EP2 receptor activates dual G protein signaling pathways that mediate contrasting proinflammatory and relaxatory responses in term pregnant human myometrium.

Prostaglandin (PG) E2 (PGE(2)) plays a central role in the regulation of smooth muscle contractions. Classically, PGE(2) stimulates contractions via EP1 and EP3 receptors, whereas EP2 and EP4 maintain quiescence. Labor involves a change from myometrial quiescence to contractions with a shift from anti- to proinflammatory pathways. EP2, a Gαs-coupled receptor, is known to mediate its actions via cAMP signaling. However, we have recently shown that EP2 also activates the proinflammatory PG G/H synthase-2 (PGHS-2). Here, we identify the mechanism underlying the ability of EP2 to maintain uterine quiescence and activate a proinflammatory/prolabor response in term-pregnant human myometrium. Human myometrial biopsies for in vivo and in vitro studies were taken at cesarean section at term, before or after the onset of labor. Activation of EP2 increased intracellular levels of cAMP and reduced contractility. Contrastingly, EP2 stimulation increased levels of PGHS-2, membrane-associated PGE synthase-1, and PGE(2). This was entirely dependent on EP2-mediated activation of calcium signaling. Both calcium signaling and up-regulation of PGHS-2 were insensitive to the Gαi inhibitor pertussis toxin but inhibited by small interfering RNA knockdown of Gαq/11. There were no differences in EP2 mRNA or protein levels between upper or lower segment myometrium or between pre- and postlabor myometrium. However, in myocytes taken after the onset of labor, cAMP signaling was markedly attenuated, whereas activation of calcium and PGHS-2 was preserved. Overall, the dual coupling of EP2 to Gαs-cAMP and Gαq/11-calcium pathways underlies its ability to mediate contrasting functions in term pregnancy and the "switching" to a prolabor receptor.

The roles of prostaglandin EP 1 and 3 receptors in the control of human myometrial contractility.

CONTEXT: Prostaglandins are central to the processes of human labor. Prostaglandin E(2) (PGE(2)) synthesized within the uterus mediates cervical ripening and uterine contractions. PGE receptors, EP1 and EP3, may each mediate contractions, and represent potential therapeutic targets in the management of preterm labor. Studies of the expression and function of EP1 and EP3 in pregnant myometrium are inconsistent. OBJECTIVE: The objective of the study was to determine the relative importance of EP1 and EP3 in human myometrial contractility. DESIGN: We studied the expression of EP1 and EP3 in upper- and lower-segment myometrium at term in vivo and the effects of specific inhibitors on contractions in vitro. PATIENTS: Myometrial biopsies for both in vivo and in vitro studies were taken at cesarean section at term before or in labor in uncomplicated pregnancies. RESULTS: We found no differences in the expression of EP1 or EP3 at mRNA or protein level between the upper and lower segment myometrium and no overall changes associated with the onset of labor. Upon labor, EP1, but not EP3, was found to relocalize to the nucleus. In studies of contractility, we found no differences in spontaneous or PGE(2)-induced contractility between the upper- and lower-segment samples. Spontaneous contractions were inhibited by acetylsalicylic acid and were rescued by PGE(2). Although an EP1 antagonist, ZD6416, had no effect, an EP3 antagonist, L798106, inhibited both spontaneous and PGE(2)-induced contractions. CONCLUSIONS: EP3 is the primary receptor subtype that mediates PGE(2) induced contractility in human pregnant myometrium at term and represents a possible therapeutic target.

NF-κB regulates a cassette of immune/inflammatory genes in human pregnant myometrium at term.

The onset of human labour resembles inflammation with increased synthesis of prostaglandins and cytokines. There is evidence from rodent models for an important role for nuclear factor-κB (NF-κB) activity in myometrium which both up-regulates contraction-associated proteins and antagonizes the relaxatory effects of progesterone. Here we show that in the human, although there are no differences in expression of NF-κB p65, or IκB-α between upper- or lower-segment myometrium or before or after labour, there is nuclear localization of serine-256-phospho-p65 and serine-536-phospho-p65 in both upper- and lower-segment myometrium both before and after the onset of labour at term. This shows that NF-κB is active in both upper and lower segment prior to the onset of labour at term. To identify the range of genes regulated by NF-κB we overexpressed p65 in myocytes in culture. This led to NF-κB activation identical to that seen following interleukin (IL)-1ß stimulation, including phosphorylation and nuclear translocation of p65 and p50. cDNA microarray analysis showed that NF-κB increased expression of 38 genes principally related to immunity and inflammation. IL-1ß stimulation also resulted in an increase in the expression of the same genes. Transfection with siRNA against p65 abolished the response to IL-1ß proving a central role for NF-κB. We conclude that NF-κB is active in myocytes in both the upper and lower segment of the uterus prior to the onset of labour at term and principally regulates a group of immune/inflammation associated genes, demonstrating that myocytes can act as immune as well as contractile cells.