[show abstract][hide abstract] ABSTRACT: Early studies suggested that TR4 nuclear receptor might play important roles in the skeletal development, yet its detailed mechanism remains unclear.
We generated TR4 knockout mice and compared skeletal development with their wild type littermates. Primary bone marrow cells were cultured and we assayed bone differentiation by alkaline phosphatase and alizarin red staining. Primary calvaria were cultured and osteoblastic marker genes were detected by quantitative PCR. Luciferase reporter assays, chromatin immunoprecipitation (ChIP) assays, and electrophoretic mobility shift assays (EMSA) were performed to demonstrate TR4 can directly regulate bone differentiation marker osteocalcin.
We first found mice lacking TR4 might develop osteoporosis. We then found that osteoblast progenitor cells isolated from bone marrow of TR4 knockout mice displayed reduced osteoblast differentiation capacity and calcification. Osteoblast primary cultures from TR4 knockout mice calvaria also showed higher proliferation rates indicating lower osteoblast differentiation ability in mice after loss of TR4. Mechanism dissection found the expression of osteoblast markers genes, such as ALP, type I collagen alpha 1, osteocalcin, PTH, and PTHR was dramatically reduced in osteoblasts from TR4 knockout mice as compared to those from TR4 wild type mice. In vitro cell line studies with luciferase reporter assay, ChIP assay, and EMSA further demonstrated TR4 could bind directly to the promoter region of osteocalcin gene and induce its gene expression at the transcriptional level in a dose dependent manner.
Together, these results demonstrate TR4 may function as a novel transcriptional factor to play pathophysiological roles in maintaining normal osteoblast activity during the bone development and remodeling, and disruption of TR4 function may result in multiple skeletal abnormalities.
Reproductive Biology and Endocrinology 06/2012; 10:43. · 2.14 Impact Factor
[show abstract][hide abstract] ABSTRACT: The testicular receptor 4 (TR4) is a member of the nuclear receptor superfamily that controls various biological activities. A protective role of TR4 against oxidative stress has recently been discovered. We here examined the protective role of TR4 against ionizing radiation (IR) and found that small hairpin RNA mediated TR4 knockdown cells were highly sensitive to IR-induced cell death. IR exposure increased the expression of TR4 in scramble control small hairpin RNA expressing cells but not in TR4 knockdown cells. Examination of IR-responsive molecules found that the expression of Gadd45a, the growth arrest and DNA damage response gene, was dramatically decreased in Tr4 deficient (TR4KO) mice tissues and could not respond to IR stimulation in TR4KO mouse embryonic fibroblast cells. This TR4 regulation of GADD45A was at the transcriptional level. Promoter analysis identified four potential TR4 response elements located in intron 3 and exon 4 of the GADD45A gene. Reporter and chromatin immunoprecipitation (ChIP) assays provided evidence indicating that TR4 regulated the GADD45A expression through TR4 response elements located in intron 3 of the GADD45A gene. Together, we find that TR4 is essential in protecting cells from IR stress. Upon IR challenges, TR4 expression is increased, thereafter inducing GADD45A through transcriptional regulation. As GADD45A is directly involved in the DNA repair pathway, this suggests that TR4 senses genotoxic stress and up-regulates GADD45A expression to protect cells from IR-induced genotoxicity.
[show abstract][hide abstract] ABSTRACT: UV irradiation is one of the major external insults to cells and can cause skin aging and cancer. In response to UV light-induced DNA damage, the nucleotide excision repair (NER) pathways are activated to remove DNA lesions. We report here that testicular nuclear receptor 4 (TR4), a member of the nuclear receptor family, modulates DNA repair specifically through the transcription-coupled (TC) NER pathway but not the global genomic NER pathway. The level of Cockayne syndrome B protein (CSB), a member of the TC-NER pathway, is 10-fold reduced in TR4-deficient mouse tissues, and TR4 directly regulates CSB at the transcriptional level. Moreover, restored CSB expression rescues UV hypersensitivity of TR4-deficient cells. Together, these results indicate that TR4 modulates UV sensitivity by promoting the TC-NER DNA repair pathway through transcriptional regulation of CSB. These results may lead to the development of new treatments for UV light-sensitive syndromes, skin cancer, and aging.
