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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 mediated phosphorylation of adherens junction proteins (VE-cadherin and β-catenin). (A) Western blot analysis to check the effect of HIV-1 Tat C protein in hBMVECs, showing a dose dependent increase in tyrosine phosphorylation of VE-cadherin (p-Y-731). (B) The graph showing the result of densitometry for western blot images showing significantly induced tyrosine phosphorylation (p-Y-731) of VE-cadherin upon HIV-1 Tat/ heat inactivated Tat treatment. Quantitation of image densities have been done by ImageJ software normalized with β-tubulin. (C) Western blot analysis showing the insignificant effect of heat inactivated HIV-1 Tat C protein on tyrosine phosphorylation (p-Y-731) of VE-cadherin (D) Western blot analysis to show dose dependent increase in tyrosine phosphorylation of β-catenin (p-Y-654) in hBMVECs exposed to HIV-1 Tat C protein. (E) Densitometry analysis of image density to show change in tyrosine phosphorylation (p-Y-654) of β-catenin. Image densities have been normalized with β-tubulin. All the experiments have been performed in three independent biological sets and results are presented as mean ± S.E., *above bars are representing the p value ≤0.05 as level of significance, n = 3. (*for p value ≤0.05, **for p value ≤0.005).
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Figure 1: HIV-1 Tat C mediated phosphorylation of adherens junction proteins (VE-cadherin and β-catenin). (A) Western blot analysis to check the effect of HIV-1 Tat C protein in hBMVECs, showing a dose dependent increase in tyrosine phosphorylation of VE-cadherin (p-Y-731). (B) The graph showing the result of densitometry for western blot images showing significantly induced tyrosine phosphorylation (p-Y-731) of VE-cadherin upon HIV-1 Tat/ heat inactivated Tat treatment. Quantitation of image densities have been done by ImageJ software normalized with β-tubulin. (C) Western blot analysis showing the insignificant effect of heat inactivated HIV-1 Tat C protein on tyrosine phosphorylation (p-Y-731) of VE-cadherin (D) Western blot analysis to show dose dependent increase in tyrosine phosphorylation of β-catenin (p-Y-654) in hBMVECs exposed to HIV-1 Tat C protein. (E) Densitometry analysis of image density to show change in tyrosine phosphorylation (p-Y-654) of β-catenin. Image densities have been normalized with β-tubulin. All the experiments have been performed in three independent biological sets and results are presented as mean ± S.E., *above bars are representing the p value ≤0.05 as level of significance, n = 3. (*for p value ≤0.05, **for p value ≤0.005).

Mentions: We investigated the tyrosine phosphorylation of VE-cadherin and β-catenin in hBMVECs treated with increasing doses from 100 ng/ml to 1 μg/ml of HIV-1 Tat C. Both the adherens junction proteins (AJPs) (VE-cadherin and β-catenin) were significantly phosphorylated up to 4 folds at their respective tyrosine positions in dose dependent manner (Figure 1). We checked the tyrosine phosphorylation (Y-731) of VE-cadherin in hBMVECs exposed to heat inactivated Tat C and couldn’t find any substantial alteration in VE-cadherin phosphorylation (Figure 1B, C).


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 mediated phosphorylation of adherens junction proteins (VE-cadherin and β-catenin). (A) Western blot analysis to check the effect of HIV-1 Tat C protein in hBMVECs, showing a dose dependent increase in tyrosine phosphorylation of VE-cadherin (p-Y-731). (B) The graph showing the result of densitometry for western blot images showing significantly induced tyrosine phosphorylation (p-Y-731) of VE-cadherin upon HIV-1 Tat/ heat inactivated Tat treatment. Quantitation of image densities have been done by ImageJ software normalized with β-tubulin. (C) Western blot analysis showing the insignificant effect of heat inactivated HIV-1 Tat C protein on tyrosine phosphorylation (p-Y-731) of VE-cadherin (D) Western blot analysis to show dose dependent increase in tyrosine phosphorylation of β-catenin (p-Y-654) in hBMVECs exposed to HIV-1 Tat C protein. (E) Densitometry analysis of image density to show change in tyrosine phosphorylation (p-Y-654) of β-catenin. Image densities have been normalized with β-tubulin. All the experiments have been performed in three independent biological sets and results are presented as mean ± S.E., *above bars are representing the p value ≤0.05 as level of significance, n = 3. (*for p value ≤0.05, **for p value ≤0.005).
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 1: HIV-1 Tat C mediated phosphorylation of adherens junction proteins (VE-cadherin and β-catenin). (A) Western blot analysis to check the effect of HIV-1 Tat C protein in hBMVECs, showing a dose dependent increase in tyrosine phosphorylation of VE-cadherin (p-Y-731). (B) The graph showing the result of densitometry for western blot images showing significantly induced tyrosine phosphorylation (p-Y-731) of VE-cadherin upon HIV-1 Tat/ heat inactivated Tat treatment. Quantitation of image densities have been done by ImageJ software normalized with β-tubulin. (C) Western blot analysis showing the insignificant effect of heat inactivated HIV-1 Tat C protein on tyrosine phosphorylation (p-Y-731) of VE-cadherin (D) Western blot analysis to show dose dependent increase in tyrosine phosphorylation of β-catenin (p-Y-654) in hBMVECs exposed to HIV-1 Tat C protein. (E) Densitometry analysis of image density to show change in tyrosine phosphorylation (p-Y-654) of β-catenin. Image densities have been normalized with β-tubulin. All the experiments have been performed in three independent biological sets and results are presented as mean ± S.E., *above bars are representing the p value ≤0.05 as level of significance, n = 3. (*for p value ≤0.05, **for p value ≤0.005).
Mentions: We investigated the tyrosine phosphorylation of VE-cadherin and β-catenin in hBMVECs treated with increasing doses from 100 ng/ml to 1 μg/ml of HIV-1 Tat C. Both the adherens junction proteins (AJPs) (VE-cadherin and β-catenin) were significantly phosphorylated up to 4 folds at their respective tyrosine positions in dose dependent manner (Figure 1). We checked the tyrosine phosphorylation (Y-731) of VE-cadherin in hBMVECs exposed to heat inactivated Tat C and couldn’t find any substantial alteration in VE-cadherin phosphorylation (Figure 1B, C).

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