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Store-operated Ca2+ entry plays a role in HMGB1-induced vascular endothelial cell hyperpermeability.

Zou M, Dong H, Meng X, Cai C, Li C, Cai S, Xue Y - PLoS ONE (2015)

Bottom Line: We have shown that human vascular endothelial cell permeability is increased, while transendothelial electrical resistance and VE-cadherin expression were reduced by HMGB1 treatment.Two SOCE inhibitors and knockdown of stromal interaction molecule 1 (STIM1), a Ca2+ sensor mediating SOCE, inhibited the HMGB1-induced influx of Ca2+ and Src activation followed by significant suppression of endothelial permeability.Moreover, knockdown of Orai1, an essential pore-subunit of SOCE channels, decreased HMGB1-induced endothelial hyperpermeability.

View Article: PubMed Central - PubMed

Affiliation: Department of Endocrinology and Metabolism, Nanfang Hospital, Southern Medical University, Guangzhou, 510515, China.

ABSTRACT

Aims: Endothelial dysfunction, including increased endothelial permeability, is considered an early marker for atherosclerosis. High-mobility group box 1 protein (HMGB1) and extracellular Ca2+ entry, primarily mediated through store-operated Ca2+ entry (SOCE), are known to be involved in increasing endothelial permeability. The aim of this study was to clarify how HMGB1 could lead to endothelia hyperpermeability.

Methods and results: We have shown that human vascular endothelial cell permeability is increased, while transendothelial electrical resistance and VE-cadherin expression were reduced by HMGB1 treatment. Two SOCE inhibitors and knockdown of stromal interaction molecule 1 (STIM1), a Ca2+ sensor mediating SOCE, inhibited the HMGB1-induced influx of Ca2+ and Src activation followed by significant suppression of endothelial permeability. Moreover, knockdown of Orai1, an essential pore-subunit of SOCE channels, decreased HMGB1-induced endothelial hyperpermeability.

Conclusions: These data suggest that SOCE, acting via STIM1, might be the predominant mechanism of Ca2+ entry in the modulation of endothelial cell permeability. STIM1 may thus represent a possible new therapeutic target against atherosclerosis.

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Related in: MedlinePlus

STIM1 knockdown decreases Ca2+ influx, HMGB1-induced permeability and Src phosphorylation.A. STIM1 protein expression after RNA inference. EA.hy926 cells were transfected for 48 h with STIM1 siRNA-1, siRNA-2 or control (scrambled) siRNA. Cells were harvested and total protein was extracted and subjected to western blotting with anti-STIM1 antibodies, with anti-GAPDH antibodies as a loading control. STIM1 expression was quantified and analyzed statistically based on three independent experiments. Transfected cells were also stimulated with 200 ng/ml HMGB1 (B) or 1 μM TG (C), followed by the addition of 2 mM CaCl2. Intracellular calcium transients were measured using an Olympus FV1000 confocal microscope. Peak intracellular Ca2+ was quantified during intracellular release or extracellular Ca2+ influx. D. HMGB1-induced permeability was inhibited by STIM1 knockdown. EA.hy926 cells were plated in the upper part of transwell chambers until the formation of a tight monolayer, then transfected with STIM1 siRNA-1, siRNA-2 or control (scrambled) siRNA. HMGB1 200 ng/ml was added and cells were incubated for an additional 24 h. After incubation, endothelial permeability was assessed, as described above. E. Representative immunoblots showing that STIM1 knockdown inhibits Src activation. Transfected cells were treated with or without 200 ng/ml HMGB1 for 2 h. Cell lysates were analyzed by SDS-PAGE followed by western blotting using antibodies against phosphorylated Src and Src. Data are presented as mean ± SD of three independent experiments. *Indicates significant difference compared with the control group (P<0.05).
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pone.0123432.g006: STIM1 knockdown decreases Ca2+ influx, HMGB1-induced permeability and Src phosphorylation.A. STIM1 protein expression after RNA inference. EA.hy926 cells were transfected for 48 h with STIM1 siRNA-1, siRNA-2 or control (scrambled) siRNA. Cells were harvested and total protein was extracted and subjected to western blotting with anti-STIM1 antibodies, with anti-GAPDH antibodies as a loading control. STIM1 expression was quantified and analyzed statistically based on three independent experiments. Transfected cells were also stimulated with 200 ng/ml HMGB1 (B) or 1 μM TG (C), followed by the addition of 2 mM CaCl2. Intracellular calcium transients were measured using an Olympus FV1000 confocal microscope. Peak intracellular Ca2+ was quantified during intracellular release or extracellular Ca2+ influx. D. HMGB1-induced permeability was inhibited by STIM1 knockdown. EA.hy926 cells were plated in the upper part of transwell chambers until the formation of a tight monolayer, then transfected with STIM1 siRNA-1, siRNA-2 or control (scrambled) siRNA. HMGB1 200 ng/ml was added and cells were incubated for an additional 24 h. After incubation, endothelial permeability was assessed, as described above. E. Representative immunoblots showing that STIM1 knockdown inhibits Src activation. Transfected cells were treated with or without 200 ng/ml HMGB1 for 2 h. Cell lysates were analyzed by SDS-PAGE followed by western blotting using antibodies against phosphorylated Src and Src. Data are presented as mean ± SD of three independent experiments. *Indicates significant difference compared with the control group (P<0.05).

