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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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Intracellular cAMP content of the MDA-MB-435  transfectants. The MDA/β4 (3A7, 5B3), MDA/β4-ΔCYT  (Δ3C12, Δ1E10) and MDA/mock transfectants (6D2, 6D7) were  plated in DME containing 10% FCS. After 18 h, cells were harvested and cAMP content was measured using a cAMP EIA protocol as described in Materials and Methods. Data shown represent the mean of 10 sample determinations ± standard error. The  difference in the [cAMP]i between the MDA/β4 and the mock  transfectants is significant (P < 0.001; asterisk), but the difference  between the mock and the β4-ΔCYT transfectants is not significant (P = 0.2). (B and C) Differential effects of forskolin stimulation on the [cAMP]i in the MDA/β4 and mock transfectants. The  [cAMP]i was assayed in the 5B3 (solid bars) and 6D7 (stippled  bars) clones plated on collagen I and treated for 15 min with either 50 μM forskolin (B) or forskolin and 1 mM IBMX (C). Note  that the MDA/β4 transfectants (5B3) are more resistant to a forskolin-stimulated increase in [cAMP]i than the mock transfectants (6D7). The inhibition of PDE activity with IBMX shown in  C reveals that α6β4 expression results in an increase in PDE activity and not a decrease in cAMP synthesis. Data shown are the  mean values ± standard error obtained from multiple experiments.
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Figure 5: Intracellular cAMP content of the MDA-MB-435 transfectants. The MDA/β4 (3A7, 5B3), MDA/β4-ΔCYT (Δ3C12, Δ1E10) and MDA/mock transfectants (6D2, 6D7) were plated in DME containing 10% FCS. After 18 h, cells were harvested and cAMP content was measured using a cAMP EIA protocol as described in Materials and Methods. Data shown represent the mean of 10 sample determinations ± standard error. The difference in the [cAMP]i between the MDA/β4 and the mock transfectants is significant (P < 0.001; asterisk), but the difference between the mock and the β4-ΔCYT transfectants is not significant (P = 0.2). (B and C) Differential effects of forskolin stimulation on the [cAMP]i in the MDA/β4 and mock transfectants. The [cAMP]i was assayed in the 5B3 (solid bars) and 6D7 (stippled bars) clones plated on collagen I and treated for 15 min with either 50 μM forskolin (B) or forskolin and 1 mM IBMX (C). Note that the MDA/β4 transfectants (5B3) are more resistant to a forskolin-stimulated increase in [cAMP]i than the mock transfectants (6D7). The inhibition of PDE activity with IBMX shown in C reveals that α6β4 expression results in an increase in PDE activity and not a decrease in cAMP synthesis. Data shown are the mean values ± standard error obtained from multiple experiments.

Mentions: To determine if α6β4 expression influences the [cAMP]i, the [cAMP]i was determined in extracts obtained from subconfluent cultures of MDA/mock, β4, and β4-ΔCYT transfectants using a cAMP enzyme-linked immunoabsorption assay. As shown in Fig. 5 A, the MDA/β4 transfectants had a 30% lower [cAMP]i (2.7 pmol cAMP per 106 cells) than either the mock (3.7 pmol cAMP per 106 cells) or β4-ΔCYT transfectants (3.8 pmol cAMP per 106 cells). This difference was statistically significant (P < 0.001). Of note, neither clustering of α6β4 using the 2B7 mAb and an appropriate secondary Ab nor LPA treatment reduced cAMP levels further (data not shown).


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)

Intracellular cAMP content of the MDA-MB-435  transfectants. The MDA/β4 (3A7, 5B3), MDA/β4-ΔCYT  (Δ3C12, Δ1E10) and MDA/mock transfectants (6D2, 6D7) were  plated in DME containing 10% FCS. After 18 h, cells were harvested and cAMP content was measured using a cAMP EIA protocol as described in Materials and Methods. Data shown represent the mean of 10 sample determinations ± standard error. The  difference in the [cAMP]i between the MDA/β4 and the mock  transfectants is significant (P < 0.001; asterisk), but the difference  between the mock and the β4-ΔCYT transfectants is not significant (P = 0.2). (B and C) Differential effects of forskolin stimulation on the [cAMP]i in the MDA/β4 and mock transfectants. The  [cAMP]i was assayed in the 5B3 (solid bars) and 6D7 (stippled  bars) clones plated on collagen I and treated for 15 min with either 50 μM forskolin (B) or forskolin and 1 mM IBMX (C). Note  that the MDA/β4 transfectants (5B3) are more resistant to a forskolin-stimulated increase in [cAMP]i than the mock transfectants (6D7). The inhibition of PDE activity with IBMX shown in  C reveals that α6β4 expression results in an increase in PDE activity and not a decrease in cAMP synthesis. Data shown are the  mean values ± standard error obtained from multiple experiments.
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Figure 5: Intracellular cAMP content of the MDA-MB-435 transfectants. The MDA/β4 (3A7, 5B3), MDA/β4-ΔCYT (Δ3C12, Δ1E10) and MDA/mock transfectants (6D2, 6D7) were plated in DME containing 10% FCS. After 18 h, cells were harvested and cAMP content was measured using a cAMP EIA protocol as described in Materials and Methods. Data shown represent the mean of 10 sample determinations ± standard error. The difference in the [cAMP]i between the MDA/β4 and the mock transfectants is significant (P < 0.001; asterisk), but the difference between the mock and the β4-ΔCYT transfectants is not significant (P = 0.2). (B and C) Differential effects of forskolin stimulation on the [cAMP]i in the MDA/β4 and mock transfectants. The [cAMP]i was assayed in the 5B3 (solid bars) and 6D7 (stippled bars) clones plated on collagen I and treated for 15 min with either 50 μM forskolin (B) or forskolin and 1 mM IBMX (C). Note that the MDA/β4 transfectants (5B3) are more resistant to a forskolin-stimulated increase in [cAMP]i than the mock transfectants (6D7). The inhibition of PDE activity with IBMX shown in C reveals that α6β4 expression results in an increase in PDE activity and not a decrease in cAMP synthesis. Data shown are the mean values ± standard error obtained from multiple experiments.
Mentions: To determine if α6β4 expression influences the [cAMP]i, the [cAMP]i was determined in extracts obtained from subconfluent cultures of MDA/mock, β4, and β4-ΔCYT transfectants using a cAMP enzyme-linked immunoabsorption assay. As shown in Fig. 5 A, the MDA/β4 transfectants had a 30% lower [cAMP]i (2.7 pmol cAMP per 106 cells) than either the mock (3.7 pmol cAMP per 106 cells) or β4-ΔCYT transfectants (3.8 pmol cAMP per 106 cells). This difference was statistically significant (P < 0.001). Of note, neither clustering of α6β4 using the 2B7 mAb and an appropriate secondary Ab nor LPA treatment reduced cAMP levels further (data not shown).

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