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HIV-1 Tat C phosphorylates VE-cadherin complex and increases human brain microvascular endothelial cell permeability.

Mishra R, Singh SK - BMC Neurosci (2014)

Bottom Line: We exposed hBMVECs to recombinant HIV-1 clade C Tat protein to study the effect of HIV-1 Tat C on permeability of hBMVECs.Redox-sensitive kinase; PYK2 activation led to increased tyrosine phosphorylation of VE-cadherin and β-catenin, leading to disruption of junctional assembly.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.

View Article: PubMed Central - HTML - PubMed

Affiliation: Laboratory of Neurovirology and Inflammation Biology, CSIR-Centre for Cellular and Molecular Biology (CCMB), Uppal Road, Hyderabad 500007, India. sunitsingh2000@gmail.com.

ABSTRACT

Background: 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.

Results: 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.

Conclusion: 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.

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Related in: MedlinePlus

HIV-1 Tat C dissociates β-catenin from the VE-cadherin/β-catenin complex and induces endothelial permeability. VE-cadherin complex were pull down from control as well as HIV-1 Tat C treated hBMVECs to examine the phosphorylation induced dissociation of β-catenin from VE-cadherin. (A) Coimmunoprecipetation of β-catenin with cadherin complex, by using monoclonal anti VE-cadherin antibody, showing a significantly decreased association of β-catenin with VE-cadherin complex in HIV-1 Tat C treated hBMVECs. (B) A significant change in association of β-catenin with VE-cadherin in HIV-1 Tat C treated hBMVECs, shown by bar diagram. The bars are representing mean ± S.E. from three independently repeated experiments. (C) The graph showing a dose dependent decrease in TEER values across the trans-well insert membrane, in HIV-1 Tat C treated hBMVECs as compared to buffer treated control cells. (D) Sodium fluorescein dye migration assay, showing a dose dependent increase in amount of flow through of fluorescent dye across the trans-well insert membrane, due to increased permeability of hBMVECs exposed to increasing dose of HIV-1 Tat C protein.
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Figure 3: HIV-1 Tat C dissociates β-catenin from the VE-cadherin/β-catenin complex and induces endothelial permeability. VE-cadherin complex were pull down from control as well as HIV-1 Tat C treated hBMVECs to examine the phosphorylation induced dissociation of β-catenin from VE-cadherin. (A) Coimmunoprecipetation of β-catenin with cadherin complex, by using monoclonal anti VE-cadherin antibody, showing a significantly decreased association of β-catenin with VE-cadherin complex in HIV-1 Tat C treated hBMVECs. (B) A significant change in association of β-catenin with VE-cadherin in HIV-1 Tat C treated hBMVECs, shown by bar diagram. The bars are representing mean ± S.E. from three independently repeated experiments. (C) The graph showing a dose dependent decrease in TEER values across the trans-well insert membrane, in HIV-1 Tat C treated hBMVECs as compared to buffer treated control cells. (D) Sodium fluorescein dye migration assay, showing a dose dependent increase in amount of flow through of fluorescent dye across the trans-well insert membrane, due to increased permeability of hBMVECs exposed to increasing dose of HIV-1 Tat C protein.

Mentions: HIV-1 Tat induced tyrosine phosphorylation of β-catenin occurs in dose dependent manner (Figures 1D, E). SHP2 and VE-PTP expression also got downregulated and dissociated from VE-cadherin complex after HIV-1 Tat treatment (500 ng/ml) in hBMVECs, leaving VE-cadherin complex prone to the phopshorylation (Figure 2). Co-immunoprecipitation of the cadherin complex by monoclonal antibody of VE-cadherin showed 60% reduction (P ≤ 0.05) in the association of β-catenin with VE-cadherin in HIV-1 Tat treated hBMVECs (Figure 3A, B). Endothelial cell permeability, assessed by TEER and sodium fluorescein migration assay showed an elevated permeability in HIV-1 Tat, treated hBMVECs (Figure 3C, D). Compared to control cells, HIV-1 Tat treated hBMVECs, has shown 40–50% decrease in TEER values, however sodium fluorescein migration assay has shown about 60–70% increase in endothelial cell permeability in trans-well membrane experiments (Figure 3C, D).


