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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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HIV-1 Tat C activates PYK-2 in dose dependent manner. (A) Western blot images showing a dose dependent increase in activity of PYK2 as increased phosphorylated form of PYK2 at Y-402 position in HIV-1 Tat C treated hBMVECs, as compared to control (buffer treated hBMVECs). (B) Densitometry of western blot images, done by ImageJ software to show quantitative changes in phosphorylation of PYK2 at different doses of HIV-1 Tat C treatment. Higher doses of HIV-1 Tat C protein significantly activated PYK2 (***shown for p value ≤0.0005) in three biological repeated experiments and results are shown as mean ± S.E. (C) Western blot analysis for phosphorylated PYK2 showing effect of ROS scavenger (DPI) on PYK2 activation. (D) The graph bars are showing densitometry to show average change in phosphorylated PYK2 after DPI treatment. P value ≤0.05 shown as *to show the significance level of change between Tat C versus DPI + Tat C treated group.
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Figure 5: HIV-1 Tat C activates PYK-2 in dose dependent manner. (A) Western blot images showing a dose dependent increase in activity of PYK2 as increased phosphorylated form of PYK2 at Y-402 position in HIV-1 Tat C treated hBMVECs, as compared to control (buffer treated hBMVECs). (B) Densitometry of western blot images, done by ImageJ software to show quantitative changes in phosphorylation of PYK2 at different doses of HIV-1 Tat C treatment. Higher doses of HIV-1 Tat C protein significantly activated PYK2 (***shown for p value ≤0.0005) in three biological repeated experiments and results are shown as mean ± S.E. (C) Western blot analysis for phosphorylated PYK2 showing effect of ROS scavenger (DPI) on PYK2 activation. (D) The graph bars are showing densitometry to show average change in phosphorylated PYK2 after DPI treatment. P value ≤0.05 shown as *to show the significance level of change between Tat C versus DPI + Tat C treated group.

Mentions: Proline-rich tyrosine kinase 2 (PYK2) is a non-receptor tyrosine kinase, belongs to the focal adhesion kinase (FAK) family [27]. HIV-1 Tat C exposed hBMVECs showed a dose dependent increase in expression level of NOX2 and NOX4 and levels of intracellular ROS. We tested the level of phospho-PYK2 in HIV-1 Tat C treated hBMVECs to delineate the role of PYK2 activation after Tat C treatment and to further find the role of PYK2 activation in tyrosine phosphorylation of β-catenin and VE-cadherin. A significant increase in the levels of phospho-PYK2 (at Y-402) was observed in hBMVECs (p ≤ .005) with increasing dose of HIV-1 Tat C protein (Figure 5A, B). Application of 100nM DPI; as ROS scavenger resulted into decreased PYK2 activation/phosphorylation (Figure 5C). ROS scavenging helped in combating the effect of HIV-1 Tat C on PYK2 activation (Figure 5C, 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 activates PYK-2 in dose dependent manner. (A) Western blot images showing a dose dependent increase in activity of PYK2 as increased phosphorylated form of PYK2 at Y-402 position in HIV-1 Tat C treated hBMVECs, as compared to control (buffer treated hBMVECs). (B) Densitometry of western blot images, done by ImageJ software to show quantitative changes in phosphorylation of PYK2 at different doses of HIV-1 Tat C treatment. Higher doses of HIV-1 Tat C protein significantly activated PYK2 (***shown for p value ≤0.0005) in three biological repeated experiments and results are shown as mean ± S.E. (C) Western blot analysis for phosphorylated PYK2 showing effect of ROS scavenger (DPI) on PYK2 activation. (D) The graph bars are showing densitometry to show average change in phosphorylated PYK2 after DPI treatment. P value ≤0.05 shown as *to show the significance level of change between Tat C versus DPI + Tat C treated group.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 5: HIV-1 Tat C activates PYK-2 in dose dependent manner. (A) Western blot images showing a dose dependent increase in activity of PYK2 as increased phosphorylated form of PYK2 at Y-402 position in HIV-1 Tat C treated hBMVECs, as compared to control (buffer treated hBMVECs). (B) Densitometry of western blot images, done by ImageJ software to show quantitative changes in phosphorylation of PYK2 at different doses of HIV-1 Tat C treatment. Higher doses of HIV-1 Tat C protein significantly activated PYK2 (***shown for p value ≤0.0005) in three biological repeated experiments and results are shown as mean ± S.E. (C) Western blot analysis for phosphorylated PYK2 showing effect of ROS scavenger (DPI) on PYK2 activation. (D) The graph bars are showing densitometry to show average change in phosphorylated PYK2 after DPI treatment. P value ≤0.05 shown as *to show the significance level of change between Tat C versus DPI + Tat C treated group.
Mentions: Proline-rich tyrosine kinase 2 (PYK2) is a non-receptor tyrosine kinase, belongs to the focal adhesion kinase (FAK) family [27]. HIV-1 Tat C exposed hBMVECs showed a dose dependent increase in expression level of NOX2 and NOX4 and levels of intracellular ROS. We tested the level of phospho-PYK2 in HIV-1 Tat C treated hBMVECs to delineate the role of PYK2 activation after Tat C treatment and to further find the role of PYK2 activation in tyrosine phosphorylation of β-catenin and VE-cadherin. A significant increase in the levels of phospho-PYK2 (at Y-402) was observed in hBMVECs (p ≤ .005) with increasing dose of HIV-1 Tat C protein (Figure 5A, B). Application of 100nM DPI; as ROS scavenger resulted into decreased PYK2 activation/phosphorylation (Figure 5C). ROS scavenging helped in combating the effect of HIV-1 Tat C on PYK2 activation (Figure 5C, 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