TR4 orphan nuclear receptor functions as an apoptosis modulator via regulation of Bcl-2 gene expression

doi:10.1016/j.bbrc.2007.06.168

Biochemical and Biophysical Research Communications

Volume 361, Issue 2, 21 September 2007, Pages 323-328

https://doi.org/10.1016/j.bbrc.2007.06.168 Get rights and content

Abstract

While Bcl-2 plays an important role in cell apoptosis, its relationship to the orphan nuclear receptors remains unclear. Here we report that mouse embryonic fibroblast (MEF) cells prepared from TR4-deficient (TR4⁻^/−) mice are more susceptible to UV-irradiation mediated apoptosis compared to TR4-Wildtype (TR4^+/+) littermates. Substantial increasing TR4⁻^/− MEF apoptosis to UV-irradiation was correlated to the down-regulation of Bcl-2 RNA and protein expression and collaterally increased caspase-3 activity. Furthermore, this TR4-induced Bcl-2 gene expression can be suppressed by co-transfection with TR4 coregulators, such as androgen receptor (AR) and receptor-interacting protein 140 (RIP140) in a dose-dependent manner. Together, our results demonstrate that TR4 might function as an apoptosis modulator through induction of Bcl-2 gene expression.

Section snippets

Materials and methods

Preparation of mouse embryonic fibroblasts, UV-irradiation and cell culture. Primary TR4^+/− and TR4^−/− MEFs were isolated from embryonic (E) 14–15 days littermates and cultured in Dulbecco’s modified Eagle’s medium (DMEM) plus 10% fetal bovine serum. TR4^+/− and TR4^−/− mice were housed and studied under the University Committee on Animal Resources-approved protocols in the animal facility of the University of Rochester Medical center. COS-1 and H1299 cells were cultured in DMEM plus 10%

Down-regulation of Bcl-2 gene expression in TR4 knockout (TR4^−/−) MEF cells increases sensitivity to UV-irradiation

We have generated TR4⁻^/⁻ mice by insertion of an IRES β-gal MCI-Neo selection cassette between exons 4 and 5 of the TR4 gene [13]. The RT-PCR analysis of total RNAs from TR4^+/⁻ and TR4⁻^/⁻ MEF cells confirmed deletion of TR4 gene in exons 4 and 5 in homozygous TR4⁻^/⁻ MEF cells (Fig. 1A). To investigate whether loss of TR4 affects MEF cell growth, MTT assays were carried out. TR4⁻^/⁻ MEF cells showed significant growth retardation as compared to TR4^+/⁻ MEF cells (Fig. 1B) with only little

Discussion

Loss of TR4 in MEF cells resulted in delayed adhesion, growth retardation, and significant sensitization to apoptosis-inducing agents. Interestingly, the Bcl-2 protein expression is nearly absent in slowly adhering cells [17]. This indicates that TR4 may participate in cell survival via regulation of Bcl-2 level. Previous reports have shown that UV-irradiation causes a decline of Bcl-2 protein level and this decline enhances caspase-3 activity leading to cell death [11]. The expression of Bcl-2

Acknowledgments

We thank Drs. M. Parker, R.H. Goodman, and J.C. Reed for kindly providing plasmids. This work was supported by the National Institutes of Health Grant and the George Whipple Professorship Endowment. The TR4⁻^/⁻ mice were generated in collaboration with Lexicon Genetics, Inc. We also thank K. Wolf for help in manuscript preparation.

