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Ectodomain shedding of L1 adhesion molecule promotes cell migration by autocrine binding to integrins.

Mechtersheimer S, Gutwein P, Agmon-Levin N, Stoeck A, Oleszewski M, Riedle S, Postina R, Fahrenholz F, Fogel M, Lemmon V, Altevogt P - J. Cell Biol. (2001)

Bottom Line: The induction of migration was blocked by alpha v beta 5-specific antibodies and required Arg-Gly-Asp sites in L1.We propose that ectodomain-released L1 promotes migration by autocrine/paracrine stimulation via alpha v beta 5.This regulatory loop could be relevant for migratory processes under physiological and pathophysiological conditions.

View Article: PubMed Central - PubMed

Affiliation: Tumor Immunology Program, G0100, German Cancer Research Center, D-69120 Heidelberg, Germany.

ABSTRACT
The L1 adhesion molecule plays an important role in axon guidance and cell migration in the nervous system. L1 is also expressed by many human carcinomas. In addition to cell surface expression, the L1 ectodomain can be released by a metalloproteinase, but the biological function of this process is unknown. Here we demonstrate that membrane-proximal cleavage of L1 can be detected in tumors and in the developing mouse brain. The shedding of L1 involved a disintegrin and metalloproteinase (ADAM)10, as transfection with dominant-negative ADAM10 completely abolishes L1 release. L1-transfected CHO cells (L1-CHO) showed enhanced haptotactic migration on fibronectin and laminin, which was blocked by antibodies to alpha v beta 5 and L1. Migration of L1-CHO cells, but not the basal migration of CHO cells, was blocked by a metalloproteinase inhibitor, indicating a role for L1 shedding in the migration process. CHO and metalloproteinase-inhibited L1-CHO cells were stimulated to migrate by soluble L1-Fc protein. The induction of migration was blocked by alpha v beta 5-specific antibodies and required Arg-Gly-Asp sites in L1. A 150-kD L1 fragment released by plasmin could also stimulate CHO cell migration. We propose that ectodomain-released L1 promotes migration by autocrine/paracrine stimulation via alpha v beta 5. This regulatory loop could be relevant for migratory processes under physiological and pathophysiological conditions.

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The L1–150-kD plasmin fragment can also stimulate cell migration. AR cell were treated with Ro 31-9790 inhibitor (Ro) in combination with plasmin and α2-antiplasmin for 6 h at 37°C. (A) Cells and supernatants were collected and analyzed by Western blotting using the indicated antibodies. (B) Supernatants of treated cultures were investigated for the ability to induce haptotactic migration of CHO cells as described in Fig. 7 A.
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fig10: The L1–150-kD plasmin fragment can also stimulate cell migration. AR cell were treated with Ro 31-9790 inhibitor (Ro) in combination with plasmin and α2-antiplasmin for 6 h at 37°C. (A) Cells and supernatants were collected and analyzed by Western blotting using the indicated antibodies. (B) Supernatants of treated cultures were investigated for the ability to induce haptotactic migration of CHO cells as described in Fig. 7 A.

Mentions: It has been shown recently that the addition of plasmin to L1-positive cells results in a dose-dependent loss of surface L1 expression with the concomitant appearance of a 140–150-kD soluble L1 species (L1–150) into the supernatant (Nayeem et al., 1999). Cleavage occurs at a dibasic site in the middle of the third FNIII repeat, thus, the released fragment contains the RGD site(s) in the 6.Ig-domain. We asked whether the plasmin fragment could substitute for the metalloproteinase-released ectodomain of L1. AR cells in serum-free medium were treated with a predetermined concentration of plasmin or plasmin plus the specific inhibitor α2-antiplasmin, and supernatants and cell pellets were taken for biochemical and functional analysis. To suppress the metalloproteinase-mediated L1 release, all supernatants were generated in the presence of Ro 31-9790 inhibitor. Fig. 10 A shows that in the presence of Ro1plasmin, a substantial loss of L1 from the cell pellet was observed which could be detected with both L1 mAbs 5G3 and UJ 127.11. The latter mAb also detected the L1-85 fragment remaining associated with the cell pellet after plasmin cleavage. This is in agreement with the notion that mAb UJ 127.11 recognizes an epitope between the dibasic cleavage site and the plasma membrane (Fig. 1 B), whereas mAb 5G3 reportedly binds to the NH2 terminus of L1 (Silletti et al., 2000). Analysis of the supernatant from treated cultures indicated that in the presence of the metalloproteinase inhibitor, the formation of soluble L1-200 was drastically reduced, and that plasmin addition resulted in the appearance of an expected 150-kD L1 fragment (Fig. 10 A). The formation of the 150-kD plasmin fragment was blocked in the presence of α2-antiplasmin.


