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

The objective of this study was to clarify the regulation of production and signal transduction of juvenile hormone (JH) in insects by experimentally examining the function and evolution of a putative receptor (USP/RXR) and a neuropeptide inhibitor (FGLamide allatostatin). To examine the role of USP/RXR, the cDNA sequence of the receptor was obtained from the cockroach Diploptera punctata. Transcript levels during developmentally critical periods for JH sensitivity may suggest USP/RXR is JH responsive. Comparative sequence analysis of evolutionary rates in the Mecopterida support current hypotheses which suggest some gain in function along this lineage, although this acquisition may have occurred more gradually than previously assumed. To examine allatostatin evolution within insects, ancestral peptides inferred using maximum likelihood ancestral reconstruction methods were assayed for in vitro inhibition of JH production in two cockroach species. Shifts in peptide potency in some ancestral peptides reconstructed may be related to peptide copy number evolution.

The objective of this study was to clarify the regulation of production and signal transduction of juvenile hormone (JH) in insects by experimentally examining the function and evolution of a putative receptor (USP/RXR) and a neuropeptide inhibitor (FGLamide allatostatin). To examine the role of USP/RXR, the cDNA sequence of the receptor was obtained from the cockroach Diploptera punctata. Transcript levels during developmentally critical periods for JH sensitivity may suggest USP/RXR is JH responsive. Comparative sequence analysis of evolutionary rates in the Mecopterida support current hypotheses which suggest some gain in function along this lineage, although this acquisition may have occurred more gradually than previously assumed. To examine allatostatin evolution within insects, ancestral peptides inferred using maximum likelihood ancestral reconstruction methods were assayed for in vitro inhibition of JH production in two cockroach species. Shifts in peptide potency in some ancestral peptides reconstructed may be related to peptide copy number evolution.

Remyelination is a regenerative process that occurs through the formation of myelin sheaths by oligodendrocytes, which are recruited as oligodendrocyte progenitor cells (OPCs) after demyelination in diseases such as Multiple Sclerosis (MS).A key environmental factor regulating OPC differentiation is the fate of myelin debris generated during demyelination. Myelin debris contains inhibitors of OPC differentiation and thus its clearance by phagocytic macrophages is an important component of creating a lesion environment conducive to remyelination. The efficiency of debris clearance declines with age, contributing to the age-associated decline in remyelination. Therefore, understanding the mechanisms of the age-related decline in myelin debris phagocytosis is important for devising means to therapeutically reverse the decline in remyelination. The aim of this study was to determine the functional/molecular differences between young and old phagocytes involved in myelin debris clearance, thereby identifying therapeutically modifiable pathways associated with efficient myelin debris phagocytosis. In this study, we show that expression of genes involved in the retinoid X receptor (RXR) and peroxisome proliferator-activated receptor (PPAR) pathways are decreased with ageing in both myelin-phagocytosing human monocytes and mouse macrophages. Disruption of RXR and PPAR using synthetic antagonists in young macrophages mimics ageing by reducing myelin debris uptake. Macrophage-specific RXR? knockout mice revealed that loss of RXR function in young mice caused delayed myelin debris uptake and slowed remyelination. Alternatively, receptor agonists partially restored myelin debris phagocytosis in aged macrophages. The FDA-approved agonists bexarotene and pioglitazone, when used in concentrations achievable in human subjects, caused a reversion of the gene expression profiles in MS patient monocytes to a more youthful profile and enhanced myelin debris phagocytosis by patient cells. Activation of these pathways also enhances immunoregulatory markers on monocytes from MS patients, further suggesting the regeneration-promoting capacity of activating these pathways in phagocytes. These results reveal the RXR/PPAR pathway as a positive regulator of myelin debris clearance and a key player in the age-related decline in remyelination that may be targeted by available or newly-developed therapeutics.

