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N-cadherin acts upstream of VE-cadherin in controlling vascular morphogenesis.

Luo Y, Radice GL - J. Cell Biol. (2005)

Bottom Line: Contrary to previous studies, we found that N-cadherin localizes to endothelial cell-cell junctions in addition to its well-known diffusive membrane expression.Loss of N-cadherin in endothelial cells results in embryonic lethality at mid-gestation due to severe vascular defects.Intriguingly, loss of N-cadherin caused a significant decrease in VE-cadherin and its cytoplasmic binding partner, p120ctn.

View Article: PubMed Central - PubMed

Affiliation: Center for Research on Reproduction and Women's Health, University of Pennsylvania School of Medicine, Philadelphia, PA 19104, USA.

ABSTRACT
Endothelial cells express two classic cadherins, VE-cadherin and N-cadherin. The importance of VE-cadherin in vascular development is well known; however, the function of N-cadherin in endothelial cells remains poorly understood. Contrary to previous studies, we found that N-cadherin localizes to endothelial cell-cell junctions in addition to its well-known diffusive membrane expression. To investigate the role of N-cadherin in vascular development, N-cadherin was specifically deleted from endothelial cells in mice. Loss of N-cadherin in endothelial cells results in embryonic lethality at mid-gestation due to severe vascular defects. Intriguingly, loss of N-cadherin caused a significant decrease in VE-cadherin and its cytoplasmic binding partner, p120ctn. The down-regulation of both VE-cadherin and p120ctn was confirmed in cultured endothelial cells using small interfering RNA to knockdown N-cadherin. We also show that N-cadherin is important for endothelial cell proliferation and motility. These findings provide a novel paradigm by which N-cadherin regulates angiogenesis, in part, by controlling VE-cadherin expression at the cell membrane.

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N-cadherin regulates VE-cadherin levels in human endothelial cells. Immunofluorescence of endothelial cells after cadherin knockdown. HUVEC were transfected with control siRNA (A–C and G–I), N-cadherin siRNA (D–F), or VE-cadherin siRNA (J–L). After 48 h, the cells were double stained for N-cadherin (A, D, H, and K) and VE-cadherin (B, E, G, and J), or p120ctn (C, F, I, and L). Note loss of VE-cadherin (E) and p120ctn (F) after knockdown of N-cadherin. In contrast, knockdown of VE-cadherin did not significantly affect N-cadherin (K) or p120ctn (L). Western analysis of HUVEC after transfection with control (con), N-cadherin (N), or VE-cadherin (VE) siRNA (M and N). Quantification of the knockdown from three independent experiments. Bars, 50 μm.
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fig4: N-cadherin regulates VE-cadherin levels in human endothelial cells. Immunofluorescence of endothelial cells after cadherin knockdown. HUVEC were transfected with control siRNA (A–C and G–I), N-cadherin siRNA (D–F), or VE-cadherin siRNA (J–L). After 48 h, the cells were double stained for N-cadherin (A, D, H, and K) and VE-cadherin (B, E, G, and J), or p120ctn (C, F, I, and L). Note loss of VE-cadherin (E) and p120ctn (F) after knockdown of N-cadherin. In contrast, knockdown of VE-cadherin did not significantly affect N-cadherin (K) or p120ctn (L). Western analysis of HUVEC after transfection with control (con), N-cadherin (N), or VE-cadherin (VE) siRNA (M and N). Quantification of the knockdown from three independent experiments. Bars, 50 μm.

Mentions: To further examine the function of N-cadherin in endothelial cells, a small interfering RNA (siRNA) approach was used to knockdown N-cadherin in HUVEC. Consistent with our in vivo findings, knockdown of N-cadherin resulted in loss of VE-cadherin from endothelial cell–cell junctions (Fig. 4 E). Furthermore, both junctional and nonjunctional p120ctn immunostaining was significantly reduced after knockdown of N-cadherin in HUVEC (Fig. 4 F). Western blot analysis confirmed the reduction of both VE-cadherin (60%) and p120ctn (65%) in cultured cells (Fig. 4 M). Beta-catenin levels also were reduced (unpublished data). In contrast, siRNA knockdown of VE-cadherin did not affect N-cadherin distribution or expression level (Fig. 4 K). In addition, p120ctn remained localized at cell–cell contacts after VE-cadherin depletion (Fig. 4 L). Western blot analysis confirmed that VE-cadherin did not affect N-cadherin expression level; however, a modest reduction (35%) of p120ctn was observed consistent with decreased VE-cadherin levels (Fig. 4 N). Thus, the ability of N-cadherin to regulate VE-cadherin and p120ctn expression was observed in both mouse and human endothelial cells, and this is a nonreciprocal relationship because VE-cadherin knockdown had no effect on N-cadherin expression.


