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Protein kinase C-dependent mobilization of the alpha6beta4 integrin from hemidesmosomes and its association with actin-rich cell protrusions drive the chemotactic migration of carcinoma cells.

Rabinovitz I, Toker A, Mercurio AM - J. Cell Biol. (1999)

Bottom Line: Using function-blocking antibodies, we show that the alpha6beta4 integrin participates in EGF-stimulated chemotaxis and is required for lamellae formation on laminin-1.At concentrations of EGF that stimulate A431 chemotaxis ( approximately 1 ng/ml), the alpha6beta4 integrin is mobilized from hemidesmosomes as evidenced by indirect immunofluorescence microscopy using mAbs specific for this integrin and hemidesmosomal components and its loss from a cytokeratin fraction obtained by detergent extraction.Importantly, we demonstrate that this mobilization of alpha6beta4 from hemidesmosomes and its redistribution to cell protrusions occurs by a mechanism that involves activation of protein kinase C-alpha and that it is associated with the phosphorylation of the beta4 integrin subunit on serine residues.

View Article: PubMed Central - PubMed

Affiliation: Department of Medicine, Beth Israel Deaconess Medical Center and Harvard Medical School, Boston, Massachusetts 02215, USA.

ABSTRACT
We explored the hypothesis that the chemotactic migration of carcinoma cells that assemble hemidesmosomes involves the activation of a signaling pathway that releases the alpha6beta4 integrin from these stable adhesion complexes and promotes its association with F-actin in cell protrusions enabling it to function in migration. Squamous carcinoma-derived A431 cells were used because they express alpha6beta4 and migrate in response to EGF stimulation. Using function-blocking antibodies, we show that the alpha6beta4 integrin participates in EGF-stimulated chemotaxis and is required for lamellae formation on laminin-1. At concentrations of EGF that stimulate A431 chemotaxis ( approximately 1 ng/ml), the alpha6beta4 integrin is mobilized from hemidesmosomes as evidenced by indirect immunofluorescence microscopy using mAbs specific for this integrin and hemidesmosomal components and its loss from a cytokeratin fraction obtained by detergent extraction. EGF stimulation also increased the formation of lamellipodia and membrane ruffles that contained alpha6beta4 in association with F-actin. Importantly, we demonstrate that this mobilization of alpha6beta4 from hemidesmosomes and its redistribution to cell protrusions occurs by a mechanism that involves activation of protein kinase C-alpha and that it is associated with the phosphorylation of the beta4 integrin subunit on serine residues. Thus, the chemotactic migration of A431 cells on laminin-1 requires not only the formation of F-actin-rich cell protrusions that mediate alpha6beta4-dependent cell movement but also the disruption of alpha6beta4-containing hemidesmosomes by protein kinase C.

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The α6β4 integrin functions in EGF-induced chemotaxis and lamellae formation of A431 cells on laminin-1. (A) The optimal concentration of EGF that stimulates chemotaxis was determined in a 2-h assay using a modified Boyden chamber coated with laminin-1 as described in Materials and Methods. Data represent the mean number of cells/microscopic field (± SE) that migrated through the separating membrane. (B) The ability of the α6-specific mAb 2B7 or IgG control to inhibit the chemotactic migration of A431 cells toward EGF (1 ng/ml) was analyzed using the method described in A. Data shown represent the percentage of cells that migrated relative to the control. (C) A431 cells were plated on laminin-1 for 1 h and either not stimulated or stimulated with EGF (1 ng/ml) for 15 min in the presence of 10 μg of either 2B7 Ab or control IgG (-). The antibodies were added 30 min before EGF stimulation. The lamellar area was analyzed using an image analysis system as described in Materials and Methods. The data shown represent the mean lamellar area ± SE.
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Figure 2: The α6β4 integrin functions in EGF-induced chemotaxis and lamellae formation of A431 cells on laminin-1. (A) The optimal concentration of EGF that stimulates chemotaxis was determined in a 2-h assay using a modified Boyden chamber coated with laminin-1 as described in Materials and Methods. Data represent the mean number of cells/microscopic field (± SE) that migrated through the separating membrane. (B) The ability of the α6-specific mAb 2B7 or IgG control to inhibit the chemotactic migration of A431 cells toward EGF (1 ng/ml) was analyzed using the method described in A. Data shown represent the percentage of cells that migrated relative to the control. (C) A431 cells were plated on laminin-1 for 1 h and either not stimulated or stimulated with EGF (1 ng/ml) for 15 min in the presence of 10 μg of either 2B7 Ab or control IgG (-). The antibodies were added 30 min before EGF stimulation. The lamellar area was analyzed using an image analysis system as described in Materials and Methods. The data shown represent the mean lamellar area ± SE.

