| Popis: |
Hypoxia is critically important to the development of the embryo and placenta. Proper placental development is critical for normal fetal growth and embryonic survival. Abnormal placental development has been implicated in numerous obstetric complications, including preeclampsia, which affects about 7% of all pregnancies and can be fatal for both mother and baby. Rodent and murine trophoblast stem cells differentiate into three distinct cell lineages: giant cells, spongiotrophoblasts, and labyrinthine cells, which form different layers and have different functions within the placenta. Recent studies in our laboratory have focused on the invasive giant cell layer using the rodent Rcho-1 choriocarcinoma stem cell-like cell line, which has been shown to be committed to differentiate into the giant cell lineage. It has been shown that chronic exposure to hypoxia inhibits Rcho-1 trophoblast differentiation. One factor that has been extensively studied in the field of hypoxia are Hypoxia-Inducible Factors (HIFs), conserved, heterodimeric proteins that bind to DNA under hypoxic conditions to up regulate oxygen-dependent gene transcription. Whereas HIF-B(ARNT) is ubiquitously expressed and stable, HIF-a is subject to rapid turnover in normoxia and has been the major focus in studies examining HIF regulation. Although HIF-1a has been widely studied, few studies have been done on the function of HIF-2a in the regulation of trophoblast differentiation. Studies in our laboratory sought to characterize the mechanism of giant cell differentiation and placental formation and have recently focused on HIF-1a. However, it is possible that HIF-1a and HIF-2a play complementary roles in controlling trophoblast differentiation and placental formation in vivo, therefore this study examined the role of HIF-2a in Rcho-1 trophoblast differentiation. Previous studies examined the transcriptional activity of HIF-1a in response to hypoxia in the Rcho-1 trophoblast placental stem cell line, using a conventional luciferase dual-reporter assay. However, it was consistently observed that the level of activation of the constitutive reporter was significantly higher in hypoxic samples than in normoxic controls; therefore, this study also examined the role of HIF-1a in the unexpected induction of luciferase constitutive reporters after exposure to hypoxia, using a more reliable transfection method. The results of this study indicate that HIF-2a protein is not detectable in Rcho-1 cells, even after exposure to hypoxia. This study also found that the hypoxic induction of luciferase constitutive reporters was a phenomenon independent of species or cell-type and sequence analysis revealed that hypoxic induction of the luciferase constitutive reporters was independent of HIF-1a. Sequence analysis also revealed the consensus sequences for several other transcription factor binding sites, including steroid hormones and NF-kB. Finally, this study demonstrates a more reliable method of controlling for transfection efficiency that negates the need for luciferase constitutive reporters, avoiding potential error caused by their hypoxic induction. |
