The retinoid X receptor (RXR) participates in a multitude of nuclear receptor signaling pathways and is induced by its highly sought-after cognate ligand, 9-cis-retinoic acid (9-cis-RA). In flies and moths, molting is mediated by the ecdysone receptor that consists of a heterodimer comprising the ecdysone receptor monomer (EcR) and the invertebrate RXR homolog ultraspiracle (USP); the latter, however, is believed to have diverged from its RXR origin. From the more evolutionarily-primitive insect Locusta migratoria (Lm), long and short RXR transcripts (LmRXR-L and LmRXR-S, respectively) were detected during embryogenesis. This thesis reports the immunochemical detection of RXR cross-reactive material throughout Locusta embryogenesis, suggesting that the protein may have another role besides ecdysone signaling. Thus, the RXR isoforms were cloned for recombinant expression and purification in order to demonstrate retinoid specificity. Both isoforms bound 9-cis-RA and all-trans-RA with high affinity. Binding was further corroborated by the identification of endogenous retinoids during embryogenesis. Embryos were first subjected to modified “Bligh and Dyer” as well as solid phase extractions to circumvent oil precipitation that rendered whole homogenates unsuitable for retinoid assay and detection. The RA-inducible Cyp26A1-promoter reporter cell line identified the presence of endogenous RAs (5.4 nM) from insect embryo extracts. Finally, high pressure liquid chromatography followed by mass spectroscopy (HPLC/MS) confirmed the identity of all-trans-RA and the more abundant 9-cis-RA (1.3 nM). These findings suggest a functional role for 9-cis-RA in the invertebrate embryo and favour signaling through the combination of 9-cis-RA and RXR in evolutionarily early RA-driven animal development. / Thesis (Master, Biology) -- Queen's University, 2007-12-13 10:14:43.474

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Cutaneous melanoma remains the deadliest form of skin cancer, with a diagnosis of metastasis indicating a median survival rate of less than a year. Solar ultraviolet (UV) radiation, especially childhood sun exposure, is an important etiological risk factor of melanoma. Previous studies determined that mice selectively lacking the nuclear hormone receptor Retinoid X Receptor α in epidermal keratinocytes (RXRα[superscript ep-/-]) developed a higher number of aggressive melanocytic tumors compared to wild type mice after two-step chemical carcinogenesis, suggesting a novel role of keratinocytic nuclear receptor signalling during melanoma progression. We then discovered a progressive loss of RXRα expression in epidermal keratinocytes during melanoma progression in humans. We also investigated the contributions of CDK4[superscript R24C/R24C] and keratinocytic RXRα to influence metastatic progression in a mouse model by generating RXRα[superscript ep-/-]/CDK4[superscript R24C/R24C] bigenic mice containing an activated cyclin dependent kinase 4 (CDK4), besides lacking RXRα in epidermal keratinocytes. Those bigenic mice developed malignant melanomas that metastasized to regional lymph nodes after carcinogen exposure. Expression of several keratinocyte-derived growth factors implicated in melanomagenesis were upregulated in the skin of bigenic mice, and recruitment of RXRα was shown on the promoters of endothelin-1 (Edn1) and hepatocyte growth factor (Hgf). We then confirmed a downregulation of factors (FAS, E-cadherin and PTEN) implicated in apoptosis, invasion and survival within the melanocytic tumors. To further evaluate the paracrine role that EDN1 has on melanocyte activation, we utilized a transgenic mouse model where the gene encoding Edn1 was selectively ablated from epidermal keratinocytes using the Cre-LoxP strategy to create the EDN1[superscript ep-/-] knockout mouse line. We discovered a direct in vivo transcriptional regulation of keratinocytic Edn1 by the tumor-suppressor p53 in epidermal keratinocytes in response to UV irradiation. We also demonstrate that in vivo disruption of keratinocyte-derived EDN1 signaling alters melanocyte proliferation and decreases epidermal and dermal melanocyte populations in both normal and UV exposed mouse skin. EDN1 also has a protective role against UVR-induced DNA damage and apoptosis and similar effects on UV-induced melanocyte proliferation and DNA damage are observed in p53-null mice. Inhibition of EDN1 signaling by topical application of an EDNRB antagonist BQ788 on mouse skin also recapitulates epidermal EDN1 ablation. Furthermore, treatment of primary murine melanocytes with BQ788 abrogates signaling downstream of this receptor. Taken together, these studies demonstrate the contribution of RXRα regulated keratinocytic paracrine signaling during the cellular transformation and malignant conversion of melanocytes. Also, they establish an essential role of EDN1 in epidermal keratinocytes to mediate UV-induced melanocyte homeostasis in vivo. / Graduation date: 2013