Cocaine abuse poses a substantial health and economic burden for which no effective treatment currently exists. Exposure to cocaine results in altered signaling in a number of central nervous system (CNS) pathways. Previous studies have primarily focused on neurotransmitter systems, such as the dopaminergic and glutamatergic systems, as well as on drug-induced neuroplasticity within the mesolimbic system, which is believed to contribute to reward, addiction, and relapse following withdrawal. Furthermore, cocaine exerts a number of effects on gene regulation that contribute to many pathological conditions commonly afflicting users such as mood disturbances, psychotic symptoms, and long-term cognitive dysfunction. While some mechanisms by which cocaine regulates gene expression have been well-characterized, a large gap in our understanding regarding its downstream actions still exists and must be elucidated in order to develop effective treatment strategies. One pathway we have discovered to be disrupted in an animal model of chronic cocaine abuse is the retinoid X receptor (RXR) signaling pathway. Retinoid X receptors serve as obligate heterodimer partners for a number of nuclear receptor transcription factors, including the thyroid hormone receptor (TR), retinoic acid receptor, vitamin D receptor, and peroxisome proliferator activated receptor. Heterodimeric complexes bind to specific recognition sequences in or around the promoter of target genes to activate, or in some cases, repress, transcriptional activity. Therefore, alterations in the levels and function of RXRs can potentially disrupt numerous signaling cascades. In this context, we observed a significant down-regulation in mRNA and protein levels of RXR-y, an isoform predominantly expressed in the CNS that is involved in dopaminergic signaling, in brains of cocaine-administered mice. Additionally, we observed significantly decreased levels of the neuroplasticity protein, neurogranin, which is regulated transcriptionally by TR/RXR heterodimers. Mechanisms underlying regulation of RXR levels in cells of the CNS are vastly unexplored. Studies in other organ systems, including liver and cardiac systems, demonstrate pro-inflammatory cytokines and cellular stress pathways exert repressive effects on RXR signaling, although these studies solely investigated regulation of the RXR-a isoform. Recently, studies have highlighted the role of the immune system during chronic drug abuse, and demonstrate that significant amounts of proinflammatory factors are produced in the brains of chronic cocaine abusers. Therefore, we hypothesized that cocaine-mediated induction of inflammatory cytokines, such as tumor necrosis factor (TNF)-a may contribute to decreased RXR-y expression within the CNS. Utilizing in vitro neuronal systems, we have demonstrated that cocaine exposure induces neuronal expression of TNF-a and that this contributes to decreased levels of RXR-y, as inhibition of TNF-a or its downstream effector c-Jun-NH2-terminal kinase (JNK) prevents cocaine-mediated reductions in RXR-y protein levels. Furthermore, treatment of neurons with TNF-a alone mimics the effects on RXR-y levels observed in cocaine-treated cells. Additionally, we show that proteasome-dependent protein degradation likely plays a role, as inhibition of the 26 S proteasome with Bortezomib during cocaine or TNF-a exposure blocks the down-regulation of RXR-y levels. Degradation of RXR-y in response to cocaine and TNF-a may involve nuclear export, as our results show an increased level of RXR-y in the cytoplasmic compartment shortly after treatment, and inhibiting nuclear export during treatment with Leptomycin B prevents decreases in whole cell protein levels of RXR-y. In addition to the effects of chronic cocaine abuse on neurons, other CNS cell types such as oligodendrocytes may be negatively impacted by exposure to cocaine. Imaging studies and post-mortem microarray data from human cocaine abuse patients reveal loss of myelin and down-regulated expression of myelin-related genes in the nucleus accumbens and frontal cortex. Altered myelin integrity likely contributes to cognitive deficits that present in many chronic cocaine abuse patients and may also exacerbate damage to neurons. However, limited investigation has been performed to evaluate the effects of cocaine on oligodendrocyte health and function. We have employed an in vivo murine model of chronic cocaine administration to evaluate the impact of cocaine on white matter protein levels. Our data reveal that cocaine induces a significant decrease in white matter protein levels, even following an extended period of withdrawal, in the nucleus accumbens. One potential mechanism for cocaine-mediated white matter damage involves perturbations of glutamate homeostasis, as glutamatergic signaling can induce excitotoxicity in CNS cells, including oligodendrocytes. In this context, we found that administration of the B-lactam antibiotic, ceftriaxone, during cocaine withdrawal ameliorates loss of white matter proteins. Ceftriaxone has previously been shown to upregulate expression and activity of the glial glutamate transporter GLT-1, lending support to the theory that cocaine-mediated myelin loss may be due, in part, to disruption of glutamatergic signaling. Ceftriaxone treatment also decreased expression of cleaved caspase-3, a pro-apoptotic signaling molecule activated during excitotoxic cell death, in cocaine-administered mice. Taken together, our studies characterize two novel consequences of cocaine exposure: (1) decreased neuronal RXR-y expression and down-regulation of RXR-target genes, such as neurogranin, and (2) loss of myelin proteins in the nucleus accumbens which can be attenuated by administration of ceftriaxone. These findings yield insight into mechanisms underlying cocaine-mediated CNS cell death, and highlight potential treatment avenues for restoring brain health. Additionally, as inflammatory processes were identified as key mediators in some of these observations, our findings likely extend to a number of neurodegenerative diseases which are characterized by a neuroinflammatory component. / Biomedical Neuroscience

