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  • EP is the major receptor to


    EP4 is the major receptor to mediate PGE2-induced inflammatory pain. EP4 is upregulated by NVP-BKM120 Hydrochloride australia in DRG neurons [16]. Intrathecal injection of the EP4 agonist produced pain responses [25]. Either systemic or local injection of EP4 antagonists relieves inflammatory pain [3], [16], [26]. However, the mechanisms of how EP4 mediates PGE2-induced inflammatory pain are poorly understood. Coupled with stimulatory Gs protein, activated EP4 increases the intracellular cyclic adenosine monophosphate (cAMP), a main signalling event responsible for PGE2-induced nociceptor sensitization [5], [28], [39], suggesting a role for EP4 in PGE2-induced sensitizing effects. Cell-surface receptor abundance controls receptor sensitivity and is dynamically regulated by receptor trafficking events. Externalization and recycling increase while internalization and degradation decrease receptor surface density. Mounting evidence shows that nociceptive receptor trafficking plays important roles in the genesis of pathological pain conditions. For example, a rapid surface insertion of GluR1 subunit of AMPA (α-amino-3-hydroxy-5-methyl-4-isoxazole propionate) receptor induced by intracolonic injection of capsaicin contributes to inflammatory pain [7]. CGRP was shown to sensitize trigeminal neurons by enhancing the externalization of purinergic P2X3 receptors [6]. Nerve growth factor (NGF) sensitized capsaicin receptor transient receptor potential V1 (TRPV1) channel by facilitating its surface insertion [52]. In DRG neurons, mustard oil sensitized its receptor TRPA1 channel by stimulating its externalization [36]. On the other hand, cannabinoid-induced internalization of CB1 receptor contributes to its analgesic tolerance [46]. All these observations imply that trafficking of nociceptive receptors contributes to the genesis of pathological pain states and analgesic tolerance. However, it is unknown whether cell-surface EP4 abundance can be modulated by its agonists or noxious stimuli and whether EP4 cell-surface trafficking contributes to PGE2-induced nociceptor sensitization and inflammatory pain. To address these issues, our first aim was to determine whether PGE2 induces EP4 externalization in cultured DRG neurons. The second aim was to investigate if activation or blockade of EP4 affects this event. Our third aim was to dissect out the roles of anterograde secretory pathway, protein synthesis, and recycling in this event. The fourth aim was to address whether PGE2-facilitated EP4 externalization is coupled with augmented EP4 activity. The last aim was to determine if inflammation increases EP4 cell-surface levels of DRG neurons. Some data have been presented in the form of an abstract [41].
    Materials and methods
    Ethics of animal experiments
    Conflict of interest
    Acknowledgements The current study was supported by the operating grants from the Canadian Institutes of Health Research (RFN. 89892) and the Discovery grant from Natural Science and Technology Research Council of Canada (RFN. 356021) to Weiya Ma.
    Introduction Preterm birth affects 5%–18% of pregnancies (Blencowe et al., 2012) and is the leading cause of neonatal death and second leading cause of childhood death below age of 5 years (Liu et al., 2012). One third of preterm labor is associated with premature rupture of the fetal membranes (PROM) (Slattery and Morrison, 2002). The human fetal membranes are composed of amnion and chorion layers. While the chorion is thicker, the amnion is significantly stronger and provides greater tensile strength for the fetal membranes (Oyen et al., 2006), as the mesenchymal layer underneath the amnion epithelium contains a compact layer of collagen-rich extracellular matrix (ECM) and abundant collagen-producing fibroblasts (Casey and MacDonald, 1996). These cross-linked collagen fibrils not only provide tensile strength but also resistance to degradation by matrix metalloproteinases (MMPs) (Buerzle et al., 2013, Stuart et al., 2005). One of the key events leading to the rupture of fetal membranes is the breakdown of uncross-linked collagen fibrils in the ECM of the amnion. Cross-linking of collagen fibrils is catalyzed by lysyl oxidase (LOX), a copper-dependent amine oxidase, expressed and secreted by fibrogenic cells (Kagan and Trackman, 1991). Lysyl oxidase oxidizes the specific lysine residues in collagen to peptidyl α-aminoadipic-δ-semialdehyde (AAS), after which peptidyl aldehyde condenses with the vicinal amino group or peptidyl aldehyde of the neighboring collagen fibrils to cross-link them to each other (Pinnell and Martin, 1968). Despite the apparent crucial role of LOX in the maintenance of the tensile strength of collagen fibrils, previous studies of fetal membrane rupture have been mostly directed to the role of MMPs (McLaren et al., 2000, Xu et al., 2002) with only a few studies addressing the role of LOX (Casey and MacDonald, 1997). Casey et al. demonstrated that LOX protein and enzymatic activity decrease dramatically with advancing gestational age in the amnion (Casey and MacDonald, 1997), however the factors initiating the down-regulation of LOX in amnion remain unknown. To truly understand the role of LOX in the maintenance of fetal membrane integrity requires identification of these factors.