Mentions: Previous reports have shown that the conserved and ubiquitously-expressed protein STIM1 plays an essential role in store-operated calcium channel (SOC)-regulated influx, and may be a common component of SOCs [21]. We therefore used STIM1 siRNA to clarify its role in HMGB1-induced permeability. As shown in Fig 6A, transfection of EA.hy926 endothelial cells with two STIM1 siRNAs, but not with scrambled siRNA, significantly blocked the expression of STIM1 protein. STIM1 protein expression levels were reduced by approximately 90% in cells treated with the STIM1 siRNAs, compared with cells treated with scrambled siRNA. HMGB1-induced Ca2+ influx was significantly reduced by around 90% in cells treated with two STIM1 siRNAs, without affecting Ca2+ store release (Fig 6B). Thapsigargin (TG) is frequently used to induce SOCE by blocking the sarcoendoplasmic reticulum Ca2+-ATPases. We therefore determined the effects of knockdown of STIM1 on TG-induced SOCE. Ca2+ influx was significantly reduced by about 80% in cells treated with two STIM1 siRNAs, without affecting Ca2+ store release (Fig 6C). We further investigated the effects of STIM1 siRNA on HMGB1-induced permeability and Src activation. siRNA knockdown of STIM1 had no effect on endothelial cell permeability, but HMGB1-induced endothelial cell hyperpermeability was significantly suppressed (Fig 6D). Src activation induced by HMGB1 was also significantly inhibited by knockdown of STIM1 (Fig 6E).


Store-operated Ca2+ entry plays a role in HMGB1-induced vascular endothelial cell hyperpermeability.

Zou M, Dong H, Meng X, Cai C, Li C, Cai S, Xue Y - PLoS ONE (2015)

