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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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Expression of the α6β4 integrin in  MDA-MB-435 carcinoma cells stimulates  chemotaxis but not haptotaxis. The migration of  the MDA/β4 (5B3 and 3A7) MDA/β4-ΔCYT  (Δ3C12, Δ1E10), and MDA/mock (6D2 and  6D7) transfectants toward laminin-1 (haptotaxis;  A), 3T3 conditioned medium (chemotaxis; B), or  LPA (chemotaxis; C and D) was assessed using a  modified Boyden chamber. The lower surfaces of  Transwell membranes were coated with either  laminin-1 (A), conditioned medium (B), or collagen I (C and D), and then either BSA (A) 3T3  conditioned medium (B) or LPA (C and D) was  added to the lower chambers. Cells (105 [A and  B] or 5 × 104 [C and D]) were placed in the upper chambers. After 4 h at 37°C, cells that did not  migrate were removed from the upper chamber  with a cotton swab and cells on the opposite side  of the membrane were fixed, stained, and quantified manually as described in the Materials and  Methods. (A) Haptotaxis toward laminin-1; (B)  chemotaxis toward NIH-3T3 conditioned medium; (C) dose response of MDA-MB-435 subclones 5B3 (β4 transfected; solid circles) and 6D7  (mock transfected; open squares) chemotaxis toward LPA; (D) Chemotaxis toward 100 nM  LPA. Data are reported as fold increases over  haptotactic migration on collagen I in the absence of LPA. Data (all panels) are shown as  mean ± standard deviation from triplicate determinations.
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Figure 1: Expression of the α6β4 integrin in MDA-MB-435 carcinoma cells stimulates chemotaxis but not haptotaxis. The migration of the MDA/β4 (5B3 and 3A7) MDA/β4-ΔCYT (Δ3C12, Δ1E10), and MDA/mock (6D2 and 6D7) transfectants toward laminin-1 (haptotaxis; A), 3T3 conditioned medium (chemotaxis; B), or LPA (chemotaxis; C and D) was assessed using a modified Boyden chamber. The lower surfaces of Transwell membranes were coated with either laminin-1 (A), conditioned medium (B), or collagen I (C and D), and then either BSA (A) 3T3 conditioned medium (B) or LPA (C and D) was added to the lower chambers. Cells (105 [A and B] or 5 × 104 [C and D]) were placed in the upper chambers. After 4 h at 37°C, cells that did not migrate were removed from the upper chamber with a cotton swab and cells on the opposite side of the membrane were fixed, stained, and quantified manually as described in the Materials and Methods. (A) Haptotaxis toward laminin-1; (B) chemotaxis toward NIH-3T3 conditioned medium; (C) dose response of MDA-MB-435 subclones 5B3 (β4 transfected; solid circles) and 6D7 (mock transfected; open squares) chemotaxis toward LPA; (D) Chemotaxis toward 100 nM LPA. Data are reported as fold increases over haptotactic migration on collagen I in the absence of LPA. Data (all panels) are shown as mean ± standard deviation from triplicate determinations.

Mentions: The possibility that expression of the α6β4 integrin influenced the rate of either haptotactic or chemotactic migration was assessed. For this purpose, stable transfectants of MDA-MB-435 cells were used that expressed either the α6β4 integrin (MDA/β4) or a deletion mutant of α6β4 (MDA/β4-ΔCYT) that retains only four amino acids of the β4 cytoplasmic domain, immediately proximal to the transmembrane domain (40). As shown in Fig. 1 A, subclones of the MDA/β4 transfectants (5B3 and 3A7) exhibited a rate of haptotactic migration toward laminin-1 that was slightly lower than the rate observed for subclones of the mock transfectants (6D7 and 6D2). In marked contrast, expression of α6β4 induced a substantial increase in the rate of chemotaxis of these cells towards conditioned medium from NIH-3T3 cells (Fig. 1 B). The rate of chemotaxis of the MDA/β4 transfectants (5B3 and 3A7) was 15– 20-fold greater than that of the mock transfectants (6D7 and 6D2) over a 4-h time period. These data indicate that expression of α6β4 potentiates chemotactic migration of MDA-MB-435 cells without substantially altering their rate of haptotaxis.


