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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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ROS scavenger (DPI) mediated rescue of SHP2 expression and downregulation in phosphorylation of β-catenin. (A) Western blot analysis for SHP2 expression level after DPI treatment and DPI treatment followed by HIV-1 Tat C treatment. DPI treatment shows significant potential for checking the HIV-1 Tat C mediated downregulation of SHP2 expression. (B) The graph bars are showing average change in SHP2 expression normalized by β-tubulin. Image densitometry analysis has been done by ImageJ software. (C) Western blot analysis showing effect of ROS inhibition on tyrosine phosphorylation of β-catenin. (D) The graph bars are representative of 3 independent experiments showing average change in tyrosine phosphorylation of β-catenin in DPI treated hBMVECs. P-value ≤0.05 has been considered as significant and shown as *between DPI + Tat C versus HIV-1 Tat C treated hBMVECs. (E) Change in TEER (Ώ/cm2) values after DPI treatment. The graph bars showing average change in endothelial permeability after scavenging ROS by DPI. All the experiments have been performed three times independently and data shown as mean ± S.E. P value ≤0.0005 shown as ***asterisk to indicate the level of significance between Tat C treated versus DPI + Tat C treated hBMVECs.
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Figure 6: ROS scavenger (DPI) mediated rescue of SHP2 expression and downregulation in phosphorylation of β-catenin. (A) Western blot analysis for SHP2 expression level after DPI treatment and DPI treatment followed by HIV-1 Tat C treatment. DPI treatment shows significant potential for checking the HIV-1 Tat C mediated downregulation of SHP2 expression. (B) The graph bars are showing average change in SHP2 expression normalized by β-tubulin. Image densitometry analysis has been done by ImageJ software. (C) Western blot analysis showing effect of ROS inhibition on tyrosine phosphorylation of β-catenin. (D) The graph bars are representative of 3 independent experiments showing average change in tyrosine phosphorylation of β-catenin in DPI treated hBMVECs. P-value ≤0.05 has been considered as significant and shown as *between DPI + Tat C versus HIV-1 Tat C treated hBMVECs. (E) Change in TEER (Ώ/cm2) values after DPI treatment. The graph bars showing average change in endothelial permeability after scavenging ROS by DPI. All the experiments have been performed three times independently and data shown as mean ± S.E. P value ≤0.0005 shown as ***asterisk to indicate the level of significance between Tat C treated versus DPI + Tat C treated hBMVECs.

Mentions: Redox-sensitive PYK2 activation and phosphorylation of β-catenin leads to conformational disorganization of cadherin complex in endothelial cells [28]. We exposed hBMVECs with ROS scavenger DPI and studied the effect of blocking ROS generation on PYK2 activation, SHP2 expression, phosphorylation of β-catenin, and endothelial permeability. We observed a sustained expression level of SHP2 in DPI treated hBMVECs (Figure 6A, B). Interestingly, DPI treatment helped in rescuing of SHP2 expression even in Tat treated hBMVECs (Figure 6A, B), which suggested the potential of blocking the ROS on kinase/phosphatase balance in hBMVECs. We checked the tyrosine phosphorylated state of the β-catenin in DPI treated hBMVECs. DPI treatment alone showed inhibition of the phosphorylation of β-catenin (Figure 6C, D) compared to highly phosphorylated state (Y-654) of β-catenin in HIV-1 Tat exposed hBMVECs. DPI treatment followed by Tat treatment resulted into decreased tyrosine phosphorylation of β-catenin. Tat induced tyrosine phosphorylation of β-catenin mediated by ROS; resulted into imbalance of kinases/phosphatases. The ROS scavenging helped in ameliorating the effect of HIV-1 Tat protein on perturbed phosphorylation of β-catenin. We observed that ROS inhibition by DPI treatment has significantly helped in mitigating the effect of HIV-1 Tat C protein on permeability of hBMVECs (p ≤0.005) (Figure 6E).


