Abstract
The placenta is susceptible to diverse insults during human pregnancy. The expression of the protein N-myc down-regulated gene 1 (NDRG1) is regulated during cell proliferation, differentiation, and in response to stress. Nevertheless, the function of this protein in humans remains unknown. We tested the hypothesis that NDRG1 is up-regulated in hypoxic primary human trophoblasts and that NDRG1 modulates trophoblast response to hypoxia. We initially demonstrated that the expression of NDRG1 is enhanced in primary human trophoblasts exposed to hypoxia. Importantly, we found a similar increase in NDRG1 expression in placental samples derived from either singleton gestations complicated by intrauterine growth restriction or from dizygotic twin gestation where one twin exhibited growth restriction. Having established efficient lentivirus-mediated transfection of primary human trophoblasts, we overexpressed NDRG1 in trophoblasts, which resulted in enhanced trophoblast differentiation. In contrast, lentivirus-driven short interfering RNA-mediated silencing of NDRG1 diminished trophoblast viability and differentiation. Consistent with these results, NDRG1 reduced the expression level of p53 in trophoblasts cultured in standard or hypoxic conditions. Furthermore, NDRG1 expression was regulated by the activity of SIRT1 (Sir2-like protein 1), which promotes cell survival. Together, our data indicate that NDRG1 interacts with SIRT1/p53 signaling to attenuate hypoxic injury in human trophoblasts.
Highlights
Using high density oligonucleotide microarrays we have previously examined differences in gene expression between placental tissues from pregnancies complicated by IUGR versus matched normal placental tissues as well as from trophoblasts cultured under hypoxic or standard culture conditions [7, 8]
The Influence of Hypoxia on the Expression of N-myc down-regulated gene 1 (NDRG1) in Trophoblasts— Using application of our novel T-score software to a series of microarray data, we previously determined that NDRG1 is one of the transcripts that was markedly up-regulated in hypoxic trophoblasts [7, 8]
To assess whether or not the expression of NDRG1 exhibits an increase in hypoxia, in vivo we determined the expression of NDRG1 in placental villi derived from either pregnancies complicated by IUGR attributed to placenta hypoperfusion or healthy controls
Summary
Tissue Procurement—The study was approved by the institutional review board of Washington University in St. Human placental tissues for cell dispersal and culture as well as for RNA analysis were obtained from normal pregnancies after a vaginal or abdominal delivery (n ϭ 20). Construction of Lentiviral NDRG1 Expression Vectors and Lentiviral siRNA and Infection of Cultured 293T Cells and Primary Term Human Trophoblasts—Lentiviral vectors FCIV, FCY-si, and FSP-si were kindly provided by Dr Jeff Milbrandt (Washington University) and described previously [35]. The caspase cleavage product cytokeratin 18, a reliable marker of apoptosis, was detected after bovine serum albumin blocking using a mouse primary antibody (1:50, M30 Cytodeath mouse monoclonal antibody, Roche Diagnostics) incubated for 2 h at room temperature followed by an AlexaFluor 488-conjugated donkey anti-mouse IgG (1:100, Molecular Probes, Eugene, OR) The nuclei were stained by 4,6-diamidino-2-phenylindole (Sigma) at 100 ng/ml in PBS for 3 min. Cell viability was estimated based on a control curve where a range of primary trophoblasts (2, 4, 6, 12 ϫ 105 cells/well in a 24-well plate) were assayed with MTS to generate a viability curve (data shown), which was consistent with the manufacturer’s assessment that absorbance at 490 nm was linear with cell number
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