The inflammatory effects of UDP-glucose in N9 microglia are not mediated by P2Y14 receptor activation
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- Brautigam, V.M., Dubyak, G.R., Crain, J.M. et al. Purinergic Signalling (2008) 4: 73. doi:10.1007/s11302-008-9095-1
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In this study we evaluated the functionality and inflammatory effects of P2Y14 receptors in murine N9 microglia. The selective P2Y14 receptor agonist UDP-glucose (UDPG) derived from microbial sources dose dependently stimulated expression of cyclooxygenase-2 and inducible nitric oxide synthase, and potentiated the effects of bacterial lipopolysaccharide on nitric oxide production. However, another selective P2Y14 receptor agonist, UDP-galactose, did not affect these endpoints either alone or in combination with lipopolysaccharide. Interestingly, synthetic UDPG also had no detectable pro-inflammatory effects, although P2Y14 receptors are both expressed and functional in N9 microglia. While synthetic UDPG decreased levels of phosphorylated cyclic AMP response element binding protein, an effect that was blocked by pertussis toxin, the pro-inflammatory effects of microbial-derived UDPG were insensitive to pertussis toxin. These data suggest that the pro-inflammatory effects of microbial-derived UDPG are independent of P2Y14 receptors and imply that microbial-derived contaminants in the UDPG preparation may be involved in the observed inflammatory effects.
KeywordsCyclic AMP response element binding protein (CREB)Cyclooxygenase-2 (COX-2)Inducible nitric oxide synthase (iNOS)MicrogliaPertussis toxin (PTX)P2Y14UDP galactose
P2 purinergic receptors potently modulate microglial inflammatory activities, but little is known concerning the effects of P2Y14 receptors in these CNS-resident innate immune cells. Previously, we and others have reported that adenine di- and triphosphate nucleotides, the endogenous ligands for P2 receptors, exert anti-inflammatory effects in microglia [3, 4]; however, the specific P2 receptors involved in these actions are not yet clear. The P2Y14 receptor, previously called GPR105 (an orphan G-protein coupled receptor), has only recently been identified as a member of the purinergic receptor family ; hence, comparatively little is known about its function in any cell type. Its newly identified presence in microglia  suggests that P2Y14 may have a role in modulating microglial responses in the CNS. Uracil-containing sugar nucleotides, such as uridine 5’ diphosphoglucose (UDPG) and UDP-galactose (UDP-gal), are selective agonists for the P2Y14 receptor , which couples intracellularly to the inhibition of adenylyl cyclase via Gi/o [1, 8, 10, 11] in many cell types. Adenylyl cyclase catalyzes the formation of cyclic AMP, enabling protein kinase A (PKA) to phosphorylate and activate the transcription factor cAMP response element binding protein (CREB), among many others. We undertook the present studies to ascertain the role of P2Y14 receptors in modulating microglial cell inflammatory functions.
Materials and methods
The following materials were purchased from Sigma Chemical (St. Louis, MO): LPS (E.coli 0111:B4), ATP, UDP-glucose (microbial-derived; catalog #U4625, multiple vials of lot #074K7024 over a period of 15 months) and UDP-galactose (catalog #U4500). Synthetic UDP-glucose was purchased from MP Biomedicals (Solon, OH) and B. pertussis toxin (PTX) was obtained from Calbiochem (San Diego, CA).
Murine N9 microglia  were cultured routinely in Dulbecco’s Modified Eagle’s Medium (DMEM; Cellgro, Herndon, VA) supplemented with 5% fetal bovine serum (Bio Whittaker, Walkersville, MD) and 100 U/ml penicillin/streptomycin (Cellgro) in 100-mm Sarstedt plates. Cells were grown to ~90% confluency and passaged every 2 days. For experimentation, cells were plated at densities of 1 × 105 cells per well in 24-well plates for nitrite measurements, or at 4 × 105 cells per well in 12-well plates for signaling studies and RT-PCR. The next day, the cells were treated as specified below.
Reverse-transcription polymerase chain reaction (RT-PCR)
RT-PCR was performed on 1 μg of total RNA from whole brain (positive control) and N9 microglia as previously reported [2, 4]. RT reactions were completed according to the manufacturer’s protocol with and without reverse transcriptase (+/-RT). The cDNA was then used for PCR using the GoTaq Green Master Mix (Promega, Madison, WI). The murine P2Y14 receptor gene was amplified using the following primers: 5’TAGAGGCCATAAACTGTGCTT and 5’AATTCTTCCTGGACTTGAGGT (expected amplimer size 742 bp). As a control, GAPDH (glyceraldehyde-3-phosphate dehydrogenase; expected amplimer size 325 bp) was amplified using the following primers: 5’CCATCACCATCTTCCAGGAG and 5’GATGGCATGGACTGTGGTC. PCR products were separated and visualized by ethidium bromide-stained 1% agarose gel electrophoresis.
