Tri-nucleotide receptors play a critical role in epithelial cell wound repair

Abstract

The cornea plays a major role in the refraction of light to the retina. Therefore, the integrity and transparency of the corneal epithelium are critical to vision. Following injury, a combination of rapid signal transduction events and longterm cell migration are essential for wound closure. We have demonstrated previously that injury resulted in the release of nucleotides that induce the propagation of a Ca 2+ wave to neighboring cells. This suggests that nucleotides and their receptors are critical components of wound healing. Epidermal growth factor (EGF) and integrins also have been shown to play a role in injury. In this study, we demonstrate that pretreatment of cells with ATP and UTP inhibited the immediate wound response, while BzATP, ADP, and UDP did not affect this response. Tri-nucleotide pretreatment also reduced the EGF induced Ca 2+ response. Additionally, lower EC 50 concentrations of ATP and UTP triggered migration of cells that was enhanced further with EGF and was inhibited by the tripeptide, RGD. Results indicate that the desensitization induced by ATP and UTP was specific. While ADP and UDP cause a homologous desensitization of their own signal, they did not cause an inhibition of the wound response nor does BzATP. Neither Ca 2+ wave propagation nor cell migration occurred in response to b,g-MeATP. Together these results lead us to hypothesize that corneal epithelial wound repair is mediated by both P2Y 2 and P2Y 4 receptors.Abbreviations: BAPTA -1,2-bis(o-aminophenoxy

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

“…Furthermore, when the wound medium or ATP is treated with apyrase and then applied to cells, both cell migration and the Ca 2+ wave are inhibited, while untreated medium elicits a typical Ca 2+ wave. ATP and UTP have been shown to play a critical role in the propagation of injury induced Ca 2+ waves while ADP, UDP and BzATP have not (Weinger et al, 2005). The data indicate that the trinucleotide P2Y receptors play a role in the Ca 2+ response that occurs immediately after injury.…”

“…Previous experiments have been performed comparing primary epithelial cells and the corneal epithelial cell line (Weinger et al, 2005). Cells were seeded at a density of 10 4 cells/cm 2 cultured in keratinocyte-SFM medium (K-SFM) supplemented with 30 μg/ml bovine pituitary extract, 0.1 ng/ml EGF, 100 U/ml penicillin and 100 μg/ml streptomycin (Gibco Invitrogen, Carlsbad,CA) (Klepeis et al, 2001).…”

“…In addition, inhibition of the EGF induced wave with AG1478 does not inhibit the injuryinduced wave (Klepeis et al, 2001). When cells were either prestimulated with nucleotides or injured and then stimulated with EGF, the Ca 2+ response was significantly lowered (Weinger et al, 2005).…”

“…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.…”

“…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). Furthermore, when the wound medium or ATP is treated with apyrase and then applied to cells, both cell migration and the Ca 2+ wave are inhibited, while untreated medium elicits a typical Ca 2+ wave.…”