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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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Forskolin stimulation of adenyl cyclase inhibits LPA-mediated chemotaxis differentially in the MDA/β4 and mock  transfectants. (A) MDA/β4 transfectants (5B3; solid circles) or  mock transfectants (6D7; open squares) were treated with the indicated concentration of forskolin for 30 min before their addition to the upper wells of the Transwell chambers. Cells were assayed for LPA-mediated chemotaxis on collagen I as described in  Fig. 1. The dashed line depicts the basal level of migration of both  subclones in the absence of LPA. (B) In a separate experiment,  the same cells were treated with forskolin for 30 min before assaying for LPA chemotaxis (solid symbols) or laminin haptotaxis  (open symbols). Data are reported as the percent migration of  cells not treated with forskolin ± standard deviation of triplicate  determinations.
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Figure 4: Forskolin stimulation of adenyl cyclase inhibits LPA-mediated chemotaxis differentially in the MDA/β4 and mock transfectants. (A) MDA/β4 transfectants (5B3; solid circles) or mock transfectants (6D7; open squares) were treated with the indicated concentration of forskolin for 30 min before their addition to the upper wells of the Transwell chambers. Cells were assayed for LPA-mediated chemotaxis on collagen I as described in Fig. 1. The dashed line depicts the basal level of migration of both subclones in the absence of LPA. (B) In a separate experiment, the same cells were treated with forskolin for 30 min before assaying for LPA chemotaxis (solid symbols) or laminin haptotaxis (open symbols). Data are reported as the percent migration of cells not treated with forskolin ± standard deviation of triplicate determinations.

Mentions: LPA is a bioactive phospholipid that can mediate its effects on cells through a receptor linked to heterotrimeric G proteins, including inhibitory type G (Gi) proteins (29). To assess the possible involvement of a Gi protein in α6β4-enhanced chemotaxis, we used pertussis toxin, which inactivates heterotrimeric Gi-proteins by ADP ribosylation (31). The LPA-stimulated chemotaxis of both the MDA/β4 and mock transfectants was inhibited by pertussis toxin with maximal inhibition observed at 100 ng/ml (data not shown). These data suggested that the α6β4 integrin enhances chemotaxis that is mediated through pertussis toxin-sensitive, Gi-linked receptors. Gi proteins are known to inhibit certain classes of adenyl cyclases and thus limit cAMP production (45). For this reason, we analyzed the impact of stimulating cAMP production on chemotaxis using forskolin. Although forskolin inhibited LPA-stimulated chemotaxis, the MDA/β4 and mock transfectants differed significantly in their response to this activator of adenyl cyclases. LPA-stimulated chemotaxis of the mock transfectants was inhibited to basal levels by 50 μM forskolin (Fig. 4 A). At this concentration of forskolin, the inhibition of chemotaxis of the MDA/β4 transfectants was only 50% and higher concentrations of forskolin (100 μM) did not abrogate chemotaxis of these cells (Fig. 4 A). Interestingly, treatment of the MDA/β4 or mock transfectants with forskolin did not inhibit haptotactic migration on laminin-1 (Fig. 4 B). These data indicate that a cAMP-sensitive pathway plays a key role in LPA-stimulated chemotaxis of MDA-MB-435 cells and they suggest that the α6β4 integrin may regulate this pathway.


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)

Forskolin stimulation of adenyl cyclase inhibits LPA-mediated chemotaxis differentially in the MDA/β4 and mock  transfectants. (A) MDA/β4 transfectants (5B3; solid circles) or  mock transfectants (6D7; open squares) were treated with the indicated concentration of forskolin for 30 min before their addition to the upper wells of the Transwell chambers. Cells were assayed for LPA-mediated chemotaxis on collagen I as described in  Fig. 1. The dashed line depicts the basal level of migration of both  subclones in the absence of LPA. (B) In a separate experiment,  the same cells were treated with forskolin for 30 min before assaying for LPA chemotaxis (solid symbols) or laminin haptotaxis  (open symbols). Data are reported as the percent migration of  cells not treated with forskolin ± standard deviation of triplicate  determinations.
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Related In: Results  -  Collection

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

Figure 4: Forskolin stimulation of adenyl cyclase inhibits LPA-mediated chemotaxis differentially in the MDA/β4 and mock transfectants. (A) MDA/β4 transfectants (5B3; solid circles) or mock transfectants (6D7; open squares) were treated with the indicated concentration of forskolin for 30 min before their addition to the upper wells of the Transwell chambers. Cells were assayed for LPA-mediated chemotaxis on collagen I as described in Fig. 1. The dashed line depicts the basal level of migration of both subclones in the absence of LPA. (B) In a separate experiment, the same cells were treated with forskolin for 30 min before assaying for LPA chemotaxis (solid symbols) or laminin haptotaxis (open symbols). Data are reported as the percent migration of cells not treated with forskolin ± standard deviation of triplicate determinations.
Mentions: LPA is a bioactive phospholipid that can mediate its effects on cells through a receptor linked to heterotrimeric G proteins, including inhibitory type G (Gi) proteins (29). To assess the possible involvement of a Gi protein in α6β4-enhanced chemotaxis, we used pertussis toxin, which inactivates heterotrimeric Gi-proteins by ADP ribosylation (31). The LPA-stimulated chemotaxis of both the MDA/β4 and mock transfectants was inhibited by pertussis toxin with maximal inhibition observed at 100 ng/ml (data not shown). These data suggested that the α6β4 integrin enhances chemotaxis that is mediated through pertussis toxin-sensitive, Gi-linked receptors. Gi proteins are known to inhibit certain classes of adenyl cyclases and thus limit cAMP production (45). For this reason, we analyzed the impact of stimulating cAMP production on chemotaxis using forskolin. Although forskolin inhibited LPA-stimulated chemotaxis, the MDA/β4 and mock transfectants differed significantly in their response to this activator of adenyl cyclases. LPA-stimulated chemotaxis of the mock transfectants was inhibited to basal levels by 50 μM forskolin (Fig. 4 A). At this concentration of forskolin, the inhibition of chemotaxis of the MDA/β4 transfectants was only 50% and higher concentrations of forskolin (100 μM) did not abrogate chemotaxis of these cells (Fig. 4 A). Interestingly, treatment of the MDA/β4 or mock transfectants with forskolin did not inhibit haptotactic migration on laminin-1 (Fig. 4 B). These data indicate that a cAMP-sensitive pathway plays a key role in LPA-stimulated chemotaxis of MDA-MB-435 cells and they suggest that the α6β4 integrin may regulate this pathway.

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