Sustained Release of Prostaglandin E₂in 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, Ca2+-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 Ca2+-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.

Prostaglandin E2 Impairs P2Y2/P2Y4 Receptor Signaling in Cerebellar Astrocytes via EP3 Receptors

“…Nevertheless, the effect of preincubation with PGE 2 was not necessary due to the simultaneous co-stimulation of astrocytes with PGE 2 and UTP reduced the calcium responses triggered by the nucleotide alone by 35%. These results were consistent with the regulatory influence of PGE 2 on P2Y signaling evident in macrophages and fibroblasts ( Ito and Matsuoka, 2008 ; Traves et al, 2013 ; Pimentel-Santillana et al, 2014 ).…”

“…We next examined whether PGE 2 modulated the migration of cerebellar astrocytes induced by UTP, as reported in macrophages and fibroblasts ( Pimentel-Santillana et al, 2014 ). In both assays used, transwell migration and wound-healing, PGE 2 attenuated the migration of astrocytes induced by UTP ( Figure 5 ).…”

“…In fact, in astrocytes in which nPKC was down-regulated by prolonged exposure to phorbol dibutyrate (PDBu) the UTP calcium responses were stronger than those obtained in control cells (results not shown). In macrophages and fibroblasts, the effect of PGE 2 was dependent on PKC and PKD activation, although it was also independent of EP receptors ( Traves et al, 2013 ; Pimentel-Santillana et al, 2014 ).…”

“…The same cross-talk between PGE 2 and P2Y signaling has been described in murine fibroblasts, suggesting that macrophages and fibroblasts contribute to the regulation of inflammatory responses and tissue damage repair through aligned mechanisms associated with P2Y signaling. Such cross-talk may open new avenues to develop future anti-inflammatory therapies ( Pimentel-Santillana et al, 2014 ).…”

The role of ATP signalling in response to mechanical stimulation studied in T24 cells using new microphysiological tools

“…The pattern of the immunoreactivity for P2X3 and P2Y 6 seen in this paper indicates that a significant part of receptors is located intracellularly; this might indicate that the cells have a continuous overexpression of these receptors which would not be surprising as the cells are derived from a urothelial cancer. Worth mentioning is that the distribution of immunoreactivity is similar to that of others who have stained P2Y receptors in cell cultures . As a counterstain, we chose to use actin‐binding phalloidin rather than antibodies against cytokeratins.…”