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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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Related in: MedlinePlus

Regulated cleavage of L1 is blocked by metalloproteinase inhibitor. Attached AR cells were treated with PMA (100 ng/ml), pervanadate (200 mmolar), or MCD for 1 h at 37°C in the absence or presence of BB-3103 inhibitor. (A) Supernatants were immunoprecipitated with mAb to L1 coupled to Sepharose and analyzed for shed L1 using blotting with mAb UJ127.11 followed by ECL detection. (B) Cell pellets were lysed and blotted with pcytL1 followed by ECL detection.
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fig3: Regulated cleavage of L1 is blocked by metalloproteinase inhibitor. Attached AR cells were treated with PMA (100 ng/ml), pervanadate (200 mmolar), or MCD for 1 h at 37°C in the absence or presence of BB-3103 inhibitor. (A) Supernatants were immunoprecipitated with mAb to L1 coupled to Sepharose and analyzed for shed L1 using blotting with mAb UJ127.11 followed by ECL detection. (B) Cell pellets were lysed and blotted with pcytL1 followed by ECL detection.

Mentions: Treatment of attached cells with PMA or pervanadate also induced shedding of L1 that can be blocked by hydroxamate-based metalloproteinase inhibitors (Gutwein et al., 2000). We examined the presence of L1-32 in AR cells after induction of cleavage in the absence or presence of BB-3103 metalloproteinase inhibitor. As shown in Fig. 3 A, dramatically increased levels of soluble L1 were detected in the supernatant after PMA (lane 2) or pervanadate treatment (lane 4) that were reduced to background levels in the presence of BB-3103 (lanes 3 and 5). Surprisingly, enhanced L1 release was observed by treatment of cells with methyl-β-cyclodextrin (MCD) (lane 6), a drug known to interfere with membrane cholesterol levels. Also, the MCD-induced release of L1 was inhibited by BB-3103 (lane 7). As shown in Fig. 3 B, the induction of L1 cleavage by PMA, pervanadate, or MCD clearly augmented the presence of L1-32 from a basal level in untreated cells. Collectively, the data suggested that L1-32 represents the portion of L1 remaining associated with the cells after shedding of the ectodomain.


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)

Regulated cleavage of L1 is blocked by metalloproteinase inhibitor. Attached AR cells were treated with PMA (100 ng/ml), pervanadate (200 mmolar), or MCD for 1 h at 37°C in the absence or presence of BB-3103 inhibitor. (A) Supernatants were immunoprecipitated with mAb to L1 coupled to Sepharose and analyzed for shed L1 using blotting with mAb UJ127.11 followed by ECL detection. (B) Cell pellets were lysed and blotted with pcytL1 followed by ECL detection.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2198870&req=5

fig3: Regulated cleavage of L1 is blocked by metalloproteinase inhibitor. Attached AR cells were treated with PMA (100 ng/ml), pervanadate (200 mmolar), or MCD for 1 h at 37°C in the absence or presence of BB-3103 inhibitor. (A) Supernatants were immunoprecipitated with mAb to L1 coupled to Sepharose and analyzed for shed L1 using blotting with mAb UJ127.11 followed by ECL detection. (B) Cell pellets were lysed and blotted with pcytL1 followed by ECL detection.
Mentions: Treatment of attached cells with PMA or pervanadate also induced shedding of L1 that can be blocked by hydroxamate-based metalloproteinase inhibitors (Gutwein et al., 2000). We examined the presence of L1-32 in AR cells after induction of cleavage in the absence or presence of BB-3103 metalloproteinase inhibitor. As shown in Fig. 3 A, dramatically increased levels of soluble L1 were detected in the supernatant after PMA (lane 2) or pervanadate treatment (lane 4) that were reduced to background levels in the presence of BB-3103 (lanes 3 and 5). Surprisingly, enhanced L1 release was observed by treatment of cells with methyl-β-cyclodextrin (MCD) (lane 6), a drug known to interfere with membrane cholesterol levels. Also, the MCD-induced release of L1 was inhibited by BB-3103 (lane 7). As shown in Fig. 3 B, the induction of L1 cleavage by PMA, pervanadate, or MCD clearly augmented the presence of L1-32 from a basal level in untreated cells. Collectively, the data suggested that L1-32 represents the portion of L1 remaining associated with the cells after shedding of the ectodomain.

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