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)

Expression of the α6β4 integrin in  MDA-MB-435 carcinoma cells stimulates  chemotaxis but not haptotaxis. The migration of  the MDA/β4 (5B3 and 3A7) MDA/β4-ΔCYT  (Δ3C12, Δ1E10), and MDA/mock (6D2 and  6D7) transfectants toward laminin-1 (haptotaxis;  A), 3T3 conditioned medium (chemotaxis; B), or  LPA (chemotaxis; C and D) was assessed using a  modified Boyden chamber. The lower surfaces of  Transwell membranes were coated with either  laminin-1 (A), conditioned medium (B), or collagen I (C and D), and then either BSA (A) 3T3  conditioned medium (B) or LPA (C and D) was  added to the lower chambers. Cells (105 [A and  B] or 5 × 104 [C and D]) were placed in the upper chambers. After 4 h at 37°C, cells that did not  migrate were removed from the upper chamber  with a cotton swab and cells on the opposite side  of the membrane were fixed, stained, and quantified manually as described in the Materials and  Methods. (A) Haptotaxis toward laminin-1; (B)  chemotaxis toward NIH-3T3 conditioned medium; (C) dose response of MDA-MB-435 subclones 5B3 (β4 transfected; solid circles) and 6D7  (mock transfected; open squares) chemotaxis toward LPA; (D) Chemotaxis toward 100 nM  LPA. Data are reported as fold increases over  haptotactic migration on collagen I in the absence of LPA. Data (all panels) are shown as  mean ± standard deviation from triplicate determinations.
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Related In: Results  -  Collection

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Figure 1: Expression of the α6β4 integrin in MDA-MB-435 carcinoma cells stimulates chemotaxis but not haptotaxis. The migration of the MDA/β4 (5B3 and 3A7) MDA/β4-ΔCYT (Δ3C12, Δ1E10), and MDA/mock (6D2 and 6D7) transfectants toward laminin-1 (haptotaxis; A), 3T3 conditioned medium (chemotaxis; B), or LPA (chemotaxis; C and D) was assessed using a modified Boyden chamber. The lower surfaces of Transwell membranes were coated with either laminin-1 (A), conditioned medium (B), or collagen I (C and D), and then either BSA (A) 3T3 conditioned medium (B) or LPA (C and D) was added to the lower chambers. Cells (105 [A and B] or 5 × 104 [C and D]) were placed in the upper chambers. After 4 h at 37°C, cells that did not migrate were removed from the upper chamber with a cotton swab and cells on the opposite side of the membrane were fixed, stained, and quantified manually as described in the Materials and Methods. (A) Haptotaxis toward laminin-1; (B) chemotaxis toward NIH-3T3 conditioned medium; (C) dose response of MDA-MB-435 subclones 5B3 (β4 transfected; solid circles) and 6D7 (mock transfected; open squares) chemotaxis toward LPA; (D) Chemotaxis toward 100 nM LPA. Data are reported as fold increases over haptotactic migration on collagen I in the absence of LPA. Data (all panels) are shown as mean ± standard deviation from triplicate determinations.
Mentions: The possibility that expression of the α6β4 integrin influenced the rate of either haptotactic or chemotactic migration was assessed. For this purpose, stable transfectants of MDA-MB-435 cells were used that expressed either the α6β4 integrin (MDA/β4) or a deletion mutant of α6β4 (MDA/β4-ΔCYT) that retains only four amino acids of the β4 cytoplasmic domain, immediately proximal to the transmembrane domain (40). As shown in Fig. 1 A, subclones of the MDA/β4 transfectants (5B3 and 3A7) exhibited a rate of haptotactic migration toward laminin-1 that was slightly lower than the rate observed for subclones of the mock transfectants (6D7 and 6D2). In marked contrast, expression of α6β4 induced a substantial increase in the rate of chemotaxis of these cells towards conditioned medium from NIH-3T3 cells (Fig. 1 B). The rate of chemotaxis of the MDA/β4 transfectants (5B3 and 3A7) was 15– 20-fold greater than that of the mock transfectants (6D7 and 6D2) over a 4-h time period. These data indicate that expression of α6β4 potentiates chemotactic migration of MDA-MB-435 cells without substantially altering their rate of haptotaxis.

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