Notably we demonstrated that EP EP
Notably we demonstrated that EP2/EP4 receptor-coupled activation of the cAMP signaling pathway accounted for both up-regulation of COX-2 and down-regulation of LOX, which is consistent with the roles of the cAMP signaling pathway in regulation of COX-2 and LOX by PGE2 in a number of other tissues or cell types (Choung et al., 1998, Steinert et al., 2009). Previously we showed that the up-regulation of COX-2 and cytosolic phospholipase A2 (cPLA2), another rate-limiting enzyme in the PGE2 synthesis cascade under wehi control, is also mediated by activation of the cAMP signaling pathway in human amnion fibroblasts (Zhu et al., 2009, Guo et al., 2010). In addition, activation of the cAMP signaling pathway in the placenta and myometrium has also been reported to be associated with a number of important events during parturition. In the placenta, activation of the cAMP signaling pathway leads to up-regulation of both corticotropin-releasing hormone (CRH) and aromatase expression (Cheng et al., 2000, Wang et al., 2012). Secretion of CRH by human placenta is believed to be associated with the timing of labor onset (McLean et al., 1995). Due to lack of P450c17, human placenta must rely on aromatase for the production of estrogen (Siiteri and MacDonald, 1966) which is known to prime the myometrium for contraction prior to parturition (Mesiano, 2001). Although there is a strong evidence to suggest that the cAMP pathway acts in synergy with the cGMP pathway during the relaxation of smooth muscle (Ishikawa et al., 1993), a recent study has demonstrated that PKA anchored in the nucleus phosphorylates transcription factors leading to increased expression of proteins associated with contraction of the myometrial smooth muscle towards the end of gestation (Fetalvero et al., 2008, Taggart et al., 2008). Previous studies using placenta (Cheng et al., 2000, Wang et al., 2012), myometrium (Fetalvero et al., 2008, Taggart et al., 2008) and amnion (Zhu et al., 2009, Guo et al., 2010) in addition to this study, point to a crucial role of activation of the cAMP signaling pathway in the rupture of fetal membranes as well as parturition in humans. There are at least four PGE2 receptor subtypes, EP1-EP4, that have been recognized so far (Narumiya et al., 1999). Despite of identification of all four PGE2 receptor subtypes in human fetal membranes (Grigsby et al., 2006), we found that EP2/EP4 rather than EP1/EP3 receptor subtypes mediated the regulation of LOX by PGE2 in human amnion fibroblasts based on data showing no effects of EP1/EP3 receptor agonists and antagonists on LOX expression and the failure of PKC inhibitors to block PGE2 inhibition of LOX expression. Neither EP2 nor EP4 blockade alone completely blocked the reduction of LOX expression by PGE2, blockade of both EP2 and EP4 receptors was required. Despite having similar nominal functions, the signaling pathway triggered by EP2 and EP4 receptors show different durations of response. Nishigaki et al. reported that responses mediated via EP4 receptor were short-lived, whereas responses mediated via EP2 receptor had a longer duration (Nishigaki et al., 1996), suggesting that activation of the cAMP/PKA pathway by EP2 and EP4 may compensate for each other resulting in long-lasting attenuation of LOX expression by PGE2. It should also be kept in mind that pharmaceutical agents as used in this study to analyze the mediating prostanoid receptors can sometimes have off-target actions which may lead to inaccurate conclusions. To avoid the possible inaccuracy, we performed comprehensive analysis of the information gathered with both agonists and antagonists to the receptors as well as the signaling pathway coupled with the receptors. Since the effects of siRNA-mediated knock-down of EP2/EP4 receptors were consistent with the antagonists, we believe that the conclusions derived from these comprehensive analyses are reliable and accurate. In summary, we have identified an alternative feed-forward mechanism by which PGE2 promotes the rupture of fetal membranes (Fig. 8). PGE2 attenuates the expression of LOX in human amnion fibroblasts thus promoting uncross-linking of collagen fibrils, which can be further amplified by simultaneous induction of COX-2 by PGE2. Uncross-linked collagen fibrils may not only reduce the tensile strength of the amnion but also predispose the fibrils to degradation by MMPs. Moreover, both of the reduction of LOX and induction of COX-2 by PGE2 are mediated by EP2/EP4 receptor-coupled activation of the cAMP/PKA signaling pathway in human amnion fibroblasts.