Orphan Receptor Nur77 Research Articles

Modulation of Orphan Nuclear Receptor NURR1 Expression by Methotrexate in Human Inflammatory Joint Disease Involves Adenosine A2A Receptor-Mediated Responses

Modulation by proinflammatory mediators indicate that NURR1 induction represents a point of convergence of distinct signaling pathways, suggesting an important common role for this transcription factor in mediating multiple inflammatory signals. The present study identifies NURR1 as a molecular target of methotrexate (MTX) action in human inflammatory joint disease and examines the mechanism through which MTX modulates NURR1 expression. MTX significantly suppresses expression of NURR1 in vivo in patients with active psoriatic arthritis (n = 10; p < 0.002) who were prescribed low-dose MTX for management of peripheral arthritis. Importantly, reduction in NURR1 levels correlate (n = 10; r = 0.57; p = 0.009) with changes in disease activity score (both clinical and laboratory parameters). MTX selectively modulates NURR1 levels induced by inflammatory stimuli and growth factors in resident cell populations of synovial tissue. In primary human synoviocytes and microvascular endothelial cells, we observe dose-dependent differential effects of MTX on steady-state and inducible NURR1 levels. Our data confirms that adenosine, and its stable analog 5'-N-ethylcarboxamideadenosine, can mimic the differential effects of MTX on NURR1 transcription. In addition, we verify that the inhibitory effect of low-dose MTX on NURR1 activation is mediated through the adenosine receptor A2. More specifically, our data distinguishes the selective involvement of the A2A receptor subtype in these responses. In summary, these findings establish the nuclear orphan receptor NURR1 as a molecular target of MTX action in human inflammatory joint disease and demonstrate that the immunomodulatory actions of MTX on NURR1 expression are mediated through adenosine release.

Retinoid X Receptor Regulates Nur77/Thyroid Hormone Receptor 3-Dependent Apoptosis by Modulating Its Nuclear Export and Mitochondrial Targeting

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 TR3 [corrected] or NGFI-B), which translocates from the nucleus to mitochondria, where it interacts with Bcl-2 to induce apoptosis. Here, we report that RXRalpha 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 RXRalpha are attributed to a putative nuclear export sequence (NES) present in its carboxyl-terminal region. RXRalpha ligands suppress NES activity by inducing RXRalpha homodimerization or altering RXRalpha/Nur77 heterodimerization. The RXRalpha NES is also silenced by RXRalpha heterodimerization with retinoic acid receptor or vitamin D receptor. Consistently, we were able to show that the mitochondrial targeting of the RXRalpha/Nur77 heterodimer and its induction of apoptosis are potently inhibited by RXR ligands. Together, our results reveal a novel nongenotropic function of RXRalpha and its involvement in the regulation of the Nur77-dependent apoptotic pathway [corrected]

Differentiation of the dopaminergic phenotype in the olfactory system of neonatal and adult mice

Olfactory bulb (OB) interneurons are derived primarily postnatally from progenitors in the anterior subventricular zone (SVZa) and migrate to the OB in the rostral migratory stream (RMS). Progenitors differentiate into phenotypically diverse granule and periglomerular cells by as yet undefined mechanisms. To visualize spatiotemporal aspects of periglomerular dopamine (DA) neuron differentiation, two independently derived transgenic mouse lines were analyzed with a 9-kb tyrosine hydroxylase (TH) promoter to drive either a LacZ or an enhanced green fluorescent protein (EGFP) reporter gene. Both reporters showed similar neonatal expression that varied from low levels in RMS, to moderate in the superficial granule cell layer, to strong in relatively large cells, possibly external tufted cells, in the periglomerular region. TH mRNA and protein were not detected in the RMS but were colocalized with the transgenes in neonatal superficial granule and periglomerular cells. By comparison, TH protein in adults was further limited to periglomerular cells. To demonstrate that transcriptional regulation was the same for EGFP and TH, expression was shown to decline similarly in the OB ipsilateral to odor deprivation produced by adult unilateral naris closure. Of two genes previously hypothesized to regulate OB DA expression, only regulated expression of the orphan receptor Nurr1, but not the homeobox-containing genes Dlx-1 and -2, was consistent with a role in regulation of the DA phenotype. These data demonstrate for the first time that DA phenotypic differentiation in neonates begins with low-level transcription in migrating progenitors in the RMS and culminates with activity-dependent protein expression in periglomerular cells innervated by olfactory receptor cells.

Rotenone induces non-specific central nervous system and systemic toxicity.

We investigated the dopaminergic (DA) neuronal degeneration in animals subjected to systemic treatment of rotenone via subcutaneous delivery. Behavioral observations revealed a hypokinetic period in rats sacrificed at 3 and 5 days, and dystonic episodes in animals sacrificed at 8 days. Less than 20% of the total number of animals given rotenone depicted brain lesions after 8 days of treatment, as demonstrated by a significant loss of DA fibers in the striatum, but not of DA nigral neurons. Tyrosine hydroxylase-negative striatal territories were characterized by post-synaptic toxicity as demonstrated by a decreased number of interneurons labeled for choline acetyltransferase, NADPH-diaphorase, parvalbumin, and projection neurons labeled for calbindin and nerve growth factor inducible-B (NGFI-B). Post-synaptic neurodegeneration was demonstrated further by abundant striatal staining for Fluoro-Jade. Decrease in the nuclear orphan receptor Nurr1 expression was the only significant change observed at the level of the substantia nigra. Autopsy reports confirmed that animals suffered from severe digestion problems. These data suggest that hypokinesia observed between 3 and 5 days is the result of general health problems rather than a specific motor deficit associated to Parkinson's disease (PD) symptoms. Overall, the effects of rotenone toxicity are widespread, and subcutaneous administration of this toxin does not provide the neuropathological and behavioral basis for a relevant and reliable PD model.