The human retinoid X receptor alpha (hRXRalpha) is a member of the nuclear receptor super-family of ligand-activated transcription factors. The Doyle laboratory has previously engineered a variety of functional hRXRalpha variants that activate gene expression in response to synthetic ligands (LG335 and γ-oxo-1-pyrenebutyric acid), compounds that are poor activators of wild-type hRXRalpha. The variants generally no longer respond to the wild-type ligand 9-cis retinoic acid. To enable targeting of these engineered receptors to arbitrary DNA sequences, we developed a program, ESPSearch, for identifying short or specific sequences in DNA or protein. ESPSearch enables identification of combinations of known zinc finger motifs to target arbitrary genes, as well having several other applications. The ability to target any DNA sequence means that the engineered receptors can be directed to control any gene. The ligand binding, self-association, coactivator interactions, and unfolding properties of the ligand binding domain of wild-type hRXRalpha were characterized. Our expression and purification protocol improves upon existing methods, providing high purity protein in a single step with more than twice prior yields. A general fluorescence-based method for measuring ligand affinity with hRXRalpha was developed, and used to determine binding constants for the small molecules. The presence of a peptide containing the binding motif from coactivator proteins (LxxLL) differentially increased the affinity of the receptor for the ligands. Assays to determine the self-association give a Kd for the dimer-tetramer equilibrium of 35 µM. hRXRalpha was found to denature irreversibly when heated, but shifts in apparent Tm due to ligands correlates strongly with the ligand binding affinities. Our results clarify disparities in existing reports and provide a benchmark for comparison. Reliable analysis of our data led to the development of a computer program for rigorous, automated data fitting. Nine functional variants of hRXRalpha were characterized to probe correlations between biophysical properties and the observed functional activity of the receptors, which differ significantly from wild-type. Although the correlation between ligand binding affinity and melting temperature was strong for all variants, there was essentially no correlation between ligand binding and activation of the variants. The mutations, which are all contained within the binding pocket, have significant long-range effects on the protein, causing changes in ligand-LxxLL interactions and oligomerization of the variants. Experimental and computational analysis of selected mutations suggests that they are highly coupled, complicating protein design. However, the large variation in properties amongst the variants also suggests that hRXRalpha can be mutated extensively while still retaining function. The long-range impact of binding pocket mutations will need to be taken into account in future engineering projects, as hRXRalpha is a flexible, dynamic protein.

No description available.

Creating receptors for control of transcription with arbitrary small molecules has widespread applications including gene therapy, biosensors, and enzyme engineering. Using the combination of high throughput docking, codon randomization, and chemical complementation, we have created new receptors to control transcription with small molecules. Chemical complementation, a new method of protein engineering, was used to discover retinoid X receptors (RXR) variants that are activated by compounds that do not activate wild-type RXR. A first library of 32,768 RXR variants was designed for the synthetic retinoid-like compound LG335. The library produced ligand-receptor pairs with LG335 that have a variety of EC50s and efficacies. One engineered variant has essentially the reverse ligand specificity of wild-type RXR and is transcriptionally active at 10 and #64979;fold lower LG335 concentration than wild-type RXR with 9cRA in yeast. The activity of this variant in mammalian cells correlates with its activity in yeast. A second library of 262,144 RXR variants was designed for two purposes: (i) to develop a high-throughput chemical complementation method to select variants that have high efficacies and low EC50s; and (ii) to find variants which are activated by small molecules not known to bind RXR variants. Selection conditions were manipulated to find only variants with high efficacies and low EC50s. This library was also selected for variants that activate transcription specifically in response to gamma-oxo-1-pyrenebutyric acid (OPBA), which is different from any known RXR ligand. OPBA was chosen as a potential ligand using high-throughput docking with the software program FlexX. Two variants are activated by OPBA with an EC50 of 5 mM. This is only ten-fold greater than the EC50 of wild type RXR with its ligand 9cRA (500 nM) in yeast. An improved method synthesizing LG335 and a method for quantifying intracellular ligand concentrations were developed. Although the LG335 synthetic method has an additional step, the overall yield was improved to 8% from 4% in the original publication. Liquid chromatography and mass spectrometry was used to quantify the intracellular concentration of LG335, which was found to be within four fold of the LG335 concentration in the media.

