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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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ADAM10 is the sheddase responsible for collagen-induced DDR1 ectodomain shedding. (A) A431 cells were transfected with siRNA for ADAM8, ADAM9, ADAM10, ADAM17, or ADAM19 or with nontargeting (NT) siRNA. After 48 h, cells were treated with collagen I (100 μg/ml) for a further 24 h. We confirmed that the level of mRNA for each enzyme was reduced by 60−99% after 72 h (right, RT-PCR). Mst (10 μM) was used as a positive control for inhibition of DDR1 shedding. Conditioned media and cell lysates were analyzed by Western blotting using anti–DDR1 ectodomain (Med), anti–DDR1 C-terminus (Cell), or anti-actin antibodies. Note that ADAM10 knockdown resulted in inhibition of endogenous DDR1 shedding. CTF, C-terminal fragment. (B) A431 cells were transfected with siRNA for MT1-MMP (si-MT1) or si-NT. Cells were then treated with collagen I for 24 h. Conditioned media and cell lysates were analyzed by Western blotting using anti-DDR1, anti-MMP14 (EP1264Y) (MT1), and anti-actin antibodies. (C) A431 cells were treated with 1 μM IM, 25 ng/ml PMA, or DMSO vehicle control (0.001%) for 1 h. Conditioned media and cell lysates were analyzed as in A. A431 cells were also treated with 1 μM IM for 1 h in the presence or absence of 10 μM Mst (right). (D) A431 cells were transiently transfected with ADAM10 or mock vector and treated with or without collagen I for 24 h. Conditioned media and cell lysates were subjected to Western blotting using anti–DDR1 ectodomain (Med), anti–DDR1 C-terminus (Cell), anti-ADAM10 (A10), and anti-actin antibodies.
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Figure 3: ADAM10 is the sheddase responsible for collagen-induced DDR1 ectodomain shedding. (A) A431 cells were transfected with siRNA for ADAM8, ADAM9, ADAM10, ADAM17, or ADAM19 or with nontargeting (NT) siRNA. After 48 h, cells were treated with collagen I (100 μg/ml) for a further 24 h. We confirmed that the level of mRNA for each enzyme was reduced by 60−99% after 72 h (right, RT-PCR). Mst (10 μM) was used as a positive control for inhibition of DDR1 shedding. Conditioned media and cell lysates were analyzed by Western blotting using anti–DDR1 ectodomain (Med), anti–DDR1 C-terminus (Cell), or anti-actin antibodies. Note that ADAM10 knockdown resulted in inhibition of endogenous DDR1 shedding. CTF, C-terminal fragment. (B) A431 cells were transfected with siRNA for MT1-MMP (si-MT1) or si-NT. Cells were then treated with collagen I for 24 h. Conditioned media and cell lysates were analyzed by Western blotting using anti-DDR1, anti-MMP14 (EP1264Y) (MT1), and anti-actin antibodies. (C) A431 cells were treated with 1 μM IM, 25 ng/ml PMA, or DMSO vehicle control (0.001%) for 1 h. Conditioned media and cell lysates were analyzed as in A. A431 cells were also treated with 1 μM IM for 1 h in the presence or absence of 10 μM Mst (right). (D) A431 cells were transiently transfected with ADAM10 or mock vector and treated with or without collagen I for 24 h. Conditioned media and cell lysates were subjected to Western blotting using anti–DDR1 ectodomain (Med), anti–DDR1 C-terminus (Cell), anti-ADAM10 (A10), and anti-actin antibodies.

