P2Y2 receptor inhibits EGF-induced MAPK pathway to stabilise keratinocyte hemidesmosomes.
α6β4 integrin is the main component of hemidesmosomes (HD) that stably anchor the epithelium to the underlying basement membrane. Epithelial cell migration requires HD remodelling, which can be promoted by epidermal growth factor (EGF). We previously showed that extracellular nucleotides inhibit growth factor-induced keratinocyte migration. Here, we investigate the effect of extracellular nucleotides on α6β4 integrin localisation in HD during EGF-induced cell migration. Using a combination of pharmacological inhibition and gene silencing approaches, we found that UTP activates the P2Y2 purinergic receptor and Gαq protein to inhibit EGF/ERK1/2-induced cell migration in keratinocytes. Using a keratinocyte cell line expressing an inducible form of the Raf kinase, we show that UTP inhibits the EGF-induced ERK1/2 pathway activation downstream of Raf. Moreover, we established that ERK1/2 activation by EGF leads to the mobilisation of α6β4 integrin from HD. Importantly, activation of P2Y2R and Gαq by UTP promotes HD formation and protects these structures from EGF-triggered dissolution as revealed by confocal analysis of the distribution of α6β4 integrin, plectin, BPAG1, BPAG2 and CD151 in keratinocytes. Finally, we demonstrated that the activation of p90RSK, downstream of ERK1/2, is sufficient to promote EGF-mediated HD dismantling and that UTP does not stabilise HD in cells expressing an activated form of p90RSK. Our data underline an unexpected role of P2Y2R and Gαq in the inhibition of the ERK1/2 signalling pathway and in the modulation of hemidesmosome dynamics and keratinocyte migration.
Aix-Marseille Université, INSERM UMR 911, Centre de Recherche en Oncologie Biologique et en Oncopharmacologie, Marseille 13005,  France.
Emilie
E
Faure
Françoise
F
Garrouste
Fabrice
F
Parat
Sylvie
S
Monferran
Ludovic
L
Leloup
Gilbert
G
Pommier
Hervé
H
Kovacic
Maxime
M
Lehmann
D002465:Cell Movement / Q000187:drug effects; D004789:Enzyme Activation / Q000187:drug effects; D004815:Epidermal Growth Factor / Q000494:pharmacology*; D048049:Extracellular Signal-Regulated MAP Kinases / Q000378:metabolism; D043802:GTP-Binding Protein alpha Subunits, Gq-G11 / Q000378:metabolism; D022002:Hemidesmosomes / Q000187:drug effects / Q000378:metabolism*; D006801:Humans; D039663:Integrin beta4 / Q000378:metabolism; D015603:Keratinocytes / Q000166:cytology* / Q000187:drug effects / Q000201:enzymology*; D020935:MAP Kinase Signaling System / Q000187:drug effects*; D008954:Models, Biological; D058473:Receptors, Purinergic P2Y2 / Q000378:metabolism*; D038744:Ribosomal Protein S6 Kinases, 90-kDa / Q000378:metabolism; D015398:Signal Transduction / Q000187:drug effects; D014544:Uridine Triphosphate / Q000494:pharmacology; D048490:raf Kinases / Q000378:metabolism
D039663:Integrin beta4; D058473:Receptors, Purinergic P2Y2; D004815:Epidermal Growth Factor; D038744:Ribosomal Protein S6 Kinases, 90-kDa; D048490:raf Kinases; D048049:Extracellular Signal-Regulated MAP Kinases; D043802:GTP-Binding Protein alpha Subunits, Gq-G11; D014544:Uridine Triphosphate