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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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Expression of L1 enhances haptotactic migration of CHO cells. (A) FN at 10 mg/ml or BSA for control were coated on the backside of Transwell chambers. CHO, h/mL1-CHO or mL1-mutIII-CHO cells stably expressing L1 devoid of RGDs were seeded into the top chamber and allowed to transmigrate for 16 h at 37°C. Each determination was done in quadruplicate and transmigrated cells were stained from the back of the filter. The dye was eluted from the filter and measured at 595 nm. The amount of dye is proportional to the number of transmigrated cells. Values for the migration on BSA were below 0.25 OD units for each cell type. (B) Staining of CHO-transfected cells used in (A) with mAbs to human or mouse L1, respectively. (C) Analysis of CHO and mL1-CHO cell migration on vitronectin or laminin (each coated at 10 mg/ml). Analysis of migrated cells was done as described in A. (D) Dose–response curve for the migration of CHO and mL1-CHO cells. The assay was done as described in A. (E) FN or BSA for control were coated on LABTEK chamber slides, and the adhesion of CHO cells and transfectants was determined in the presence of 2 mM Ca 21 and 2 mM Mg21. Binding to BSA was below 50 cells/area. (F) Cells were grown on coverslips, stained with mAb 324 to mouse L1, and analyzed by fluorescence microscopy. (G) Cells were cultivated for 24 h and TCA-precipitated supernatants and cell lysates were analyzed. After SDS-PAGE, L1 was detected by Western blotting and ECL detection using anti L1 antibodies.
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fig5: Expression of L1 enhances haptotactic migration of CHO cells. (A) FN at 10 mg/ml or BSA for control were coated on the backside of Transwell chambers. CHO, h/mL1-CHO or mL1-mutIII-CHO cells stably expressing L1 devoid of RGDs were seeded into the top chamber and allowed to transmigrate for 16 h at 37°C. Each determination was done in quadruplicate and transmigrated cells were stained from the back of the filter. The dye was eluted from the filter and measured at 595 nm. The amount of dye is proportional to the number of transmigrated cells. Values for the migration on BSA were below 0.25 OD units for each cell type. (B) Staining of CHO-transfected cells used in (A) with mAbs to human or mouse L1, respectively. (C) Analysis of CHO and mL1-CHO cell migration on vitronectin or laminin (each coated at 10 mg/ml). Analysis of migrated cells was done as described in A. (D) Dose–response curve for the migration of CHO and mL1-CHO cells. The assay was done as described in A. (E) FN or BSA for control were coated on LABTEK chamber slides, and the adhesion of CHO cells and transfectants was determined in the presence of 2 mM Ca 21 and 2 mM Mg21. Binding to BSA was below 50 cells/area. (F) Cells were grown on coverslips, stained with mAb 324 to mouse L1, and analyzed by fluorescence microscopy. (G) Cells were cultivated for 24 h and TCA-precipitated supernatants and cell lysates were analyzed. After SDS-PAGE, L1 was detected by Western blotting and ECL detection using anti L1 antibodies.

Mentions: Having established that ectodomain cleavage of L1 occurs in human tumor cells and during mouse brain development, we set out to explore the physiological role of the shedding process. In the brain, L1 plays an important role in the migration of neural cells. Therefore, we examined the role of L1 and its ectodomain cleavage process in cell migration. As shown in Fig. 5 A, we observed that CHO cells stably transfected with mouse or human L1 (mL1-CHO or hL1-CHO cells) showed a three- to fivefold increase in haptotactic migration on fibronectin compared with CHO cells. In contrast, CHO cells expressing L1 in which both RGD sites were mutated to RGE (mL1-mutIII-CHO) or those expressing mutant forms of L1 containing either the cytoplasmic and transmembrane portion of L-selectin (L1/L-selectin) or the ectodomain of L-selectin (L-selectin/L1) did not show increased migration (Fig. 5 A). The transfected CHO cells expressed L1 or the chimeric forms of L1 at comparable level as detected by FACS analysis (Fig. 5 B).