HIV-1 Tat C phosphorylates VE-cadherin complex and increases human brain microvascular endothelial cell permeability.

Mishra R, Singh SK - BMC Neurosci (2014)

HIV-1 Tat C dissociates β-catenin from the VE-cadherin/β-catenin complex and induces endothelial permeability. VE-cadherin complex were pull down from control as well as HIV-1 Tat C treated hBMVECs to examine the phosphorylation induced dissociation of β-catenin from VE-cadherin. (A) Coimmunoprecipetation of β-catenin with cadherin complex, by using monoclonal anti VE-cadherin antibody, showing a significantly decreased association of β-catenin with VE-cadherin complex in HIV-1 Tat C treated hBMVECs. (B) A significant change in association of β-catenin with VE-cadherin in HIV-1 Tat C treated hBMVECs, shown by bar diagram. The bars are representing mean ± S.E. from three independently repeated experiments. (C) The graph showing a dose dependent decrease in TEER values across the trans-well insert membrane, in HIV-1 Tat C treated hBMVECs as compared to buffer treated control cells. (D) Sodium fluorescein dye migration assay, showing a dose dependent increase in amount of flow through of fluorescent dye across the trans-well insert membrane, due to increased permeability of hBMVECs exposed to increasing dose of HIV-1 Tat C protein.
© Copyright Policy - open-access
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC4230799&req=5

Figure 3: HIV-1 Tat C dissociates β-catenin from the VE-cadherin/β-catenin complex and induces endothelial permeability. VE-cadherin complex were pull down from control as well as HIV-1 Tat C treated hBMVECs to examine the phosphorylation induced dissociation of β-catenin from VE-cadherin. (A) Coimmunoprecipetation of β-catenin with cadherin complex, by using monoclonal anti VE-cadherin antibody, showing a significantly decreased association of β-catenin with VE-cadherin complex in HIV-1 Tat C treated hBMVECs. (B) A significant change in association of β-catenin with VE-cadherin in HIV-1 Tat C treated hBMVECs, shown by bar diagram. The bars are representing mean ± S.E. from three independently repeated experiments. (C) The graph showing a dose dependent decrease in TEER values across the trans-well insert membrane, in HIV-1 Tat C treated hBMVECs as compared to buffer treated control cells. (D) Sodium fluorescein dye migration assay, showing a dose dependent increase in amount of flow through of fluorescent dye across the trans-well insert membrane, due to increased permeability of hBMVECs exposed to increasing dose of HIV-1 Tat C protein.
Mentions: HIV-1 Tat induced tyrosine phosphorylation of β-catenin occurs in dose dependent manner (Figures 1D, E). SHP2 and VE-PTP expression also got downregulated and dissociated from VE-cadherin complex after HIV-1 Tat treatment (500 ng/ml) in hBMVECs, leaving VE-cadherin complex prone to the phopshorylation (Figure 2). Co-immunoprecipitation of the cadherin complex by monoclonal antibody of VE-cadherin showed 60% reduction (P ≤ 0.05) in the association of β-catenin with VE-cadherin in HIV-1 Tat treated hBMVECs (Figure 3A, B). Endothelial cell permeability, assessed by TEER and sodium fluorescein migration assay showed an elevated permeability in HIV-1 Tat, treated hBMVECs (Figure 3C, D). Compared to control cells, HIV-1 Tat treated hBMVECs, has shown 40–50% decrease in TEER values, however sodium fluorescein migration assay has shown about 60–70% increase in endothelial cell permeability in trans-well membrane experiments (Figure 3C, D).

Bottom Line: We exposed hBMVECs to recombinant HIV-1 clade C Tat protein to study the effect of HIV-1 Tat C on permeability of hBMVECs.Redox-sensitive kinase; PYK2 activation led to increased tyrosine phosphorylation of VE-cadherin and β-catenin, leading to disruption of junctional assembly.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.

View Article: PubMed Central - HTML - PubMed

Affiliation: Laboratory of Neurovirology and Inflammation Biology, CSIR-Centre for Cellular and Molecular Biology (CCMB), Uppal Road, Hyderabad 500007, India. sunitsingh2000@gmail.com.

ABSTRACT

Background: 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.

Results: 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.

Conclusion: 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.

Show MeSH
Related in: MedlinePlus