Transcription factors E2F1 and E2F3 are expressed in placenta but do not regulate MMP14
Introduction
Preeclampsia (PE) is a serious complication of pregnancy. It is a multi-system disorder affecting the maternal vasculature, kidneys, liver, hematological system and fetoplacental unit. It can also affect the nervous system causing cerebral edema, seizures, and rarely death. Currently the only efficacious treatment for preeclampsia is delivery of the placenta and baby, which imposes risks of disability and death on the baby if the disease is severe and early onset (less than 34 weeks gestation) [1].
Soluble fms-like tyrosine kinase-1 (sFlt-1) and soluble endoglin (sEng) are anti-angiogenic factors that play a key role in causing endothelial dysfunction seen in preeclampsia [2]. Both factors are increased in women with preeclampsia, and levels correlate with disease severity [1]. Previous studies have demonstrated that whilst over-expression of sFlt1 alone in pregnant rats produced hallmarks of preeclampsia including hypertension and proteinuria, adenoviral co-expression of sEng and sFlt1 in combination phenocopied severe preeclampsia, with hemolysis, liver dysfunction, thrombocytopenia, cerebral edema and fetal growth restriction observed [1], [2]. Such studies highlight the critical role of sEng in severe preeclampsia.
In 2010, Hawinkels [3] demonstrated using an over-expression system in COS-7 cells that matrix metalloproteinase 14 (MMP-14; MT1-MMP) cleaved membrane bound endoglin to produce sEng [3]. We subsequently published data to demonstrate that the same mechanism occurs in placental tissue [4].
Intriguingly, although MMP14 is believed to play important roles in cancer development and metastasis, its regulation is still poorly understood. Given the lack of treatment options for preeclampsia, an understanding of the signaling pathways governing the release of sEng may uncover new therapeutic targets.
The E2Fs are a large family of transcription factors containing conserved DNA binding domains that bind promoters to regulate gene expression [5]. Whilst E2F transcription factors are most commonly known for their role in regulating the mammalian cell cycle, recent studies have suggested they also play important roles in development, differentiation, apoptosis and DNA repair [6], [7], [8]. The promoters of MMPs, including MMP-14, contain multiple E2F-binding sites. Excitingly, a recent report has shown that E2F transcription factors directly regulate MMPs in metastatic disease by binding to their promoter regions [9]. More specifically, in non-small cell lung cancer cells, MMP-9, -14 and −15 expression were up regulated by E2F binding to the promoter, and E2F1 and E2F3 inhibition significantly reduced MMP-14 mRNA expression. Together, these data provide clear evidence for a role of E2Fs in regulating MMP expression.
Given our recent data [4] to demonstrate that MMP14 is a key protease involved in the secretion of sEng from placenta, we sought to assess whether E2F transcription factors are present in placenta, altered in preeclampsia and whether they regulate MMP14, and thus sEng release from primary human trophoblast and endothelial cells.
Section snippets
Tissue collection
Women presenting to the Mercy Hospital for Women gave informed written consent for placental tissue collection. Placenta was obtained from preterm pregnancies not complicated by PE (n = 18) and those complicated by severe early-onset PE (n = 28). Severe preeclamptics were diagnosed in accordance with ACOG guidelines and included the presence of hypertension >160/110 on two occasions greater than 6 h apart, proteinuria >5 g/day, oliguria <500 ml/day, visual disturbance, pulmonary edema, right
E2F1 and E2F3 are expressed in placenta
Given that E2F transcription factors have not been characterized in placenta, we initially set out to examine their localization in severe preeclamptic (PE) and preterm (PT) control placenta by immunohistochemistry (Fig. 1A, B). Consistent with their roles as transcription factors, both E2F1 and E2F3 localized to the nuclei of the syncytiotrophoblast and stromal cells of the villous tips in placental sections. Nuclear staining was also observed within the endothelial cells lining the villous
Discussion
We have previously identified MMP14 as a key protease involved in the release of sEng from placenta [4]. However, the molecular mechanisms regulating MMP14, and thus sEng release, remain elusive. In this study, we assessed E2F transcription factors that are known to regulate MMP14 in cancer [9]. We demonstrate that both E2F1 and 3 are expressed in placenta, and that E2F1 is down-regulated in preeclampsia. Whilst we found no effect of E2F silencing on MMP14 expression in primary trophoblast, our
Sources of funding
The National Health and Medical Research Council of Australia provided salary (#1062418 to T.K, #628927 to N.H, #1050765 to S.T.) and project support (#1048707).
Acknowledgments
The authors acknowledge Clinical Research midwives Gabrielle Pell, Debra Jinks, Rachel Murdoch and Genevieve Christophers and the Obstetrics midwifery staff and patients at the Mercy Hospital for Women (Heidelberg) for their provision of placental tissue.
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