UDP induces intestinal epithelial migration via the P2Y₆ receptor

Abstract

BACKGROUND AND PURPOSEExtracellular 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. EXPERIMENTAL APPROACHWound-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. KEY RESULTSScratching 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. CONCLUSION AND IMPLICATIONSWound-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. AbbreviationsUDP, uridine 5'-diphosphate; UTP, uridine 5'-triphosphate

P2Y6 Receptors Regulate CXCL10 Expression and Secretion in Mouse Intestinal Epithelial Cells

“…We noted also the expression of P2Y 1 , P2Y 2 , P2X2, P2X4, and of the adenosine receptor A 2A in IEC. Previous studies also showed the presence of P2Y 6 in Caco2/15 and IEC-6, which are human and rat intestinal cell lines, respectively ( Grbic et al, 2008 ; Nakamura et al, 2013 ).…”

Thymidine 5'-O-monophosphorothioate induces HeLa cell migration by activation of the P2Y6 receptor

“…Among those three receptors, activation of P2Y6 seemed be the most probable because UDPβS was previously shown to be a specific agonist for P2Y6 receptors with no effect on P2Y2 and P2Y4 [20]. Besides, it was shown that UDP induced intestinal epithelial migration via the P2Y6 receptor [17]. Thus, we examined next whether the enhancement of HeLa migration by TMPS and UMPS was specifically mediated via the P2Y6 receptor using knockdown by siRNA.…”

“…Thus, we further examined if tested compounds regulated the migratory potency of these cells with scratch wound migration assay. Recent data suggest that UDP plays a key role in promoting the migration of intestinal epithelium via activation of P2Y6 receptors [17]. [23].…”

“…We tested a series of pyrimidine nucleotides and their phosphorothioate analogs and revealed that besides stimulation of cellular viability, pyrimidine nucleoside 5′-O-monophosphorothioates are able to potentiate migratory potency of the cervical epithelial carcinoma HeLa cells, representing the Bwork horse^of many cell biology laboratories. Since it was shown that UDP induced intestinal epithelial migration via the P2Y6 receptor [17], we aimed to investigate the possible modulation of the P2Y6 receptor by pyrimidine nucleoside 5′-Omonophosphorothioates as well. We adopted in vitro scratch wound assay linked to specific inhibition of P2Y6 gene expression by small interfering RNAs and diisothiocyanate derivative MRS 2578, which is a selective antagonist of the P2Y6R.…”

Anti-proliferative Effects of Nucleotides on Gastric Cancer via a Novel P2Y6/SOCE/Ca2+/β-catenin Pathway

“…The latter are further divided into the ligand-gated ion channel P2X receptors and G-protein-coupled receptors (GPCRs) P2Y, which are subdivided into eight subtypes (P2Y1, 2, 4, 6 and P2Y11–14). P2Y6 receptors, one member of P2Y receptors highly activated by UTP and UDP, mediate various human physiological and pathological processes 8 – 13 . Over recent years, nucleotides and purinergic receptors have been the focus of increasing attention by immunologists and oncologists since ATP and UTP are found to modulate tumor growth in various tumor models 14 , 15 .…”