Limits...
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.

Show MeSH

Related in: MedlinePlus

(A) Evaluation of PI3-K involvement in PDE activity.  The MDA/β4 transfectants were incubated for 30 min in the  presence of either forskolin or wortmannin, or both in combination, before assay of PDE activity as described in Materials and  Methods. (B) Evaluation of the cAMP regulation of PI3-K activity. The MDA/β4 and mock transfectants were incubated in suspension with either forskolin or IBMX or both for 10 min. Subsequently, these cells were either maintained in suspension or  incubated with a β4 integrin-specific antibody and allowed to adhere to anti-mouse IgG-coated plates or laminin-1–coated plates  for 30 min. Aliquots of cell extracts that contained equivalent  amounts of protein were incubated with the anti-phosphotyrosine  mAb 4G10 and protein A–Sepharose for 3 h. After washing, the  beads were resuspended in kinase buffer and incubated for 10  min at room temperature. The phosphorylated lipids were resolved by thin layer chromatography. Arrows, position of the D3-phosphoinositides.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC2132981&req=5

Figure 10: (A) Evaluation of PI3-K involvement in PDE activity. The MDA/β4 transfectants were incubated for 30 min in the presence of either forskolin or wortmannin, or both in combination, before assay of PDE activity as described in Materials and Methods. (B) Evaluation of the cAMP regulation of PI3-K activity. The MDA/β4 and mock transfectants were incubated in suspension with either forskolin or IBMX or both for 10 min. Subsequently, these cells were either maintained in suspension or incubated with a β4 integrin-specific antibody and allowed to adhere to anti-mouse IgG-coated plates or laminin-1–coated plates for 30 min. Aliquots of cell extracts that contained equivalent amounts of protein were incubated with the anti-phosphotyrosine mAb 4G10 and protein A–Sepharose for 3 h. After washing, the beads were resuspended in kinase buffer and incubated for 10 min at room temperature. The phosphorylated lipids were resolved by thin layer chromatography. Arrows, position of the D3-phosphoinositides.

Mentions: A possible relationship between cAMP metabolism and PI3-K signaling is of interest given our recent finding that α6β4 stimulates the preferential activation of PI3-K and that this activity is required for invasion and the formation of lamellae (40). To determine if PI3-K activity is required for the cAMP-specific PDE activity we observed in the MDA/β4 transfectants, these cells were incubated in the presence of wortmannin, a specific inhibitor of PI3-K, before extraction and assay of PDE activity. As shown in Fig. 10 A, wortmannin had no effect on PDE activity in these cells and it did not inhibit the marked induction of PDE activity that we had observed in response to forskolin stimulation. The possibility also existed that cAMP influences the α6β4-mediated activation of PI3-K. To address this issue, we used the α6-specific mAb G0H3 to cluster α6 integrins on the MDA/β4 transfectants in the presence of the PDE inhibitor IBMX and forskolin. As shown in Fig. 10 B, mAb-mediated clustering of α6β4 in MDA/β4 transfectants activated PI3-K markedly compared with cells maintained in suspension, in agreement with our previous results (40). However, treatment of MDA/β4 transfectants with either IBMX or forskolin did not inhibit α6β4-mediated activation of PI3-K (Fig. 10 B). In fact, no inhibition of PI3-K was observed when both of these inhibitors were used in combination, a treatment that increases the [cAMP]i from 4 to 120 pmoles per 106 cells (Fig. 5).


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)

(A) Evaluation of PI3-K involvement in PDE activity.  The MDA/β4 transfectants were incubated for 30 min in the  presence of either forskolin or wortmannin, or both in combination, before assay of PDE activity as described in Materials and  Methods. (B) Evaluation of the cAMP regulation of PI3-K activity. The MDA/β4 and mock transfectants were incubated in suspension with either forskolin or IBMX or both for 10 min. Subsequently, these cells were either maintained in suspension or  incubated with a β4 integrin-specific antibody and allowed to adhere to anti-mouse IgG-coated plates or laminin-1–coated plates  for 30 min. Aliquots of cell extracts that contained equivalent  amounts of protein were incubated with the anti-phosphotyrosine  mAb 4G10 and protein A–Sepharose for 3 h. After washing, the  beads were resuspended in kinase buffer and incubated for 10  min at room temperature. The phosphorylated lipids were resolved by thin layer chromatography. Arrows, position of the D3-phosphoinositides.
© Copyright Policy
Related In: Results  -  Collection

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

Figure 10: (A) Evaluation of PI3-K involvement in PDE activity. The MDA/β4 transfectants were incubated for 30 min in the presence of either forskolin or wortmannin, or both in combination, before assay of PDE activity as described in Materials and Methods. (B) Evaluation of the cAMP regulation of PI3-K activity. The MDA/β4 and mock transfectants were incubated in suspension with either forskolin or IBMX or both for 10 min. Subsequently, these cells were either maintained in suspension or incubated with a β4 integrin-specific antibody and allowed to adhere to anti-mouse IgG-coated plates or laminin-1–coated plates for 30 min. Aliquots of cell extracts that contained equivalent amounts of protein were incubated with the anti-phosphotyrosine mAb 4G10 and protein A–Sepharose for 3 h. After washing, the beads were resuspended in kinase buffer and incubated for 10 min at room temperature. The phosphorylated lipids were resolved by thin layer chromatography. Arrows, position of the D3-phosphoinositides.
Mentions: A possible relationship between cAMP metabolism and PI3-K signaling is of interest given our recent finding that α6β4 stimulates the preferential activation of PI3-K and that this activity is required for invasion and the formation of lamellae (40). To determine if PI3-K activity is required for the cAMP-specific PDE activity we observed in the MDA/β4 transfectants, these cells were incubated in the presence of wortmannin, a specific inhibitor of PI3-K, before extraction and assay of PDE activity. As shown in Fig. 10 A, wortmannin had no effect on PDE activity in these cells and it did not inhibit the marked induction of PDE activity that we had observed in response to forskolin stimulation. The possibility also existed that cAMP influences the α6β4-mediated activation of PI3-K. To address this issue, we used the α6-specific mAb G0H3 to cluster α6 integrins on the MDA/β4 transfectants in the presence of the PDE inhibitor IBMX and forskolin. As shown in Fig. 10 B, mAb-mediated clustering of α6β4 in MDA/β4 transfectants activated PI3-K markedly compared with cells maintained in suspension, in agreement with our previous results (40). However, treatment of MDA/β4 transfectants with either IBMX or forskolin did not inhibit α6β4-mediated activation of PI3-K (Fig. 10 B). In fact, no inhibition of PI3-K was observed when both of these inhibitors were used in combination, a treatment that increases the [cAMP]i from 4 to 120 pmoles per 106 cells (Fig. 5).

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