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

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.

Figure 1.

UTP inhibits the IGF-I–induced PI3K signaling pathway through P2Y2/P2Y4 receptors. (A) HaCaT keratinocytes were stimulated for the indicated times with UTP (100 μM), IGF-I (50 ng/ml), or both. (B) Cells were pretreated with increasing doses of p110α inhibitor (PIK-75) or p110β inhibitor (TGX-211) for 5 min and then stimulated with IGF-I (50 ng/ml, 5 min). (C) Cells were stimulated with IGF-I (IGF; 50 ng/ml) either alone or supplemented with UTP (100 μM; IGF+UTP) for 1, 4, and 10 min. Cells were lysed and PI3K was immunoprecipitated using an anti-p85 antibody. p110α catalytic activity, i.e., production of PIP3, was measured in the immunoprecipitated material by inverted ELISA assay. p85 immunoblot shows that equivalent amount of PI3K were analyzed. For more details, see Materials and Methods. Data are expressed as a mean ± SD from two independent experiments made in triplicates. (D) Cells were stimulated for 5 min with IGF-I (50 ng/ml) either alone (Ctrl) or in the presence of various purinergic receptor agonists at 10 μM and 100 μM as indicated; (−), untreated cells. Cell lysates were analyzed by Western blot using anti-phospho-Akt (p-Akt), anti-phospho-GSK3 (p-GSK-3), and anti-Akt (Akt) antibodies as indicated. Data shown are representative of three independent experiments.

Figure 2.

UTP inhibits IGF-I–induced PI3K signaling in a Gα_(q/11) but not a G_(i/0)-dependent manner. (A) HaCaT keratinocytes were transiently nucleofected with Gα_q siRNA or nontargeting siRNA as a control (Ctrl siRNA) as described in Materials and Methods. Forty-eight hours after transfection, cells were stimulated with IGF-I, either alone or in presence of UTP for the indicated times. (B) HaCaT keratinocytes were pretreated with YM-254890 (YM, 3 μM, 5 min) or pertussis toxin (PTX; 100 ng/ml, 18 h) to selectively inhibit Gα_(q/11) and G_(i/0), respectively. Cells were then stimulated with IGF-I (50 ng/ml), alone or supplemented with UTP (100 μM) for the indicated times. (C) Cells were treated with YM-254890 as in B, and p110α-PI3K activity was measured as described in Figure 1C. Data are representative of two independent experiments. (D) To inhibit the PLC_β, cells were pretreated with U73122 (5 μM, 30 min) and then treated as in A. Cell lysates were analyzed by Western blot using anti-phospho-Akt (p-Akt) and anti-Akt (Akt) antibodies as indicated. Data shown are representative of three independent experiments.

Figure 3.

UTP inhibits the PI3K signaling pathway downstream of p110α-PI3K membrane recruitment. (A) HaCaT keratinocytes were stimulated for 2- and 5-min with UTP (100 μM), IGF-I (50 ng/ml), or both. Immunoprecipitations were performed on cell lysates with an anti-IGF-I receptor (IGF-IR) antibody (top) or anti-IRS1 antibody (bottom). Immunoprecipitates were analyzed by Western blot using anti-phospho-tyrosine antibody (PY) and anti-p85-PI3K antibody (p85), as indicated. Anti-IRS1 (IRS1) and anti-IGF-IR (IGF-IR) antibodies were used as controls. (B) Myr-p110α*-mER–expressing HaCaT clone and vector-transfected clone were treated with either 4-hydroxytamoxifen (4-OHT) or solvent (Ctrl) and then stimulated with IGF-I (50 ng/ml) (IGF) and/or UTP (100 μM; UTP) for 5 min. Cell lysates were analyzed by Western blot using anti-phospho-Akt (p-Akt) and anti-Akt antibody (Akt) as loading control. Data shown are representative of three independent experiments.

Figure 4.

UTP blocks PI3K-dependent cortactin membrane translocation. HaCaT keratinocytes were treated or not with LY294002 (LY; 30 μM, 60 min) and then stimulated with IGF-I (IGF; 50 ng/ml), either alone or in the presence of UTP (100 μM) for the indicated period of time. Untreated cells were used as a control (Ctrl). Before confocal analysis, filamentous actin organization was revealed by phalloidin staining (red), and cortactin was labeled using an anti-cortactin antibody (green). Scale bar, 20 μm.

Figure 5.

UTP inhibits IGF-I–induced PI3K-dependent cell spreading through Gαq activation. (A) HaCaT keratinocytes were allowed to spread on LM-5–enriched matrix in the presence of IGF-I 50 ng/ml (IGF), either alone or supplemented with UTP 100 μM (IGF+UTP). Top, phase-contrast microphotographs show cell-spreading inhibition by UTP 20 min after seeding. Bottom, a time-course spreading of HaCat cells on LM-5. When indicated, cells were preincubated with LY294002 (LY; 30 μM, 60 min). Cell surface was quantified as described in Materials and Methods. Data are expressed as the mean ± SEM (n = 100) and are representative of three independent experiments. (B) Cells were pretreated with YM-254890 (YM; 3 μM, 5 min), and then spreading assays were performed as described in A. (C) Cells were nucleofected with a nontargeting siRNA (Ctrl siRNA) or a Gq siRNA. Forty-eight hours after transfection, spreading assays were performed as described in A. Data were statistically analyzed using the Student's t test. **p < 0.001.

Figure 6.

Effect of UTP on IGF-I–induced keratinocyte random migration. (A) Presentation of 10 HaCaT keratinocyte migration paths during 120 min: untreated cells (Ctrl), cells stimulated by IGF-I (50 ng/ml), either alone (IGF) or in combination with UTP (100 μM) (IGF+UTP). (B and C) Motility assays were performed with HaCat cells (B) or normal human keratinocytes (C; NHK) as described in A. In each experimental condition, trajectory of at least 40 individual cells was analyzed. Cell velocity and directional persistence were calculated from time-lapse movies as described in Materials and Methods. Data are expressed as the mean ± SD and are representative of three independent experiments. Data are statistically analyzed using the Student's t test. **p < 0.001; NS, p > 0.5.

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.