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)

Expression of L1 enhances haptotactic migration of CHO cells. (A) FN at 10 mg/ml or BSA for control were coated on the backside of Transwell chambers. CHO, h/mL1-CHO or mL1-mutIII-CHO cells stably expressing L1 devoid of RGDs were seeded into the top chamber and allowed to transmigrate for 16 h at 37°C. Each determination was done in quadruplicate and transmigrated cells were stained from the back of the filter. The dye was eluted from the filter and measured at 595 nm. The amount of dye is proportional to the number of transmigrated cells. Values for the migration on BSA were below 0.25 OD units for each cell type. (B) Staining of CHO-transfected cells used in (A) with mAbs to human or mouse L1, respectively. (C) Analysis of CHO and mL1-CHO cell migration on vitronectin or laminin (each coated at 10 mg/ml). Analysis of migrated cells was done as described in A. (D) Dose–response curve for the migration of CHO and mL1-CHO cells. The assay was done as described in A. (E) FN or BSA for control were coated on LABTEK chamber slides, and the adhesion of CHO cells and transfectants was determined in the presence of 2 mM Ca 21 and 2 mM Mg21. Binding to BSA was below 50 cells/area. (F) Cells were grown on coverslips, stained with mAb 324 to mouse L1, and analyzed by fluorescence microscopy. (G) Cells were cultivated for 24 h and TCA-precipitated supernatants and cell lysates were analyzed. After SDS-PAGE, L1 was detected by Western blotting and ECL detection using anti L1 antibodies.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2198870&req=5

fig5: Expression of L1 enhances haptotactic migration of CHO cells. (A) FN at 10 mg/ml or BSA for control were coated on the backside of Transwell chambers. CHO, h/mL1-CHO or mL1-mutIII-CHO cells stably expressing L1 devoid of RGDs were seeded into the top chamber and allowed to transmigrate for 16 h at 37°C. Each determination was done in quadruplicate and transmigrated cells were stained from the back of the filter. The dye was eluted from the filter and measured at 595 nm. The amount of dye is proportional to the number of transmigrated cells. Values for the migration on BSA were below 0.25 OD units for each cell type. (B) Staining of CHO-transfected cells used in (A) with mAbs to human or mouse L1, respectively. (C) Analysis of CHO and mL1-CHO cell migration on vitronectin or laminin (each coated at 10 mg/ml). Analysis of migrated cells was done as described in A. (D) Dose–response curve for the migration of CHO and mL1-CHO cells. The assay was done as described in A. (E) FN or BSA for control were coated on LABTEK chamber slides, and the adhesion of CHO cells and transfectants was determined in the presence of 2 mM Ca 21 and 2 mM Mg21. Binding to BSA was below 50 cells/area. (F) Cells were grown on coverslips, stained with mAb 324 to mouse L1, and analyzed by fluorescence microscopy. (G) Cells were cultivated for 24 h and TCA-precipitated supernatants and cell lysates were analyzed. After SDS-PAGE, L1 was detected by Western blotting and ECL detection using anti L1 antibodies.
Mentions: Having established that ectodomain cleavage of L1 occurs in human tumor cells and during mouse brain development, we set out to explore the physiological role of the shedding process. In the brain, L1 plays an important role in the migration of neural cells. Therefore, we examined the role of L1 and its ectodomain cleavage process in cell migration. As shown in Fig. 5 A, we observed that CHO cells stably transfected with mouse or human L1 (mL1-CHO or hL1-CHO cells) showed a three- to fivefold increase in haptotactic migration on fibronectin compared with CHO cells. In contrast, CHO cells expressing L1 in which both RGD sites were mutated to RGE (mL1-mutIII-CHO) or those expressing mutant forms of L1 containing either the cytoplasmic and transmembrane portion of L-selectin (L1/L-selectin) or the ectodomain of L-selectin (L-selectin/L1) did not show increased migration (Fig. 5 A). The transfected CHO cells expressed L1 or the chimeric forms of L1 at comparable level as detected by FACS analysis (Fig. 5 B).

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