Journal of Biological Chemistry 09/2011; 286(44):38103-8. · 4.65 Impact Factor
[show abstract][hide abstract] ABSTRACT: The nuclear receptor TR4 is a key regulator for many physiological processes, including growth, development, and metabolism. However, how the transcriptional activity of TR4 is regulated in the absence of ligand(s) remains largely unknown. Here we found that an androgen receptor (AR) coactivator, ARA55, might function as a corepressor to suppress TR4 transactivation. Molecular mechanistic dissection with mutation analysis found that ARA55 could enhance TR4 acetylation at the conserved acetylation sites of lysine 175 and lysine 176 in the DNA-binding domain via recruiting proteins with histone acetyl transferase activity, which might then reduce significantly the TR4 DNA binding activity that resulted in the suppression of TR4 transactivation. These results are in contrast to the classic ARA55 coactivator function to enhance AR transactivation partially via increased AR acetylation in the hinge/ligand-binding domain. Together, these results not only provide a novel functional mechanism showing that acetylation of different nuclear receptors at different domains by coregulator may lead to differential receptor transactivation activity but also provide a new way for small molecules to control TR4 transactivation via altering TR4 acetylation levels, and such small molecules may have potential therapeutic applications in the future.
Journal of Biological Chemistry 06/2011; 286(24):21129-36. · 4.65 Impact Factor
[show abstract][hide abstract] ABSTRACT: The estimated incidence of mitochondrial diseases in humans is approximately 1:5000 to 1:10,000, whereas the molecular mechanisms for more than 50% of human mitochondrial disease cases still remain unclear. Here we report that mice lacking testicular nuclear receptor 4 (TR4(-/-)) suffered mitochondrial myopathy, and histological examination of TR4(-/-) soleus muscle revealed abnormal mitochondrial accumulation. In addition, increased serum lactate levels, decreased mitochondrial ATP production, and decreased electron transport chain complex I activity were found in TR4(-/-) mice. Restoration of TR4 into TR4(-/-) myoblasts rescued mitochondrial ATP generation capacity and complex I activity. Further real-time PCR quantification and promoter studies found TR4 could modulate complex I activity via transcriptionally regulating the complex I assembly factor NDUFAF1, and restoration of NDUFAF1 level in TR4(-/-) myoblasts increased mitochondrial ATP generation capacity and complex I activity. Together, these results suggest that TR4 plays vital roles in mitochondrial function, which may help us to better understand the pathogenesis of mitochondrial myopathy, and targeting TR4 via its ligands/activators may allow us to develop better therapeutic approaches.
[show abstract][hide abstract] ABSTRACT: Early studies suggest that TR4 nuclear receptor is a key transcriptional factor regulating various biological activities, including reproduction, cerebella development, and metabolism. Here we report that mice lacking TR4 (TR4(-/-)) exhibited increasing genome instability and defective oxidative stress defense, which are associated with premature aging phenotypes. At the cellular level, we observed rapid cellular growth arrest and less resistance to oxidative stress and DNA damage in TR4(-/-) mouse embryonic fibroblasts (MEFs) in vitro. Restoring TR4 or supplying the antioxidant N-acetyl-l-cysteine (NAC) to TR4(-/-) MEFs reduced the DNA damage and slowed down cellular growth arrest. Focused qPCR array revealed alteration of gene profiles in the DNA damage response (DDR) and anti-reactive oxygen species (ROS) pathways in TR4(-/-) MEFs, which further supports the hypothesis that the premature aging in TR4(-/-) mice might stem from oxidative DNA damage caused by increased oxidative stress or compromised genome integrity. Together, our finding identifies a novel role of TR4 in mediating the interplay between oxidative stress defense and aging.