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Liver X receptor (LXR) is a member of the nuclear hormone receptor superfamily that is activated by hydroxylated cholesterol derivatives referred to as oxysterols. It has also been shown to play a crucial role in regulating cholesterol trafficking and lipid metabolism in liver and macrophages. Furthermore, LXR. has also been directly implicated in the reduction of atherosclerosis in several murine models of the disease by virtue of its ability to promote reverse cholesterol efllux from intima-resident lipid-loaded macrophages. While roles for LXR in monocyte biology have focused primarily on cholesterol trafficking, evidence for other functions for the receptor outside of its traditional role as a mediator of cholesterol homeostasis is lacking. Presented herein is evidence that LXR also serves as a mediator of cytokine expression. This work has shown that treatment of human peripheral blood monocytes or monocytic THP-1 cells with the LXR ligand 22(R)-hydroxycholesterol (22R-HC), in combination with 9-cis-retinoic acid (9cRA), a ligand for the LXR. heterodimerization partner retinoid X receptor (RXR), results in the specific induction of the potent pro-apoptotic and pro-inflammatory cytokine tumor necrosis factor-alpha (TNF-α.). Promoter analysis, inhibitor studies, and order-of-addition experiments demonstrated that TNF-α. induction by 22R-HC and 9cRA occurs by a novel two-step process. The initial step involves 22R-HC-dependent induction of TNF-α. mRNA, and intracellular accumulation of TNF-alpha protein, mediated by binding of LXRα/RXRα to an LXR response element at position -879 of the TNF-α promoter. Subsequent cell release of TNF-alpha protein occurs via a separable RXR-dependent step that requires de novo transcription and protein synthesis. Furthermore, the RXR-dependent secretory event can be mimicked by agents that induce monocytic differentiation like phorbol esters that culminate in RXR activation by a pathway that does not require exogenous ligand. In this context, RXR was also shown to be a down stream target of the protein kinase C (PKC) signal transduction cascade, that results in the activation of RXR and the induction of secretory factor(s) which facilitate secretion of LXR-derived TNF-α. These studies have provided evidence that should help to expand the currently known role for LXR in monocyte biology and have furthermore identified a new role for RXRs in promoting the secretion of soluble factors like cytokines. Furthermore, in light of reports that show LXR activity promotes a reduction in atherosclerosis, it stands to reason that this regulatory circuit of LXR-dependent production of TNF -α from monocytes would similarly contribute to the attenuation of atherosclerosis 𝘪𝘯 𝘷𝘪𝘷𝘰. / Thesis / Master of Science (MSc)

Section I: This section is an introduction to the field of nuclear receptors. A general overview of nuclear receptor-mediated transcriptional regulation is followed by a review of literature on retinoid and thyroid receptor-mediated signaling. Section II: An introduction to NPDC-1 (neural proliferation, differentiation, and control), its discovery and characterization with regards to developmental expression and cellular localization. In addition NPDC-1 has been found to associate with a number of cell cycle regulatory proteins. NPDC-1 is characterized as a regulator of nuclear receptor-mediated transcriptional regulation. NPDC-1 was also demonstrated to be regulated post-transcriptionally through the ubiquitin/proteosome degradation pathway. Section III: Axolotl NPDC-1 (aNPDC-1) was cloned from axolotl brain and analyzed for homology to NPDC-1 from higher vertebrates. The tissue distribution and developmental expression of axolotl NPDC-1 were also examined. Section IV: The axolotl homolog for RAR (aRAR) was isolated from axolotl brain. Axolotl NPDC-1 and aRAR were then examined in a series of assays for interactions. Axolotl NPDC-1 was found to repress transcription mediated by aRAR to a smaller extent than the repression observed in higher vertebrates. The DNA binding of aRAR-RXR was increased in the presence of aNPDC-1 and complex mobility was also observed. The domain of interaction between aNPDC-1, aRAR and hRXR was localized in the amino terminus of aNPDC-1. Axolotl NPDC-1 was also demonstrated to repress proliferation as measured by reduced [3H] thymidine incorporation. Section V: The axolotl homologs of TR and TR (aTR) genes were isolated and utilized in a series of experiments to demonstrate an interaction between aTRs and aNPDC-1. As observed for RE, aNPDC-1 increases the binding of aTR-RXR heterodimer to xDR4, but no change in the mobility of the complex was observed. Interaction between aNPDC-1, aTR and aTR was localized to the amino terminus of aNPDC-1. In contrast to previous observations for other nuclear receptors, aNPDC-1 was found to stimulate transcription mediated by axolotl TRs, suggesting a role for aNPDC-1 in axolotl metamorphosis. Section VI: A summary of data presented in the previous sections as well as a presentation of future directions and a proposed model for NPDC-1 actions in retinoid and thyroid-receptor mediated signaling in axolotl.

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Age-related macular degeneration (AMD) is the leading cause of blindness in the United States of America. The pyridinium bis-retinoid A2-ethanolamine (A2E) has been implicated to play a role in AMD. We have observed novel pyridinium bis-retinoids through melanolipofuscin and human RPE extractions that may also play a role in the pathology of AMD. We have begun the construction of an amino-retinoid library in order to identify these ocular compounds. The compounds from the amino-retinoid library are also used in a targeted and triggered drug delivery system for treating cancer. Folic acid is coupled with the amino-retinoids to specifically target cancer cells. The first two amino-retinoids to be synthesized and characterized were A2-dopamine (A2D) and A2-cadaverine (A2C). Both pyridinium bis-retinoids were shown to generate cytotoxic oxidation products similar to A2E. Successful coupling of folic acid to A2C was achieved to form the folic acid-A2-cadaverine (FA-A2C) product. Preliminary irradiation results suggest that the FA-A2C product may be more photoreactive than initially anticipated. This could mean less drug and light exposure required to induce apoptosis and could eventually lead to a less invasive and toxic cancer treatment.