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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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Domain structure of laminin γ2 (Lmγ2) and its biological activities toward endothelial cells. (a) Domain structure of Lmγ2 chain. Left, full-length; right, short arm (γ2SA). Laminin γ2 consists of domains I/II, III, IV (globular domain), and V. Arrow, proteolytic cleavage site. Domain V (γ2dV) consists of three N-terminal epidermal growth factor-like repeats (NE1–3). (b) SDS-PAGE profiles of purified γ2 full-length (left panel) and γ2pf and γ2dV (right panel) as detected by Coomassie Brilliant Blue staining. Approximately 2 μg protein was run in each lane, as reported previously.(28) A 50-kDa protein (arrowhead) in γ2 full-length seems to be γ2pf. M, molecular weight markers and their size. (c) Effect of γ2pf on proliferation of vascular endothelial cells. Cells (HUVECs) were incubated with PBS as vehicle control (Control) or with 2 μg/mL γ2pf or 10 ng/mL basic fibroblast growth factor (bFGF) in MCDB131 medium supplemented with 1% FCS and 10 ng/mL epidermal growth factor on collagen-coated 96-well plates for 3 days. The relative number of grown cells was determined by MTT formazan assay with Cell Counting Kit-8 (Dojindo, Kumamoto, Japan). Each point represents the mean ± SD (bar) of triplicate wells. *P < 0.05. (d) Morphological change of HUVECs by γ2pf treatment. A confluent culture of HUVECs in serum-free basal medium was treated with PBS (Control) or 2 μg/mL γ2pf for 2 h. Phase-contrast micrographs were taken at an original magnification of ×200. Scale bar = 20 μm.
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fig01: Domain structure of laminin γ2 (Lmγ2) and its biological activities toward endothelial cells. (a) Domain structure of Lmγ2 chain. Left, full-length; right, short arm (γ2SA). Laminin γ2 consists of domains I/II, III, IV (globular domain), and V. Arrow, proteolytic cleavage site. Domain V (γ2dV) consists of three N-terminal epidermal growth factor-like repeats (NE1–3). (b) SDS-PAGE profiles of purified γ2 full-length (left panel) and γ2pf and γ2dV (right panel) as detected by Coomassie Brilliant Blue staining. Approximately 2 μg protein was run in each lane, as reported previously.(28) A 50-kDa protein (arrowhead) in γ2 full-length seems to be γ2pf. M, molecular weight markers and their size. (c) Effect of γ2pf on proliferation of vascular endothelial cells. Cells (HUVECs) were incubated with PBS as vehicle control (Control) or with 2 μg/mL γ2pf or 10 ng/mL basic fibroblast growth factor (bFGF) in MCDB131 medium supplemented with 1% FCS and 10 ng/mL epidermal growth factor on collagen-coated 96-well plates for 3 days. The relative number of grown cells was determined by MTT formazan assay with Cell Counting Kit-8 (Dojindo, Kumamoto, Japan). Each point represents the mean ± SD (bar) of triplicate wells. *P < 0.05. (d) Morphological change of HUVECs by γ2pf treatment. A confluent culture of HUVECs in serum-free basal medium was treated with PBS (Control) or 2 μg/mL γ2pf for 2 h. Phase-contrast micrographs were taken at an original magnification of ×200. Scale bar = 20 μm.

Mentions: The Lmγ2 chain is cleaved at a specific site of domain III in the short arm (γ2SA) by some proteinases, releasing the N-terminal fragment, named γ2pf (Fig. 1a). It was previously found that γ2SA has tumor-promoting activity in vivo.(28) To examine the biological activities of the γ2 chain toward vascular endothelial cells, we prepared the full-length γ2 and its various N-terminal fragments, including γ2pf, γ2dV, and γ2SA (Fig. 1b). Among them, γ2pf and γ2dV were mainly used in this study because of their high purity and relative importance.(36) When the growth effect of γ2pf on endothelial cells (HUVECs) was examined, it showed a statistically significant but faint growth-stimulatory effect (Fig. 1c). In the two-chamber assay, γ2pf scarcely stimulated the migration of HUVECs (Fig. S1a). However, when γ2pf was added to the monolayer culture of HUVECs, these cells became markedly retracted or shrunken compared with untreated cells (Fig. 1d). Such a morphological change was not observed when the epithelial cell line MDCK was treated with γ2pf (data not shown). We also confirmed that γ2SA induced similar morphological changes in endothelial cells (Fig. S1b,c). However, γ2SA showed little growth effect on endothelial cells (data not shown).


