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Release of cAMP gating by the alpha6beta4 integrin stimulates lamellae formation and the chemotactic migration of invasive carcinoma cells.

O'Connor KL, Shaw LM, Mercurio AM - J. Cell Biol. (1998)

Bottom Line: Mercurio.Both lamellae formation and chemotactic migration are inhibited or "gated" by cAMP and our results reveal that a critical function of alpha6beta4 is to suppress the intracellular cAMP concentration by increasing the activity of a rolipram-sensitive, cAMP-specific phosphodiesterase (PDE).Although PI3-K and cAMP-specific PDE activities are both required to promote lamellae formation and chemotactic migration, our data indicate that they are components of distinct signaling pathways.

View Article: PubMed Central - PubMed

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

ABSTRACT
The alpha6beta4 integrin promotes carcinoma in-vasion by its activation of a phosphoinositide 3-OH (PI3-K) signaling pathway (Shaw, L.M., I. Rabinovitz, H.H.-F. Wang, A. Toker, and A.M. Mercurio. Cell. 91: 949-960). We demonstrate here using MDA-MB-435 breast carcinoma cells that alpha6beta4 stimulates chemotactic migration, a key component of invasion, but that it has no influence on haptotaxis. Stimulation of chemotaxis by alpha6beta4 expression was observed in response to either lysophosphatidic acid (LPA) or fibroblast conditioned medium. Moreover, the LPA-dependent formation of lamellae in these cells is dependent upon alpha6beta4 expression. Both lamellae formation and chemotactic migration are inhibited or "gated" by cAMP and our results reveal that a critical function of alpha6beta4 is to suppress the intracellular cAMP concentration by increasing the activity of a rolipram-sensitive, cAMP-specific phosphodiesterase (PDE). This PDE activity is essential for lamellae formation, chemotactic migration and invasion based on data obtained with PDE inhibitors. Although PI3-K and cAMP-specific PDE activities are both required to promote lamellae formation and chemotactic migration, our data indicate that they are components of distinct signaling pathways. The essence of our findings is that alpha6beta4 stimulates the chemotactic migration of carcinoma cells through its ability to influence key signaling events that underlie this critical component of carcinoma invasion.

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Inhibition of α6β4-stimulated migration by integrin-specific antibodies. MDA/β4 (5B3; A, gray bars) or mock transfectants (6D7; B, stippled bars) were incubated with the indicated  function blocking mAbs for 30 min before their use in a chemotaxis assay using 100 nM LPA on collagen I (A) or a haptotaxis  assay on laminin-1–coated wells (B) as described in Fig. 1. Nonspecific mouse IgG was used as a negative control. Data are reported as the percentage of migration observed for the IgG control ± standard deviation from triplicate determinations.
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Figure 2: Inhibition of α6β4-stimulated migration by integrin-specific antibodies. MDA/β4 (5B3; A, gray bars) or mock transfectants (6D7; B, stippled bars) were incubated with the indicated function blocking mAbs for 30 min before their use in a chemotaxis assay using 100 nM LPA on collagen I (A) or a haptotaxis assay on laminin-1–coated wells (B) as described in Fig. 1. Nonspecific mouse IgG was used as a negative control. Data are reported as the percentage of migration observed for the IgG control ± standard deviation from triplicate determinations.

Mentions: The increased chemotaxis observed for the MDA/β4 transfectants in response to LPA was evident on both collagen I (Fig. 1, C and D) and laminin-1 (data not shown), indicating that α6β4-enhanced migration is independent of the matrix protein used for traction. This possibility was examined further by preincubating the MDA/β4 transfectants with function-blocking mAbs before their use in the chemotaxis assays. As shown in Fig. 2 A, inhibition of α6 integrin function with the mAb 2B7 did not block the chemotaxis of the MDA/β4 transfectants on collagen I towards LPA. However, this mAb inhibited the haptotaxis of MDA-MB-435 cells toward a laminin-1 gradient (Fig. 2 B), a process that is dependent on the α6β1 integrin (38). Chemotaxis toward LPA was inhibited completely, however, by preincubating the cells with the β1 integrin-specific mAb 13 (Fig. 2 A). Collectively, these data indicate that the stimulation of chemotaxis by expression of α6β4 can be independent of the adhesive functions of α6β4, and that the adhesive interactions required for α6β4-enhanced chemotaxis on collagen I are mediated through β1 integrins.


