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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 upregulates NOX2 and NOX4 expression and increases ROS production in hBMVECs. (A) Western blot analysis to monitor the changes in expression levels of NOX2 and NOX4 in hBMVECs exposed to HIV-1 Tat C protein. The level of expression of both the NOX proteins increased after HIV-1 Tat C treatment, and led to enhanced ROS production by hBMVECs in a dose–dependent manner. (B, C) Densitometry for western images for NOX2 and NOX4 normalized with image density of β-tubulin. The graphs are representative of three independent experiments and results are shown as mean ± S.E. The levels of significance in the changes of the gene expression; compared to control groups, the p values ≤0.05 are indicated as *above the bar, (*for p value ≤0.05, **for p value ≤0.005). (D) The figure showing fluorescence microscopic images of intracellular ROS level in hBMVECs detected via DCFDA staining. Increasing doses of HIV-1 Tat C treatments are showing increase in ROS level in linear fashion. hBMVECs were treated with HIV-1 Tat C protein, washed with HBSS and incubated with DCFDA reagent (10 μM final concentration) for 15 minutes, washed and visualized under microscope. Images were captured by Axio Observer-A1 at 20 × magnification with GFP filters.
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Figure 4: HIV-1 Tat C upregulates NOX2 and NOX4 expression and increases ROS production in hBMVECs. (A) Western blot analysis to monitor the changes in expression levels of NOX2 and NOX4 in hBMVECs exposed to HIV-1 Tat C protein. The level of expression of both the NOX proteins increased after HIV-1 Tat C treatment, and led to enhanced ROS production by hBMVECs in a dose–dependent manner. (B, C) Densitometry for western images for NOX2 and NOX4 normalized with image density of β-tubulin. The graphs are representative of three independent experiments and results are shown as mean ± S.E. The levels of significance in the changes of the gene expression; compared to control groups, the p values ≤0.05 are indicated as *above the bar, (*for p value ≤0.05, **for p value ≤0.005). (D) The figure showing fluorescence microscopic images of intracellular ROS level in hBMVECs detected via DCFDA staining. Increasing doses of HIV-1 Tat C treatments are showing increase in ROS level in linear fashion. hBMVECs were treated with HIV-1 Tat C protein, washed with HBSS and incubated with DCFDA reagent (10 μM final concentration) for 15 minutes, washed and visualized under microscope. Images were captured by Axio Observer-A1 at 20 × magnification with GFP filters.

Mentions: NADPH oxidases regulate ROS production; which can affect the PTK (protein tyrosine kinase) and PTP (protein tyrosine phosphates) balance in cells [26]. Intracellular ROS level can regulate PYK2 activation and might play role in VE-PTP dissociation from cadherin complex [16]. We checked the expression level of NOX2 and NOX4 and overall intracellular ROS production by DCFDA staining. We observed the significant (p <0.005) induction in the expression levels of NOX2 and NOX4 in HIV-1 Tat C exposed hBMVECs (Figure 4A-C) and increase in the ROS production in dose dependent manner (Figure 4D).


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 upregulates NOX2 and NOX4 expression and increases ROS production in hBMVECs. (A) Western blot analysis to monitor the changes in expression levels of NOX2 and NOX4 in hBMVECs exposed to HIV-1 Tat C protein. The level of expression of both the NOX proteins increased after HIV-1 Tat C treatment, and led to enhanced ROS production by hBMVECs in a dose–dependent manner. (B, C) Densitometry for western images for NOX2 and NOX4 normalized with image density of β-tubulin. The graphs are representative of three independent experiments and results are shown as mean ± S.E. The levels of significance in the changes of the gene expression; compared to control groups, the p values ≤0.05 are indicated as *above the bar, (*for p value ≤0.05, **for p value ≤0.005). (D) The figure showing fluorescence microscopic images of intracellular ROS level in hBMVECs detected via DCFDA staining. Increasing doses of HIV-1 Tat C treatments are showing increase in ROS level in linear fashion. hBMVECs were treated with HIV-1 Tat C protein, washed with HBSS and incubated with DCFDA reagent (10 μM final concentration) for 15 minutes, washed and visualized under microscope. Images were captured by Axio Observer-A1 at 20 × magnification with GFP filters.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 4: HIV-1 Tat C upregulates NOX2 and NOX4 expression and increases ROS production in hBMVECs. (A) Western blot analysis to monitor the changes in expression levels of NOX2 and NOX4 in hBMVECs exposed to HIV-1 Tat C protein. The level of expression of both the NOX proteins increased after HIV-1 Tat C treatment, and led to enhanced ROS production by hBMVECs in a dose–dependent manner. (B, C) Densitometry for western images for NOX2 and NOX4 normalized with image density of β-tubulin. The graphs are representative of three independent experiments and results are shown as mean ± S.E. The levels of significance in the changes of the gene expression; compared to control groups, the p values ≤0.05 are indicated as *above the bar, (*for p value ≤0.05, **for p value ≤0.005). (D) The figure showing fluorescence microscopic images of intracellular ROS level in hBMVECs detected via DCFDA staining. Increasing doses of HIV-1 Tat C treatments are showing increase in ROS level in linear fashion. hBMVECs were treated with HIV-1 Tat C protein, washed with HBSS and incubated with DCFDA reagent (10 μM final concentration) for 15 minutes, washed and visualized under microscope. Images were captured by Axio Observer-A1 at 20 × magnification with GFP filters.
Mentions: NADPH oxidases regulate ROS production; which can affect the PTK (protein tyrosine kinase) and PTP (protein tyrosine phosphates) balance in cells [26]. Intracellular ROS level can regulate PYK2 activation and might play role in VE-PTP dissociation from cadherin complex [16]. We checked the expression level of NOX2 and NOX4 and overall intracellular ROS production by DCFDA staining. We observed the significant (p <0.005) induction in the expression levels of NOX2 and NOX4 in HIV-1 Tat C exposed hBMVECs (Figure 4A-C) and increase in the ROS production in dose dependent manner (Figure 4D).

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