Nuclear receptors (NRs) are important transcription factors involved in the regulation of a variety of physiological processes such as embryonic development, cell differentiation and homeostasis (for review, see Mangelsdorf et al., 1995 TenBaum and Baniahrned, 1997). In contrast to membrane bound receptors, they bind small lipophilic ligands and function in the nucleus as ligand-modulated transcription factors. The ligands for nuclear receptors include steroids (glucocorticoids, progestins, mineralocorticoids, androgens and estrogens), vitamin D3, retinoids, thyroid hormone, prostaglandins, farnesoids etc. Several other nuclear receptors are classified as orphan receptors for which no ligand has yet been identified. More than 300 nuclear receptors have now been identified and together these proteins comprise the single largest family of metazoan transcription factors, the nuclear receptor superfamily. Recently, a unified nomenclature has been evolved (nuclear receptor nomenclature committee, 1999), a summary of which is presented in Table 1.

Marine environments are under constant pressure from anthropogenic pollution. Chemical pollutants are introduced into the aquatic environment through waste disposal, sewage, land runoff and environmental exploitation (harbours, fisheries, tourism) leading to disastrous effects on the marine wildlife. Developmental malformations, reproduction failure including sex changes and high death rates are commonly observed in aquatic animal populations around the world. Unfortunately, the underlying molecular mechanisms of these pollution effects, in particular for marine invertebrate species, are often unknown. One proposed mechanism through which environmental pollution affects wildlife, is the disruption of nuclear receptors (NRs), ligand-binding transcription factors in animals. Environmental pollutants can directly interact with nuclear receptors, inducing incorrect signals for gene expression and subsequently disrupt developmental and physiological processes. Elucidation of the exact mechanism in invertebrates, however, is sparse due to limited understanding of invertebrate endocrinology and molecular regulatory mechanisms. Here, I have investigated the presence, expression and function of NRs in the Pacific oyster, Crassostrea gigas, and explored their interrelation with known environmental pollutants. Using a suite of molecular techniques and bioinformatics tools I demonstrate that the Pacific oyster possesses a large variety of NR homologs (43 NRs), which display individual expression profiles during embryo/larval development and supposedly fulfil distinct functions in developmental and physiological processes. Functional studies on a small subset of oyster NRs provided evidence for their ability to regulate gene expression, including interactions with DNA, other NRs or small molecules (ligand-binding). Oyster receptors also show a high likeliness to be disrupted by environmental pollutants. Computational docking showed that the retinoid X receptor ortholog, CgRXR, is able to bind and be activated by 9-cis retinoic acid and by the well-known environmental contaminant tributyltin. A potential interaction between tributyltin and the peroxisome proliferator-activated receptor ortholog CgPPAR has also been found. In addition, exposure of oyster embryos to retinoic acids and tributyltin resulted in shell deformations and developmental failure. In contrast, computer modelling of another putative target for pollutants, the retinoic acid receptor ortholog CgRAR, did not indicate interactions with common retinoic acids, supporting a recently developed theory of loss of retinoid binding in molluscan RARs. Sequence analyses revealed six residues in the receptor sequence, which prevent the successful interaction with retinoid ligands. In conclusion, this investigative work aids the understanding of fundamental processes in invertebrates, such as gene expression and endocrinology, as well as further understanding and prediction of effects of environmental pollutants on marine invertebrates.