Endoplasmic Reticulum/Golgi Nucleotide Sugar Transporters Contribute to the Cellular Release of UDP-sugar Signaling Molecules

Abstract

Extracellular UDP-sugars promote cellular responses by interacting with widely distributed P2Y 14 receptors, but the mechanisms by which these molecules are released from cells are poorly understood. Given the active role of UDP-sugars in glycosylation reactions within the secretory pathway, we hypothesized that UDP-sugar release includes an exocytotic component. This hypothesis was tested by assessing the contribution of endoplasmic reticulum (ER)/Golgi-resident UDPGlcNAc transporters to the cellular release of their cognate substrates. A sensitive and highly selective assay for UDP-GlcNAc mass was developed using purified AGX2, an isoenzyme of human UDP-GlcNAc pyrophosphorylase. Robust constitutive release of UDP-GlcNAc was observed in yeast as well as in well differentiated human airway epithelial cells. The human UDPGlcNAc transporter HFRC1 was overexpressed in human bronchial epithelial cells and was shown to localize in the Golgi and to enhance the surface expression of N-acetylglucosamine-rich glycans. HFRC1-overexpressing cells also displayed increased constitutive and hypotonic stress-stimulated release of UDPGlcNAc. Yeast mutants lacking Yea4 (the ER UDP-GlcNAc transporter endogenously expressed in Saccharomyces cerevisiae) showed reduced UDP-GlcNAc release. Yea4-deficient cells complemented with Yea4 showed UDP-GlcNAc release rates at levels similar to or higher than wild type cells. Our results illustrate that ER/Golgi lumen constitutes a significant source of extracellular UDP-sugars and therefore plays a critical role in nucleotide sugar-promoted cell signaling.Extracellular nucleotides perform important signaling functions via activation of broadly distributed P2X and P2Y purinergic receptors (1, 2). P2X receptors comprise seven species (P2X 1 -P2X 7 ) of ATP-gated ion channels. P2Y receptors belong to the superfamily of G protein-coupled receptors. At least eight human P2Y receptor species have been identified, seven of which (P2Y 1 , P2Y 2 , P2Y 4 , P2Y 6 , P2Y 11 , P2Y 12 , and P2Y 13 ) are activated by adenine and/or uridine nucleoside di-and triphos-

UDP-glucose promotes neutrophil recruitment in the lung

“…Previous studies indicated robust levels of UDP-sugars in in vivo airway surface liquid (BALF) from chronically diseased lungs (e.g., cystic fibrosis) [24], but the extent to which concentrations of UDP-sugars in CF airway secretions differ from healthy controls is not known. Therefore, we examined the nucleotide content in sputum samples (believed to reflect secretions from the large airways) [38] obtained from CF patients with moderate or severe lung disease and from healthy control subjects.…”

“…Previous studies indicated that UDP-N-acetylglucosamine is present in BALF isolated from patients with CF and other inflammatory diseases at concentrations similar to UDPglucose concentrations [24]. On a speculative basis, UDP-Nacetylglucosamine and potentially UDP-galactose and UDPglucuronic acid may contribute to P2Y 14 R-dependent lung phenotypes.…”

“…Thus, one mechanism potentially contributing to UDPglucose accumulation in ASL is its release from vesicles as co-cargo with exported glycoconjugates. Indeed, we have shown that, by controlling the entry of UDP-sugars into the Golgi, SLC35 nucleotide-sugar transporters contribute to the vesicular release of UDP-sugars from cultured airway epithelial cells [24]. Furthermore, goblet cell-rich airway epithelial cell models exhibit enhanced release of UDP-glucose concomitantly with mucins, heavily glycosylated proteins.…”

“…Studies from our lab have illustrated the occurrence of regulated release of UDP-sugars from several cell types, including airway epithelial cells [22][23][24][25][26][27][28]. Notably, enhanced UDPsugar release was observed in mucin-secreting cells.…”

“…For example: (i) primary cultures of human bronchial epithelial (HBE) cells induced to develop goblet cell metaplasia by infection with respiratory syncytial virus or treatment with interleukin 13 (IL-13) exhibited enhanced release of UDPglucose concomitantly with increased mucin secretion [22]; and (ii) goblet cell-like Calu-3 cells exhibited enhanced Ca 2+ -regulated exocytosis of mucin granules, which was accompanied by increased release of UDP-glucose [27]. Bronchoalveolar lavage fluids (BALF) from patients with CF and control diseases exhibit robust accumulation of UDP-sugars [24]. In spite of the apparent association between UDP-sugar levels in airway surface liquids and mucin hypersecretion and inflammation, the pathophysiological consequences of UDP-glucose release into the airways are not well understood.…”