Single-cell analysis of prostaglandin E2-induced decidual cell differentiation: does extracellular 8-Br-cAMP cause artifacts?

Abstract

Development of the uterine decidua, the transient maternal tissue contacting the fetus during extended gestation, is the hallmark of reproduction in many placental mammals. Differentiation of decidual stromal cells is known to be induced by stimuli that activate the nuclear progesterone receptor and the cyclic AMP/protein kinase A (cAMP/PKA) pathways. The nature of the stimulus upstream of PKA has not been clearly defined, although a number of candidates have been proposed. To bypass this uncertainty for in vitro experiments, direct addition of membrane-permeable cAMP along with progestin has been the prevailing method. Phylogenetic inference suggests that the inflammatory eicosanoid prostaglandin E2 (PGE2) was the stimulus that ancestrally induced decidualization. Accordingly, we developed a protocol to decidualize human endometrial stromal fibroblasts using progestin and PGE2 and analyzed the response in comparison with a cAMP-based protocol. Transcriptomic comparison reveals a common activation of core decidual cell genes between both treatments, and a set of senescence-related genes exaggerated under cAMP treatment. Single-cell transcriptomic analysis of PGE2-mediated decidualization revealed a major transcriptomic transition between an early activated cell state and a differentiated decidual state, but notably did not identify a developmental trajectory representing a distinct senescent decidual state as reported in recent literature. Furthermore, investigation of the signal transduction process underlying PGE2-mediated decidualization showed that it depends upon progestin-dependent induction of PGE2 receptor 2 (PTGER2 aka EP2) and PKA, the kinase activated by PTGER2. This progesterone-dependent induction of PTGER2 is absent in the opossum, a species incapable of decidualization. Together, these findings suggest that the origin of the decidual cell type involved the evolution of progesterone-dependent activation of the PGE2/EP2/PKA axis. We propose the use of PGE2 for in vitro decidualization studies as a potentially more physiological model than 8-Br-cAMP.