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

Mishra R, Singh SK - BMC Neurosci (2014)

ROS scavenger (DPI) mediated rescue of SHP2 expression and downregulation in phosphorylation of β-catenin. (A) Western blot analysis for SHP2 expression level after DPI treatment and DPI treatment followed by HIV-1 Tat C treatment. DPI treatment shows significant potential for checking the HIV-1 Tat C mediated downregulation of SHP2 expression. (B) The graph bars are showing average change in SHP2 expression normalized by β-tubulin. Image densitometry analysis has been done by ImageJ software. (C) Western blot analysis showing effect of ROS inhibition on tyrosine phosphorylation of β-catenin. (D) The graph bars are representative of 3 independent experiments showing average change in tyrosine phosphorylation of β-catenin in DPI treated hBMVECs. P-value ≤0.05 has been considered as significant and shown as *between DPI + Tat C versus HIV-1 Tat C treated hBMVECs. (E) Change in TEER (Ώ/cm2) values after DPI treatment. The graph bars showing average change in endothelial permeability after scavenging ROS by DPI. All the experiments have been performed three times independently and data shown as mean ± S.E. P value ≤0.0005 shown as ***asterisk to indicate the level of significance between Tat C treated versus DPI + Tat C treated hBMVECs.
© Copyright Policy - open-access
Related In: Results  -  Collection

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Figure 6: ROS scavenger (DPI) mediated rescue of SHP2 expression and downregulation in phosphorylation of β-catenin. (A) Western blot analysis for SHP2 expression level after DPI treatment and DPI treatment followed by HIV-1 Tat C treatment. DPI treatment shows significant potential for checking the HIV-1 Tat C mediated downregulation of SHP2 expression. (B) The graph bars are showing average change in SHP2 expression normalized by β-tubulin. Image densitometry analysis has been done by ImageJ software. (C) Western blot analysis showing effect of ROS inhibition on tyrosine phosphorylation of β-catenin. (D) The graph bars are representative of 3 independent experiments showing average change in tyrosine phosphorylation of β-catenin in DPI treated hBMVECs. P-value ≤0.05 has been considered as significant and shown as *between DPI + Tat C versus HIV-1 Tat C treated hBMVECs. (E) Change in TEER (Ώ/cm2) values after DPI treatment. The graph bars showing average change in endothelial permeability after scavenging ROS by DPI. All the experiments have been performed three times independently and data shown as mean ± S.E. P value ≤0.0005 shown as ***asterisk to indicate the level of significance between Tat C treated versus DPI + Tat C treated hBMVECs.
Mentions: Redox-sensitive PYK2 activation and phosphorylation of β-catenin leads to conformational disorganization of cadherin complex in endothelial cells [28]. We exposed hBMVECs with ROS scavenger DPI and studied the effect of blocking ROS generation on PYK2 activation, SHP2 expression, phosphorylation of β-catenin, and endothelial permeability. We observed a sustained expression level of SHP2 in DPI treated hBMVECs (Figure 6A, B). Interestingly, DPI treatment helped in rescuing of SHP2 expression even in Tat treated hBMVECs (Figure 6A, B), which suggested the potential of blocking the ROS on kinase/phosphatase balance in hBMVECs. We checked the tyrosine phosphorylated state of the β-catenin in DPI treated hBMVECs. DPI treatment alone showed inhibition of the phosphorylation of β-catenin (Figure 6C, D) compared to highly phosphorylated state (Y-654) of β-catenin in HIV-1 Tat exposed hBMVECs. DPI treatment followed by Tat treatment resulted into decreased tyrosine phosphorylation of β-catenin. Tat induced tyrosine phosphorylation of β-catenin mediated by ROS; resulted into imbalance of kinases/phosphatases. The ROS scavenging helped in ameliorating the effect of HIV-1 Tat protein on perturbed phosphorylation of β-catenin. We observed that ROS inhibition by DPI treatment has significantly helped in mitigating the effect of HIV-1 Tat C protein on permeability of hBMVECs (p ≤0.005) (Figure 6E).

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