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Gq-coupled purinergic receptors inhibit insulin-like growth factor-I/phosphoinositide 3-kinase pathway-dependent keratinocyte migration.

Taboubi S, Garrouste F, Parat F, Pommier G, Faure E, Monferran S, Kovacic H, Lehmann M - Mol. Biol. Cell (2010)

Bottom Line: Purinergic signaling does not affect the formation of the IGF-I receptor/insulin receptor substrate-I/p85 complex, but blocks the activity of a membrane-targeted active p110alpha mutant, indicating that UTP acts downstream of PI3K membrane recruitment.UTP was also found to efficiently attenuate, within few minutes, the IGF-I-induced PI3K-controlled translocation of the actin-nucleating protein cortactin to the plasma membrane.These findings provide new insight into the signaling cross-talk between receptor tyrosine kinase and Galpha((q/11))-coupled receptors, which mediate opposite effects on p110alpha-PI3K activity and keratinocyte migration.

View Article: PubMed Central - PubMed

Affiliation: INSERM UMR 911, Centre de Recherche en Oncologie Biologique et en Oncopharmacologie, Université Aix-Marseille, Marseille 13005, France.

ABSTRACT
Insulin-like growth factor-I (IGF-I) activation of phosphoinositol 3-kinase (PI3K) is an essential pathway for keratinocyte migration that is required for epidermis wound healing. We have previously reported that activation of Galpha((q/11))-coupled-P2Y(2) purinergic receptors by extracellular nucleotides delays keratinocyte wound closure. Here, we report that activation of P2Y(2) receptors by extracellular UTP inhibits the IGF-I-induced p110alpha-PI3K activation. Using siRNA and pharmacological inhibitors, we demonstrate that the UTP antagonistic effects on PI3K pathway are mediated by Galpha((q/11))-and not G((i/o))-independently of phospholipase Cbeta. Purinergic signaling does not affect the formation of the IGF-I receptor/insulin receptor substrate-I/p85 complex, but blocks the activity of a membrane-targeted active p110alpha mutant, indicating that UTP acts downstream of PI3K membrane recruitment. UTP was also found to efficiently attenuate, within few minutes, the IGF-I-induced PI3K-controlled translocation of the actin-nucleating protein cortactin to the plasma membrane. This supports the UTP ability to alter later migratory events. Indeed, UTP inhibits keratinocyte spreading and migration promoted by either IGF-I or a membrane-targeted active p110alpha mutant, in a Galpha(q/11)-dependent manner both. These findings provide new insight into the signaling cross-talk between receptor tyrosine kinase and Galpha((q/11))-coupled receptors, which mediate opposite effects on p110alpha-PI3K activity and keratinocyte migration.

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Gα(q/11) activation inhibits p110α-PI3K–dependent keratinocyte motility. (A) HaCaT keratinocytes were pretreated with or without LY294002 (LY; 10 μM) and then with IGF-I (50 ng/ml) and assayed for 2D random migration. (B) Random motility assays were performed with Myr-p110α*-mER HaCaT clone (□) and vector-transfected clone (■). PI3K activation was induced by 4-OHT treatment (4-OHT); solvent-treated cells were used as control (Ctrl). Cells were treated with UTP (100 μM) as indicated. (C) HaCaT cells were pretreated with YM-254890 (YM; 3 μM, 5 min) and then stimulated with IGF-I (50 ng/ml; IGF) with or without UTP (100 μM; UTP) as indicated, and motility assays were performed as described in A. In each experimental condition, trajectories of 40 cells were analyzed, and cell velocity was calculated as described in Materials and Methods. Data are expressed as the mean ± SD and are representative of three independent experiments. Data were statistically analyzed using the Student's t test. **p < 0.001.
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Figure 7: Gα(q/11) activation inhibits p110α-PI3K–dependent keratinocyte motility. (A) HaCaT keratinocytes were pretreated with or without LY294002 (LY; 10 μM) and then with IGF-I (50 ng/ml) and assayed for 2D random migration. (B) Random motility assays were performed with Myr-p110α*-mER HaCaT clone (□) and vector-transfected clone (■). PI3K activation was induced by 4-OHT treatment (4-OHT); solvent-treated cells were used as control (Ctrl). Cells were treated with UTP (100 μM) as indicated. (C) HaCaT cells were pretreated with YM-254890 (YM; 3 μM, 5 min) and then stimulated with IGF-I (50 ng/ml; IGF) with or without UTP (100 μM; UTP) as indicated, and motility assays were performed as described in A. In each experimental condition, trajectories of 40 cells were analyzed, and cell velocity was calculated as described in Materials and Methods. Data are expressed as the mean ± SD and are representative of three independent experiments. Data were statistically analyzed using the Student's t test. **p < 0.001.

