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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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Exogenous addition of soluble L1 enhances migration of CHO cells. (A) CHO cells in the presence of the indicated conditioned media (1:10 final dilution) were seeded into the upper compartment of the Transwell chamber coated with FN from the backside and allowed to transmigrate for 16 h at 37°C. (B) Schematic representation of the L1-Fc and L1-Fc mut III fusion proteins. (C) CHO cells in the presence of the indicated Fc fusion protein (final concentration 0.67 mg/ml) were seeded into the upper compartment of the Transwell chamber. (D) ML1-CHO cells were pretreated with BB-3103 to prevent L1 shedding and haptotactic migration. Cells were then seeded together with the indicated Fc fusion proteins into the upper compartment of the Transwell chamber. Each determination was done in quadruplicates and transmigrated cells were stained from the backside of the filter as described in Fig. 5 A.
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fig7: Exogenous addition of soluble L1 enhances migration of CHO cells. (A) CHO cells in the presence of the indicated conditioned media (1:10 final dilution) were seeded into the upper compartment of the Transwell chamber coated with FN from the backside and allowed to transmigrate for 16 h at 37°C. (B) Schematic representation of the L1-Fc and L1-Fc mut III fusion proteins. (C) CHO cells in the presence of the indicated Fc fusion protein (final concentration 0.67 mg/ml) were seeded into the upper compartment of the Transwell chamber. (D) ML1-CHO cells were pretreated with BB-3103 to prevent L1 shedding and haptotactic migration. Cells were then seeded together with the indicated Fc fusion proteins into the upper compartment of the Transwell chamber. Each determination was done in quadruplicates and transmigrated cells were stained from the backside of the filter as described in Fig. 5 A.

Mentions: We hypothesized that cleaved L1 might deliver an autocrine signal to the cells and thereby promote enhanced migration. According to this hypothesis, the addition of soluble L1 should enhance the migration of CHO cells. To test this assumption, CHO cells were first exposed to conditioned medium from L1-positive cells, and the effect on the haptotactic migration was analyzed. As shown in Fig. 7 A, the medium from L1-CHO but not CHO cells could stimulate cell migration that was of similar magnitude as L1-CHO cells. The stimulatory activity was also present in the conditioned medium of AR tumor cells but was absent when AR cells or L1-CHO cells were cultivated in the presence of BB-3103 in order to block shedding. To further prove that the activity was mediated by soluble L1, we used a recombinant L1-Fc protein (Fig. 7 B). As shown in Fig. 7 C, the L1-Fc protein but not a control protein (SART-1-Fc) could stimulate CHO migration like conditioned medium containing native L1. We concluded that soluble L1 can stimulate cell migration.


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)

Exogenous addition of soluble L1 enhances migration of CHO cells. (A) CHO cells in the presence of the indicated conditioned media (1:10 final dilution) were seeded into the upper compartment of the Transwell chamber coated with FN from the backside and allowed to transmigrate for 16 h at 37°C. (B) Schematic representation of the L1-Fc and L1-Fc mut III fusion proteins. (C) CHO cells in the presence of the indicated Fc fusion protein (final concentration 0.67 mg/ml) were seeded into the upper compartment of the Transwell chamber. (D) ML1-CHO cells were pretreated with BB-3103 to prevent L1 shedding and haptotactic migration. Cells were then seeded together with the indicated Fc fusion proteins into the upper compartment of the Transwell chamber. Each determination was done in quadruplicates and transmigrated cells were stained from the backside of the filter as described in Fig. 5 A.
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Related In: Results  -  Collection

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

fig7: Exogenous addition of soluble L1 enhances migration of CHO cells. (A) CHO cells in the presence of the indicated conditioned media (1:10 final dilution) were seeded into the upper compartment of the Transwell chamber coated with FN from the backside and allowed to transmigrate for 16 h at 37°C. (B) Schematic representation of the L1-Fc and L1-Fc mut III fusion proteins. (C) CHO cells in the presence of the indicated Fc fusion protein (final concentration 0.67 mg/ml) were seeded into the upper compartment of the Transwell chamber. (D) ML1-CHO cells were pretreated with BB-3103 to prevent L1 shedding and haptotactic migration. Cells were then seeded together with the indicated Fc fusion proteins into the upper compartment of the Transwell chamber. Each determination was done in quadruplicates and transmigrated cells were stained from the backside of the filter as described in Fig. 5 A.
Mentions: We hypothesized that cleaved L1 might deliver an autocrine signal to the cells and thereby promote enhanced migration. According to this hypothesis, the addition of soluble L1 should enhance the migration of CHO cells. To test this assumption, CHO cells were first exposed to conditioned medium from L1-positive cells, and the effect on the haptotactic migration was analyzed. As shown in Fig. 7 A, the medium from L1-CHO but not CHO cells could stimulate cell migration that was of similar magnitude as L1-CHO cells. The stimulatory activity was also present in the conditioned medium of AR tumor cells but was absent when AR cells or L1-CHO cells were cultivated in the presence of BB-3103 in order to block shedding. To further prove that the activity was mediated by soluble L1, we used a recombinant L1-Fc protein (Fig. 7 B). As shown in Fig. 7 C, the L1-Fc protein but not a control protein (SART-1-Fc) could stimulate CHO migration like conditioned medium containing native L1. We concluded that soluble L1 can stimulate cell migration.

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