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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

Downregulation and dissociation of VE-PTP and SHP2 from VE-cadherin in HIV-1 Tat C treated BMVECs. (A) Downregulation of VE-PTP expression in HIV-1 Tat C treated hBMVECs. HIV-1 Tat C treatment has been given for 12 hours on hBMVECs and western blot analysis was done by using anti VE-PTP antibody to show decrease in expression level of phosphatase VE-PTP. (B) Immnunoblot images of co-immunoprecipitaion of VE-PTP along with VE-cadherin complex showing the decreased association of VE-PTP with VE-cadherin. Same blots have been re-probed with VE-cadherin antibody to exhibit equal loading of pulled down cadherin complex. Monoclonal VE-cadherin antibody has been used for pull down of VE-cadherin complex by using NP-40 mild lysis buffer in cold conditions. (C) Compared to control hBMVECs (buffer treated), HIV-1 Tat C treated hBMVECs are showing a dose-dependent decrease in SHP2 expression levels. (D) SHP2 dissociation from cadherin complex has been shown by immunoblotting against SHP2 in pulled down cadherin complex by using anti VE-cadherin antibody. (E) The densitometry graph is showing almost 60–70% decrease in VE-PTP expression at higher doses of HIV-1 Tat C treatment. (F) Quantitation of image density by ImageJ software normalized by VE-cadherin image density. Pull-down experiment has been repeated three times and results are shown as mean ± S.E. (*p value ≤0.05). (G) Quantitation of image density of SHP2 showing a significant dissociation (**p value ≤0.005) from cadherin complex, normalized with amount of VE-cadherin pull down. All the results shown here are representative of three independently repeated experiments and shown as mean ± S.E.
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Figure 2: Downregulation and dissociation of VE-PTP and SHP2 from VE-cadherin in HIV-1 Tat C treated BMVECs. (A) Downregulation of VE-PTP expression in HIV-1 Tat C treated hBMVECs. HIV-1 Tat C treatment has been given for 12 hours on hBMVECs and western blot analysis was done by using anti VE-PTP antibody to show decrease in expression level of phosphatase VE-PTP. (B) Immnunoblot images of co-immunoprecipitaion of VE-PTP along with VE-cadherin complex showing the decreased association of VE-PTP with VE-cadherin. Same blots have been re-probed with VE-cadherin antibody to exhibit equal loading of pulled down cadherin complex. Monoclonal VE-cadherin antibody has been used for pull down of VE-cadherin complex by using NP-40 mild lysis buffer in cold conditions. (C) Compared to control hBMVECs (buffer treated), HIV-1 Tat C treated hBMVECs are showing a dose-dependent decrease in SHP2 expression levels. (D) SHP2 dissociation from cadherin complex has been shown by immunoblotting against SHP2 in pulled down cadherin complex by using anti VE-cadherin antibody. (E) The densitometry graph is showing almost 60–70% decrease in VE-PTP expression at higher doses of HIV-1 Tat C treatment. (F) Quantitation of image density by ImageJ software normalized by VE-cadherin image density. Pull-down experiment has been repeated three times and results are shown as mean ± S.E. (*p value ≤0.05). (G) Quantitation of image density of SHP2 showing a significant dissociation (**p value ≤0.005) from cadherin complex, normalized with amount of VE-cadherin pull down. All the results shown here are representative of three independently repeated experiments and shown as mean ± S.E.

Mentions: VE-PTPs are essentially associated with VE-cadherin and perform the function of keeping VE-cadherin in dephosphorylated state in quiescent cells [10]. We observed a dose dependent increase in tyrosine phosphorylation of VE-cadherin (Y731 position) in HIV-1 Tat C exposed hBMVECs (Figure 1A, B). We further investigated the levels of VE-PTP and their association with VE-cadherin in HIV-1 Tat exposed hBMVECs. We observed decreased expression of total VE-PTP (Figure 2A, E), along with the significant dissociation (p ≤ .05) of VE-PTP from VE-cadherin complex in HIV-1 Tat treated hBMVECs (Figure 2B, F). SHP2, a non-receptor protein-tyrosine phosphatase co-precipitates with VE-cadherin complex in basal state of cells [12]. SHP2 phosphatase maintains the integrity of VE-cadherin complex, by dephosphorylating VE-cadherin-associated β-catenin as well as VE-Cadherin [13,18]. SHP2 expression levels decreased significantly (p ≤0.005) along with increasing dose of HIV-1 Tat (Figure 2C). The pull down experiment with unit amount of VE-cadherin complex by using monoclonal VE-cadherin antibody has shown significant reduction (p ≤0.005) in SHP2 association with VE-cadherin complex (Figure 2D, G). We observed that both the tyrosine phosphatases regulating the tyrosine phosphorylation, get dissociated from the VE-cadherin complex in HIV-1 Tat C exposed hBMVECs.


