PKA and EPAC: cyclic AMP effectors in the regulation of myometrial inflammatory responses in parturition

Abstract

The timing of parturition is key to a successful conclusion of pregnancy. For births that occur before 37 weeks, which is defined as preterm birth (PTB), the baby is at increased risk of neonatal morbidity and mortality. PTB remains a significant public health concern, affecting around 1 in 10 births worldwide. The initiation of parturition is caused by an accumulation of factors that promote myometrial contractions, and inflammation is often proposed to be one such factor. At the same time, it is also proposed that a withdrawal of signalling pathways that support myometrial quiescence is sufficient to trigger parturition. Progesterone is most recognised for this role in maintaining myometrial quiescence. There is also ample evidence to suggest that cAMP signalling also plays a role. Recent published studies have suggested that PKA can enhance the effects of progesterone and suppress myometrial inflammatory responses. Additionally, a pro-contractile role for cAMP via EPAC-mediated pathways has also been proposed. The overall aim for this thesis was to investigate the role of cAMP in the regulation of myometrial inflammatory responses in the context of parturition. Using primary myometrial cells cultured from human myometrial biopsies, which were all obtained from term gestation non-labouring study participants at time of elective/planned Caesarean section, my studies demonstrated that cAMP’s anti-inflammatory effects are mediated by PKA. PKA-specific activator N6-benzoyl-cAMP (6-Bnz-cAMP) suppressed myometrial expression of IL-8 in response to IL-1β stimuli. Furthermore, siRNA knockdown of catalytic PKA subunit (PKAc) partially reduced forskolin’s ability to suppress IL-8 expression. My data also showed forskolin’s ability to reduce NF-κB translocation is lost in the event of PKAc knockdown. The role of EPAC was investigated using EPAC activator 8-pCPT-2'-O-Me-cAMP and inhibitor ESI-09. My experiments showed EPAC activation was able to increase phosphorylation of MEK1/2, and downstream p65 Ser 536 phosphorylation. However, this did not further increase IL-8 expression. Most interestingly, the EPAC inhibitor reduced extracellular IL-8 concentrations but increased levels of IL-8 mRNA and intracellular protein, which suggests EPAC has a role in regulating IL-8 secretion. Finally, using a genetically modified hTERT-HM myometrial cell line that can express inducible progesterone receptor A (PR-A) and B (PR-B), I examined the impact of EPAC signalling on PR-A phosphorylation and total protein abundance. Using 8-pCPT-2'-O-Me-cAMP, the findings from my data suggested that an EPAC-mediated increase in JNK activity can result in both an increase in PR-A Ser 345 phosphorylation and total protein levels. This suggests EPAC can promote PR-A dominance, which is a well-recognised feature of the progesterone functional withdrawal concept that is often used to explain how human parturition is initiated. In summary, the work presented here provides evidence that PKA and EPAC have distinct contributions to myometrial inflammatory response at cellular level. Further study of how their roles coordinate with each other to control the dynamics of myometrial cAMP signally will add depth to our understanding of the molecular processes that support myometrial quiescence or enhance myometrial contractility.