I. RORalpha and gamma: The retinoid-related orphan receptor a and g (RORa and RORg) are members of the nuclear receptor superfamily. To identify the physiological functions of RORa and g, mice deficient in RORa and g function were analyzed. RORg exhibit several functions in the immune system. RORg expression is indispensable for lymph node organogenesis and plays a critical role in thymocyte homeostasis. Recently a role for RORg in Th17 cell differentiation was identified. We demonstrated that both RORa and RORg are induced during Th17 cells differentiation and double knockouts mice are resistant to experimental autoimmune encephalomyelitis, a model for multiple sclerosis. In addition, RORg-deficient mice are less susceptible to aovalbumin (OVA)-induced inflammation in mice, a model for allergic airway disease. Retinoid-related orphan receptors alpha (RORa) and gamma (RORg) are both expressed in liver;however, their physiological functions in this tissue have not yet been clearly defined. RORa1 and RORg1 show an oscillatory pattern of expression during circadian rhythm. Comparison of gene expression profiles of livers from WT, RORa-deficient staggerer mice (RORasg/sg), RORg-/-, and RORasg/sgRORg-/- double knockout (DKO) mice by microarray analysis demonstrated that RORa and RORg are particularly important in the regulation of genes encoding several Phase I and Phase II metabolic enzymes, including several 3b-hydroxysteroid dehydrogenases (Hsd3b), cytochrome P450 (Cyp) enzymes, and sulfotransferases. In addition, our results indicate that RORa and RORg each affect the expression of a specific set of genes but also exhibit functional redundancy. Our study shows that RORa and RORg receptors influence the regulation of several metabolic pathways, including those involved in the metabolism of steroids, bile acids, and xenobiotics, suggesting that RORs are important in the control of metabolic homeostasis. RORa plays an important role in the regulation of energy homeostasis. Mice deficient in RORa are resistant to diet-induced obesity and reduced inflammation in adipose tissue. A number of lipogenic genes were identified that are positively regulated by RORa. Because RORs function as ligand-dependent transcription factors, RORa might be a therapeutic target for the management of obesity. RORg was found to play an important role in the circadian regulation of several genes downstream of the circadian clock. Our studies further demonstrated that Prox1 interacts with ROR nuclear receptors and represses the transcriptional activity of RORγand expression of RORγ, thereby contributing to the down-regulation of several ROR target genes. The AF2 in the LBD of RORs and the homeo/propero-like domain of Prox1 are required for the interaction as well as repression. Our study identifies Prox1 as a novel modulator of ROR transcriptional regulation and as such is an integral part of the circadian clock and metabolic regulatory networks. II. TAK1: The nuclear orphan receptor TAK1 functions as a positive as well as a negative regulator of transcription;however little is know about factors mediating its activity. Yeast two-hybrid analysis using the ligand binding domain of TAK1 as bait identified a novel TAK1-interacting protein, referred to as TIP27. Our studies indicate that TIP27 is an effective repressor of transcriptional activation by TAK1 and, therefore, may play a critical role in the regulation of several physiological functions by TAK1. Generation of TAK1 knockout mice revealed several phenotypes that are currently being investigated. TAK1-deficient (TAK1-/-) mice and report that these mice exhibit a smaller cerebellum and deficit in foliation of lobules VI-VII. The absence of TAK1 results in a coordinated deficit in cerebellar granule neurons, Purkinje cells, and radial glia during development altering long-term neurobehavioral functioning. These data indicate that TAK1 is an important transcriptional modulator of neurodevelopmentally-regulated behavior. In addition, we found that TAK1 plays a role in the regulation of energy homeostasis. Mice deficient in TAK1 are protected against the development of obesity,hepatic steatosis,and insulin resistance. A number of lipogenic genes were identified that are positively regulated by TAK1. III. Receptor associated protein (RAP80), a nuclear protein containing two ubiquitin-interacting motifs (UIMs), interacts with the esstrogen receptor alpha (ERa) in an agonist dependent manner. In addition, RAP80 is implicated in DNA repair and is associated with the tumor suppressor Breast cancer-1 (BRCA1) protein complex and mediates BRCA1 translocation to sites of DNA damage. We showed that this translocation is dependent on the UIMs of RAP80. We demonstrated that the ataxia-telangiectasia mutated protein kinase (ATM) can phosphorylate RAP80 in vitro at Ser205. Using an anti-RAP80Ser205P antibody that specifically recognizes RAP80 phosphorylated at Ser205 we demonstrated that RAP80Ser205P translocates to sites of DNA damage. We show that this phosphorylation is mediated by ATM and does not require a functional BRCA1. Ultraviolet (UV) irradiation also induces translocation of RAP80 to DNA damage foci that co-localize with γ-H2AX. We further show that this translocation is also dependent on the UIMs of RAP80 and that the UV-induced phosphorylation of RAP80 at Ser205 is mediated by ATR, not ATM. Our findings suggest that RAP80 has a more general role in different types of DNA damage response signaling pathways. Using genomic and functional analysis we established that the expression of the RAP80 gene is regulated in a DNA damage-responsive manner by the master regulator p53. This regulation occurs at the transcriptional level through a noncanonical p53 response element in the RAP80 promoter. While it is inducible by p53, RAP80 is also able to regulate p53 through an association with both p53 and the E3 ubiquitin ligase HDM2, providing HDM2-dependent enhancement of p53 polyubiquitination. Thus, we provide evidence that RAP80 can function in an autoregulatory loop consisting of RAP80, HDM2 and the p53 master regulatory network, implying an important role for this loop in genome stability and oncogenesis. RAP80 knockout mice were generated to determine the effect of loss of RAP80 on DNA stability and susceptibility to cancer. . RAP80-/- mice were more susceptible to spontaneous lymphoma development and the development of DMBA-induced mammary gland tumors. Moreover, the loss of RAP80 accelerated tumor formation in both p53-/- and p53+/- mice. Our data indicate that RAP80-deficiency promotes genomic instability and causes an increase in cancer risk consistent with the concept that RAP80 exhibits a tumor suppressor function. III. Our studies further demonstrate that mice deficient in CD44 are considerably resistant to diet-induced hepatic steatosis, fibrogenesis, and inflammation, adipose-associated infiltration of M1 macrophages, glucose intolerance, and insulin resistance. These observations suggest that CD44 provides a critical link between metabolic changes and the development of inflammation and insulin resistance. Because CD44 functions as a receptor, it may provide a convenient therapeutic target in the management of lipid dysregulation in diet-induced liver disease and type 2 diabetes.

Project Start
Project End
Budget Start
Budget End
Support Year
11
Fiscal Year
2013
Total Cost
$1,197,737
Indirect Cost
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Teng, Christina T; Beames, Burton; Alex Merrick, B et al. (2014) Development of a stable cell line with an intact PGC-1?/ERR? axis for screening environmental chemicals. Biochem Biophys Res Commun 444:177-81
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