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

Mishra R, Singh SK - BMC Neurosci (2014)

Downregulation and dissociation of VE-PTP and SHP2 from VE-cadherin in HIV-1 Tat C treated BMVECs. (A) Downregulation of VE-PTP expression in HIV-1 Tat C treated hBMVECs. HIV-1 Tat C treatment has been given for 12 hours on hBMVECs and western blot analysis was done by using anti VE-PTP antibody to show decrease in expression level of phosphatase VE-PTP. (B) Immnunoblot images of co-immunoprecipitaion of VE-PTP along with VE-cadherin complex showing the decreased association of VE-PTP with VE-cadherin. Same blots have been re-probed with VE-cadherin antibody to exhibit equal loading of pulled down cadherin complex. Monoclonal VE-cadherin antibody has been used for pull down of VE-cadherin complex by using NP-40 mild lysis buffer in cold conditions. (C) Compared to control hBMVECs (buffer treated), HIV-1 Tat C treated hBMVECs are showing a dose-dependent decrease in SHP2 expression levels. (D) SHP2 dissociation from cadherin complex has been shown by immunoblotting against SHP2 in pulled down cadherin complex by using anti VE-cadherin antibody. (E) The densitometry graph is showing almost 60–70% decrease in VE-PTP expression at higher doses of HIV-1 Tat C treatment. (F) Quantitation of image density by ImageJ software normalized by VE-cadherin image density. Pull-down experiment has been repeated three times and results are shown as mean ± S.E. (*p value ≤0.05). (G) Quantitation of image density of SHP2 showing a significant dissociation (**p value ≤0.005) from cadherin complex, normalized with amount of VE-cadherin pull down. All the results shown here are representative of three independently repeated experiments and shown as mean ± S.E.
© Copyright Policy - open-access
Related In: Results  -  Collection

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Figure 2: Downregulation and dissociation of VE-PTP and SHP2 from VE-cadherin in HIV-1 Tat C treated BMVECs. (A) Downregulation of VE-PTP expression in HIV-1 Tat C treated hBMVECs. HIV-1 Tat C treatment has been given for 12 hours on hBMVECs and western blot analysis was done by using anti VE-PTP antibody to show decrease in expression level of phosphatase VE-PTP. (B) Immnunoblot images of co-immunoprecipitaion of VE-PTP along with VE-cadherin complex showing the decreased association of VE-PTP with VE-cadherin. Same blots have been re-probed with VE-cadherin antibody to exhibit equal loading of pulled down cadherin complex. Monoclonal VE-cadherin antibody has been used for pull down of VE-cadherin complex by using NP-40 mild lysis buffer in cold conditions. (C) Compared to control hBMVECs (buffer treated), HIV-1 Tat C treated hBMVECs are showing a dose-dependent decrease in SHP2 expression levels. (D) SHP2 dissociation from cadherin complex has been shown by immunoblotting against SHP2 in pulled down cadherin complex by using anti VE-cadherin antibody. (E) The densitometry graph is showing almost 60–70% decrease in VE-PTP expression at higher doses of HIV-1 Tat C treatment. (F) Quantitation of image density by ImageJ software normalized by VE-cadherin image density. Pull-down experiment has been repeated three times and results are shown as mean ± S.E. (*p value ≤0.05). (G) Quantitation of image density of SHP2 showing a significant dissociation (**p value ≤0.005) from cadherin complex, normalized with amount of VE-cadherin pull down. All the results shown here are representative of three independently repeated experiments and shown as mean ± S.E.
Mentions: VE-PTPs are essentially associated with VE-cadherin and perform the function of keeping VE-cadherin in dephosphorylated state in quiescent cells [10]. We observed a dose dependent increase in tyrosine phosphorylation of VE-cadherin (Y731 position) in HIV-1 Tat C exposed hBMVECs (Figure 1A, B). We further investigated the levels of VE-PTP and their association with VE-cadherin in HIV-1 Tat exposed hBMVECs. We observed decreased expression of total VE-PTP (Figure 2A, E), along with the significant dissociation (p ≤ .05) of VE-PTP from VE-cadherin complex in HIV-1 Tat treated hBMVECs (Figure 2B, F). SHP2, a non-receptor protein-tyrosine phosphatase co-precipitates with VE-cadherin complex in basal state of cells [12]. SHP2 phosphatase maintains the integrity of VE-cadherin complex, by dephosphorylating VE-cadherin-associated β-catenin as well as VE-Cadherin [13,18]. SHP2 expression levels decreased significantly (p ≤0.005) along with increasing dose of HIV-1 Tat (Figure 2C). The pull down experiment with unit amount of VE-cadherin complex by using monoclonal VE-cadherin antibody has shown significant reduction (p ≤0.005) in SHP2 association with VE-cadherin complex (Figure 2D, G). We observed that both the tyrosine phosphatases regulating the tyrosine phosphorylation, get dissociated from the VE-cadherin complex in HIV-1 Tat C exposed hBMVECs.

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