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ADAM10 controls collagen signaling and cell migration on collagen by shedding the ectodomain of discoidin domain receptor 1 (DDR1).

Shitomi Y, Thøgersen IB, Ito N, Leitinger B, Enghild JJ, Itoh Y - Mol. Biol. Cell (2014)

Bottom Line: DDR1 shedding is not a result of an activation of its signaling pathway, since DDR1 mutants defective in signaling were shed in an efficient manner.DDR1 and ADAM10 were found to be in a complex on the cell surface, but shedding did not occur unless collagen bound to DDR1.Using a shedding-resistant DDR1 mutant, we found that ADAM10-dependent DDR1 shedding regulates the half-life of collagen-induced phosphorylation of the receptor.

View Article: PubMed Central - PubMed

Affiliation: Kennedy Institute of Rheumatology, Nuffield Department of Orthopaedics, Rheumatology and Musculoskeletal Sciences, University of Oxford, Oxford OX3 7FY, United Kingdom.

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DDR1 ectodomain shedding is required for efficient cell migration on a collagen matrix. (A) A431 cells transfected with siRNAs for DDR1 (si-D1), ADAM10 (si-A10), or both DDR1 and ADAM10 (si-A10+si-D1) were subjected to wound-closure assay on a collagen matrix. Control cells were also treated with 50 μM Mst to inhibit DDR1 shedding. si-NT, nontargeting siRNA. Scale bar, 300 μm. Wound-healing edges of cells were traced and indicated with dashed lines. (B) Relative cell migration of each treatment in A. The data are presented as mean ± SEM (n = 6). **p < 0.01; ***p < 0.005 (one-way ANOVA) compared with each si-NT. p < 0.05 is considered statistically significant. Statistical analyses were performed with Prism, version 6 (GraphPad). (C) Knockdown levels of each protein were confirmed by Western blotting with anti-DDR1, anti-ADAM10, and anti-actin antibodies. Pro, proform of ADAMs; Active, active form of ADAMs. (D) A431 cells stably expressing DDR1-wild type (WT), -6xD, ΔC, and ΔC-6xD were subjected to wound-closure assay on a collagen matrix. Representative images from each treatment. Wound-healing edges of cells were traced and are indicated with dashed lines. (E) Data from D were analyzed as in B (n = 16). *p < 0.05; **p < 0.01; ****p < 0.0001 (two-tailed Student's t test). (F) A431 cells stably expressing DDR1 mutants were subjected to Western blotting to confirm the expression levels of each mutant. All constructs contain an N-terminal HA tag and a C-terminal FLAG tag. Asterisk indicates nonspecific band.
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Figure 9: DDR1 ectodomain shedding is required for efficient cell migration on a collagen matrix. (A) A431 cells transfected with siRNAs for DDR1 (si-D1), ADAM10 (si-A10), or both DDR1 and ADAM10 (si-A10+si-D1) were subjected to wound-closure assay on a collagen matrix. Control cells were also treated with 50 μM Mst to inhibit DDR1 shedding. si-NT, nontargeting siRNA. Scale bar, 300 μm. Wound-healing edges of cells were traced and indicated with dashed lines. (B) Relative cell migration of each treatment in A. The data are presented as mean ± SEM (n = 6). **p < 0.01; ***p < 0.005 (one-way ANOVA) compared with each si-NT. p < 0.05 is considered statistically significant. Statistical analyses were performed with Prism, version 6 (GraphPad). (C) Knockdown levels of each protein were confirmed by Western blotting with anti-DDR1, anti-ADAM10, and anti-actin antibodies. Pro, proform of ADAMs; Active, active form of ADAMs. (D) A431 cells stably expressing DDR1-wild type (WT), -6xD, ΔC, and ΔC-6xD were subjected to wound-closure assay on a collagen matrix. Representative images from each treatment. Wound-healing edges of cells were traced and are indicated with dashed lines. (E) Data from D were analyzed as in B (n = 16). *p < 0.05; **p < 0.01; ****p < 0.0001 (two-tailed Student's t test). (F) A431 cells stably expressing DDR1 mutants were subjected to Western blotting to confirm the expression levels of each mutant. All constructs contain an N-terminal HA tag and a C-terminal FLAG tag. Asterisk indicates nonspecific band.

Mentions: During cell migration, cell adhesion and detachment of the cells from the matrix have to be coordinated with each other. It has been shown that DDR1 can facilitate cell adhesion to collagen matrices (Xu et al., 2012), whereas DDR1 ectodomain shedding would disengage cell–collagen interaction through DDR1. We therefore investigated whether shedding of DDR1 has any role in cell migration on a collagen matrix. First, ADAM10 and/or DDR1 were knocked down in A431 cells and cells subjected to a wound-healing assay on a collagen matrix. We confirmed that ADAM10 and DDR1 proteins were effectively down-regulated by siRNA (Figure 9C). As shown in Figure 9, A and B, knockdown of ADAM10 significantly reduced wound closure by 45%, suggesting that ADAM10-dependent DDR1 shedding may play a role in cell migration. Of interest, DDR1 knockdown also resulted in a similar reduction of wound closure. This suggests that DDR1 positively promotes cell migration. Cells silenced for both ADAM10 and DDR1 did not show any additional effect on cell migration (Figure 9, A and B), suggesting that decreased cell migration by ADAM10 knockdown depended on the presence of DDR1. When cells transfected with si-NT were incubated with Mst, wound closure was also inhibited to similar levels (Figure 9, A and B).


