Ectodomain shedding of L1 adhesion molecule promotes cell migration by autocrine binding to integrins - PubMed (original) (raw)

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

S Mechtersheimer et al. J Cell Biol. 2001.

Erratum in

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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Figures

Figure 1.

Figure 1.

L1 is shed without cytoplasmic tail. (A) CHO or L1-transfected CHO cells were incubated at 37°C in suspension in medium without FCS for 1 h. The cell pellet was lysed and analyzed by Western blot with mAb UJ127.11 to the ectodomain or pcytL1 to the cytoplasmic domain of L1. (B) Schematic representation of L1 cleavage fragments and their relative localization. The location of antibody recognition sites are indicated. (1), mAb 5G3; (2), mAb UJ 127.11; (3) pcytL1.

Figure 2.

Figure 2.

Presence of a L1-32 indicates membrane-proximal cleavage. Cells were incubated at 37°C in suspension in medium without FCS for 1 h (A) Cell pellets were lysed and blotted with pcytL1 followed by ECL detection. Note that a parallel blot using the secondary antibody only was negative. (B) Supernatants were analyzed for the presence of shed L1 using blotting with mAb UJ127.11 to the ectodomain of human L1 followed by ECL detection. (C) Supernatants were also probed for the presence of soluble L1 using a capture ELISA.

Figure 3.

Figure 3.

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.

Figure 4.

Figure 4.

L1-32 can be detected in the developing mouse brain. Mouse brains of different ages were homogenized in lysis buffer, and equal amounts of protein were analyzed by SDS-PAGE and Western blotting using pcytL1 followed by peroxidase-conjugated secondary antibodies and ECL detection.

Figure 5.

Figure 5.

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.

Figure 5.

Figure 5.

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.

Figure 5.

Figure 5.

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.

Figure 5.

Figure 5.

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.

Figure 5.

Figure 5.

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.

Figure 5.

Figure 5.

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.

Figure 6.

Figure 6.

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.

Figure 6.

Figure 6.

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.

Figure 6.

Figure 6.

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.

Figure 6.

Figure 6.

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.

Figure 7.

Figure 7.

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.

Figure 7.

Figure 7.

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.

Figure 7.

Figure 7.

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.

Figure 8.

Figure 8.

The level of L1 expression affects cell migration. SKOV3 cell variants differing in the expression levels of L1 (SKOV3hi or SKOV3lo) were established by repeated FACS sorting. (A) Cytofluorographic staining of SKOV3hi or SKOV3lo cells with mAb UJ 127.11. (B) Analysis of cell adhesion and haptotactic cell migration of SKOV3hi or SKOV3lo cells. The assays were done as described in Figs. 5 E and 7 C, respectively. (C) Cells were preincubated with the indicated mAbs at 10 mg/ml and transmigration on FN was tested as described in Fig. 6 A. (D) Cells were preincubated with the indicated mAbs at 10 mg/ml and transmigration on laminin was tested as described in Fig. 6 A.

Figure 8.

Figure 8.

The level of L1 expression affects cell migration. SKOV3 cell variants differing in the expression levels of L1 (SKOV3hi or SKOV3lo) were established by repeated FACS sorting. (A) Cytofluorographic staining of SKOV3hi or SKOV3lo cells with mAb UJ 127.11. (B) Analysis of cell adhesion and haptotactic cell migration of SKOV3hi or SKOV3lo cells. The assays were done as described in Figs. 5 E and 7 C, respectively. (C) Cells were preincubated with the indicated mAbs at 10 mg/ml and transmigration on FN was tested as described in Fig. 6 A. (D) Cells were preincubated with the indicated mAbs at 10 mg/ml and transmigration on laminin was tested as described in Fig. 6 A.

Figure 8.

Figure 8.

The level of L1 expression affects cell migration. SKOV3 cell variants differing in the expression levels of L1 (SKOV3hi or SKOV3lo) were established by repeated FACS sorting. (A) Cytofluorographic staining of SKOV3hi or SKOV3lo cells with mAb UJ 127.11. (B) Analysis of cell adhesion and haptotactic cell migration of SKOV3hi or SKOV3lo cells. The assays were done as described in Figs. 5 E and 7 C, respectively. (C) Cells were preincubated with the indicated mAbs at 10 mg/ml and transmigration on FN was tested as described in Fig. 6 A. (D) Cells were preincubated with the indicated mAbs at 10 mg/ml and transmigration on laminin was tested as described in Fig. 6 A.

Figure 9.

Figure 9.

ADAM10 is involved in L1 shedding. (A) AR cells were transiently transfected with 10 mg plasmid DNA encoding HA-tagged ADAM10 or Flag-tagged dominant-negative ADAM10 (ADAM10 DN). 48 h after transfection, cells were stimulated with PMA or pervanadate to induce shedding. After 2 h, the supernatants were harvested and the cells lysed in lysis buffer. Cell lysates and conditioned media were analysed by ELISA for soluble L1. (B) Detection of HA-tagged ADAM10 or FLAG-tagged ADAM10 DN in the lysate of transfected cells.

Figure 10.

Figure 10.

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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