Investigations of cellular stress-responsive proteins and PGF gene expression in human trophoblast

Author

Jonathan Dean Leavenworth, Southern Illinois University CarbondaleFollow

Date of Award

5-2009

Degree Name

Doctor of Philosophy

Department

Molecular Biology, Microbiology and Biochemistry

First Advisor

Torry, Donald

Abstract

Placenta growth factor (PGF) expression is downregulated in preeclampsia (PE), a leading cause of maternal morbidity and mortality. The pathophysiology of PE is thought to be manifested by a poorly perfused placenta hampered by hypoxic stress. Two stress-mediate angiogenic responses include post-transcriptional regulation of mRNA stability, and regulation of PML sequestering protein. We investigate whether these mechanisms occur in hypoxic stressed trophoblast and preeclamptic placenta. Methods: To determine transcript stability, PGF mRNA was measured in normal vs. stressed conditions, and the PGF 3'UTR was analyzed for consensus 3'AREs. To characterize stability regulation, the PGF 3'UTR was cloned into a reporter construct. To investigate the association between PGF mRNA and RNA binding proteins, an RNA-Immunoprecipitation assay was performed on trophoblast. PML study: Normal (n=6) and preeclamptic (n=6) placentae were assessed for PML expression. Immunoblot, qRT-PCR and immunohistochemistry techniques were used to determine PML gene expression and localization in placental tissue and primary cells. Results: Two consensus ARE motifs were detected within the human PGF 3'UTR at the 42nd nucleotide and the 91st nucleotide downstream from the PGF coding region. Identical and spatially conserved ARE motifs were found in bovine, rat and mouse PGF 3'UTR. Actinomycin D transcription inhibitor ii studies that were used to measure RNA decay rates in hypoxic and normal conditions, demonstrated a transcriptional response of PGF mRNA to hypoxic stress. Additionally, the PGF 3'UTR did not alter gene expression significantly relative to a site-directed mutant after 24 hours hypoxia. However, PGF 3'UTR decreased reporter activity relative to parental control, suggesting that it could function to regulate stability, but not in hypoxic stress. Western blots and immunohistochemical analyses showed the presence of three potential ARE binding proteins in trophoblast. RNA-Immunoprecipitation assays suggest that PGF mRNA may interact weakly with HuR protein. Upregulation of RNABP expression by insulin was investigated for its effects to upregulate RNABP, and was found to transcriptionally downregulate PGF mRNA. Tumor necrosis factor alpha (TNFa) treatment initiated a short-term upregulation of PGF mRNA. PML Study Results: PML protein was immunolocalized within nuclei of villus mesenchyme, but largely absent in trophoblast nuclei. A trend for increased PML reactivity in placenta of preeclamptic patients was observed. Immunoblot analyses of nuclear extracts confirmed relative increases (~3 fold) of PML expression in preeclamptic placentae (p< 0.05). Conversely, less PML mRNA (~2 fold) was detected in preeclamptic versus normal placental samples. In vitro, PML expression could be increased by hypoxia in cultured endothelial cells but not trophoblast. Conclusion: These results suggest that a post-transcriptional mechanism directed through the 3'UTR does not regulate PGF mRNA expression in stressed trophoblast. PML Study conclusion: Increased PML protein expression in preeclamptic villi suggests it could contribute to decreased vascularity and placental growth and/or function.