The Impact of the Ubiquitin-Proteasome Pathway and CRM1-Independent Exportins on Thyroid Hormone Receptor Localization and the Expression of T3-Responsive Genes

AbstractThyroid hormone is critical to the regulation of development, growth, and metabolism and is released from the thyroid gland in two forms, T3 and T4. T3 movement into the cell allows it to bind thyroid hormone receptor (TR) in the cytoplasm or nucleus, and this hormone-receptor complex can regulate gene expression by enhancing or repressing transcription at thyroid hormone response elements (TREs). Alternative splicing creates six specific isoforms of TR that vary in their localization and ability to bind T3 and TREs. The two main isoforms of TR are TRα and TRβ. The necessity of TR for hormone-dependent regulation is complicated by TR nucleocytoplasmic shuttling; proper TR shuttling dynamics allow for the nuanced patterns of gene expression observed in development, growth and metabolism. TR shuttling into and out of the nucleus is mediated by importins that recognize nuclear localization signal (NLS) motifs and facilitate nuclear import, and exportins that recognize nuclear export signal (NES) motifs and facilitate nuclear export. While TR may be partially dependent on the general CRM1/calreticulin cooperative nuclear export pathway, previous studies have suggested that TR uses an alternative exportin 5-mediated nuclear export pathway as well. Elucidating the impact of exportin 7 (XPO7), a recently discovered exportin with a broad substrate specificity, on TR nuclear export was one of the primary objectives of this thesis research. To assess the impact XPO7 had on TR localization, transient transfections of HeLa cells with XPO7 were performed and TR distribution was examined by fluorescence microscopy. CAT ELISAs and measurement of TRE-CAT reporter gene expression under conditions of XPO7 overexpression also served as an indicator of TR presence in the nucleus. Localization data from fluorescence microscopy showed that overexpression of XPO7 shifted TR from a predominantly nuclear to either a nuclear and cytoplasmic or whole cell distribution, suggesting that XPO7 plays a central role in TR cellular localization. Analysis of transcriptional activity from CAT ELISAs proved less direct as TRα-transfected cells overexpressing XPO7 had a large amount of variability, with XPO7 overexpressing cells demonstrating both higher and lower levels of CAT reporter gene expression under control of a TRE relative to control cells, in contrast TRβ-transfected cells overexpressing XPO7 having significantly higher CAT reporter gene expression under control of a TRE relative to control cells. Another aspect of this thesis research involved assessing the possible regulatory role of the Ubiquitin-Proteasome Pathway (UPP) on T3-responsive gene expression, as preliminary studies indicated that ubiquitinated and liganded TRα is bound to chromatin. CAT ELISAs performed under conditions of proteasome inhibition and ubiquitin overexpression proved inconclusive. Further investigation is required to better comprehend the relationship between the UPP and T3–mediated gene expression.