Mentions: As shown on Figure 7A, PI3K inhibition by LY294002 abrogated the IGF-I–induced increase of cell velocity. To confirm the role of p110α-PI3K, we conducted a second series of motility assays with vector-transfected or Myr-p110α*-mER HaCat clones. We first verified that these cells behave like untransfected HaCat cells upon stimulation by IGF-I and UTP (data not shown). As already reported, activation of Myr-p110α*-mER by 4-OHT promotes HaCat migration in wound healing assays (Pankow et al., 2006). Figure 7B shows that, in random motility assays, activation of the p110α-PI3K mutant by 4-OHT–induced an increase in cell velocity (1.2 ± 0.09 μm/min, □) compared with empty vector–transfected cells (0.47 ± 0.02 μm/min, ■). As reported above for IGF-I (see Figure 6), no increase in cell migration persistence was noted (not shown). Importantly, UTP was able to inhibit cell velocity of cells expressing activated Myr-p110α*-mER (Figure 7B). Using YM-254890 (3 μM), we showed that Gα(q/11) also plays a crucial role in the signaling pathway driven by extracellular UTP to slow down keratinocyte migration (Figure 7C). Taken together, these results clearly show that activation of Gα(q/11)by extracellular nucleotides inhibited p110α-dependent keratinocyte velocity.


Gq-coupled purinergic receptors inhibit insulin-like growth factor-I/phosphoinositide 3-kinase pathway-dependent keratinocyte migration.

Taboubi S, Garrouste F, Parat F, Pommier G, Faure E, Monferran S, Kovacic H, Lehmann M - Mol. Biol. Cell (2010)

Gα(q/11) activation inhibits p110α-PI3K–dependent keratinocyte motility. (A) HaCaT keratinocytes were pretreated with or without LY294002 (LY; 10 μM) and then with IGF-I (50 ng/ml) and assayed for 2D random migration. (B) Random motility assays were performed with Myr-p110α*-mER HaCaT clone (□) and vector-transfected clone (■). PI3K activation was induced by 4-OHT treatment (4-OHT); solvent-treated cells were used as control (Ctrl). Cells were treated with UTP (100 μM) as indicated. (C) HaCaT cells were pretreated with YM-254890 (YM; 3 μM, 5 min) and then stimulated with IGF-I (50 ng/ml; IGF) with or without UTP (100 μM; UTP) as indicated, and motility assays were performed as described in A. In each experimental condition, trajectories of 40 cells were analyzed, and cell velocity was calculated as described in Materials and Methods. Data are expressed as the mean ± SD and are representative of three independent experiments. Data were statistically analyzed using the Student's t test. **p < 0.001.
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Figure 7: Gα(q/11) activation inhibits p110α-PI3K–dependent keratinocyte motility. (A) HaCaT keratinocytes were pretreated with or without LY294002 (LY; 10 μM) and then with IGF-I (50 ng/ml) and assayed for 2D random migration. (B) Random motility assays were performed with Myr-p110α*-mER HaCaT clone (□) and vector-transfected clone (■). PI3K activation was induced by 4-OHT treatment (4-OHT); solvent-treated cells were used as control (Ctrl). Cells were treated with UTP (100 μM) as indicated. (C) HaCaT cells were pretreated with YM-254890 (YM; 3 μM, 5 min) and then stimulated with IGF-I (50 ng/ml; IGF) with or without UTP (100 μM; UTP) as indicated, and motility assays were performed as described in A. In each experimental condition, trajectories of 40 cells were analyzed, and cell velocity was calculated as described in Materials and Methods. Data are expressed as the mean ± SD and are representative of three independent experiments. Data were statistically analyzed using the Student's t test. **p < 0.001.
Mentions: As shown on Figure 7A, PI3K inhibition by LY294002 abrogated the IGF-I–induced increase of cell velocity. To confirm the role of p110α-PI3K, we conducted a second series of motility assays with vector-transfected or Myr-p110α*-mER HaCat clones. We first verified that these cells behave like untransfected HaCat cells upon stimulation by IGF-I and UTP (data not shown). As already reported, activation of Myr-p110α*-mER by 4-OHT promotes HaCat migration in wound healing assays (Pankow et al., 2006). Figure 7B shows that, in random motility assays, activation of the p110α-PI3K mutant by 4-OHT–induced an increase in cell velocity (1.2 ± 0.09 μm/min, □) compared with empty vector–transfected cells (0.47 ± 0.02 μm/min, ■). As reported above for IGF-I (see Figure 6), no increase in cell migration persistence was noted (not shown). Importantly, UTP was able to inhibit cell velocity of cells expressing activated Myr-p110α*-mER (Figure 7B). Using YM-254890 (3 μM), we showed that Gα(q/11) also plays a crucial role in the signaling pathway driven by extracellular UTP to slow down keratinocyte migration (Figure 7C). Taken together, these results clearly show that activation of Gα(q/11)by extracellular nucleotides inhibited p110α-dependent keratinocyte velocity.

