Functional evidence for retinoid X receptor (RXR) as a nonsilent partner in the thyroid hormone receptor/RXR heterodimer

Many members of the thyroid hormone/retinoid receptor subfamily (type II nuclear receptors) function as heterodimers with the retinoid X receptor (RXR). In heterodimers which are referred to as permissive, such as peroxisome proliferator activated receptor/RXR, both partners can bind cognate ligands and elicit ligand-dependent transactivation. In contrast, the thyroid hormone receptor (TR)/RXR heterodimer is believed to be nonpermissive, where RXR is thought to be incapable of ligand binding and is often referred to as a silent partner. In this report, we used a sensitive derepression assay system that we developed previously to reexamine the TR/RXR interrelationship. We provide functional evidence suggesting that in a TR/RXR heterodimer, the RXR component can bind its ligand in vivo. Ligand binding by RXR does not appear to directly activate the TR/RXR heterodimer; instead, it leads to a (at least transient or dynamic) dissociation of a cellular inhibitor(s)/corepressor(s) from its TR partner and thus may serve to modulate unliganded TR-mediated repression and/or liganded TR-mediated activation. Our results argue against the current silent-partner model for RXR in the TR/RXR heterodimer and reveal an unexpected aspect of cross regulation between TR and RXR.     

Novel roles of retinoid X receptor (RXR) and RXR ligand in dynamically modulating the activity of the thyroid hormone receptor/RXR heterodimer

Many members of the type II nuclear receptor subfamily function as heterodimers with the retinoid X receptor (RXR). A permissive heterodimer (e.g. peroxisome proliferator-activated receptor/RXR) allows for ligand binding by both partners of the receptor complex. In contrast, RXR has been thought to be incapable of ligand binding in a nonpermissive heterodimer, such as that of thyroid hormone receptor (TR)/RXR, where it has been referred to as a silent partner. However, we recently presented functional evidence suggesting that RXR in the TR/RXR heterodimer can bind its natural ligand 9-cis-RA in cells. Here we extended our study of the interrelationship of TR and RXR. We examined the potential modulatory effect of RXR and its ligand on the activity of TR, primarily using a Gal4-TR chimera. This study led to several novel and unexpected findings: 1) heterodimerization of apo-RXRalpha (in the absence of 9-cis-RA) with Gal4-TR inhibits T3-mediated transactivation; 2) the inhibition of Gal4-TR activity by RXRalpha is further enhanced by 9-cis-RA; 3) two different RXR subtypes (alpha and beta) differentially modulate the activity of Gal4-TR; 4) the N-terminal A/B domains of RXR alpha and beta are largely responsible for their differential modulation of TR activity; and 5) the RXR ligand 9-cis-RA appears to differentially affect T3-mediated transactivation from the Gal4-TR/RXRalpha (which is inhibited by 9-cis-RA) and TRE-bound TR/RXRalpha (which is further activated by 9-cis-RA) heterodimers. Taken together, these results further support our recent proposal that the RXR component in a TR/RXR heterodimer is not silent and, more importantly, reveal novel aspects of regulation of the activity of the TR/RXR heterodimer by RXR and RXR ligand.     

The p21(WAF1/CIP1) promoter is methylated in Rat-1 cells: stable restoration of p53-dependent p21(WAF1/CIP1) expression after transfection of a genomic clone containing the p21(WAF1/CIP1) gene

Rat-1 cells are used in many studies on transformation, cell cycle, and apoptosis. Whereas UV treatment of Rat-1 cells results in apoptosis, X-ray treatment does not induce either apoptosis or a cell cycle block. X-ray treatment of Rat-1 cells results in both an increase of p53 protein and expression of the p53-inducible gene MDM2 but not the protein or mRNA of the p53-inducible p21(WAF1/CIP1) gene, which in other cells plays an important role in p53-mediated cell cycle block. The lack of p21(WAF1/CIP1) expression appears to be the result of hypermethylation of the p21(WAF1/CIP1) promoter region, as p21(WAF1/CIP1) protein expression could be induced by growth of Rat-1 cells in the presence of 5-aza-2-deoxycytidine. Furthermore, sequence analysis of bisulfite-treated DNA demonstrated extensive methylation of cytosine residues in CpG dinucleotides in a CpG-rich island in the promoter region of the p21(WAF1/CIP1) gene. Stable X-ray-induced p53-dependent p21(WAF1/CIP1) expression and cell cycle block were restored to a Rat-1 clone after transfection with a P1 artificial chromosome (PAC) DNA clone containing a rat genomic copy of the p21(WAF1/CIP1) gene. The absence of expression of the p21(WAF1/CIP1) gene may contribute to the suitability of Rat-1 cells for transformation, cell cycle, and apoptosis studies.     

