P2Y receptors play a critical role in epithelial cell communication and migration

Abstract

Cellular injury induces a complex series of events that involves Ca2+ signaling, cell communication, and migration. One of the first responses following mechanical injury is the propagation of a Ca2+ wave (Klepeis et al. [2001] J Cell Sci 114(Pt 23):4185-4195). The wave is generated by the extracellular release of ATP, which also induces phosphorylation of ERK (Yang et al. [2004] J Cell Biochem 91(5):938-950). ATP and other nucleotides, which bind to and activate specific purinergic receptors were used to mimic injury. Our goal was to determine which of the P2Y purinergic receptors are expressed and stimulated in corneal epithelial cells and which signaling pathways are activated leading to changes in cell migration, an event critical for wound closure. In this study, we demonstrated that the P2Y1, P2Y2, P2Y4, P2Y6, and P2Y11 receptors were present in corneal epithelial cells. A potency profile was determined by Ca2+ imaging for nucleotide agonists as follows: ATP > or = UTP > ADP > or = UDP. In contrast, negligible responses were seen for beta,gamma-meATP, a general P2X receptor agonist and adenosine, a P1 receptor agonist. Homologous desensitization of the Ca2+ response was observed for the four nucleotides. However, P2Y receptor internalization and degradation was not detected following stimulation with ATP, which is in contrast to EGFR internalization observed in response to EGF. ATP induced cell migration was comparable to that of EGF and was maximal at 1 microM. Cells exposed to ATP, UTP, ADP, and UDP demonstrated a rapid twofold increase in phosphorylation of paxillin at Y31 and Y118, however, there was no activation elicited by beta,gamma-meATP or adenosine. Additional studies demonstrated that wound closure was inhibited by reactive blue 2. These results indicate that P2Y receptors play a critical role in the injury repair process.

NADPH oxidases in lung biology and pathology: Host defense enzymes, and more

“…Purinergic receptors that are activated by ATP include P2X receptor channels and P2Y G-protein coupled receptors that mediate the activation of phospholipase C, intracellular Ca 2+ mobilization and activation of PKC (139). Several P2X and P2Y isoforms are expressed in airway epithelial cells and are involved in regulating a number of epithelial properties including epithelial anion transport (139,147), ciliary beat frequency (139), mucin secretion (148-151), production of inflammatory mediators such as CCL20, IL-8, and MMP-9 (87,(152)(153)(154), as well as epithelial cell migration and wound repair (155)(156)(157). Since P2 receptor stimulation induces Ca 2+ mobilization, it is not surprising that extracellular ATP is capable of stimulating DUOX-mediated H 2 O 2 production by airway epithelial cells.…”

Injury and nucleotides induce phosphorylation of epidermal growth factor receptor: MMP and HB-EGF dependent pathway

“…The transwell migration assay was performed as described (Weinger et al 2005, Klepeis et al 2004. Briefly, cells were pre-incubated in the presence or absence of AG1478 (10 nM), trypsinized, and resuspended to a concentration of 125,000 cells/100 μl in binding buffer in the presence or absence of AG1478.…”

“…After 5 min, cells were rinsed with PBS, fixed for 20 min with 3.7% formalin (pH 7.2). Cells were prepared for immunohistochemical staining as described previously (Klepeis et al, 2004). Cells were blocked with 5% BSA/PBS and then incubated overnight at 4°C with the antibody of interest in 3% BSA/PBS.…”

“…Nucleotides may be released from cells via physical injury, pores in channels, or exocytotic release upon ligand binding causing a rapid elevation in intracellular Ca 2+ (Burnstock 1997;Abbracchio and Burnstock, 1998;Neary et al,1999;Schwiebert and Zsembery, 2003;Yang et al, 2004;Klepeis et al, 2001;Klepeis et al, 2004;Weinger et al, 2005;Pintor et al, 2004). The extracellular nucleotides stimulate cells by activation of P2 purinergic receptors present on the cell surface.…”

“…The P2Y receptors are G-protein coupled receptors (GPCRs) that stimulate the release of intracellular Ca 2+ by phospholipase C β-mediated hydrolysis and activation of the IP 3 pathway. We have shown that epithelial cells express P2Y receptors (1,2,4,6 and 11) and that Reactive Blue 2 (RB-2), an antagonist of P2YR, impairs wound closure (Klepeis et al, 2004). In live cell imaging experiments, injury induces the propagation of a Ca 2+ wave, which occurs in the absence of extracellular Ca 2+ but is inhibited with thapsigargin or BAPTA (Klepeis et al, 2001;Weinger et al, 2005).…”