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)

Domain structure of laminin γ2 (Lmγ2) and its biological activities toward endothelial cells. (a) Domain structure of Lmγ2 chain. Left, full-length; right, short arm (γ2SA). Laminin γ2 consists of domains I/II, III, IV (globular domain), and V. Arrow, proteolytic cleavage site. Domain V (γ2dV) consists of three N-terminal epidermal growth factor-like repeats (NE1–3). (b) SDS-PAGE profiles of purified γ2 full-length (left panel) and γ2pf and γ2dV (right panel) as detected by Coomassie Brilliant Blue staining. Approximately 2 μg protein was run in each lane, as reported previously.(28) A 50-kDa protein (arrowhead) in γ2 full-length seems to be γ2pf. M, molecular weight markers and their size. (c) Effect of γ2pf on proliferation of vascular endothelial cells. Cells (HUVECs) were incubated with PBS as vehicle control (Control) or with 2 μg/mL γ2pf or 10 ng/mL basic fibroblast growth factor (bFGF) in MCDB131 medium supplemented with 1% FCS and 10 ng/mL epidermal growth factor on collagen-coated 96-well plates for 3 days. The relative number of grown cells was determined by MTT formazan assay with Cell Counting Kit-8 (Dojindo, Kumamoto, Japan). Each point represents the mean ± SD (bar) of triplicate wells. *P < 0.05. (d) Morphological change of HUVECs by γ2pf treatment. A confluent culture of HUVECs in serum-free basal medium was treated with PBS (Control) or 2 μg/mL γ2pf for 2 h. Phase-contrast micrographs were taken at an original magnification of ×200. Scale bar = 20 μm.
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fig01: Domain structure of laminin γ2 (Lmγ2) and its biological activities toward endothelial cells. (a) Domain structure of Lmγ2 chain. Left, full-length; right, short arm (γ2SA). Laminin γ2 consists of domains I/II, III, IV (globular domain), and V. Arrow, proteolytic cleavage site. Domain V (γ2dV) consists of three N-terminal epidermal growth factor-like repeats (NE1–3). (b) SDS-PAGE profiles of purified γ2 full-length (left panel) and γ2pf and γ2dV (right panel) as detected by Coomassie Brilliant Blue staining. Approximately 2 μg protein was run in each lane, as reported previously.(28) A 50-kDa protein (arrowhead) in γ2 full-length seems to be γ2pf. M, molecular weight markers and their size. (c) Effect of γ2pf on proliferation of vascular endothelial cells. Cells (HUVECs) were incubated with PBS as vehicle control (Control) or with 2 μg/mL γ2pf or 10 ng/mL basic fibroblast growth factor (bFGF) in MCDB131 medium supplemented with 1% FCS and 10 ng/mL epidermal growth factor on collagen-coated 96-well plates for 3 days. The relative number of grown cells was determined by MTT formazan assay with Cell Counting Kit-8 (Dojindo, Kumamoto, Japan). Each point represents the mean ± SD (bar) of triplicate wells. *P < 0.05. (d) Morphological change of HUVECs by γ2pf treatment. A confluent culture of HUVECs in serum-free basal medium was treated with PBS (Control) or 2 μg/mL γ2pf for 2 h. Phase-contrast micrographs were taken at an original magnification of ×200. Scale bar = 20 μm.
Mentions: The Lmγ2 chain is cleaved at a specific site of domain III in the short arm (γ2SA) by some proteinases, releasing the N-terminal fragment, named γ2pf (Fig. 1a). It was previously found that γ2SA has tumor-promoting activity in vivo.(28) To examine the biological activities of the γ2 chain toward vascular endothelial cells, we prepared the full-length γ2 and its various N-terminal fragments, including γ2pf, γ2dV, and γ2SA (Fig. 1b). Among them, γ2pf and γ2dV were mainly used in this study because of their high purity and relative importance.(36) When the growth effect of γ2pf on endothelial cells (HUVECs) was examined, it showed a statistically significant but faint growth-stimulatory effect (Fig. 1c). In the two-chamber assay, γ2pf scarcely stimulated the migration of HUVECs (Fig. S1a). However, when γ2pf was added to the monolayer culture of HUVECs, these cells became markedly retracted or shrunken compared with untreated cells (Fig. 1d). Such a morphological change was not observed when the epithelial cell line MDCK was treated with γ2pf (data not shown). We also confirmed that γ2SA induced similar morphological changes in endothelial cells (Fig. S1b,c). However, γ2SA showed little growth effect on endothelial cells (data not shown).

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