The Bcl-2 family proteins are key regulators of apoptosis in human diseases and cancers. Though known to block apoptosis, Bcl-2 promotes cell death through an undefined mechanism. Here, we show that Bcl-2 interacts with orphan nuclear receptor Nur77 (also known as TR3), which is required for cancer cell apoptosis induced by many antineoplastic agents. The interaction is mediated by the N-terminal loop region of Bcl-2 and is required for Nur77 mitochondrial localization and apoptosis. Nur77 binding induces a Bcl-2 conformational change that exposes its BH3 domain, resulting in conversion of Bcl-2 from a protector to a killer. These findings establish the coupling of Nur77 nuclear receptor with the Bcl-2 apoptotic machinery and demonstrate that Bcl-2 can manifest opposing phenotypes, induced by interactions with proteins such as Nur77, suggesting novel strategies for regulating apoptosis in cancer and other diseases.
TR3, also known as NGFI-B or nur77, is an immediate-early response gene and an orphan member of the steroid/thyroid/retinoid receptor superfamily. We previously reported that TR3 expression was induced by apoptotic stimuli and was required for their apoptotic effect in lung cancer cells. Here, we present evidence that TR3 was also induced by epidermal growth factor (EGF) and serum and was required for their mitogenic effect in lung cancer cells. Ectopic expression of TR3 in both H460 and Calu-6 lung cancer cell lines promoted their cell cycle progression and BrdU incorporation, while inhibition of TR3 expression by the small interfering RNA approach suppressed the mitogenic effect of EGF and serum. Analysis of TR3 mutants showed that both TR3 DNA binding and transactivation were required for its mitogenic effect. In contrast, they were dispensable for its apoptotic activity. Furthermore, confocal microscopy analysis demonstrated that TR3 functioned in the nucleus to induce cell proliferation, whereas it acted on mitochondria to induce apoptosis. In examining the signaling that regulates the mitogenic function of TR3, we observed that coexpression of constitutive-active MEKK1 inhibited TR3 transcriptional activity and TR3-induced proliferation. The inhibitory effect of MEKK1 was mediated through activation of Jun N-terminal kinase, which efficiently phosphorylated TR3, resulting in loss of its DNA binding. Together, our results demonstrate that TR3 is capable of inducing both proliferation and apoptosis in the same cells depending on the stimuli and its cellular localization.TR3 (also known as NGFI-B and nur77) (6,17,44), an immediate-early response gene, is an orphan member of the steroid/thyroid/retinoid receptor superfamily, whose members mainly act as transcriptional factors to positively or negatively regulate gene expression (22,41,67). The role of TR3 in cell proliferation was suggested by numerous observations showing that its expression is rapidly induced by several mitogenic inducers, including serum growth factor, epidermal growth factor (EGF), and fibroblast growth factor (6,9,13,17,35,44,58). However, whether TR3 expression has a causal role in promoting cell proliferation remains to be illustrated. Recent evidence also indicates that the expression of TR3 is required for apoptosis. TR3 was rapidly induced by T-cell receptor signaling in immature thymocytes and T-cell hybridomas (39, 60). Overexpression of a dominant-negative TR3 protein (60) or inhibition of TR3 expression by antisense TR3 mRNA (39) inhibited T-cell-receptor-induced apoptosis, whereas constitutive expression of TR3 resulted in massive cell death (57, 64).