Mentions: Our data indicated that the enzyme responsible for collagen-induced DDR1 shedding is a metalloproteinase insensitive to TIMP-1 and TIMP-2 and partially sensitive or insensitive to TIMP-3 (Figure 2, B and C). It has been shown that TIMP-insensitive metalloproteinases include ADAM8, ADAM9 (Amour et al., 2002), and ADAM19 (Chesneau et al., 2003; Shiomi et al., 2010). We therefore knocked down ADAM8, ADAM9, and ADAM19, along with well-characterized sheddases, ADAM10 and ADAM17, in A431 cells. The mRNA levels of each ADAM in the respective siRNA treated cells were significantly down-regulated compared with the nontargeting small interfering RNA (siRNA; si-NT)–treated cells by 60% (ADAM8), 86% (ADAM9), 95% (ADAM10), 89% (ADAM17), and 99% (ADAM19; Figure 3A). Under these conditions, only ADAM10 knockdown abolished collagen-induced DDR1 shedding (Figure 3A, DDR1, Med). It was noted that although Mst and ADAM10 knockdown inhibited shedding of DDR1, cellular level of DDR1 did not recover to the level of non–collagen-treated cells in A431 cells (Figure 3A). This is likely due to down-regulation of DDR1 production upon collagen treatment during 24-h incubation. We also confirmed the effect of ADAM10 knockdown in MCF-7 (Supplemental Figure S2) and HEK293 cells (see later discussion of Figure 7E, WT). Recently it was reported that overexpression of MT1-matrix metalloproteinase (MMP) induced spontaneous DDR1 shedding in COS1 cells and T47D breast cancer cells, although the authors could not confirm a role of endogenous MT1-MMP as a constitutive DDR1 sheddase in HCC1806 breast cancer cell (Fu et al., 2013). Because A431 cells express endogenous MT1-MMP along with endogenous DDR1, we next examined whether MT1-MMP is involved in DDR1 shedding in our experimental setting. As shown in Figure 3B, knockdown of MT1-MMP in A431 cells did not affect spontaneous or collagen-induced DDR1 shedding, suggesting that MT1-MMP is not involved in DDR1 shedding in A431 cells, supporting the data on HCC1806 in the report by Fu et al. (2013). MT1-MMP is expressed in neither HEK293 nor MCF7 cells (unpublished data). Thus MT1-MMP is unlikely to be involved in DDR1 shedding in our experimental conditions.


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)

ADAM10 is the sheddase responsible for collagen-induced DDR1 ectodomain shedding. (A) A431 cells were transfected with siRNA for ADAM8, ADAM9, ADAM10, ADAM17, or ADAM19 or with nontargeting (NT) siRNA. After 48 h, cells were treated with collagen I (100 μg/ml) for a further 24 h. We confirmed that the level of mRNA for each enzyme was reduced by 60−99% after 72 h (right, RT-PCR). Mst (10 μM) was used as a positive control for inhibition of DDR1 shedding. Conditioned media and cell lysates were analyzed by Western blotting using anti–DDR1 ectodomain (Med), anti–DDR1 C-terminus (Cell), or anti-actin antibodies. Note that ADAM10 knockdown resulted in inhibition of endogenous DDR1 shedding. CTF, C-terminal fragment. (B) A431 cells were transfected with siRNA for MT1-MMP (si-MT1) or si-NT. Cells were then treated with collagen I for 24 h. Conditioned media and cell lysates were analyzed by Western blotting using anti-DDR1, anti-MMP14 (EP1264Y) (MT1), and anti-actin antibodies. (C) A431 cells were treated with 1 μM IM, 25 ng/ml PMA, or DMSO vehicle control (0.001%) for 1 h. Conditioned media and cell lysates were analyzed as in A. A431 cells were also treated with 1 μM IM for 1 h in the presence or absence of 10 μM Mst (right). (D) A431 cells were transiently transfected with ADAM10 or mock vector and treated with or without collagen I for 24 h. Conditioned media and cell lysates were subjected to Western blotting using anti–DDR1 ectodomain (Med), anti–DDR1 C-terminus (Cell), anti-ADAM10 (A10), and anti-actin antibodies.
