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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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Assay of cAMP-specific PDE activity. (A) MDA/β4  (3A7 and 5B3) or mock transfectants (6D7) plated on collagen I  were treated with 50 μM forskolin or 100 nM LPA as noted. Cells  were harvested and the cytosolic fraction was assayed for PDE  activity as described in Materials and Methods. The PDE activity  of the MDA/β4 transfectants was compared with the MDA/mock  transfectants for statistical significance: *, P < 0.002; †, P < 0.01.  (B) Extracts from cells treated as in A were incubated with  100 μM rolipram before assaying for PDE activity to determine  how much of the activity in A constitutes cAMP-specific PDE  (PDE 4). Data shown are mean ± standard error of four separate  determinations (A and B). ns, not significant; ⋄, P = 0.02. (C)  Relative expression of PDE 4B in the MDA-MB-435 transfectants. Extracts (40 μg protein) obtained from the MDA/β4 (3A7  and 5B3) and mock (6D2 and 6D7) transfectants, as well as purified PDE 4 proteins (short form of variants A, B, and D; 10 ng  each; provided by M. Conti) were resolved by SDS-PAGE and  immunoblotted with a PDE 4B-specific Ab. Arrows, long and  short forms of PDE 4B.
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Figure 6: Assay of cAMP-specific PDE activity. (A) MDA/β4 (3A7 and 5B3) or mock transfectants (6D7) plated on collagen I were treated with 50 μM forskolin or 100 nM LPA as noted. Cells were harvested and the cytosolic fraction was assayed for PDE activity as described in Materials and Methods. The PDE activity of the MDA/β4 transfectants was compared with the MDA/mock transfectants for statistical significance: *, P < 0.002; †, P < 0.01. (B) Extracts from cells treated as in A were incubated with 100 μM rolipram before assaying for PDE activity to determine how much of the activity in A constitutes cAMP-specific PDE (PDE 4). Data shown are mean ± standard error of four separate determinations (A and B). ns, not significant; ⋄, P = 0.02. (C) Relative expression of PDE 4B in the MDA-MB-435 transfectants. Extracts (40 μg protein) obtained from the MDA/β4 (3A7 and 5B3) and mock (6D2 and 6D7) transfectants, as well as purified PDE 4 proteins (short form of variants A, B, and D; 10 ng each; provided by M. Conti) were resolved by SDS-PAGE and immunoblotted with a PDE 4B-specific Ab. Arrows, long and short forms of PDE 4B.

Mentions: To establish more directly that expression of the α6β4 integrin can regulate cAMP-dependent PDE activity, the activity of this enzyme was assayed in cell extracts obtained from the MDA/mock and β4 transfectants. As shown in Fig. 6 A, the MDA/β4 transfectants exhibited a significantly higher rate of PDE activity than the mock transfected cells. Moreover, the PDE activity of the MDA/ β4 transfectants was markedly increased (51% for 5B3 and 45% for 3A7) in response to forskolin stimulation compared with the mock transfectants (29% for 6D7; Fig. 6 A). The difference in PDE activity between the MDA/β4 and mock transfectants was eliminated by rolipram, a type IV PDE-specific (PDE 4) inhibitor (Fig. 6 B). These data indicate that a cAMP-dependent PDE 4 activity is influenced by α6β4 expression in MDA-MB-435 cells. Also, this activity is likely responsible for the observed decrease in [cAMP]i and the resistance to forskolin-mediated inhibition of LPA chemotaxis observed in the MDA/β4 transfectants.


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)

Assay of cAMP-specific PDE activity. (A) MDA/β4  (3A7 and 5B3) or mock transfectants (6D7) plated on collagen I  were treated with 50 μM forskolin or 100 nM LPA as noted. Cells  were harvested and the cytosolic fraction was assayed for PDE  activity as described in Materials and Methods. The PDE activity  of the MDA/β4 transfectants was compared with the MDA/mock  transfectants for statistical significance: *, P < 0.002; †, P < 0.01.  (B) Extracts from cells treated as in A were incubated with  100 μM rolipram before assaying for PDE activity to determine  how much of the activity in A constitutes cAMP-specific PDE  (PDE 4). Data shown are mean ± standard error of four separate  determinations (A and B). ns, not significant; ⋄, P = 0.02. (C)  Relative expression of PDE 4B in the MDA-MB-435 transfectants. Extracts (40 μg protein) obtained from the MDA/β4 (3A7  and 5B3) and mock (6D2 and 6D7) transfectants, as well as purified PDE 4 proteins (short form of variants A, B, and D; 10 ng  each; provided by M. Conti) were resolved by SDS-PAGE and  immunoblotted with a PDE 4B-specific Ab. Arrows, long and  short forms of PDE 4B.
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Figure 6: Assay of cAMP-specific PDE activity. (A) MDA/β4 (3A7 and 5B3) or mock transfectants (6D7) plated on collagen I were treated with 50 μM forskolin or 100 nM LPA as noted. Cells were harvested and the cytosolic fraction was assayed for PDE activity as described in Materials and Methods. The PDE activity of the MDA/β4 transfectants was compared with the MDA/mock transfectants for statistical significance: *, P < 0.002; †, P < 0.01. (B) Extracts from cells treated as in A were incubated with 100 μM rolipram before assaying for PDE activity to determine how much of the activity in A constitutes cAMP-specific PDE (PDE 4). Data shown are mean ± standard error of four separate determinations (A and B). ns, not significant; ⋄, P = 0.02. (C) Relative expression of PDE 4B in the MDA-MB-435 transfectants. Extracts (40 μg protein) obtained from the MDA/β4 (3A7 and 5B3) and mock (6D2 and 6D7) transfectants, as well as purified PDE 4 proteins (short form of variants A, B, and D; 10 ng each; provided by M. Conti) were resolved by SDS-PAGE and immunoblotted with a PDE 4B-specific Ab. Arrows, long and short forms of PDE 4B.
Mentions: To establish more directly that expression of the α6β4 integrin can regulate cAMP-dependent PDE activity, the activity of this enzyme was assayed in cell extracts obtained from the MDA/mock and β4 transfectants. As shown in Fig. 6 A, the MDA/β4 transfectants exhibited a significantly higher rate of PDE activity than the mock transfected cells. Moreover, the PDE activity of the MDA/ β4 transfectants was markedly increased (51% for 5B3 and 45% for 3A7) in response to forskolin stimulation compared with the mock transfectants (29% for 6D7; Fig. 6 A). The difference in PDE activity between the MDA/β4 and mock transfectants was eliminated by rolipram, a type IV PDE-specific (PDE 4) inhibitor (Fig. 6 B). These data indicate that a cAMP-dependent PDE 4 activity is influenced by α6β4 expression in MDA-MB-435 cells. Also, this activity is likely responsible for the observed decrease in [cAMP]i and the resistance to forskolin-mediated inhibition of LPA chemotaxis observed in the MDA/β4 transfectants.

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