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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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Inhibition of cell migration by antibodies and metalloproteinase inhibitor. (A) ML1-CHO cells or CHO cells were preincubated with the indicated mAbs at 10 mg/ml and added to the upper compartment of the Transwell chamber coated with FN from the backside. Note that antibody was present during the assay time. (B) Staining of CHO and mL1-CHO cells with a mAb to αvβ5 integrin. (C) ML1-CHO cells or CHO cells were preincubated with the indicated amount of BB-3103 inhibitor for 2 h and then transferred to the upper compartment of the Transwell chamber. Each determination was done in quadruplicate and transmigrated cells were stained from the backside of the filter as described in Fig. 5 A. (D) Inhibition of cell migration by a mAb to the α5 integrin. ML1-CHO or CHO cells were preincubated with the mAbs at 10 mg/ml and added to the upper compartment of the Transwell chamber coated with FN.
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fig6: Inhibition of cell migration by antibodies and metalloproteinase inhibitor. (A) ML1-CHO cells or CHO cells were preincubated with the indicated mAbs at 10 mg/ml and added to the upper compartment of the Transwell chamber coated with FN from the backside. Note that antibody was present during the assay time. (B) Staining of CHO and mL1-CHO cells with a mAb to αvβ5 integrin. (C) ML1-CHO cells or CHO cells were preincubated with the indicated amount of BB-3103 inhibitor for 2 h and then transferred to the upper compartment of the Transwell chamber. Each determination was done in quadruplicate and transmigrated cells were stained from the backside of the filter as described in Fig. 5 A. (D) Inhibition of cell migration by a mAb to the α5 integrin. ML1-CHO or CHO cells were preincubated with the mAbs at 10 mg/ml and added to the upper compartment of the Transwell chamber coated with FN.

Mentions: We investigated the difference in migration between mL1-CHO cells and CHO cells in more detail using blocking mAbs. As shown in Fig. 6 A, mAb P1F6 to human αvβ5 crossreacted with hamster integrins and could inhibit the migration of mL1-CHO cells to the level of CHO cells. Importantly, there was no effect of the αvβ5-specific mAb on the migration of CHO cells. Additional FACS analysis with mAb P1F6 showed no difference in the expression level of the αvβ5 integrin (Fig. 6 B). The migration of mL1-CHO was also efficiently blocked in the presence of mAb 324 against mouse L1, but not by the isotype-matched control mAb 79 (Fig. 6 A).


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)

Inhibition of cell migration by antibodies and metalloproteinase inhibitor. (A) ML1-CHO cells or CHO cells were preincubated with the indicated mAbs at 10 mg/ml and added to the upper compartment of the Transwell chamber coated with FN from the backside. Note that antibody was present during the assay time. (B) Staining of CHO and mL1-CHO cells with a mAb to αvβ5 integrin. (C) ML1-CHO cells or CHO cells were preincubated with the indicated amount of BB-3103 inhibitor for 2 h and then transferred to the upper compartment of the Transwell chamber. Each determination was done in quadruplicate and transmigrated cells were stained from the backside of the filter as described in Fig. 5 A. (D) Inhibition of cell migration by a mAb to the α5 integrin. ML1-CHO or CHO cells were preincubated with the mAbs at 10 mg/ml and added to the upper compartment of the Transwell chamber coated with FN.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2198870&req=5

fig6: Inhibition of cell migration by antibodies and metalloproteinase inhibitor. (A) ML1-CHO cells or CHO cells were preincubated with the indicated mAbs at 10 mg/ml and added to the upper compartment of the Transwell chamber coated with FN from the backside. Note that antibody was present during the assay time. (B) Staining of CHO and mL1-CHO cells with a mAb to αvβ5 integrin. (C) ML1-CHO cells or CHO cells were preincubated with the indicated amount of BB-3103 inhibitor for 2 h and then transferred to the upper compartment of the Transwell chamber. Each determination was done in quadruplicate and transmigrated cells were stained from the backside of the filter as described in Fig. 5 A. (D) Inhibition of cell migration by a mAb to the α5 integrin. ML1-CHO or CHO cells were preincubated with the mAbs at 10 mg/ml and added to the upper compartment of the Transwell chamber coated with FN.
Mentions: We investigated the difference in migration between mL1-CHO cells and CHO cells in more detail using blocking mAbs. As shown in Fig. 6 A, mAb P1F6 to human αvβ5 crossreacted with hamster integrins and could inhibit the migration of mL1-CHO cells to the level of CHO cells. Importantly, there was no effect of the αvβ5-specific mAb on the migration of CHO cells. Additional FACS analysis with mAb P1F6 showed no difference in the expression level of the αvβ5 integrin (Fig. 6 B). The migration of mL1-CHO was also efficiently blocked in the presence of mAb 324 against mouse L1, but not by the isotype-matched control mAb 79 (Fig. 6 A).

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