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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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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.
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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.

Mentions: Like other GPCRs, P2Y receptors can activate PI3K pathway (Irino et al., 2008). Here, we stimulated HaCat cells with 100 μM of extracellular UTP, a noncytotoxic concentration widely used to activate P2YRs in keratinocytes (Dixon et al., 1999; Lee et al., 2001; Koizumi et al., 2004; Yoshida et al., 2006; Inoue et al., 2007; Taboubi et al., 2007). Similarly, we found that UTP stimulated the phosphorylation of the ser/thr protein kinase Akt and the glycogene synthase kinase-3 (GSK-3), two PI3K effectors (Figure 1A, left). However, phosphorylation of both proteins was detectable only 15 min after UTP treatment. On the other hand, IGF-I (50 ng/ml) activated the PI3K pathway earlier; within 2 min of treatment (Figure 1A, middle). Noteworthy, costimulation of keratinocytes by IGF-I and UTP resulted in a notable delay in the onset of both Akt and GSK-3 phosphorylation (15 vs. 2 min, Figure 1A, right). Similar results were obtained using ATP instead of UTP (data not shown). Thus, ATP and UTP elicited opposite dual regulatory signals toward PI3K/Akt pathway, i.e., an already reported activating signal and an unusual inhibitory signal revealed when the PI3K pathway was activated by growth factors such as IGF-I.


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)

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.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2836975&req=5

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.
Mentions: Like other GPCRs, P2Y receptors can activate PI3K pathway (Irino et al., 2008). Here, we stimulated HaCat cells with 100 μM of extracellular UTP, a noncytotoxic concentration widely used to activate P2YRs in keratinocytes (Dixon et al., 1999; Lee et al., 2001; Koizumi et al., 2004; Yoshida et al., 2006; Inoue et al., 2007; Taboubi et al., 2007). Similarly, we found that UTP stimulated the phosphorylation of the ser/thr protein kinase Akt and the glycogene synthase kinase-3 (GSK-3), two PI3K effectors (Figure 1A, left). However, phosphorylation of both proteins was detectable only 15 min after UTP treatment. On the other hand, IGF-I (50 ng/ml) activated the PI3K pathway earlier; within 2 min of treatment (Figure 1A, middle). Noteworthy, costimulation of keratinocytes by IGF-I and UTP resulted in a notable delay in the onset of both Akt and GSK-3 phosphorylation (15 vs. 2 min, Figure 1A, right). Similar results were obtained using ATP instead of UTP (data not shown). Thus, ATP and UTP elicited opposite dual regulatory signals toward PI3K/Akt pathway, i.e., an already reported activating signal and an unusual inhibitory signal revealed when the PI3K pathway was activated by growth factors such as IGF-I.

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