBMVECs treated with PYK2 inhibiter (PF-431396). PF-431396 abrogates the kinase activity of PYK2/FAK family kinases. A significant decrease in p-Y-β-catenin was observed in the hBMVECs treated with PF-431396 (Figure 7A, B). Our data are in accordance with the previous reports; where β-catenin has been reported to act as a substrate of PYK2 kinase . Phosphorylation of β-catenin was also checked in the hBMVECs, treated with PF-431396 followed by similar amount of HIV-1 Tat protein exposure. The effect of HIV-1 Tat on tyrosine phosphorylation of β-catenin was significantly reduced in hBMVECs treated with PF-431396 as well as in ROS scavenger treated hBMVECs, which suggested that the β-catenin phosphorylation can be controlled by manipulating PYK2 kinase activity and well as intracellular ROS levels. Pharmacological inhibition of PYK2 has been reported as a potential therapeutic strategy to combat acute lung injury in patients as well as mice model of asthma [41, 47, 48]. PYK2 inhibition rescued the permeability of hBMVECs exposed to HIV-1 Tat protein (Figure 7C). Inhibition of the PYK2 activity by PF-431396 had a partial effect on tyrosine phosphorylation of VE-cadherin (data not shown). This suggested that the phosphorylation events of VE-cadherin are also partially regulated through Src and Rac mediated kinase cascades .
RM is currently working as CSIR-Senior Research Fellow and pursuing her Ph.D at CCMB, Hyderabad. SKS is leading a research group in the area of Neurovirology at CCMB, Hyderabad.
Authors are highly thankful to Prof. Joan W. Berman, Dept. of Pathology, Albert Einstein College of Medicine (AECOM), New York, for her kind gift of human brain microvascular endothelial cells. We thankfully acknowledge to Astrid F. Nottebaum, Max Planck Institute of Molecular Medicine, Muenster, Germany for sharing the VE-PTP antibody. We acknowledge to NIH AIDS Reagent program for sharing CEM-GFP cells. Authors also acknowledge the support of Director, CSIR-Centre for Cellular and Molecular Biology (CCMB), Hyderabad. The work was supported through-CSIR Network Project (BSC0115) and Indo-Swiss Grant DST/INT/SWISS/P-44/2012.
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