N-cadherin acts upstream of VE-cadherin in controlling vascular morphogenesis.

Luo Y, Radice GL - J. Cell Biol. (2005)

N-cadherin regulates VE-cadherin levels in human endothelial cells. Immunofluorescence of endothelial cells after cadherin knockdown. HUVEC were transfected with control siRNA (A–C and G–I), N-cadherin siRNA (D–F), or VE-cadherin siRNA (J–L). After 48 h, the cells were double stained for N-cadherin (A, D, H, and K) and VE-cadherin (B, E, G, and J), or p120ctn (C, F, I, and L). Note loss of VE-cadherin (E) and p120ctn (F) after knockdown of N-cadherin. In contrast, knockdown of VE-cadherin did not significantly affect N-cadherin (K) or p120ctn (L). Western analysis of HUVEC after transfection with control (con), N-cadherin (N), or VE-cadherin (VE) siRNA (M and N). Quantification of the knockdown from three independent experiments. Bars, 50 μm.
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Related In: Results  -  Collection

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fig4: N-cadherin regulates VE-cadherin levels in human endothelial cells. Immunofluorescence of endothelial cells after cadherin knockdown. HUVEC were transfected with control siRNA (A–C and G–I), N-cadherin siRNA (D–F), or VE-cadherin siRNA (J–L). After 48 h, the cells were double stained for N-cadherin (A, D, H, and K) and VE-cadherin (B, E, G, and J), or p120ctn (C, F, I, and L). Note loss of VE-cadherin (E) and p120ctn (F) after knockdown of N-cadherin. In contrast, knockdown of VE-cadherin did not significantly affect N-cadherin (K) or p120ctn (L). Western analysis of HUVEC after transfection with control (con), N-cadherin (N), or VE-cadherin (VE) siRNA (M and N). Quantification of the knockdown from three independent experiments. Bars, 50 μm.
Mentions: To further examine the function of N-cadherin in endothelial cells, a small interfering RNA (siRNA) approach was used to knockdown N-cadherin in HUVEC. Consistent with our in vivo findings, knockdown of N-cadherin resulted in loss of VE-cadherin from endothelial cell–cell junctions (Fig. 4 E). Furthermore, both junctional and nonjunctional p120ctn immunostaining was significantly reduced after knockdown of N-cadherin in HUVEC (Fig. 4 F). Western blot analysis confirmed the reduction of both VE-cadherin (60%) and p120ctn (65%) in cultured cells (Fig. 4 M). Beta-catenin levels also were reduced (unpublished data). In contrast, siRNA knockdown of VE-cadherin did not affect N-cadherin distribution or expression level (Fig. 4 K). In addition, p120ctn remained localized at cell–cell contacts after VE-cadherin depletion (Fig. 4 L). Western blot analysis confirmed that VE-cadherin did not affect N-cadherin expression level; however, a modest reduction (35%) of p120ctn was observed consistent with decreased VE-cadherin levels (Fig. 4 N). Thus, the ability of N-cadherin to regulate VE-cadherin and p120ctn expression was observed in both mouse and human endothelial cells, and this is a nonreciprocal relationship because VE-cadherin knockdown had no effect on N-cadherin expression.

Bottom Line: Contrary to previous studies, we found that N-cadherin localizes to endothelial cell-cell junctions in addition to its well-known diffusive membrane expression.Loss of N-cadherin in endothelial cells results in embryonic lethality at mid-gestation due to severe vascular defects.Intriguingly, loss of N-cadherin caused a significant decrease in VE-cadherin and its cytoplasmic binding partner, p120ctn.

View Article: PubMed Central - PubMed

Affiliation: Center for Research on Reproduction and Women's Health, University of Pennsylvania School of Medicine, Philadelphia, PA 19104, USA.

ABSTRACT
Endothelial cells express two classic cadherins, VE-cadherin and N-cadherin. The importance of VE-cadherin in vascular development is well known; however, the function of N-cadherin in endothelial cells remains poorly understood. Contrary to previous studies, we found that N-cadherin localizes to endothelial cell-cell junctions in addition to its well-known diffusive membrane expression. To investigate the role of N-cadherin in vascular development, N-cadherin was specifically deleted from endothelial cells in mice. Loss of N-cadherin in endothelial cells results in embryonic lethality at mid-gestation due to severe vascular defects. Intriguingly, loss of N-cadherin caused a significant decrease in VE-cadherin and its cytoplasmic binding partner, p120ctn. The down-regulation of both VE-cadherin and p120ctn was confirmed in cultured endothelial cells using small interfering RNA to knockdown N-cadherin. We also show that N-cadherin is important for endothelial cell proliferation and motility. These findings provide a novel paradigm by which N-cadherin regulates angiogenesis, in part, by controlling VE-cadherin expression at the cell membrane.

Show MeSH