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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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PYK2 inhibiter abrogates the phosphorylation of β-catenin and rescues endothelial permeability. (A) Tyrosine phosphorylation of β-catenin demonstrated after PYK2 inhibition by applying chemical inhibiter PF-431396. PYK2 inhibition significantly abrogated the tyrosine phosphorylation of β-catenin even in HIV-1 Tat C exposed hBMVECs. Cells were treated with PF-431396 followed by treatment of HIV-1 Tat C protein and treated cells were harvested after 12 hours for western blot analysis. (B) Densitometry analysis showing the significantly diminished tyrosine phosphorylation of β-catenin in PF-431396 treated cells. All the experiments have been repeated as three independent biological sets and results are shown as mean ± S.E. (*p value ≤0.05). (C) The graph bars are showing consequences of inhibiting PYK2 kinase activity on endothelial permeability. PYK2 inhibiter provide significant rescuing of endothelial permeability as compared to HIV-1 Tat C treated human BMVECs (**p value ≤0.005). Results are shown as mean ± S.E of three biological independent experiments.
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Figure 7: PYK2 inhibiter abrogates the phosphorylation of β-catenin and rescues endothelial permeability. (A) Tyrosine phosphorylation of β-catenin demonstrated after PYK2 inhibition by applying chemical inhibiter PF-431396. PYK2 inhibition significantly abrogated the tyrosine phosphorylation of β-catenin even in HIV-1 Tat C exposed hBMVECs. Cells were treated with PF-431396 followed by treatment of HIV-1 Tat C protein and treated cells were harvested after 12 hours for western blot analysis. (B) Densitometry analysis showing the significantly diminished tyrosine phosphorylation of β-catenin in PF-431396 treated cells. All the experiments have been repeated as three independent biological sets and results are shown as mean ± S.E. (*p value ≤0.05). (C) The graph bars are showing consequences of inhibiting PYK2 kinase activity on endothelial permeability. PYK2 inhibiter provide significant rescuing of endothelial permeability as compared to HIV-1 Tat C treated human BMVECs (**p value ≤0.005). Results are shown as mean ± S.E of three biological independent experiments.

Mentions: hBMVECs were treated with PYK2 inhibiter PF-431396 (Inhibiter of kinase activity) (Tocris Bioscience) and checked for tyrosine phosphorylation of β-catenin. Significant reduction in tyrosine phosphorylation of β-catenin resulted into an accumulation of total β-catenin (Figure 7A, B). No significant effect on tyrosine phosphorylation of β-catenin was observed in hBMVECs treated with PYK2 inhibiter (PF-431396), followed by HIV-1 Tat treatment. The level of total β-catenin was comparable to PF-431396 treated hBMVECs (Figure 7A, B). The effect of PF-431396 was also investigated on the permeability of hBMVECs. The inhibition of PYK2 significantly abrogated the effect of HIV-1 Tat and maintained the endothelial permeability (p ≤0.005) (Figure 7C).


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

Mishra R, Singh SK - BMC Neurosci (2014)

PYK2 inhibiter abrogates the phosphorylation of β-catenin and rescues endothelial permeability. (A) Tyrosine phosphorylation of β-catenin demonstrated after PYK2 inhibition by applying chemical inhibiter PF-431396. PYK2 inhibition significantly abrogated the tyrosine phosphorylation of β-catenin even in HIV-1 Tat C exposed hBMVECs. Cells were treated with PF-431396 followed by treatment of HIV-1 Tat C protein and treated cells were harvested after 12 hours for western blot analysis. (B) Densitometry analysis showing the significantly diminished tyrosine phosphorylation of β-catenin in PF-431396 treated cells. All the experiments have been repeated as three independent biological sets and results are shown as mean ± S.E. (*p value ≤0.05). (C) The graph bars are showing consequences of inhibiting PYK2 kinase activity on endothelial permeability. PYK2 inhibiter provide significant rescuing of endothelial permeability as compared to HIV-1 Tat C treated human BMVECs (**p value ≤0.005). Results are shown as mean ± S.E of three biological independent experiments.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 7: PYK2 inhibiter abrogates the phosphorylation of β-catenin and rescues endothelial permeability. (A) Tyrosine phosphorylation of β-catenin demonstrated after PYK2 inhibition by applying chemical inhibiter PF-431396. PYK2 inhibition significantly abrogated the tyrosine phosphorylation of β-catenin even in HIV-1 Tat C exposed hBMVECs. Cells were treated with PF-431396 followed by treatment of HIV-1 Tat C protein and treated cells were harvested after 12 hours for western blot analysis. (B) Densitometry analysis showing the significantly diminished tyrosine phosphorylation of β-catenin in PF-431396 treated cells. All the experiments have been repeated as three independent biological sets and results are shown as mean ± S.E. (*p value ≤0.05). (C) The graph bars are showing consequences of inhibiting PYK2 kinase activity on endothelial permeability. PYK2 inhibiter provide significant rescuing of endothelial permeability as compared to HIV-1 Tat C treated human BMVECs (**p value ≤0.005). Results are shown as mean ± S.E of three biological independent experiments.
Mentions: hBMVECs were treated with PYK2 inhibiter PF-431396 (Inhibiter of kinase activity) (Tocris Bioscience) and checked for tyrosine phosphorylation of β-catenin. Significant reduction in tyrosine phosphorylation of β-catenin resulted into an accumulation of total β-catenin (Figure 7A, B). No significant effect on tyrosine phosphorylation of β-catenin was observed in hBMVECs treated with PYK2 inhibiter (PF-431396), followed by HIV-1 Tat treatment. The level of total β-catenin was comparable to PF-431396 treated hBMVECs (Figure 7A, B). The effect of PF-431396 was also investigated on the permeability of hBMVECs. The inhibition of PYK2 significantly abrogated the effect of HIV-1 Tat and maintained the endothelial permeability (p ≤0.005) (Figure 7C).

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