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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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The α6β4 integrin  is required for the LPA-dependent formation of  lamellae in MDA-MB-435  cells. MDA/β4 (A and B) and  mock transfectants (C) were  plated onto coverslips that  had been coated with 20 μg/ ml collagen I. Cells were allowed to adhere for 2 h at  37°C and then treated with  LPA for 5 min. (B and C) or  left untreated (A). The cells  were visualized using Nomarski DIC optics. Note the large  lamellae that are formed in  response to LPA stimulation  of the MDA/β4 transfectants.  (D) The effect of LPA on  lamellar area was quantified  using IPLab Spectrum imaging software. Data are shown  as mean lamellar area ± standard error in which n > 20.  Bar, 10 μm.
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Figure 3: The α6β4 integrin is required for the LPA-dependent formation of lamellae in MDA-MB-435 cells. MDA/β4 (A and B) and mock transfectants (C) were plated onto coverslips that had been coated with 20 μg/ ml collagen I. Cells were allowed to adhere for 2 h at 37°C and then treated with LPA for 5 min. (B and C) or left untreated (A). The cells were visualized using Nomarski DIC optics. Note the large lamellae that are formed in response to LPA stimulation of the MDA/β4 transfectants. (D) The effect of LPA on lamellar area was quantified using IPLab Spectrum imaging software. Data are shown as mean lamellar area ± standard error in which n > 20. Bar, 10 μm.

Mentions: Chemotactic migration frequently involves the formation of broad sheets of polymerized actin at the leading edge of the cell termed lamellae (27). To determine if expression of the α6β4 integrin influenced the formation of such motile structures, we analyzed the morphology of the MDA-MB-435 transfectants plated on collagen I (Fig. 3). Prominent lamellae were not evident in the mock transfectants and treatment with 100 nM LPA did not stimulate a significant increase in lamellar area (Fig. 3, C and D). The MDA/β4 transfectants exhibited a similar morphology to that of the mock transfectants when plated on collagen I (Fig. 3, compare A with C) or laminin-1 (data not shown). Within minutes after LPA treatment, however, the MDA/ β4 transfectants formed large, ruffling lamellae (Fig. 3 C). Quantification of these cells by digital image analysis indicated that LPA stimulated a dramatic increase in the lamellar area of the two subclones of the MDA/β4 transfectants (Fig. 3 D). In contrast, no increase in the lamellar area of the mock transfectants in response to LPA was detected by this analysis (Fig. 3 D).


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)

The α6β4 integrin  is required for the LPA-dependent formation of  lamellae in MDA-MB-435  cells. MDA/β4 (A and B) and  mock transfectants (C) were  plated onto coverslips that  had been coated with 20 μg/ ml collagen I. Cells were allowed to adhere for 2 h at  37°C and then treated with  LPA for 5 min. (B and C) or  left untreated (A). The cells  were visualized using Nomarski DIC optics. Note the large  lamellae that are formed in  response to LPA stimulation  of the MDA/β4 transfectants.  (D) The effect of LPA on  lamellar area was quantified  using IPLab Spectrum imaging software. Data are shown  as mean lamellar area ± standard error in which n > 20.  Bar, 10 μm.
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Related In: Results  -  Collection

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Figure 3: The α6β4 integrin is required for the LPA-dependent formation of lamellae in MDA-MB-435 cells. MDA/β4 (A and B) and mock transfectants (C) were plated onto coverslips that had been coated with 20 μg/ ml collagen I. Cells were allowed to adhere for 2 h at 37°C and then treated with LPA for 5 min. (B and C) or left untreated (A). The cells were visualized using Nomarski DIC optics. Note the large lamellae that are formed in response to LPA stimulation of the MDA/β4 transfectants. (D) The effect of LPA on lamellar area was quantified using IPLab Spectrum imaging software. Data are shown as mean lamellar area ± standard error in which n > 20. Bar, 10 μm.
Mentions: Chemotactic migration frequently involves the formation of broad sheets of polymerized actin at the leading edge of the cell termed lamellae (27). To determine if expression of the α6β4 integrin influenced the formation of such motile structures, we analyzed the morphology of the MDA-MB-435 transfectants plated on collagen I (Fig. 3). Prominent lamellae were not evident in the mock transfectants and treatment with 100 nM LPA did not stimulate a significant increase in lamellar area (Fig. 3, C and D). The MDA/β4 transfectants exhibited a similar morphology to that of the mock transfectants when plated on collagen I (Fig. 3, compare A with C) or laminin-1 (data not shown). Within minutes after LPA treatment, however, the MDA/ β4 transfectants formed large, ruffling lamellae (Fig. 3 C). Quantification of these cells by digital image analysis indicated that LPA stimulated a dramatic increase in the lamellar area of the two subclones of the MDA/β4 transfectants (Fig. 3 D). In contrast, no increase in the lamellar area of the mock transfectants in response to LPA was detected by this analysis (Fig. 3 D).

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