Sustained release of prostaglandin E₂ in fibroblasts expressing ectopically cyclooxygenase 2 impairs P2Y-dependent Ca²⁺-mobilization

Abstract

The nucleotide uridine trisphosphate (UTP) released to the extracellular milieu acts as a signaling molecule via activation of specific pyrimidine receptors (P2Y). P2Y receptors are G protein-coupled receptors expressed in many cell types. These receptors mediate several cell responses and they are involved in intracellular calcium mobilization. We investigated the role of the prostanoid PGE2 in P2Y signaling in mouse embryonic fibroblasts (MEFs), since these cells are involved in different ontogenic and physiopathological processes, among them is tissue repair following proinflammatory activation. Interestingly, Ca(2+)-mobilization induced by UTP-dependent P2Y activation was reduced by PGE2 when this prostanoid was produced by MEFs transfected with COX-2 or when PGE2 was added exogenously to the culture medium. This Ca(2+)-mobilization was important for the activation of different metabolic pathways in fibroblasts. Moreover, inhibition of COX-2 with selective coxibs prevented UTP-dependent P2Y activation in these cells. The inhibition of P2Y responses by PGE2 involves the activation of PKCs and PKD, a response that can be suppressed after pharmacological inhibition of these protein kinases. In addition to this, PGE2 reduces the fibroblast migration induced by P2Y-agonists such as UTP. Taken together, these data demonstrate that PGE2 is involved in the regulation of P2Y signaling in these cells.

Figure 1

PGE₂ released in MEFs overexpressing COX-2 and effect on P2Y-dependent Ca²⁺-mobilization. WT and KI (COX-2-deficient MEFs overexpressing COX-2) MEFs, treated in the absence or presence of LPS (200 ng/mL) plus cytokines (IFN-γ, TNF-α, and IL-1β, 20 ng/mL), were used. The protein levels of COX-1 and COX-2 and the PGE₂ released into the culture medium were determined by immunoblot and ELISA, respectively (a). The percentage of cells showing Ca²⁺-mobilization in response to the P2Y agonist UTP (100 μM) was determined using the nonratiometric Fluo-4 assay (b). Results show a representative blot (a) and the mean + SD of three experiments for release of PGE₂ to the culture medium and Ca²⁺-mobilization. *P < 0.05, **P < 0.001 versus the corresponding control.

Figure 2

Characterization of targeting of PKC, PKD, and PKA on the effect of PGE₂ on the UTP-dependent Ca²⁺-mobilization. WT or KI MEFs were washed with fresh medium and maintained in culture for 1 h to remove PGE₂ accumulated and then treated for 10 min with the indicated effectors, except for DFU that was added immediately after washing (1 μM DFU, an inhibitor of COX-2; 5 μM PGE₂; 100 nM staurosporine; 100 nM Gö6976; 5 μM Gö6850; 10 nM Gö6983, a selective inhibitor of classic PKCs; 200 nM PDBU; 200 nM αPDD; 200 nM CID755376, a selective inhibitor of PKD; 5 μM dibutyryl cAMP) and the percentage of cells showing Ca²⁺-mobilization in response to UTP (100 μM) was determined using the nonratiometric Fluo-4 assay. Results show the mean + SD of three experiments for Ca²⁺-mobilization. *P < 0.05, **P < 0.001 versus the same condition in the WT cells.

Figure 3

Subcellular distribution of P2Y2, P2Y4, and P2Y6 receptors in MEFs. WT or KI MEFs were cultured and, after changing the medium, were maintained in the absence or presence of 1 μM DFU for 2 h. Cells were fixed with paraformaldehyde (4%; pH 7.2) and permeabilized with cold methanol at RT. After incubation with anti-P2Y2, anti-P2Y4 and anti-P2Y6 antibodies (1 : 500) overnight at 4°C, cells were visualized by confocal microscopy using a FITC-conjugated secondary Ab (Alexa-Fluor 488, 1 : 1000). Nuclei were stained with Hoechst 33258. Coverslips were mounted in Prolong Gold antifade reagent (Molecular Probes) and the intensity of the fluorescence was measured using Image J software (NIH, Bethesda, MD, USA). Results show the mean + SD of three experiments. **P < 0.001 versus the same condition in the WT cells.

Figure 4

Characterization of EP1–4 and P2Y2–P2Y4 expression and effect of ionophores on Ca²⁺-mobilization in MEF cells. The expression levels of the prostaglandin receptors EP1–4 and the levels of P2Y2 and P2Y4 were determined by qPCR (a-b). The response to 1 μM ionomycin (c) and 500 nM thapsigargin (d) on Ca²⁺-mobilization was determined in MEFs overexpressing COX-2, using the dual excitation 340/380 nm protocol as described in Section 2. MEFs KI were washed with fresh medium to remove PGE₂ accumulated and maintained in the absence or presence of 1 μM DFU and 5 μM PGE₂. Different extracellular concentrations of calcium were used. Results show the mean + SD of three experiments (a-b) or a representative trace (c-d). *P < 0.05 versus the same condition in WT cells.

Figure 5

Effect of thapsigargin on Ca²⁺-mobilization and migration of MEFs in response to UTP and chemotactic stimuli. WT or COX-2 KI cells were activated, or not, for 24 h with LPS (200 ng/mL) plus cytokines (IFN-γ, TNF-α, and IL-1β, 20 ng/mL) and then treated for 5 min with 500 nM thapsigargin and in the absence or presence of 5 μM PGE₂. The levels of the indicated phosphoproteins were determined by Western blot (a). The capacity of these cells to migrate in transwell was determined after incubation with 5 μM PGE₂ and/or 100 μM UTP. The migration was measured after 24 h of incubation in the absence or presence of different combinations of 10% FBS, PGE₂ (5 μM), or UTP (100 nM) in the lower wells (b). Results show a representative blot (a) out of three or the mean + SD of four experiments (b). *P < 0.05; **P < 0.001 versus the same condition in the absence of treatment in the upper chamber.