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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 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.
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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.

Mentions: In keratinocytes, lamellipodium extension is driven by p110α-PI3K activation (Haase et al., 2003; Pankow et al., 2006). In our previous work, we showed that within few minutes UTP treatment induces actin cytoskeleton disorganization and a transient lamellipodium withdrawal (Taboubi et al., 2007). Among the large array of proteins involved in cortical actin regulation, cortactin plays a key role as a promoter of actin filament branching and subsequent lamellipodium extension (Bryce et al., 2005; Kowalski et al., 2005; Ammer and Weed, 2008). Growth factors lead to cortactin relocalization from internal cytoplasmic region to the cortical actin network. Although the precise mechanism controlling cortactin translocation is not clearly established, it has been reported that, in keratinocytes, PI3K is involved in this process (Ceccarelli et al., 2007). In HaCat cells, we observed a clear cortactin translocation from the cytosol to the rim of membrane ruffles, with a maximal recruitment at early time points of IGF-I stimulation (1 and 5 min) (Figure 4). In LY294002-treated cells, cortactin redistribution was potently blocked, confirming the involvement of PI3K in this process. As shown in Figure 4, UTP (100 μM) transiently perturbed the IGF-I–induced cortactin relocalization. Although we observed a short delay, UTP-induced inhibition of both cortactin recruitment and p110α-PI3K function were timely correlated (see also Figures 1 and 2). Moreover, the kinetics of the cortactin translocation inhibition induced by UTP is very similar to that of lamellipodium withdrawal described elsewhere (Taboubi et al., 2007), suggesting that cortactin is oppositely regulated by UTP and IGF-I to control lamellipodium formation. Thus, we identified cortactin recruitment to the plasma membrane as a critical early event downstream of the PI3K activation by IGF-I that is inhibited by extracellular UTP and may affect later processes such as cell spreading and motility (see below).


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 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.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2836975&req=5

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.
Mentions: In keratinocytes, lamellipodium extension is driven by p110α-PI3K activation (Haase et al., 2003; Pankow et al., 2006). In our previous work, we showed that within few minutes UTP treatment induces actin cytoskeleton disorganization and a transient lamellipodium withdrawal (Taboubi et al., 2007). Among the large array of proteins involved in cortical actin regulation, cortactin plays a key role as a promoter of actin filament branching and subsequent lamellipodium extension (Bryce et al., 2005; Kowalski et al., 2005; Ammer and Weed, 2008). Growth factors lead to cortactin relocalization from internal cytoplasmic region to the cortical actin network. Although the precise mechanism controlling cortactin translocation is not clearly established, it has been reported that, in keratinocytes, PI3K is involved in this process (Ceccarelli et al., 2007). In HaCat cells, we observed a clear cortactin translocation from the cytosol to the rim of membrane ruffles, with a maximal recruitment at early time points of IGF-I stimulation (1 and 5 min) (Figure 4). In LY294002-treated cells, cortactin redistribution was potently blocked, confirming the involvement of PI3K in this process. As shown in Figure 4, UTP (100 μM) transiently perturbed the IGF-I–induced cortactin relocalization. Although we observed a short delay, UTP-induced inhibition of both cortactin recruitment and p110α-PI3K function were timely correlated (see also Figures 1 and 2). Moreover, the kinetics of the cortactin translocation inhibition induced by UTP is very similar to that of lamellipodium withdrawal described elsewhere (Taboubi et al., 2007), suggesting that cortactin is oppositely regulated by UTP and IGF-I to control lamellipodium formation. Thus, we identified cortactin recruitment to the plasma membrane as a critical early event downstream of the PI3K activation by IGF-I that is inhibited by extracellular UTP and may affect later processes such as cell spreading and motility (see below).

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