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TR2 and TR4 Orphan Nuclear Receptors: An Overview
2017, Current Topics in Developmental Biology
Citation Excerpt :
The recruitment of RIP140 then suppresses TR4 transactivation activity (Huq et al., 2006). Kim et al. also reported that TR4-induced Bcl-2 gene expression can be suppressed by cotransfection with RIP140 in a dose-dependent manner (Kim, Ma, et al., 2007). Interestingly, Xie et al. found that the ARA55 coregulator might play dual roles in regulating AR vs TR4.
Testicular nuclear receptors 2 and 4 (TR2, TR4), also known as NR2C1 and NR2C2, belong to the nuclear receptor superfamily and were first cloned in 1989 and 1994, respectively. Although classified as orphan receptors, several natural molecules, their metabolites, and synthetic compounds including polyunsaturated fatty acids (PUFAs), PUFA metabolites 13-hydroxyoctadecadienoic acid, 15-hydroxyeicosatetraenoic acid, and the antidiabetic drug thiazolidinediones can transactivate TR4. Importantly, many of these ligands/activators can also transactivate peroxisome proliferator-activated receptor gamma (PPARγ), also known as NR1C3 nuclear receptor. Both TR4 and PPARγ can bind to similar hormone response elements (HREs) located in the promoter of their common downstream target genes. However, these two nuclear receptors, even with shared ligands/activators and shared binding ability for similar HREs, have some distinct functions in many diseases they influence. In cancer, PPARγ inhibits thyroid, lung, colon, and prostate cancers but enhances bladder cancer. In contrast, TR4 inhibits liver and prostate cancer initiation but enhances pituitary corticotroph, liver, and prostate cancer progression. In type 2 diabetes, PPARγ increases insulin sensitivity but TR4 decreases insulin sensitivity. In cardiovascular disease, PPARγ inhibits atherosclerosis but TR4 enhances atherosclerosis through increasing foam cell formation. In bone physiology, PPARγ inhibits bone formation but TR4 increases bone formation. Together, the contrasting impact of TR4 and PPARγ on different diseases may raise a critical issue about drug used to target any one of these nuclear receptors.
Di-(2-ethylhexyl) phthalate induces apoptosis of GC-2spd cells via TR4/Bcl-2 pathway
2016, Environmental Toxicology and Pharmacology
Citation Excerpt :
Bcl-2 gene expression of embryo fibroblasts separated and cultured from TR4(-/-) mice is down regulated and further decreased after exposure to ultraviolet rays. Activated caspase-3 can degrade Bcl-2 to accelerate cell apoptosis, indicating that TR4 regulates cell apoptosis through inducing Bcl-2 gene expression (Kim et al., 2007). Studies have shown that the testicular tissues of 6-week-old mice showed significant cell apoptosis and reduced expression of Bcl-2 mRNA after one oral exposure to DEHP 2000 mg/kg (Kijima et al., 2004).
Di-(2-ethylhexyl) phthalate (DEHP) is a widely used environmental endocrine disruptor. Many studies have reported that DEHP exposure causes reproductive toxicity and cells apoptosis. However, the mechanism by which DEHP exposure causes male reproductive toxicity remains unknown. This study investigated the role of the testicular orphan nuclear receptor4 (TR4)/Bcl-2 pathway in apoptosis induced by DEHP, which resulted in reproductive damage. To elucidate the mechanism underpinning the male reproductive toxicity of DEHP, we sought to investigate apoptotic effects, expression levels of TR4/Bcl-2 pathway in GC-2spd cells, including TR4, Bcl-2 and caspase-3. GC-2spd cells were exposed to various concentrations of DEHP (0, 50, 100, or 200 μM). The results indicated that, with the increase of the concentrations of DEHP, the survival rate of cell decreased gradually. DEHP exposure at over 100 μM significantly induced apoptotic cell death. DEHP decreased SOD and GSH-Px activity in 200 μM group. Compared to the control group, the mRNA levels of caspase-3 increased significantly, however, Bcl-2 mRNA decreased (P < 0.05). In addition, there was a significant reduction in TR4, Bcl-2 and procaspase-3 protein levels. Taken together, these results lead us to speculate that in vitro exposure to DEHP might induce apoptosis in GC-2spd cells through the TR4/Bcl-2 pathway.