AJP Endocrinology and Metabolism 04/2011; 301(1):E91-8. · 4.51 Impact Factor
[show abstract][hide abstract] ABSTRACT: The human testicular receptor 2 and 4 (TR2 and TR4) are two evolutionarily related orphan nuclear receptors belonging to the
same nuclear receptor subfamily (Lee et al. 2002, J Steroid Biochem Mol Biol 81(4–5), 291–308). They regulate gene expression by binding to DNA as homodimers or a heterodimer with each other. TR4 may
also cross-talk with other nuclear receptors, to control its target genes. In vitro and in vivo studies have identified several
TR4 target genes, including ciliary neurotrophic factor alpha (CNTFRα) (Young et al. 1997, J Biol Chem 272(5), 3109–3116), apolipoprotein E (ApoE) (Kim et al. 2003, J Biol Chem 278(47), 46919–46926) and phosphenolpyruvate carboxykinase (PEPCK) (Liu et al. 2007, Diabetes 56(12), 2901–2909). Recent studies using TR4 knockout (TR4–/–) mice suggested that TR4 may play essential roles in growth, development, and metabolism (Zhang et al. 2007, Mol Endocrinol 21(4), 908–920; Kim et al. 2005, Biochem Biophys Res Commun 328(1), 85–90; Chen et al. 2005, Mol Cell Biol 25(7), 2722–2732; Mu et al. 2004, Mol Cell Biol 24(13), 5887–5899; Collins et al. 2004, Proc Natl Acad Sci U S A 101(42), 15058–15063). Mice with a germline deletion of TR4 are viable but have high early postnatal mortality, growth retardation,
and profound reduction in body weight. Further studies showed that TR4 plays essential roles in the development and functioning
in the central nervous system (Chen et al. 2005, Mol Cell Biol 25(7), 2722–2732), such as proper myelination and oligodendrocyte differentiation (Zhang et al. 2007, Mol Endocrinol 21(4), 908–920). Studies also showed that TR4 is important for spermatogenesis in male mice (Mu et al. 2004, Mol Cell Biol 24(13), 5887–5899) and folliculogenesis in female mice (Chen et al. 2008, Mol Endocrinol 22, 858–867). In addition, TR4 might be involved in skeletal muscle function and bone remodeling. TR4 and TR2 also regulate
embryonic and fetal globin gene transcription (Tanabe et al. 2002, EMBO J 21(13), 3434–3442; Tanabe et al. 2007, EMBO J 26(9), 2295–2306). Surprisingly, mice lacking TR2 are viable and have no serious developmental defects. Thus, TR2 may either
not be important in spermatogenesis and testis development, or its roles may be compensated by other closely related proteins
such as TR4. Therefore, this chapter will focus on the in vivo roles of TR4.
[show abstract][hide abstract] ABSTRACT: Testicular orphan nuclear receptor 4 (TR4) is an orphan member of the nuclear receptor superfamily with diverse physiological functions. Using TR4 knockout (TR4(-/-)) mice to study its function in cardiovascular diseases, we found reduced cluster of differentiation (CD)36 expression with reduced foam cell formation in TR4(-/-) mice. Mechanistic dissection suggests that TR4 induces CD36 protein and mRNA expression via a transcriptional regulation. Interestingly, we found this TR4-mediated CD36 transactivation can be further enhanced by polyunsaturated fatty acids (PUFAs), such as omega-3 and -6 fatty acids, and their metabolites such as 15-hydroxyeico-satetraonic acid (15-HETE) and 13-hydroxy octa-deca dieonic acid (13-HODE) and thiazolidinedione (TZD)-rosiglitazone. Both electrophoretic mobility shift assays (EMSA) and chromatin immunoprecipitation (ChIP) assays demonstrate that TR4 binds to the TR4 response element located on the CD36 5'-promoter region for the induction of CD36 expression. Stably transfected TR4-siRNA or functional TR4 cDNA in the RAW264.7 macrophage cells resulted in either decreased or increased CD36 expression with decreased or increased foam cell formation. Restoring functional CD36 cDNA in the TR4 knockdown macrophage cells reversed the decreased foam cell formation. Together, these results reveal an important signaling pathway controlling CD36-mediated foam cell formation/cardiovascular diseases, and findings that TR4 transactivation can be activated via its ligands/activators, such as PUFA metabolites and TZD, may provide a platform to screen new drug(s) to battle the metabolism syndrome, diabetes, and cardiovascular diseases.