Mentions: Our previous data established that α6β4 participates in carcinoma migration through its ability to interact with the actin cytoskeleton 44. For this reason, we examined the involvement of this integrin in the chemotactic migration of A431 cells towards EGF because these cells express high levels of the EGF receptor and this growth factor is known to stimulate their migration 3253. As shown in Fig. 2 A, relatively low concentrations of EGF (1 ng/ml) stimulated a robust migration response in A431 cells. However, EGF did not stimulate significant migration when used at higher concentrations (>10 ng/ml) (Fig. 2 A). The effect of EGF on cell migration was mostly chemotactic in nature because the chemokinetic element represented <20% of the total migration (measured as the migration occurring in the presence of EGF in both chambers to disrupt gradients at the optimal dose of 1 ng/ml; data not shown). The fact that A431 cells express α6β4 and no α6β1 integrin (1, 17, and data not shown) enabled us to use function-blocking, α6-subunit specific antibodies to examine the contribution of α6β4 to A431 chemotaxis. Treatment of A431 cells with the 2B7 mAb inhibited chemotaxis toward EGF on laminin-1 by 60% (Fig. 2 B). This mAb did not inhibit the attachment of the cells to laminin-1 (data not shown), indicating a distinct function for α6β4 in the chemotactic migration of A431 cells. Lamellipodial protrusions are thought to be critical in cell migration and are the basis for generating lamellae, which are larger protrusions that are associated with the direction the cell migrates 3652. Indeed, A431 cells displayed a striking increase in lamellipodia and ruffle formation for sustained periods of time in response to treatment with EGF at a concentration that stimulate optimal chemotaxis (1 ng/ml) (Fig. 1 E). High concentrations of EGF (>5 ng/ml), which were inefficient in stimulating chemotaxis, caused the cells to round up quickly after a short period of protrusive activity, leaving behind numerous retraction fibers (data not shown). For this reason, we used low concentrations of EGF to stimulate A431 cells in subsequent experiments (0.5–2 ng/ml). The importance of α6β4 in the formation of such protrusions is supported by the fact that the lamellar area of A431 cells was substantially reduced by pretreatment with the 2B7 mAb before plating on laminin-1 (Fig. 2 C).


Protein kinase C-dependent mobilization of the alpha6beta4 integrin from hemidesmosomes and its association with actin-rich cell protrusions drive the chemotactic migration of carcinoma cells.

Rabinovitz I, Toker A, Mercurio AM - J. Cell Biol. (1999)

The α6β4 integrin functions in EGF-induced chemotaxis and lamellae formation of A431 cells on laminin-1. (A) The optimal concentration of EGF that stimulates chemotaxis was determined in a 2-h assay using a modified Boyden chamber coated with laminin-1 as described in Materials and Methods. Data represent the mean number of cells/microscopic field (± SE) that migrated through the separating membrane. (B) The ability of the α6-specific mAb 2B7 or IgG control to inhibit the chemotactic migration of A431 cells toward EGF (1 ng/ml) was analyzed using the method described in A. Data shown represent the percentage of cells that migrated relative to the control. (C) A431 cells were plated on laminin-1 for 1 h and either not stimulated or stimulated with EGF (1 ng/ml) for 15 min in the presence of 10 μg of either 2B7 Ab or control IgG (-). The antibodies were added 30 min before EGF stimulation. The lamellar area was analyzed using an image analysis system as described in Materials and Methods. The data shown represent the mean lamellar area ± SE.
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Related In: Results  -  Collection