Retinoid X receptor (RXR) plays a central role in the regulation of intracellular receptor signaling pathways by acting as a ubiquitous heterodimerization partner of many nuclear receptors, including the orphan receptor Nur77 (also known as thyroid hormone receptor 3 or NGFI-B), which translocates from the nucleus to mitochondria, where it interacts with Bcl-2 to induce apoptosis. Here, we report that RXR␣ is required for nuclear export and mitochondrial targeting of Nur77 through their unique heterodimerization that is mediated by dimerization interfaces located in their DNA-binding domain. The effects of RXR␣ are attributed to a putative nuclear export sequence (NES) present in its carboxyl-terminal region. RXR␣ ligands suppress NES activity by inducing RXR␣ homodimerization or altering RXR␣/Nur77 heterodimerization. The RXR␣ NES is also silenced by RXR␣ heterodimerization with retinoic acid receptor or vitamin D receptor. Consistently, we were able to show that the mitochondrial targeting of the RXR␣/Nur77 heterodimer and its induction of apoptosis are potently inhibited by RXR ligands. Together, our results reveal a novel nongenotropic function of RXR␣ and its involvement in the regulation of the Nur77-dependent apoptotic pathway.Retinoid X receptors (RXRs) belong to the nuclear receptor superfamily, consisting of a large number of ligand-regulated transcription factors that mediate the diverse physiological functions of their ligands, such as steroid hormones, retinoids, thyroid hormone, and vitamin D 3 , in embryonic development, growth, differentiation, apoptosis, and homeostasis (29, 46). The superfamily also includes many orphan receptors whose ligands remain to be identified. All nuclear receptors consist of three major domains: the variable length N-terminal domain, the well-conserved DNA-binding domain (DBD), and the ligand-binding domain (LBD) (29,46). The C-terminal LBD is multifunctional and, in addition to harboring a ligand-binding site, contains regions for receptor dimerization and the ligand-dependent transactivation function (AF-2). The DBD also contains a dimerization interface that determines target gene specificity (38,55,60,85). RXRs mediate retinoid signaling through the RXR/retinoic acid receptor (RAR) heterodimer and the RXR/RXR homodimer (29,46,89). In addition, RXRs form heterodimers with many members of the subfamily 1 nuclear receptors, including vitamin D receptor (VDR), peroxisome proliferator-activated receptor (PPAR), and thyroid hormone receptor (TR), as well as several orphan receptors, such as liver X receptor, pregnane X receptor, constitutively activated receptor, and Nur77 (TR3 or NGFI-B) (29, 46). RXRs, therefore, play an essential role in the regulation of multiple nuclear hormone-signaling pathways through their unique and potent dimerization capacity. The vitamin A metabolite, 9-cis-retinoic acid (9-cis-RA), is a high-affinity ligand for RXRs (22,39). It induces transactivation of the RXR homodimer (26,29,46,88,90) and certain RXR heterodimers, such as the RXR/Nur77 ...
The functions of microRNAs (miRNAs) as either oncogenes or tumor suppressors in regulating cancer-related events have been established. We analyzed the alterations in the miRNA expression profile of the glioma cell line U-251 caused by ionizing radiation (IR) by using an miRNA array and identified several miRNAs whose expression was significantly affected by IR. Among the IR-responsive miRNAs, we further examined the function of miR-193a-3p, which exhibited the most significant growth-inhibiting effect. miR-193a-3p was observed to induce apoptosis in both U-251 and HeLa cells. We also demonstrated that miR-193a-3p induces the accumulation of intracellular reactive oxygen species (ROS) and DNA damage as determined by the level of γH2AX and by performing the comet assay. The induction of both apoptosis and DNA damage by miR-193a-3p was blocked by antioxidant treatment, indicating the crucial role of ROS in the action of miR-193a-3p. Among the putative target proteins, the expression of Mcl-1, an anti-apoptotic Bcl-2 family member, decreased because of miR-193a-3p transfection. A reporter assay using a luciferase construct containing the 3'-untranslated region of Mcl-1 confirmed that Mcl-1 is a direct target of miR-193a-3p. Down-regulation of Mcl-1 by siRNA transfection closely mimicked the outcome of miR-193a-3p transfection showing increased ROS, DNA damage, cytochrome c release, and apoptosis. Ectopic expression of Mcl-1 suppressed the pro-apoptotic action of miR-193a-3p, suggesting that Mcl-1 depletion is critical for miR-193a-3p induced apoptosis. Collectively, our results suggest a novel function for miR-193a-3p and its potential application in cancer therapy.
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