ADAM10 controls collagen signaling and cell migration on collagen by shedding the ectodomain of discoidin domain receptor 1 (DDR1).

Shitomi Y, Thøgersen IB, Ito N, Leitinger B, Enghild JJ, Itoh Y - Mol. Biol. Cell (2014)

DDR1 ectodomain shedding is required for efficient cell migration on a collagen matrix. (A) A431 cells transfected with siRNAs for DDR1 (si-D1), ADAM10 (si-A10), or both DDR1 and ADAM10 (si-A10+si-D1) were subjected to wound-closure assay on a collagen matrix. Control cells were also treated with 50 μM Mst to inhibit DDR1 shedding. si-NT, nontargeting siRNA. Scale bar, 300 μm. Wound-healing edges of cells were traced and indicated with dashed lines. (B) Relative cell migration of each treatment in A. The data are presented as mean ± SEM (n = 6). **p < 0.01; ***p < 0.005 (one-way ANOVA) compared with each si-NT. p < 0.05 is considered statistically significant. Statistical analyses were performed with Prism, version 6 (GraphPad). (C) Knockdown levels of each protein were confirmed by Western blotting with anti-DDR1, anti-ADAM10, and anti-actin antibodies. Pro, proform of ADAMs; Active, active form of ADAMs. (D) A431 cells stably expressing DDR1-wild type (WT), -6xD, ΔC, and ΔC-6xD were subjected to wound-closure assay on a collagen matrix. Representative images from each treatment. Wound-healing edges of cells were traced and are indicated with dashed lines. (E) Data from D were analyzed as in B (n = 16). *p < 0.05; **p < 0.01; ****p < 0.0001 (two-tailed Student's t test). (F) A431 cells stably expressing DDR1 mutants were subjected to Western blotting to confirm the expression levels of each mutant. All constructs contain an N-terminal HA tag and a C-terminal FLAG tag. Asterisk indicates nonspecific band.
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Figure 9: DDR1 ectodomain shedding is required for efficient cell migration on a collagen matrix. (A) A431 cells transfected with siRNAs for DDR1 (si-D1), ADAM10 (si-A10), or both DDR1 and ADAM10 (si-A10+si-D1) were subjected to wound-closure assay on a collagen matrix. Control cells were also treated with 50 μM Mst to inhibit DDR1 shedding. si-NT, nontargeting siRNA. Scale bar, 300 μm. Wound-healing edges of cells were traced and indicated with dashed lines. (B) Relative cell migration of each treatment in A. The data are presented as mean ± SEM (n = 6). **p < 0.01; ***p < 0.005 (one-way ANOVA) compared with each si-NT. p < 0.05 is considered statistically significant. Statistical analyses were performed with Prism, version 6 (GraphPad). (C) Knockdown levels of each protein were confirmed by Western blotting with anti-DDR1, anti-ADAM10, and anti-actin antibodies. Pro, proform of ADAMs; Active, active form of ADAMs. (D) A431 cells stably expressing DDR1-wild type (WT), -6xD, ΔC, and ΔC-6xD were subjected to wound-closure assay on a collagen matrix. Representative images from each treatment. Wound-healing edges of cells were traced and are indicated with dashed lines. (E) Data from D were analyzed as in B (n = 16). *p < 0.05; **p < 0.01; ****p < 0.0001 (two-tailed Student's t test). (F) A431 cells stably expressing DDR1 mutants were subjected to Western blotting to confirm the expression levels of each mutant. All constructs contain an N-terminal HA tag and a C-terminal FLAG tag. Asterisk indicates nonspecific band.
Mentions: During cell migration, cell adhesion and detachment of the cells from the matrix have to be coordinated with each other. It has been shown that DDR1 can facilitate cell adhesion to collagen matrices (Xu et al., 2012), whereas DDR1 ectodomain shedding would disengage cell–collagen interaction through DDR1. We therefore investigated whether shedding of DDR1 has any role in cell migration on a collagen matrix. First, ADAM10 and/or DDR1 were knocked down in A431 cells and cells subjected to a wound-healing assay on a collagen matrix. We confirmed that ADAM10 and DDR1 proteins were effectively down-regulated by siRNA (Figure 9C). As shown in Figure 9, A and B, knockdown of ADAM10 significantly reduced wound closure by 45%, suggesting that ADAM10-dependent DDR1 shedding may play a role in cell migration. Of interest, DDR1 knockdown also resulted in a similar reduction of wound closure. This suggests that DDR1 positively promotes cell migration. Cells silenced for both ADAM10 and DDR1 did not show any additional effect on cell migration (Figure 9, A and B), suggesting that decreased cell migration by ADAM10 knockdown depended on the presence of DDR1. When cells transfected with si-NT were incubated with Mst, wound closure was also inhibited to similar levels (Figure 9, A and B).

Bottom Line: DDR1 shedding is not a result of an activation of its signaling pathway, since DDR1 mutants defective in signaling were shed in an efficient manner.DDR1 and ADAM10 were found to be in a complex on the cell surface, but shedding did not occur unless collagen bound to DDR1.Using a shedding-resistant DDR1 mutant, we found that ADAM10-dependent DDR1 shedding regulates the half-life of collagen-induced phosphorylation of the receptor.

View Article: PubMed Central - PubMed

Affiliation: Kennedy Institute of Rheumatology, Nuffield Department of Orthopaedics, Rheumatology and Musculoskeletal Sciences, University of Oxford, Oxford OX3 7FY, United Kingdom.

Show MeSH
Related in: MedlinePlus