Overexpression of JAZF1 protected ApoE-deficient mice from atherosclerosis by inhibiting hepatic cholesterol synthesis via CREB-dependent mechanisms
2014, International Journal of Cardiology
Citation Excerpt :
NR2C2, also known as TAK1/TR4, is an orphan nuclear receptor targeting many genes important in metabolism [9]. NR2C2 has been reported to modulate apoptosis [6,10] and its loss in mice is associated with reduced mitochondrial function and increased oxidative stress, and conversely its increase is associated with reduced adipose tissue inflammation, hepatic steatosis and insulin resistance [11,12]. As an inhibitor of the TAK1/TR4 signaling pathway, JAZF1 has been speculated to be involved in gluconeogenesis and lipid metabolism [6,13–16].
Genome wide association studies have suggested an association of Juxtaposed with another zinc finger gene 1(JAZF1) with type 2 diabetes mellitus (T2DM). As an inhibitor of the TAK1/TR4 signaling pathway, JAZF1 has been shown to be involved in gluconeogenesis, lipid metabolism and insulin sensitivity. However, its role in insulin resistance and atherosclerosis in vivo remains unknown. The present study was designed to investigate in vivo the impact of JAZF1 on insulin resistance-associated dyslipidemia and atherosclerosis. Adenovirus-mediated JAZF1 overexpression was used to characterize the role of JAZF1 in the regulation of lipid metabolism and the development of atherosclerosis in normal chow- or HFD-fed ApoE KO mice. Insulin sensitivity was examined by EHC. Cholesterol de novo synthesis was measured by intraperitoneal [1-¹⁴C] acetate injection and atherosclerotic plaques were quantified by histological analysis. A dual-luciferase reporter assay was used to assess the ability of JAZF1 to regulate HMGCR transcriptional activity. JAZF1 overexpression improved HFD-induced hepatic insulin resistance in C57BL/6J mice. In HFD-fed ApoE KO mice, JAZF1 overexpression decreased serum cholesterol levels and hepatic cholesterol synthesis by inhibiting CREB-dependent HMGCR promoter transcriptional activity. Analysis of atherosclerotic lesion showed that JAZF1 overexpression had significantly reduced aortic and aortic sinus en face and cross-sectional plaque areas in HFD-fed ApoE KO mice. These data provide the first evidence for an important role of JAZF1 in increasing hepatic insulin sensitivity and preventing atherosclerosis.
Increased chemosensitivity via targeting testicular nuclear receptor 4 (TR4)-Oct4-interleukin 1 receptor antagonist (IL1Ra) axis in prostate cancer CD133<sup>+</sup> stem/progenitor cells to battle prostate cancer
2013, Journal of Biological Chemistry
Citation Excerpt :
The testicular nuclear receptor 4 (TR4) belongs to the nuclear receptor superfamily and was first cloned from human prostate and testis cDNA libraries (23). It has been known to modulate many signaling pathways by interacting with the thyroid receptor, androgen receptor, retinoic acid receptor/retinoid X receptor, and estrogen receptor (24–26). TR4 knock-out mouse studies have shown that TR4 knockout results in defects in development and abnormalities in spermatogenesis and reproductive systems in both genders, which indicates that TR4 might play important roles in stem/progenitor cell differentiation (24).
Prostate cancer (PCa) stem/progenitor cells are known to have higher chemoresistance than non-stem/progenitor cells, but the underlying molecular mechanism remains unclear. We found the expression of testicular nuclear receptor 4 (TR4) is significantly higher in PCa CD133⁺ stem/progenitor cells compared with CD133⁻ non-stem/progenitor cells. Knockdown of TR4 levels in the established PCa stem/progenitor cells and the CD133⁺ population of the C4-2 PCa cell line with lentiviral TR4 siRNA led to increased drug sensitivity to the two commonly used chemotherapeutic drugs, docetaxel and etoposide, judging from significantly reduced IC₅₀ values and increased apoptosis in the TR4 knockdown cells. Mechanism dissection studies found that suppression of TR4 in these stem/progenitor cells led to down-regulation of Oct4 expression, which, in turn, down-regulated the IL-1 receptor antagonist (IL1Ra) expression. Neutralization experiments via adding these molecules into the TR4 knockdown PCa stem/progenitor cells reversed the chemoresistance, suggesting that the TR4-Oct4-IL1Ra axis may play a critical role in the development of chemoresistance in the PCa stem/progenitor cells. Together, these studies suggest that targeting TR4 may alter chemoresistance of PCa stem/progenitor cells, and this finding provides the possibility of targeting TR4 as a new and better approach to overcome the chemoresistance problem in PCa therapeutics.