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Figure 2: The α6β4 integrin functions in EGF-induced chemotaxis and lamellae formation of A431 cells on laminin-1. (A) The optimal concentration of EGF that stimulates chemotaxis was determined in a 2-h assay using a modified Boyden chamber coated with laminin-1 as described in Materials and Methods. Data represent the mean number of cells/microscopic field (± SE) that migrated through the separating membrane. (B) The ability of the α6-specific mAb 2B7 or IgG control to inhibit the chemotactic migration of A431 cells toward EGF (1 ng/ml) was analyzed using the method described in A. Data shown represent the percentage of cells that migrated relative to the control. (C) A431 cells were plated on laminin-1 for 1 h and either not stimulated or stimulated with EGF (1 ng/ml) for 15 min in the presence of 10 μg of either 2B7 Ab or control IgG (-). The antibodies were added 30 min before EGF stimulation. The lamellar area was analyzed using an image analysis system as described in Materials and Methods. The data shown represent the mean lamellar area ± SE.
Mentions: Our previous data established that α6β4 participates in carcinoma migration through its ability to interact with the actin cytoskeleton 44. For this reason, we examined the involvement of this integrin in the chemotactic migration of A431 cells towards EGF because these cells express high levels of the EGF receptor and this growth factor is known to stimulate their migration 3253. As shown in Fig. 2 A, relatively low concentrations of EGF (1 ng/ml) stimulated a robust migration response in A431 cells. However, EGF did not stimulate significant migration when used at higher concentrations (>10 ng/ml) (Fig. 2 A). The effect of EGF on cell migration was mostly chemotactic in nature because the chemokinetic element represented <20% of the total migration (measured as the migration occurring in the presence of EGF in both chambers to disrupt gradients at the optimal dose of 1 ng/ml; data not shown). The fact that A431 cells express α6β4 and no α6β1 integrin (1, 17, and data not shown) enabled us to use function-blocking, α6-subunit specific antibodies to examine the contribution of α6β4 to A431 chemotaxis. Treatment of A431 cells with the 2B7 mAb inhibited chemotaxis toward EGF on laminin-1 by 60% (Fig. 2 B). This mAb did not inhibit the attachment of the cells to laminin-1 (data not shown), indicating a distinct function for α6β4 in the chemotactic migration of A431 cells. Lamellipodial protrusions are thought to be critical in cell migration and are the basis for generating lamellae, which are larger protrusions that are associated with the direction the cell migrates 3652. Indeed, A431 cells displayed a striking increase in lamellipodia and ruffle formation for sustained periods of time in response to treatment with EGF at a concentration that stimulate optimal chemotaxis (1 ng/ml) (Fig. 1 E). High concentrations of EGF (>5 ng/ml), which were inefficient in stimulating chemotaxis, caused the cells to round up quickly after a short period of protrusive activity, leaving behind numerous retraction fibers (data not shown). For this reason, we used low concentrations of EGF to stimulate A431 cells in subsequent experiments (0.5–2 ng/ml). The importance of α6β4 in the formation of such protrusions is supported by the fact that the lamellar area of A431 cells was substantially reduced by pretreatment with the 2B7 mAb before plating on laminin-1 (Fig. 2 C).

Bottom Line: Using function-blocking antibodies, we show that the alpha6beta4 integrin participates in EGF-stimulated chemotaxis and is required for lamellae formation on laminin-1.At concentrations of EGF that stimulate A431 chemotaxis ( approximately 1 ng/ml), the alpha6beta4 integrin is mobilized from hemidesmosomes as evidenced by indirect immunofluorescence microscopy using mAbs specific for this integrin and hemidesmosomal components and its loss from a cytokeratin fraction obtained by detergent extraction.Importantly, we demonstrate that this mobilization of alpha6beta4 from hemidesmosomes and its redistribution to cell protrusions occurs by a mechanism that involves activation of protein kinase C-alpha and that it is associated with the phosphorylation of the beta4 integrin subunit on serine residues.

View Article: PubMed Central - PubMed

Affiliation: Department of Medicine, Beth Israel Deaconess Medical Center and Harvard Medical School, Boston, Massachusetts 02215, USA.

ABSTRACT
We explored the hypothesis that the chemotactic migration of carcinoma cells that assemble hemidesmosomes involves the activation of a signaling pathway that releases the alpha6beta4 integrin from these stable adhesion complexes and promotes its association with F-actin in cell protrusions enabling it to function in migration. Squamous carcinoma-derived A431 cells were used because they express alpha6beta4 and migrate in response to EGF stimulation. Using function-blocking antibodies, we show that the alpha6beta4 integrin participates in EGF-stimulated chemotaxis and is required for lamellae formation on laminin-1. At concentrations of EGF that stimulate A431 chemotaxis ( approximately 1 ng/ml), the alpha6beta4 integrin is mobilized from hemidesmosomes as evidenced by indirect immunofluorescence microscopy using mAbs specific for this integrin and hemidesmosomal components and its loss from a cytokeratin fraction obtained by detergent extraction. EGF stimulation also increased the formation of lamellipodia and membrane ruffles that contained alpha6beta4 in association with F-actin. Importantly, we demonstrate that this mobilization of alpha6beta4 from hemidesmosomes and its redistribution to cell protrusions occurs by a mechanism that involves activation of protein kinase C-alpha and that it is associated with the phosphorylation of the beta4 integrin subunit on serine residues. Thus, the chemotactic migration of A431 cells on laminin-1 requires not only the formation of F-actin-rich cell protrusions that mediate alpha6beta4-dependent cell movement but also the disruption of alpha6beta4-containing hemidesmosomes by protein kinase C.

Show MeSH
Related in: MedlinePlus