P2Y2 receptor inhibits EGF-induced MAPK pathway to stabilise keratinocyte hemidesmosomes.

Abstract

Summary a6b4 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 a6b4 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 Gaq 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 a6b4 integrin from HD. Importantly, activation of P2Y2R and Gaq by UTP promotes HD formation and protects these structures from EGF-triggered dissolution as revealed by confocal analysis of the distribution of a6b4 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 Gaq in the inhibition of the ERK1/2 signalling pathway and in the modulation of hemidesmosome dynamics and keratinocyte migration.

β-Neoendorphin Enhances Wound Healing by Promoting Cell Migration in Keratinocyte

“…As shown in Figure 2 , Erk1/2 was activated by β-NEP treatment ( Figure 2 A). Previous findings suggested that the Erk1/2 activation and its effector P90 RSK phosphorylate β4 integrin and plectin, which is required for hemidesmosome disruption leading to cell migration [ 27 , 28 ]. Moreover, Erk1/2 phosphorylates transcription factors, including Elk-1, which is mainly engaged in cell migration by regulating matrix metalloproteinase (MMP)-2 and -9 expression [ 29 ].…”

Human Keratinocytes Respond to Extracellular UTP by Induction of Hyaluronan Synthase 2 Expression and Increased Hyaluronan Synthesis

“…UTP stimulates the migration of endothelial cells, arterial smooth muscle cells, corneal epithelial cells, prostate cancer cells, and schwannoma cells (80 -83), whereas in keratinocytes it inhibits cell spreading and motility (84,85). The influence of hyaluronan on cell motility is obviously complex.…”

“…Moreover, the expression of hyaluronidases was not changed. However, because the inhibition of cell motility occurs already within 15 to 30 min after the UTP exposure (84,85), it unlikely relates to hyaluronan synthesis, which takes place later.…”

Networking galore: intermediate filaments and cell migration

“…The knockdown of actinin-4, an actin-binding protein, results in a loss of directionality during the migration of individual keratinocytes, correlating with a mislocalization of α6β4 integrin and BPAG1e (Figure 2A) and defects in cell polarity and lamellipodial dynamics [31]. The p90 ribosomal protein S6 kinase (RSK) has been implicated in hemidesmosome remodeling [32,33] and in the regulation of the wound-inducible keratin 17 [34], raising the issue of its influence in complete keratin-null and/or actinin-4 knockdown keratinocytes. Besides, Bordeleau et al showed that the knockdown of keratin 8 (K8) in cultured hepatoma cells impaired cell migration in a scratch-wound assay [35], decreased cell surface area upon spreading, altered Rho-dependent actin fiber organization, and decreased local stiffness at focal adhesions, reflecting an interplay between K8/K18 IFs and Rho-mediated actin dynamics occurring through plectin, RACK1 and Src [36] (see below).…”