p21(WAF1/CIP1) inhibits initiator caspase cleavage by TRAIL death receptor DR4

Death receptors of the Tumor Necrosis Factor (TNF) family form membrane-bound self-activating signaling complexes that initiate apoptosis through cleavage of proximal caspases including CASP8 and 10. Here we show that overexpression of the cytoplasmic domain (CD) of the DR4 TRAIL receptor (TNFRSF10A, TRAIL R1) in human breast, lung, and colon cancer cell lines, using an adenovirus vector (Ad-DR4-CD), leads to p53-independent apoptotic cell death involving cleavage of CASP8 and 10 proximally and CASP3, 6, and 7 distally. DR4-CD overexpression also leads to cleavage of poly(ADP-ribose) polymerase (PARP) and the DNA fragmentation factor (DFF45; ICAD). Importantly, normal lung fibroblasts are resistant to DR4-CD overexpression and show no evidence of PARP-, CASP8- or CASP3-cleavage despite similar levels of adenovirus-delivered DR4-CD protein as the cancer cells. These results suggest that DR4 may signal death through known caspases and that further studies are required to evaluate Ad-DR4-CD as a novel anti-cancer agent. Finally, we show that overexpression of the cyclin-dependent kinase inhibitor p21(WAF1/CIP1) (CDKN1A), or its N-terminal 91 amino acids containing cell cycle-inhibitory activity, inhibits DR4-CD-dependent proximal caspase cleavage. The blockage of initiator caspase activation provides a novel insight into how p21 may suppress apoptosis and enhance cell survival.     

Flt3 mutation activates p21WAF1/CIP1 gene expression through the action of STAT5

Metastin, a post-translationally modified variant of KiSS1, was recently identified as an endogenous peptide agonist for a novel G-protein coupled receptor, hOT7T175 (AXOR12, GPR54). In this study, we analyzed the role of KiSS1 and hOT7T175 in both pancreatic cancer tissues and pancreatic cancer cell lines. Furthermore, we synthesized novel short variant forms of metastin and tested the inhibitory effect of those variants on in vitro cell functions that are relevant to metastasis. Pancreatic cancer tissues showed significantly lower expression of KiSS1 mRNA than normal tissues (p=0.018), while cancer tissues showed significantly higher expression of hOT7T175 mRNA than normal pancreatic tissues (p=0.027). In human pancreatic cancer cell lines, KiSS1 mRNA was highly expressed in 2 out of 6 pancreatic cancer cell lines, while hOT7T175 mRNA was expressed in all cell lines at various degrees. PANC-1 cells showed the highest expression of hOT7T175. Exogenous metastin did not suppress cell proliferation but significantly reduced the in vitro migration of PANC-1 cells (p<0.01). Metastin induced activation of ERK1 in PANC-1 and AsPC-1 cells. Finally, we synthesized 3 novel short variant forms of metastin, FM053a2TFA, FM059a2TFA, and FM052a4TFA. These metastin variants significantly suppressed the migration of PANC-1 cells and activated ERK1. These data suggest that the metastin receptor, hOT7T175, is one of the promising targets for suppression of metastasis, and that small metastin variants could be an anti-metastatic agent to pancreatic cancer.     

Targeted p21(WAF1/CIP1) Activation by RNAa Inhibits Hepatocellular Carcinoma Cells

