The UDP-sugar-sensing P2Y₁₄ receptor promotes Rho-mediated signaling and chemotaxis in human neutrophils

Abstract

The G(i)-coupled P2Y(14) receptor (P2Y(14)-R) is potently activated by UDP-sugars and UDP. Although P2Y(14)-R mRNA is prominently expressed in circulating neutrophils, the signaling pathways and functional responses associated with this receptor are undefined. In this study, we illustrate that incubation of isolated human neutrophils with UDP-glucose resulted in cytoskeleton rearrangement, change of cell shape, and enhanced cell migration. We also demonstrate that UDP-glucose promotes rapid, robust, and concentration-dependent activation of RhoA in these cells. Ecto-nucleotidases expressed on neutrophils rapidly hydrolyzed extracellular ATP, but incubation with UDP-glucose for up to 1 h resulted in negligible metabolism of the nucleotide-sugar. HL60 human promyelocytic leukemia cells do not express the P2Y(14)-R, but neutrophil differentiation of HL60 cells with DMSO resulted in markedly enhanced P2Y(14)-R expression. Accordingly, UDP-glucose, UDP-galactose, and UDP-N-acetylglucosamine promoted Rho activation in differentiated but not in undifferentiated HL60 cells. Stable expression of recombinant human P2Y(14)-R conferred UDP-sugar-promoted responses to undifferentiated HL60 cells. UDP-glucose-promoted RhoA activation also was accompanied by enhanced cell migration in differentiated HL60 cells, and these responses were blocked by Rho kinase inhibitors. These results support the notion that UDP-glucose is a stable and potent proinflammatory mediator that promotes P2Y(14)-R-mediated neutrophil motility via Rho/Rho kinase activation.

UDP-glucose promotes neutrophil recruitment in the lung

“…3a, b). Similar effects of UDP-glucose were also observed after pre-treatment of UDP-glucose with apyrase to eliminate potential contamination with ATP/UTP [29] (not shown). Time-course experiments indicated that maximal PMN recruitment occurred within 12 h following UDPglucose instillation; neutrophil levels declined considerably after 24 h (Fig.…”

“…UDP-glucose concentrations in control sputa were in the low nanomolar range (UDP-glucose 9.5 ± 2.1 nM) but increased up to 11-fold in CF (Fig. 1a), i.e., reaching concentration values capable of promoting P2Y 14 R-mediated responses [29]. Although the small sample size prevented us from establishing a correlation between UDP-glucose levels and lung function or sputum neutrophils within the CF group (Suppl.…”

“…1). Ecto-nucleotidases expressed on (or secreted/shed from) epithelial cells and inflammatory cells [29,39,40] could have contributed to the pattern of nucleotide accumulation observed in control and CF sputum. To gain an insight into potential metabolism differences between ATP and UDPglucose in lung secretions, rates of ATP and UDP-glucose hydrolysis were assessed in sputum supernatants.…”

“…Materials UDP-glucose (Fluka catalog # 94335) was purchased from Sigma-Aldrich (St. Louis, MO); highperformance liquid chromatography (HPLC) analysis indicated that UDP-glucose was not contaminated with ATP, UTP, or other nucleotide-signaling molecules [29]. PPTN was synthesized as previously described [30] and used as a dimethylsulfoxide (DMSO) stock solution (100 mM), which was stored at −20°C.…”

“…In addition, we recently demonstrated that UDP-glucose promotes P2Y 14 R-mediated chemotaxis of freshly isolated human neutrophils. Enhanced PMN migration was observed in response to UDP-glucose gradients, and this activity was abolished when the P2Y 14 R antagonist PPTN [4-(piperidin-4-yl)-phenyl)-7-(4-(trifluoromethyl)-phenyl-2-naphthoic acid] was included in the cell suspension buffer [29,30]. We also showed that UDP-glucose-promoted chemotaxis in neutrophil-like HL60 cells was absolutely dependent on the expression of P2Y 14 R in these cells and was abolished by PPTN [29,30].…”