Access Type

Open Access Dissertation

Date of Award

January 2011

Degree Type

Dissertation

Degree Name

Ph.D.

Department

Anatomy and Cell Biology

First Advisor

David R. Armant

Abstract

HBEGF is a multifunctional protein in early pregnancy that induces cytotrophoblast (CTB) cell differentiation to an invasive phenotype, protects against apoptosis, and is involved in an autocrine signaling mechanism that leads to its own protein synthesis. CTBs exist in a low O2 environment during the first 10 weeks of implantation, during which they invade the decidualized uterine stroma. Inhibitors of intracellular signaling pathways demonstrated that at 20% O2 HBEGF induces an increase in cell migration through the ERK, MAPK14, JNK, or PIK3 pathways downstream of signaling through its ERBB receptors. Also downstream of these four pathways, HBEGF induces a post-transcriptional increase in ITGA1 and decrease in ITGA6 expression (i.e., integrin-switching). This phenotype is characteristic of invasive CTBs and is also induced by culture on Matrigel, but not on growth factor-reduced Matrigel. TUNEL assays indicate that HBEGF signals through the MAPK14 pathway to inhibit apoptosis in CTBs induced by 2% O2 or reoxygenation injury. Previous studies have identified that HBEGF is specifically translated in CTBs at low O2 (2%), as compared to 20% O2. Utilizing a chemical mimic of low oxygen (CoCl2) that stabilizes HIFα at 20% O2 and an inhibitor of nascent transcription, HBEGF protein was found to be upregulated downstream of HIF-mediated transcription of metalloproteinases. In addition, a cascade involving MMP2 exists that leads to HBEGF shedding and its autocrine signaling at 2% O2. It also signals through ERBB receptors to induce an autocrine post-transcriptional increase in its own protein levels through the ERK, MAPK14 or JNK pathways. HBEGF mRNA is stable at both 20% and 2% O2 and its protein appears to be specifically regulated by O2. Dual luciferase vectors containing various fragments of the HBEGF 3'UTR identified regions that may post-transcriptionally regulate HBEGF mRNA. Regions flanking the 3'UTR may be bound by miRNA at 2% O2, leading to increased protein synthesis. At 20% O2, sequences in the central portion of the 3'UTR mediate translational suppression and may override the influence of the flanking regions, leading to translational suppression of HBEGF at high oxygen concentrations. These studies identify several mechanisms of HBEGF action in CTBs during early pregnancy. In addition, they begin to address the rapid post-transcriptional regulation of HBEGF by O2, a phenomena that is important in the survival and successful implantation of CTBs during implantation.

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