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Figure 3: ADAM10 is the sheddase responsible for collagen-induced DDR1 ectodomain shedding. (A) A431 cells were transfected with siRNA for ADAM8, ADAM9, ADAM10, ADAM17, or ADAM19 or with nontargeting (NT) siRNA. After 48 h, cells were treated with collagen I (100 μg/ml) for a further 24 h. We confirmed that the level of mRNA for each enzyme was reduced by 60−99% after 72 h (right, RT-PCR). Mst (10 μM) was used as a positive control for inhibition of DDR1 shedding. Conditioned media and cell lysates were analyzed by Western blotting using anti–DDR1 ectodomain (Med), anti–DDR1 C-terminus (Cell), or anti-actin antibodies. Note that ADAM10 knockdown resulted in inhibition of endogenous DDR1 shedding. CTF, C-terminal fragment. (B) A431 cells were transfected with siRNA for MT1-MMP (si-MT1) or si-NT. Cells were then treated with collagen I for 24 h. Conditioned media and cell lysates were analyzed by Western blotting using anti-DDR1, anti-MMP14 (EP1264Y) (MT1), and anti-actin antibodies. (C) A431 cells were treated with 1 μM IM, 25 ng/ml PMA, or DMSO vehicle control (0.001%) for 1 h. Conditioned media and cell lysates were analyzed as in A. A431 cells were also treated with 1 μM IM for 1 h in the presence or absence of 10 μM Mst (right). (D) A431 cells were transiently transfected with ADAM10 or mock vector and treated with or without collagen I for 24 h. Conditioned media and cell lysates were subjected to Western blotting using anti–DDR1 ectodomain (Med), anti–DDR1 C-terminus (Cell), anti-ADAM10 (A10), and anti-actin antibodies.
Mentions: Our data indicated that the enzyme responsible for collagen-induced DDR1 shedding is a metalloproteinase insensitive to TIMP-1 and TIMP-2 and partially sensitive or insensitive to TIMP-3 (Figure 2, B and C). It has been shown that TIMP-insensitive metalloproteinases include ADAM8, ADAM9 (Amour et al., 2002), and ADAM19 (Chesneau et al., 2003; Shiomi et al., 2010). We therefore knocked down ADAM8, ADAM9, and ADAM19, along with well-characterized sheddases, ADAM10 and ADAM17, in A431 cells. The mRNA levels of each ADAM in the respective siRNA treated cells were significantly down-regulated compared with the nontargeting small interfering RNA (siRNA; si-NT)–treated cells by 60% (ADAM8), 86% (ADAM9), 95% (ADAM10), 89% (ADAM17), and 99% (ADAM19; Figure 3A). Under these conditions, only ADAM10 knockdown abolished collagen-induced DDR1 shedding (Figure 3A, DDR1, Med). It was noted that although Mst and ADAM10 knockdown inhibited shedding of DDR1, cellular level of DDR1 did not recover to the level of non–collagen-treated cells in A431 cells (Figure 3A). This is likely due to down-regulation of DDR1 production upon collagen treatment during 24-h incubation. We also confirmed the effect of ADAM10 knockdown in MCF-7 (Supplemental Figure S2) and HEK293 cells (see later discussion of Figure 7E, WT). Recently it was reported that overexpression of MT1-matrix metalloproteinase (MMP) induced spontaneous DDR1 shedding in COS1 cells and T47D breast cancer cells, although the authors could not confirm a role of endogenous MT1-MMP as a constitutive DDR1 sheddase in HCC1806 breast cancer cell (Fu et al., 2013). Because A431 cells express endogenous MT1-MMP along with endogenous DDR1, we next examined whether MT1-MMP is involved in DDR1 shedding in our experimental setting. As shown in Figure 3B, knockdown of MT1-MMP in A431 cells did not affect spontaneous or collagen-induced DDR1 shedding, suggesting that MT1-MMP is not involved in DDR1 shedding in A431 cells, supporting the data on HCC1806 in the report by Fu et al. (2013). MT1-MMP is expressed in neither HEK293 nor MCF7 cells (unpublished data). Thus MT1-MMP is unlikely to be involved in DDR1 shedding in our experimental conditions.

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