Prostaglandin E2 activates complement protein CD55 to enhance cell adhesion in endometriosis

Abstract

Endometriosis is a leading cause of pelvic pain and infertility. Prostaglandin E2 (PGE2) is widely regarded to be central to its pathogenesis.1 A promising novel target of PGE2 signaling is decay accelerating factor (CD55) which has increased RNA expression in endometriotic stromal cells.2 CD55 has a well described role in the complement pathway, but emerging evidence indicates other functions in cell proliferation and activation of cellular signaling. The objective of this work was to determine whether CD55 protein is expressed in ectopic endometrium, whether PGE2 is a regulator of CD55 expression, and whether CD55 is necessary and sufficient for endometriosis pathogenesis via a non-canonical pathway. Laboratory investigation. To study the principal cellular compartments of endometriotic lesions in vitro, immortalized endometrial stromal (T-HESC) and endometriotic epithelial (12Z) cell lines were studied. Immunohistochemistry was performed to localize the presence of CD55 in a random sample of eight human pathology specimens of eutopic and ectopic endometrium. CD55 expression analysis was performed on cell lines (RNA expression by quantitative real-time PCR; protein expression by immunoblotting and flow cytometry) treated first with 0, 1, 3, 10, 30, and 100 uM of PGE2 then with 10uM PGE2 for 0, 1, 2, 6, and 24 hours. Overexpression of CD55 in 12Z cells was achieved using lentiviral transduction with an empty vector control. Cell adhesion was determined by plating cells on cell culture plates coated with fibronectin, collagen I, or bovine serum albumin at different time points, washing the plates, then quantifying remaining adherent cells using a luminescent ATP detection assay. Intra- and inter-experimental variation was addressed by multiple wells per condition and ≥2 replicates per experiment. Both 12Z and T-HESC lines demonstrated CD55 expression at baseline, as did histologic specimens of eutopic and ectopic endometrium. Treatment with PGE2 of 12Z and T-HESC lines resulted in increased CD55 expression in a dose dependent fashion until maximal treatment effect (3-fold) was achieved at 10 uM. In time course experiments, CD55 RNA expression was maximal at 6 hours and protein expression at 18 hours. 12Z cells overexpressing CD55 demonstrated more than 2-fold number of adherent cells as compared to wildtype and empty vector controls. These preliminary cellular studies indicate that PGE2 induction of CD55 can lead to promotion of adhesion of endometriotic cells, a necessary step in the pathogenesis of endometriosis. Future in vivo studies using CD55-/- mice will investigate CD55 function in adhesion in mouse models of endometriosis. Understanding this downstream pathway of PGE2 signaling may identify points of fragility for future translational therapies.