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Amino-terminal fragments of laminin γ2 chain retract vascular endothelial cells and increase vascular permeability.

Sato H, Oyanagi J, Komiya E, Ogawa T, Higashi S, Miyazaki K - Cancer Sci. (2014)

Bottom Line: Such effects of γ2pf led to prominent actin stress fiber formation in HUVECs, which was blocked by a ROCK inhibitor.As possible receptors, γ2pf interacted with heparan sulfate proteoglycans on the surface of HUVECs.Moreover, we localized the active site of γ2pf to the N-terminal epidermal growth factor-like repeat.

View Article: PubMed Central - PubMed

Affiliation: Department of Genome Science, Graduate School of Integrated Science and Nanobioscience, Yokohama City University, Yokohama, Japan; Division of Cell Biology, Kihara Institute for Biological Research, Yokohama City University, Yokohama, Japan.

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Effect of γ2 proteolytic fragment (γ2pf) on localization of cytoskeletal and intercellular junctional proteins. (a) F-actin and tubulin. Cells (HUVECs) in sparse culture were incubated for 2 days with PBS (Control) or γ2pf (2 μg/mL) in a factor-free and 1% FCS-supplemented medium. Green, tubulin; red, F-actin. (b) Effect of ROCK inhibitor (Y-27632) on γ2pf-induced actin stress fiber formation. Cells were pretreated with 10 μM Y-27632 for 30 min then treated with γ2pf as described above. (c, d) Effect of γ2pf on VE-cadherin (c, green) and β-catenin (d, red). Confluent cultures of HUVECs were treated with PBS (Control) or γ2pf. Other experimental conditions were the same as above. Scale bars = 20 μm.
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fig06: Effect of γ2 proteolytic fragment (γ2pf) on localization of cytoskeletal and intercellular junctional proteins. (a) F-actin and tubulin. Cells (HUVECs) in sparse culture were incubated for 2 days with PBS (Control) or γ2pf (2 μg/mL) in a factor-free and 1% FCS-supplemented medium. Green, tubulin; red, F-actin. (b) Effect of ROCK inhibitor (Y-27632) on γ2pf-induced actin stress fiber formation. Cells were pretreated with 10 μM Y-27632 for 30 min then treated with γ2pf as described above. (c, d) Effect of γ2pf on VE-cadherin (c, green) and β-catenin (d, red). Confluent cultures of HUVECs were treated with PBS (Control) or γ2pf. Other experimental conditions were the same as above. Scale bars = 20 μm.

Mentions: To elucidate the mechanism by which the laminin γ2 chain disrupts the barrier function of vascular endothelial cells, we next examined effect of γ2pf on the localization of cytoskeletal proteins and adherence junction proteins (Fig. 6). In a sparse culture of HUVECs, double fluorescence staining of F-actin and microtubules showed that γ2pf strongly induced actin stress fibers in the cytoplasm, while it reduced or disassembled microtubule structures (Fig. 6a). As expected, the γ2pf-induced actin stress fiber formation was effectively blocked by the treatment with the ROCK inhibitor Y-27632 (Fig. 6b).


Amino-terminal fragments of laminin γ2 chain retract vascular endothelial cells and increase vascular permeability.

Sato H, Oyanagi J, Komiya E, Ogawa T, Higashi S, Miyazaki K - Cancer Sci. (2014)

Effect of γ2 proteolytic fragment (γ2pf) on localization of cytoskeletal and intercellular junctional proteins. (a) F-actin and tubulin. Cells (HUVECs) in sparse culture were incubated for 2 days with PBS (Control) or γ2pf (2 μg/mL) in a factor-free and 1% FCS-supplemented medium. Green, tubulin; red, F-actin. (b) Effect of ROCK inhibitor (Y-27632) on γ2pf-induced actin stress fiber formation. Cells were pretreated with 10 μM Y-27632 for 30 min then treated with γ2pf as described above. (c, d) Effect of γ2pf on VE-cadherin (c, green) and β-catenin (d, red). Confluent cultures of HUVECs were treated with PBS (Control) or γ2pf. Other experimental conditions were the same as above. Scale bars = 20 μm.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4317827&req=5

fig06: Effect of γ2 proteolytic fragment (γ2pf) on localization of cytoskeletal and intercellular junctional proteins. (a) F-actin and tubulin. Cells (HUVECs) in sparse culture were incubated for 2 days with PBS (Control) or γ2pf (2 μg/mL) in a factor-free and 1% FCS-supplemented medium. Green, tubulin; red, F-actin. (b) Effect of ROCK inhibitor (Y-27632) on γ2pf-induced actin stress fiber formation. Cells were pretreated with 10 μM Y-27632 for 30 min then treated with γ2pf as described above. (c, d) Effect of γ2pf on VE-cadherin (c, green) and β-catenin (d, red). Confluent cultures of HUVECs were treated with PBS (Control) or γ2pf. Other experimental conditions were the same as above. Scale bars = 20 μm.
Mentions: To elucidate the mechanism by which the laminin γ2 chain disrupts the barrier function of vascular endothelial cells, we next examined effect of γ2pf on the localization of cytoskeletal proteins and adherence junction proteins (Fig. 6). In a sparse culture of HUVECs, double fluorescence staining of F-actin and microtubules showed that γ2pf strongly induced actin stress fibers in the cytoplasm, while it reduced or disassembled microtubule structures (Fig. 6a). As expected, the γ2pf-induced actin stress fiber formation was effectively blocked by the treatment with the ROCK inhibitor Y-27632 (Fig. 6b).

Bottom Line: Such effects of γ2pf led to prominent actin stress fiber formation in HUVECs, which was blocked by a ROCK inhibitor.As possible receptors, γ2pf interacted with heparan sulfate proteoglycans on the surface of HUVECs.Moreover, we localized the active site of γ2pf to the N-terminal epidermal growth factor-like repeat.

View Article: PubMed Central - PubMed

Affiliation: Department of Genome Science, Graduate School of Integrated Science and Nanobioscience, Yokohama City University, Yokohama, Japan; Division of Cell Biology, Kihara Institute for Biological Research, Yokohama City University, Yokohama, Japan.

Show MeSH
Related in: MedlinePlus