RNA activation (RNAa) is a mechanism of gene activation triggered by promoter-targeted small double-stranded RNA (dsRNA), also known as small activating RNA (saRNA). p21(WAF1/CIP1) (p21) is a putative tumor suppressor gene due to its role as a key negative regulator of the cell cycle and cell proliferation. It is frequently downregulated in cancer including hepatocellular carcinoma (HCC), but is rarely mutated or deleted, making it an ideal target for RNAa-based overexpression to restore its tumor suppressor function. In the present study, we investigated the antigrowth effects of p21 RNAa in HCC cells. Transfection of a p21 saRNA (dsP21-322) into HepG2 and Hep3B cells significantly induced the expression of p21 at both the mRNA and protein levels, and inhibited cell proliferation and survival. Further analysis of dsP21-322 transfected cells revealed that dsP21-322 arrested the cell cycle at the G(0)/G(1) phase in HepG2 cells but at G(2)/M phase in Hep3B cells which lack functional p53 and Rb genes, and induced both early and late stage apoptosis by activating caspase 3 in both cell lines. These results demonstrated that RNAa of p21 has in vitro antigrowth effects on HCC cells via impeding cell cycle progression and inducing apoptotic cell death. This study suggests that targeted activation of p21 by RNAa may be explored as a novel therapy for the treatment of HCC.     

Mitochondrial ribosomal protein L41 mediates serum starvation-induced cell-cycle arrest through an increase of p21(WAF1/CIP1)

Ribosomal proteins not only act as components of the translation apparatus but also regulate cell proliferation and apoptosis. A previous study reported that MRPL41 plays an important role in p53-dependent apoptosis. It also showed that MRPL41 arrests the cell cycle by stabilizing p27(Kip1) in the absence of p53. This study found that MRPL41 mediates the p21(WAF1/CIP1)-mediated G1 arrest in response to serum starvation. The cells were released from serum starvation-induced G1 arrest via the siRNA-mediated blocking of MRPL41 expression. Overall, these results suggest that MRPL41 arrests the cell cycle by increasing the p21(WAF1/CIP1) and p27(Kip1) levels under the growth inhibitory conditions.     

Identification of promoter elements responsible for transcriptional inhibition of polo-like kinase 1 and topoisomerase IIalpha genes by p21(WAF1/CIP1/SDI1)

Induction of p21 (WAF1/CIP1/SDI1), a physiological mediator of cell cycle arrest, inhibits multiple genes involved in cell division. We have investigated the determinants of p21- mediated inhibition of two of these genes, polo-like kinase 1 (PLK1) and topoisomerase IIalpha (TOPO IIalpha) p21 expression from an inducible promoter in human HT1080 cells rapidly decreases cellular levels of PLK1 and TOPO IIalpha promoters in transient and stable transfection assays. Promoter mutagenesis studies show that inhibition of the PLK1 promoter by p21 is mediated in part by tandem sequences CDE (cell cycle-dependent element) and CHR (cell cycle genes homology region). p21 response of the TOPO IIalpha promoter inhibition and the effects of promoter mutations differ under the conditions of growth arrest produced by p21 induction or by mimosine, a cell cycle inhibitor that increases p21 RNA but not protein expression in HT1080 cells. These results indicate that inhibition of cell division-associated genes by p21 is mediated by different but overlapping mechanisms, which are not a general con-sequence of cell cycle arrest.     

DNA损伤生物学反应中ATM对p21~(WAF1/CIP1)蛋白的直接磷酸化

毛细血管扩张性共济失调症突变蛋白 (mutatedinataxiatelangiectasia ,ATM)是直接感受DNA双链断裂损伤并起始诸多DNA损伤信号反应通路的主开关分子 .已有研究发现 ,DNA损伤生物学反应中 ,ATM可通过磷酸化活化p5 3,继而转录活化细胞周期检查点蛋白p2 1WAF1 CIP1的表达 ,而对于ATM是否直接参与p2 1WAF1 CIP1的早期活化迄今尚无实验证明 .通过免疫共沉淀反应 ,检测到细胞电离辐射 (ionizingradiation ,IR)反应早期ATM与p2 1WAF1 CIP1蛋白存在相互作用 .将p2 1WAF1 CIP1蛋白编码基因全长克隆入原核表达载体pGEX4T 2 ,经诱导表达及亲和层析纯化获取GST p2 1融合蛋白作为磷酸化底物 .体外磷酸化实验检测证明 ,IR活化的ATM具磷酸化p2 1WAF1 CIP1蛋白的功能 ,并且此磷酸化功能可被PI3K家族特异性抑制剂Wortmannin所抑制 .结果揭示了IR后ATM可通过直接磷酸化p2 1WAF1 CIP1蛋白 ,在IR致DNA损伤生物学反应早期调控p2 1WAF1 CIP1蛋白的快速活化过程