Bottom Line: Purinergic signaling does not affect the formation of the IGF-I receptor/insulin receptor substrate-I/p85 complex, but blocks the activity of a membrane-targeted active p110alpha mutant, indicating that UTP acts downstream of PI3K membrane recruitment.UTP was also found to efficiently attenuate, within few minutes, the IGF-I-induced PI3K-controlled translocation of the actin-nucleating protein cortactin to the plasma membrane.These findings provide new insight into the signaling cross-talk between receptor tyrosine kinase and Galpha((q/11))-coupled receptors, which mediate opposite effects on p110alpha-PI3K activity and keratinocyte migration.

View Article: PubMed Central - PubMed

Affiliation: INSERM UMR 911, Centre de Recherche en Oncologie Biologique et en Oncopharmacologie, Université Aix-Marseille, Marseille 13005, France.

ABSTRACT
Insulin-like growth factor-I (IGF-I) activation of phosphoinositol 3-kinase (PI3K) is an essential pathway for keratinocyte migration that is required for epidermis wound healing. We have previously reported that activation of Galpha((q/11))-coupled-P2Y(2) purinergic receptors by extracellular nucleotides delays keratinocyte wound closure. Here, we report that activation of P2Y(2) receptors by extracellular UTP inhibits the IGF-I-induced p110alpha-PI3K activation. Using siRNA and pharmacological inhibitors, we demonstrate that the UTP antagonistic effects on PI3K pathway are mediated by Galpha((q/11))-and not G((i/o))-independently of phospholipase Cbeta. Purinergic signaling does not affect the formation of the IGF-I receptor/insulin receptor substrate-I/p85 complex, but blocks the activity of a membrane-targeted active p110alpha mutant, indicating that UTP acts downstream of PI3K membrane recruitment. UTP was also found to efficiently attenuate, within few minutes, the IGF-I-induced PI3K-controlled translocation of the actin-nucleating protein cortactin to the plasma membrane. This supports the UTP ability to alter later migratory events. Indeed, UTP inhibits keratinocyte spreading and migration promoted by either IGF-I or a membrane-targeted active p110alpha mutant, in a Galpha(q/11)-dependent manner both. These findings provide new insight into the signaling cross-talk between receptor tyrosine kinase and Galpha((q/11))-coupled receptors, which mediate opposite effects on p110alpha-PI3K activity and keratinocyte migration.

Show MeSH
Related in: MedlinePlus