Ectodomain shedding of L1 adhesion molecule promotes cell migration by autocrine binding to integrins.

Mechtersheimer S, Gutwein P, Agmon-Levin N, Stoeck A, Oleszewski M, Riedle S, Postina R, Fahrenholz F, Fogel M, Lemmon V, Altevogt P - J. Cell Biol. (2001)

The L1–150-kD plasmin fragment can also stimulate cell migration. AR cell were treated with Ro 31-9790 inhibitor (Ro) in combination with plasmin and α2-antiplasmin for 6 h at 37°C. (A) Cells and supernatants were collected and analyzed by Western blotting using the indicated antibodies. (B) Supernatants of treated cultures were investigated for the ability to induce haptotactic migration of CHO cells as described in Fig. 7 A.
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Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2198870&req=5

fig10: The L1–150-kD plasmin fragment can also stimulate cell migration. AR cell were treated with Ro 31-9790 inhibitor (Ro) in combination with plasmin and α2-antiplasmin for 6 h at 37°C. (A) Cells and supernatants were collected and analyzed by Western blotting using the indicated antibodies. (B) Supernatants of treated cultures were investigated for the ability to induce haptotactic migration of CHO cells as described in Fig. 7 A.
Mentions: It has been shown recently that the addition of plasmin to L1-positive cells results in a dose-dependent loss of surface L1 expression with the concomitant appearance of a 140–150-kD soluble L1 species (L1–150) into the supernatant (Nayeem et al., 1999). Cleavage occurs at a dibasic site in the middle of the third FNIII repeat, thus, the released fragment contains the RGD site(s) in the 6.Ig-domain. We asked whether the plasmin fragment could substitute for the metalloproteinase-released ectodomain of L1. AR cells in serum-free medium were treated with a predetermined concentration of plasmin or plasmin plus the specific inhibitor α2-antiplasmin, and supernatants and cell pellets were taken for biochemical and functional analysis. To suppress the metalloproteinase-mediated L1 release, all supernatants were generated in the presence of Ro 31-9790 inhibitor. Fig. 10 A shows that in the presence of Ro1plasmin, a substantial loss of L1 from the cell pellet was observed which could be detected with both L1 mAbs 5G3 and UJ 127.11. The latter mAb also detected the L1-85 fragment remaining associated with the cell pellet after plasmin cleavage. This is in agreement with the notion that mAb UJ 127.11 recognizes an epitope between the dibasic cleavage site and the plasma membrane (Fig. 1 B), whereas mAb 5G3 reportedly binds to the NH2 terminus of L1 (Silletti et al., 2000). Analysis of the supernatant from treated cultures indicated that in the presence of the metalloproteinase inhibitor, the formation of soluble L1-200 was drastically reduced, and that plasmin addition resulted in the appearance of an expected 150-kD L1 fragment (Fig. 10 A). The formation of the 150-kD plasmin fragment was blocked in the presence of α2-antiplasmin.

Bottom Line: The induction of migration was blocked by alpha v beta 5-specific antibodies and required Arg-Gly-Asp sites in L1.We propose that ectodomain-released L1 promotes migration by autocrine/paracrine stimulation via alpha v beta 5.This regulatory loop could be relevant for migratory processes under physiological and pathophysiological conditions.

View Article: PubMed Central - PubMed

Affiliation: Tumor Immunology Program, G0100, German Cancer Research Center, D-69120 Heidelberg, Germany.

ABSTRACT
The L1 adhesion molecule plays an important role in axon guidance and cell migration in the nervous system. L1 is also expressed by many human carcinomas. In addition to cell surface expression, the L1 ectodomain can be released by a metalloproteinase, but the biological function of this process is unknown. Here we demonstrate that membrane-proximal cleavage of L1 can be detected in tumors and in the developing mouse brain. The shedding of L1 involved a disintegrin and metalloproteinase (ADAM)10, as transfection with dominant-negative ADAM10 completely abolishes L1 release. L1-transfected CHO cells (L1-CHO) showed enhanced haptotactic migration on fibronectin and laminin, which was blocked by antibodies to alpha v beta 5 and L1. Migration of L1-CHO cells, but not the basal migration of CHO cells, was blocked by a metalloproteinase inhibitor, indicating a role for L1 shedding in the migration process. CHO and metalloproteinase-inhibited L1-CHO cells were stimulated to migrate by soluble L1-Fc protein. The induction of migration was blocked by alpha v beta 5-specific antibodies and required Arg-Gly-Asp sites in L1. A 150-kD L1 fragment released by plasmin could also stimulate CHO cell migration. We propose that ectodomain-released L1 promotes migration by autocrine/paracrine stimulation via alpha v beta 5. This regulatory loop could be relevant for migratory processes under physiological and pathophysiological conditions.

Show MeSH
Related in: MedlinePlus