Release of cAMP gating by the alpha6beta4 integrin stimulates lamellae formation and the chemotactic migration of invasive carcinoma cells.

O'Connor KL, Shaw LM, Mercurio AM - J. Cell Biol. (1998)

Inhibition of α6β4-stimulated migration by integrin-specific antibodies. MDA/β4 (5B3; A, gray bars) or mock transfectants (6D7; B, stippled bars) were incubated with the indicated  function blocking mAbs for 30 min before their use in a chemotaxis assay using 100 nM LPA on collagen I (A) or a haptotaxis  assay on laminin-1–coated wells (B) as described in Fig. 1. Nonspecific mouse IgG was used as a negative control. Data are reported as the percentage of migration observed for the IgG control ± standard deviation from triplicate determinations.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2132981&req=5

Figure 2: Inhibition of α6β4-stimulated migration by integrin-specific antibodies. MDA/β4 (5B3; A, gray bars) or mock transfectants (6D7; B, stippled bars) were incubated with the indicated function blocking mAbs for 30 min before their use in a chemotaxis assay using 100 nM LPA on collagen I (A) or a haptotaxis assay on laminin-1–coated wells (B) as described in Fig. 1. Nonspecific mouse IgG was used as a negative control. Data are reported as the percentage of migration observed for the IgG control ± standard deviation from triplicate determinations.
Mentions: The increased chemotaxis observed for the MDA/β4 transfectants in response to LPA was evident on both collagen I (Fig. 1, C and D) and laminin-1 (data not shown), indicating that α6β4-enhanced migration is independent of the matrix protein used for traction. This possibility was examined further by preincubating the MDA/β4 transfectants with function-blocking mAbs before their use in the chemotaxis assays. As shown in Fig. 2 A, inhibition of α6 integrin function with the mAb 2B7 did not block the chemotaxis of the MDA/β4 transfectants on collagen I towards LPA. However, this mAb inhibited the haptotaxis of MDA-MB-435 cells toward a laminin-1 gradient (Fig. 2 B), a process that is dependent on the α6β1 integrin (38). Chemotaxis toward LPA was inhibited completely, however, by preincubating the cells with the β1 integrin-specific mAb 13 (Fig. 2 A). Collectively, these data indicate that the stimulation of chemotaxis by expression of α6β4 can be independent of the adhesive functions of α6β4, and that the adhesive interactions required for α6β4-enhanced chemotaxis on collagen I are mediated through β1 integrins.

Bottom Line: Mercurio.Both lamellae formation and chemotactic migration are inhibited or "gated" by cAMP and our results reveal that a critical function of alpha6beta4 is to suppress the intracellular cAMP concentration by increasing the activity of a rolipram-sensitive, cAMP-specific phosphodiesterase (PDE).Although PI3-K and cAMP-specific PDE activities are both required to promote lamellae formation and chemotactic migration, our data indicate that they are components of distinct signaling pathways.

View Article: PubMed Central - PubMed

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

ABSTRACT
The alpha6beta4 integrin promotes carcinoma in-vasion by its activation of a phosphoinositide 3-OH (PI3-K) signaling pathway (Shaw, L.M., I. Rabinovitz, H.H.-F. Wang, A. Toker, and A.M. Mercurio. Cell. 91: 949-960). We demonstrate here using MDA-MB-435 breast carcinoma cells that alpha6beta4 stimulates chemotactic migration, a key component of invasion, but that it has no influence on haptotaxis. Stimulation of chemotaxis by alpha6beta4 expression was observed in response to either lysophosphatidic acid (LPA) or fibroblast conditioned medium. Moreover, the LPA-dependent formation of lamellae in these cells is dependent upon alpha6beta4 expression. Both lamellae formation and chemotactic migration are inhibited or "gated" by cAMP and our results reveal that a critical function of alpha6beta4 is to suppress the intracellular cAMP concentration by increasing the activity of a rolipram-sensitive, cAMP-specific phosphodiesterase (PDE). This PDE activity is essential for lamellae formation, chemotactic migration and invasion based on data obtained with PDE inhibitors. Although PI3-K and cAMP-specific PDE activities are both required to promote lamellae formation and chemotactic migration, our data indicate that they are components of distinct signaling pathways. The essence of our findings is that alpha6beta4 stimulates the chemotactic migration of carcinoma cells through its ability to influence key signaling events that underlie this critical component of carcinoma invasion.

Show MeSH
Related in: MedlinePlus