STIM1 knockdown decreases Ca2+ influx, HMGB1-induced permeability and Src phosphorylation.A. STIM1 protein expression after RNA inference. EA.hy926 cells were transfected for 48 h with STIM1 siRNA-1, siRNA-2 or control (scrambled) siRNA. Cells were harvested and total protein was extracted and subjected to western blotting with anti-STIM1 antibodies, with anti-GAPDH antibodies as a loading control. STIM1 expression was quantified and analyzed statistically based on three independent experiments. Transfected cells were also stimulated with 200 ng/ml HMGB1 (B) or 1 μM TG (C), followed by the addition of 2 mM CaCl2. Intracellular calcium transients were measured using an Olympus FV1000 confocal microscope. Peak intracellular Ca2+ was quantified during intracellular release or extracellular Ca2+ influx. D. HMGB1-induced permeability was inhibited by STIM1 knockdown. EA.hy926 cells were plated in the upper part of transwell chambers until the formation of a tight monolayer, then transfected with STIM1 siRNA-1, siRNA-2 or control (scrambled) siRNA. HMGB1 200 ng/ml was added and cells were incubated for an additional 24 h. After incubation, endothelial permeability was assessed, as described above. E. Representative immunoblots showing that STIM1 knockdown inhibits Src activation. Transfected cells were treated with or without 200 ng/ml HMGB1 for 2 h. Cell lysates were analyzed by SDS-PAGE followed by western blotting using antibodies against phosphorylated Src and Src. Data are presented as mean ± SD of three independent experiments. *Indicates significant difference compared with the control group (P<0.05).
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pone.0123432.g006: STIM1 knockdown decreases Ca2+ influx, HMGB1-induced permeability and Src phosphorylation.A. STIM1 protein expression after RNA inference. EA.hy926 cells were transfected for 48 h with STIM1 siRNA-1, siRNA-2 or control (scrambled) siRNA. Cells were harvested and total protein was extracted and subjected to western blotting with anti-STIM1 antibodies, with anti-GAPDH antibodies as a loading control. STIM1 expression was quantified and analyzed statistically based on three independent experiments. Transfected cells were also stimulated with 200 ng/ml HMGB1 (B) or 1 μM TG (C), followed by the addition of 2 mM CaCl2. Intracellular calcium transients were measured using an Olympus FV1000 confocal microscope. Peak intracellular Ca2+ was quantified during intracellular release or extracellular Ca2+ influx. D. HMGB1-induced permeability was inhibited by STIM1 knockdown. EA.hy926 cells were plated in the upper part of transwell chambers until the formation of a tight monolayer, then transfected with STIM1 siRNA-1, siRNA-2 or control (scrambled) siRNA. HMGB1 200 ng/ml was added and cells were incubated for an additional 24 h. After incubation, endothelial permeability was assessed, as described above. E. Representative immunoblots showing that STIM1 knockdown inhibits Src activation. Transfected cells were treated with or without 200 ng/ml HMGB1 for 2 h. Cell lysates were analyzed by SDS-PAGE followed by western blotting using antibodies against phosphorylated Src and Src. Data are presented as mean ± SD of three independent experiments. *Indicates significant difference compared with the control group (P<0.05).
Mentions: Previous reports have shown that the conserved and ubiquitously-expressed protein STIM1 plays an essential role in store-operated calcium channel (SOC)-regulated influx, and may be a common component of SOCs [21]. We therefore used STIM1 siRNA to clarify its role in HMGB1-induced permeability. As shown in Fig 6A, transfection of EA.hy926 endothelial cells with two STIM1 siRNAs, but not with scrambled siRNA, significantly blocked the expression of STIM1 protein. STIM1 protein expression levels were reduced by approximately 90% in cells treated with the STIM1 siRNAs, compared with cells treated with scrambled siRNA. HMGB1-induced Ca2+ influx was significantly reduced by around 90% in cells treated with two STIM1 siRNAs, without affecting Ca2+ store release (Fig 6B). Thapsigargin (TG) is frequently used to induce SOCE by blocking the sarcoendoplasmic reticulum Ca2+-ATPases. We therefore determined the effects of knockdown of STIM1 on TG-induced SOCE. Ca2+ influx was significantly reduced by about 80% in cells treated with two STIM1 siRNAs, without affecting Ca2+ store release (Fig 6C). We further investigated the effects of STIM1 siRNA on HMGB1-induced permeability and Src activation. siRNA knockdown of STIM1 had no effect on endothelial cell permeability, but HMGB1-induced endothelial cell hyperpermeability was significantly suppressed (Fig 6D). Src activation induced by HMGB1 was also significantly inhibited by knockdown of STIM1 (Fig 6E).

Bottom Line: We have shown that human vascular endothelial cell permeability is increased, while transendothelial electrical resistance and VE-cadherin expression were reduced by HMGB1 treatment.Two SOCE inhibitors and knockdown of stromal interaction molecule 1 (STIM1), a Ca2+ sensor mediating SOCE, inhibited the HMGB1-induced influx of Ca2+ and Src activation followed by significant suppression of endothelial permeability.Moreover, knockdown of Orai1, an essential pore-subunit of SOCE channels, decreased HMGB1-induced endothelial hyperpermeability.

View Article: PubMed Central - PubMed

Affiliation: Department of Endocrinology and Metabolism, Nanfang Hospital, Southern Medical University, Guangzhou, 510515, China.

ABSTRACT

Aims: Endothelial dysfunction, including increased endothelial permeability, is considered an early marker for atherosclerosis. High-mobility group box 1 protein (HMGB1) and extracellular Ca2+ entry, primarily mediated through store-operated Ca2+ entry (SOCE), are known to be involved in increasing endothelial permeability. The aim of this study was to clarify how HMGB1 could lead to endothelia hyperpermeability.

Methods and results: We have shown that human vascular endothelial cell permeability is increased, while transendothelial electrical resistance and VE-cadherin expression were reduced by HMGB1 treatment. Two SOCE inhibitors and knockdown of stromal interaction molecule 1 (STIM1), a Ca2+ sensor mediating SOCE, inhibited the HMGB1-induced influx of Ca2+ and Src activation followed by significant suppression of endothelial permeability. Moreover, knockdown of Orai1, an essential pore-subunit of SOCE channels, decreased HMGB1-induced endothelial hyperpermeability.

Conclusions: These data suggest that SOCE, acting via STIM1, might be the predominant mechanism of Ca2+ entry in the modulation of endothelial cell permeability. STIM1 may thus represent a possible new therapeutic target against atherosclerosis.

Show MeSH
Related in: MedlinePlus