The role of proline rich 15 in trophoblast cell development

Abstract

Maintenance of pregnancy in eutherian mammals requires a sophisticated and tightly regulated program of gene expression in order to develop a fully functional placenta. This transient organ mediates nutrient and gas exchange between the mother and fetus while protecting the fetus from the maternal immune system. Deviations from the normal regulation of gene expression during early pregnancy can lead to early embryonic loss as well as dysfunctional placentation, which can cause significant maternal and fetal morbidity and mortality. Proline rich 15 (PRR15) is a low molecular weight nuclear protein expressed by the trophoblast during early gestation in several mammalian species, including humans, mice, cattle, sheep, and horses. Immunohistochemistry revealed localization of PRR15 to the trophectoderm and extraembryonic endoderm of day 15 sheep conceptuses. In humans, PRR15 is localized in the nuclei of both first and second trimester trophoblast cells. Additional research has shown increased PRR15 transcription in colorectal cancers with mutations in the adenomatous polyposis coli (Apc) protein, suggesting a link to the Wnt signaling pathway. PRR15 mRNA concentrations increase when trophoblast cells, both sheep (oTR) and human (ACH-3P), are cultured on Matrigel, a basement membrane matrix. The expression profile in the sheep conceptus during pregnancy revealed a rise in PRR15 mRNA concentrations during the period of conceptus elongation with a peak in expression at day 16 of gestation, followed by a decline to day 30 of gestation. This peak coincides with a halt in elongation of the conceptus, and the initial period of apposition to the uterine luminal epithelium. Lentiviral-mediated knockdown of PRR15 in ovine trophectoderm at the blastocyst stage led to demise of the embryo by day 15 of gestation. This provides compelling evidence that PRR15 is a critical factor during this precarious window of development when initial attachment and implantation begin. The first aim of this research was to determine the effect of PRR15 deficiency on trophoblast gene expression, as well as trophoblast proliferation and survival. The human first trimester trophoblast cell line, ACH-3P, was infected with control lentivirus (LL3.7) and lentivirus expressing a short hairpin (sh)RNA to target PRR15 mRNA for degradation, resulting in a 68% decrease in PRR15 mRNA (p<0.01). Microarray analysis of these cell lines revealed differential expression of genes related to cancer, focal adhesion, and p53 signaling. We selected 21 genes for validation of mRNA levels by quantitative real-time RT-PCR, 18 (86%) of which gave results consistent with the microarray analysis, with similar direction and magnitude fold changes. This included significant up-regulation of GDF15, a cytokine increased in pregnancies with preeclampsia. GDF15 mRNA concentrations were examined more extensively during early ovine gestation, which revealed that GDF15 was low during peak PRR15 expression, then increased significantly at day 30 when PRR15 was nearly undetectable. Proliferation, as measured by cell metabolic activity and bromodeoxyuridine (BrdU) uptake, decreased in the PRR15-deficient cells, which was consistent with a decrease observed in cell cycle-related genes CCND1 and CDK6, and an increase in CCNG2 and CDKN1A in the PRR15-deficient cells. TNFSF10, a tumor necrosis factor superfamily member known to induce apoptosis, and its receptor, TNFRSF10b, increased significantly in the PRR15-deficient cells, suggesting trophoblast cells may be more susceptible to apoptosis when depleted of PRR15. Assays for caspase activity and annexin V staining revealed an increased population of apoptotic cells when treated with shRNA to target PRR15. These results suggest that PRR15 is required for driving trophoblast proliferation and survival during early development of the placenta, functions that are critical to early embryonic survival and successful placentation. The second experimental aim was to examine regions of the PRR15 promoter that are necessary for regulating its expression in trophoblast cells and to identify the role of Wnt signaling in PRR15 transcription. The 5'-flanking sequences from -824, -640, -424, -326, and -284 bp to +7 bp relative to the annotated transcription start site were amplified by PCR and ligated into the pGL3-Basic plasmid. These vectors were co-transfected into the first trimester human trophoblast cell line, ACH-3P, HT29 (human colorectal carcinoma), oTR, and BHK-21 (hamster kidney fibroblast) cells with a RSV-β-galactosidase vector control. In ACH-3P cells, transactivation of the luciferase reporter was maximal following transfections with the -326 construct (15.4 ± 4.8-fold). Significant promoter activity was absent in the -284, -424, and -640 constructs, but regained with the -824 construct (14.8 ± 5.8-fold). These results suggest that cis-acting elements within the proximal promoter of the PRR15 gene are essential for expression in trophoblast cells, requiring the regions from -284 to -326 and -640 to -824. DNase I footprinting and electrophoretic mobility shift assays were performed to identify transcription factor binding sites within these regions. Due to the potential link to the Wnt signaling pathway, cells were treated with an inhibitor to GSK3β, the kinase responsible for phosphorylation and proteasomal degradation of β-catenin. Inhibition of GSK3β decreased PRR15 mRNA concentrations and decreased transactivation of the luciferase reporter in all proximal promoter reporter constructs; this effect was mediated through β-catenin activity in the proximal 284 bases of the PRR15 5'-flanking region. Furthermore, trophoblast cell proliferation decreased after treatment with the GSK3β inhibitor. Electrophoretic mobility shift assays on the region from -98 to -68 revealed differential binding of nuclear proteins derived from ACH-3P cells grown in the presence or absence of the GSK3β inhibitor. These results reveal that canonical Wnt signaling inhibits the transcription of PRR15, mediated in part through the -98 to -68 region of the 5'-flanking region, and decreases proliferation in trophoblast cells. This indicates that suppression of Wnt signaling may be crucial during early trophectoderm outgrowth in order to allow significant transcriptional activation of PRR15 and conceptus survival.