Measurement of nitrite production
Cells were stimulated with LPS (1 μg/mL) either alone or together with UDPG for 18–22 h at the concentrations indicated. In some experiments, cells were treated with increasing concentrations of UDPG or UDP-gal alone (50–300 μM), or with UDPG that had been boiled at 105°C for 30–60 min. In other experiments, microglia were pretreated with PTX (100 ng/mL) for 18 h, followed by stimulation with UDPG. Nitric oxide (NO) levels were measured in the culture medium using the Greiss reagent to analyze nitrite production, a stable breakdown product of NO generation . The cells were collected for immunoblot analyses.
Whole-cell lysates were prepared and total protein content determined as previously described . Equal amounts of protein (~25 μg) were loaded per lane and separated by 10% SDS-PAGE . Proteins in the gels were transferred to Immobilon polyvinylidene difluoride (PVDF) membrane (Millipore, Bedford, MA), and the membranes were subjected to immunoblot analyses for iNOS, COX-2 and phospho-CREB as described previously . To confirm equal protein loading, membranes were probed with antibodies recognizing the cytosolic proteins Grb-2 or GAPDH (Santa Cruz Biotechnology, Santa Cruz, CA). The data shown are representative of at least three independent experiments performed in triplicate.
Statistical analyses were performed using an ANOVA pre-hoc test and Tukey-Kramer, Dunnett or Bonferroni multiple comparison post-hoc analyses. Statistical significance was set at the 95% confidence limit (P < 0.05). Quantitative data are expressed as the mean ± SD of three to six independent experiments.
UDP-glucose stimulates nitric oxide (NO) production in N9 microglia
P2Y14 receptors are functionally expressed in N9 microglia
Microbial-derived UDPG-stimulated NO production is insensitive to pertussis toxin
To ascertain if the pro-inflammatory effects of microbial-derived UDPG in N9 microglia were mediated by activation of P2Y14 receptors, we used PTX to prevent Gi/o proteins from interacting with the P2Y14 receptor. As shown in Fig. 3c, the presence of PTX did not interfere with the ability of UDPG to stimulate nitrite accumulation, suggesting a P2Y14 receptor-independent effect of microbial-derived UDPG.
UDP-galactose is without effect on inflammatory gene expression in N9 microglia
Synthetic UDP-glucose lacks pro-inflammatory activities in N9 microglia
These studies indicate that UDPG preparations derived from microbial sources may have pro-inflammatory activities that are independent of P2Y14 receptor activation. In addition, our data suggest that selective activation of P2Y14 receptors (by synthetic UDPG or by UDP-gal) has no effect on the production of the inflammatory mediators iNOS/NO and COX-2 by N9 microglia. Because synthetic UDPG and UDP-gal inhibit the phosphorylation of CREB in a manner that is sensitive to pertussis toxin, P2Y14 receptors appear to be expressed and functional in N9 microglia. However, because the pro-inflammatory effects of microbial-derived UDPG on NO production were not blocked by PTX, it is likely that these stimulatory effects in microglia do not involve P2Y14 receptors.
Although the function of P2Y14 in any cell type remains unclear, in immature dendritic cells, activation of P2Y14 receptors increases expression of CD86, suggesting their involvement in dendritic cell maturation . Also, pro-inflammatory stimuli such as LPS  and interferon-γ (JMC and JJW, unpublished observations) increase the expression levels of GPR105 in glial cells and in N9 microglia, respectively, supporting the idea that this receptor plays a role in the microglial response to pro-inflammatory stimuli. While we did not observe any modulatory effects of selective P2Y14 receptor activation on iNOS or COX-2 pathways in microglia, it remains probable that P2Y14 receptors alter some other microglial inflammatory function(s) not measured here.
It is important to note that in addition to our study, other literature reports also indicate different responses to UDPG and UDP-gal. Whereas both UDPG and UDP-gal are agonists of P2Y14 receptors [1, 14], in human neutrophils  and in murine T-lymphocytes  UDP-gal does not mimic the effects of UDPG. Coincidentally, the source of UDPG used in the above studies was also microbial-derived. In a separate set of studies, microbial-derived UDPG had effects in a cell line in which P2Y14 receptors were not detectably expressed , supporting the idea that UDPG preparations from microbial sources may contain some other activity that is independent of P2Y14 receptors. Because many studies on P2Y14 receptor function are performed in transfected cell systems, potential microbial contaminants in the nucleotide preparations are admittedly not as large a concern as when studies are performed in immune cells, which can strongly respond to microbial products. However, based on the data presented here, the presence of microbial products in the nucleotide preparation may significantly alter data interpretation.
We would like to thank Dr. Paula Ricciardi-Castagnoli for providing the murine microglial N9 cell line used in these studies. This work was supported by NIH grant NS049033 (JJW).