UDP induces intestinal epithelial migration via the P2Y6 receptor.
OBJECTIVE
Extracellular nucleotides are released at high concentrations from damaged cells and function through P2 receptor activation. Intestinal epithelial restitution, which is defined as cell migration independent of cell proliferation, is an important initial step in the process of wound healing. In this study, we investigated the role of extracellular nucleotides in intestinal epithelial migratory responses.


METHODS
Wound-healing and trans-well migration assays were performed with a rat intestinal epithelial cell line (IEC-6). The concentrations of extracellular nucleotides released from injured IEC-6 cells were measured by HPLC. TGF-β expression was assessed by RT-PCR and elisa.


RESULTS
Scratching the monolayer of IEC-6 cells induced cell migration. Pretreatment with apyrase or MRS2578, a selective P2Y6 antagonist, inhibited the wound-induced cell migration. Among the cellular nucleotides, only ATP and uridine 5'-diphosphate (UDP) were detected in the culture medium after cell wounding. Exogenously applied UDP dose-dependently enhanced the migration more effectively than ATP but did not induce proliferation. In addition, cell wounding and UDP increased the expression of TGF-β, and both the wound-induced and UDP-enhanced migration were inhibited by MRS2578 or ALK5Inhibitor (ALK5i), a TGF-β receptor blocker. Furthermore, cell wounding and UDP stimulation up-regulated the expression of P2Y6 receptor mRNA, and this effect was suppressed by MRS2578 or ALK5i.


CONCLUSIONS
Wound-induced UDP evokes intestinal epithelial restitution by activation of P2Y6 receptors, which mediates de novo synthesis of TGF-β. In addition, the expression of P2Y6 receptors is increased by cell wounding and UDP, which constitutes a positive-feedback loop for mucosal repair.
Department of Veterinary Pharmacology, Graduate School of Agriculture and Life Sciences, The University of Tokyo, Tokyo, 113-8657, Japan.
Tatsuro
T
Nakamura
Takahisa
T
Murata
Masatoshi
M
Hori
Hiroshi
H
Ozaki
D000818:Animals; D002460:Cell Line; D002465:Cell Movement / Q000187:drug effects*; D004305:Dose-Response Relationship, Drug; D004847:Epithelial Cells / Q000187:drug effects* / Q000378:metabolism; D007413:Intestinal Mucosa / Q000166:cytology / Q000187:drug effects* / Q000378:metabolism; D047428:Protein Kinase Inhibitors / Q000494:pharmacology; D017346:Protein Serine-Threonine Kinases / Q000037:antagonists & inhibitors / Q000378:metabolism; D058910:Purinergic P2 Receptor Agonists / Q000494:pharmacology*; D058919:Purinergic P2 Receptor Antagonists / Q000494:pharmacology; D051381:Rats; D000077293:Receptor, Transforming Growth Factor-beta Type I; D018048:Receptors, Purinergic P2 / Q000187:drug effects* / Q000378:metabolism; D018125:Receptors, Transforming Growth Factor beta / Q000037:antagonists & inhibitors / Q000378:metabolism; D015398:Signal Transduction / Q000187:drug effects; D013997:Time Factors; D016212:Transforming Growth Factor beta / Q000378:metabolism; D014530:Uridine Diphosphate / Q000378:metabolism / Q000494:pharmacology*; D014945:Wound Healing / Q000187:drug effects*
D047428:Protein Kinase Inhibitors; D058910:Purinergic P2 Receptor Agonists; D058919:Purinergic P2 Receptor Antagonists; D018048:Receptors, Purinergic P2; D018125:Receptors, Transforming Growth Factor beta; D016212:Transforming Growth Factor beta; C099845:purinoceptor P2Y6; D014530:Uridine Diphosphate; D017346:Protein Serine-Threonine Kinases; D000077293:Receptor, Transforming Growth Factor-beta Type I; C000625843:Tgfbr1 protein, rat
P2Y6 receptor; TGF-β; UDP; extracellular nucleotides; intestinal epithelial migration