Background: PCa stem/progenitor cells develop higher chemoresistance.
Results: High TR4 levels in PCa stem/progenitor cells were shown to be critical in conferring chemoresistance to these cells.
Conclusion: TR4-Oct4-IL1Ra signaling is important in conferring chemoresistance to PCa stem/progenitor cells.
Significance: This finding suggests that targeting TR4 and its downstream molecules may be a better therapeutic approach to battle PCa stem/progenitor cell-originated chemoresistance.
Testicular nuclear receptor 4 (TR4) regulates UV light-induced responses via cockayne syndrome B protein-mediated transcription-coupled DNA repair
2011, Journal of Biological Chemistry
Citation Excerpt :
Our data demonstrated that TR4 directly modulates CSB transcription and thus provides a novel pathway in CSB functional regulation. Our results might also add one more dimension of mechanism to our previous finding that TR4 regulates the apoptotic response upon UV overexposure via Bcl-2 (8) because CSB-deficient cells also have increased apoptosis (31). p53 has been previously linked to the cellular UV response (39), and p53 up-regulation was observed in CSBm/m mice (40), but the TR4-regulated UV response is p53-independent because TR4 functions in the p53-null cell line H1299 (supplemental Fig. S1B).
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.
Stearoyl CoA desaturase (SCD) facilitates proliferation of prostate cancer cells through enhancement of androgen receptor transactivation
2011, Molecules and Cells
Stearoyl-CoA desaturase (SCD), the rate-limiting enzyme in the biosynthesis of monounsaturated fatty acids, is highly expressed in prostate cancer although the SCD protein has been known to be rapidly turned over by proteolytic cleavage. The present data demonstrate that SCD can promote proliferation of androgen receptor (AR)-positive LNCaP prostate cancer cells and enhance dihydrotestosterone (DHT)-induced AR transcriptional activity, resulting in increased expression of prostate-specific antigen (PSA) and kallikrein-related peptidase 2 (KLK2). Interestingly, among the previously reported SCD-derived peptides produced by proteolytic cleavage of SCD, a peptide spanning amino acids 130-162 of SCD (SCD-CoRNR) contained the CoRNR box motif (LFLII) and enhanced AR transcriptional activity. In contrast, a mutant SCD-CoRNR in which Leu¹³⁶ was replaced by Ala had no effect on AR transcriptional activity. Moreover, SCD-CoRNR directly interacted with AR and inhibited RIP140 suppression of AR transactivation. Knockdown of the SCD gene by SCD microRNA suppressed AR transactivation with decreased cell proliferation, suggesting that SCD may regulate the proliferation of LNCaP cells via modulation of AR transcriptional activity. Moreover, ectopic expression of SCD in LNCaP cells facilitated LNCaP tumor formation and growth in nude mice. Together, the data indicate that SCD plays a key role in the regulation of AR transcriptional activity in prostate cancer cells.

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¹: These authors contributed equally to this work.

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TR4 orphan nuclear receptor functions as an apoptosis modulator via regulation of Bcl-2 gene expression

Abstract

Section snippets

Materials and methods

Down-regulation of Bcl-2 gene expression in TR4 knockout (TR4−/−) MEF cells increases sensitivity to UV-irradiation

Discussion

Acknowledgments

Cell

J. Biol. Chem.

J. Biol. Chem.

J. Biol. Chem.

J. Biol. Chem.

J. Biol. Chem.

J. Biol. Chem.

FEBS Lett.

Mol. Cell. Proteomics

J. Biol. Chem.

The t(14;18) chromosome translocations involved in B-cell neoplasms result from mistakes in VDJ joining

Science

Bcl-2 is an inner mitochondrial membrane protein that blocks programmed cell death

Nature

Down-regulation of Bcl-2 gene expression in TR4 knockout (TR4^−/−) MEF cells increases sensitivity to UV-irradiation