Proceedings of the National Academy of Sciences 08/2009; 106(32):13353-8. · 9.74 Impact Factor
[show abstract][hide abstract] ABSTRACT: In earlier studies, we had suggested that the fasting signal induces TR4 orphan nuclear receptor expression in vivo. The detailed mechanism(s), however, remain unclear. In this study, we found that cAMP/PKA, the mediator of fasting and glucagon signals, could induce TR4 gene expression that in turn modulates gluconeogenesis. Mechanistic dissection by in vitro studies in hepatocytes demonstrated that cAMP/PKA might trigger C/EBP alpha and beta binding to the selective cAMP response element, which is located at the TR4 promoter, thus inducing TR4 transcription. We also demonstrated that the binding activity of C/EBPs to the TR4 promoter is increased in response to cAMP treatment. Together, our data identified a new signaling pathway from the fasting signal --> cAMP/PKA --> C/EBP alpha and beta --> TR4 --> gluconeogenesis in hepatocytes; and suggested that TR4 could be an important regulator to control glucose homeostasis. The identification of activator(s)/inhibitor(s) or ligand(s) of TR4 may provide us an alternative way to control gluconeogenesis.
[show abstract][hide abstract] ABSTRACT: Prostate cancer is initially responsive to hormonal therapy, but cancers inevitably progress in an androgen-independent fashion with virtually all tumors evolving into more aggressive androgen refractory disease. Immunohistological comparisons of cyclooxygenase 2 (COX-2) expressions in 3 pairs of prostate cancer patients before and after the combined androgen blockade (CAB) therapy show elevated COX-2 expressions. This observation from clinical specimens is further supported by in vitro laboratory data using human prostate cancer cells in which the antiandrogen hydroxyflutamide (HF) induced COX-2 expression, and androgen suppressed COX-2 expression. By applying knockdown and overexpression strategies to modulate AR expression in prostate cancer cells, we confirmed that androgen/AR signal suppressed, and HF induced COX-2 expression at both protein and mRNA levels. COX-2 promoter reporter assay indicated that the suppression of COX-2 by androgen/AR is at the transcriptional level via modulation of NF-kappaB signals. Treatment of LNCaP and LAPC4 cells with 1 microM HF in the presence of 1 nM DHT, which mimics the CAB therapy condition, promotes cell growth, and this growth induction can be suppressed via adding the COX-2 specific inhibitor, NS398. This suggests that HF promoted prostate cancer cell growth is COX-2 dependent and this HF-COX-2 activation pathway can account for one reason of CAB therapy failure. Together, these findings provide a possible explanation how CAB with antiandrogen HF therapy might fail and provide a potential new therapeutic approach to battle prostate cancer via combination of CAB therapy with COX-2 inhibitor(s).
International Journal of Cancer 08/2008; 123(1):195-201. · 6.20 Impact Factor
[show abstract][hide abstract] ABSTRACT: Early studies reveal that testicular orphan nuclear receptor 4 (TR4) modulates signaling pathways that control various cell functions. However, how TR4 activity is regulated without the involvement of specific ligand(s) remains unclear. Here we identify a daf-16 family protein-binding element (DBE; 5'-TGTTTAC-3') in the TR4 promoter that can be recognized by the forkhead transcriptional factor FOXO3a, a key stress-responsive factor, through which TR4 gene expression is activated. The interaction between DBE and FOXO3a was confirmed using EMSA and chromatin immunoprecipitation assays. Activation of FOXO3a by oxidative stress and phosphatidylinositol 3-kinase inhibitor induced TR4 expression; in contrast, suppression of FOXO3a by small interfering RNA can reduce oxidative stress-induced TR4 expression. The biological consequence of the FOXO3a-induced TR4 by oxidative stress is to protect against stress-induced cell death in which cells with reduced FOXO3a are less resistant to oxidative stress, and addition of functional TR4 can increase stress resistance. These results suggest that this new identified oxidative stress-FOXO3a-TR4 pathway is a fundamentally important mechanism regulating stress resistance and cell survival.