Orphan nuclear receptor Nur77 translocates to mitochondria in the early phase of apoptosis induced by synthetic chenodeoxycholic acid derivatives in human stomach cancer cell line SNU-1.

Apoptosis-inducing activity of synthetic CDCA derivatives, HS-1199 and HS-1200, on gastric cancer cell line SNU-1 cells was explored. CDCA derivatives demonstrated various apoptosis hallmarks, such as mitochondrial changes, activation of caspase, DNA fragmentation, and nuclear condensation. Importantly, the orphan receptor Nur77 (TR3) was shown to translocate from the nucleus to mitochondria at the early time points after CDCA derivatives treatment. These data support the theory that CDCA derivatives-induced apoptosis of SNU-1 gastric cancer cell lines is mediated by mitochondria and caspase, and, at least in part, by Nur77.

New and old regulators of uterine leiomyoma growth from screening with DNA arrays

New and old regulators of uterine leiomyoma growth from screening with DNA arrays

Structure and function of Nurr1 identifies a class of ligand-independent nuclear receptors.

Members of the nuclear receptor (NR) superfamily of transcription factors modulate gene transcription in response to small lipophilic molecules. Transcriptional activity is regulated by ligands binding to the carboxy-terminal ligand-binding domains (LBDs) of cognate NRs. A subgroup of NRs referred to as 'orphan receptors' lack identified ligands, however, raising issues about the function of their LBDs. Here we report the crystal structure of the LBD of the orphan receptor Nurr1 at 2.2 A resolution. The Nurr1 LBD adopts a canonical protein fold resembling that of agonist-bound, transcriptionally active LBDs in NRs, but the structure has two distinctive features. First, the Nurr1 LBD contains no cavity as a result of the tight packing of side chains from several bulky hydrophobic residues in the region normally occupied by ligands. Second, Nurr1 lacks a 'classical' binding site for coactivators. Despite these differences, the Nurr1 LBD can be regulated in mammalian cells. Notably, transcriptional activity is correlated with the Nurr1 LBD adopting a more stable conformation. Our findings highlight a unique structural class of NRs and define a model for ligand-independent NR function.

Orphan nuclear receptors in T lymphocyte development

Lymphocyte development is initiated from hematopoietic stem cells and can be divided into multiple phenotypically distinct stages. Transcription factors play important roles in programming the developmental process of lymphocytes. Recent studies have identified key roles of several orphan nuclear receptors in T lymphocyte development. The orphan nuclear receptor RORgamma has been shown to promote thymocyte survival by activating the expression of antiapoptotic protein Bcl-x(L). RORgamma is also required for the development of lymph nodes and Peyer's patches. The orphan receptors Nur77 and Nor1 are involved in TCR-mediated cell death and thymocyte-negative selection. These studies provide novel insights into the molecular mechanisms of T lymphocyte development.

Activation of nuclear orphan receptor NURR1 transcription by NF-kappa B and cyclic adenosine 5'-monophosphate response element-binding protein in rheumatoid arthritis synovial tissue.

Modulation of the NURR subfamily of nuclear receptors may be an important mechanism regulating pathways associated with inflammatory joint disease. We examined the signaling mechanisms through which inflammatory mediators, produced by rheumatoid arthritis (RA) synovial tissue, contribute to the regulation of the NURR subfamily. Markedly enhanced expression of NURR1 is observed in synovial tissue of patients with RA compared with normal subjects. Modulation by proinflammatory mediators in primary RA and normal synoviocytes shows that PGE(2), IL-1beta, and TNF-alpha markedly enhance NURR1 mRNA and protein levels in contrast to other subfamily members, NUR77 and NOR-1. We have established that transcriptional activation of the NURR1 gene by IL-1beta and TNF-alpha requires a proximal promoter region that contains a consensus NF-kappaB DNA-binding motif. IL-1beta- and TNF-alpha-induced NF-kappaB binding to this site is due predominantly to p65-p50 heterodimer and p50 homodimer subunit protein complexes. We further demonstrate a direct CREB-1-dependent regulation by PGE(2) situated at promoter region -171/-163. Moreover, analyses confirm the presence of CREB-1 and NF-kappaB p50 and p65 subunit binding to the NURR1 promoter under basal conditions in freshly explanted RA synovial tissue. In summary, enhanced NF-kappaB- and CREB-1-binding activity on the NURR1 promoter by inflammatory mediators delineates novel mechanisms in the regulation of NURR1 transcription. PGE(2)-, TNF-alpha-, and IL-1beta-dependent stimulation of the NURR1 gene implies that NURR1 induction represents a point of convergence of at least two distinct signaling pathways, suggesting an important common role for this transcription factor in mediating multiple inflammatory signals.

Retinoic acid prevents experimental Cushing syndrome.

Cushing syndrome is caused by an excess of adrenocorticotropic hormone (ACTH) production by neuroendocrine tumors, which subsequently results in chronic glucocorticoid excess. We found that retinoic acid inhibits the transcriptional activity of AP-1 and the orphan receptors Nur77 and Nurr1 in ACTH-secreting tumor cells. Retinoic acid treatment resulted in reduced pro-opiomelanocortin transcription and ACTH production. ACTH inhibition was also observed in human pituitary ACTH-secreting tumor cells and a small-cell lung cancer cell line, but not in normal cells. This correlated with the expression of the orphan receptor COUP-TFI, which was found in normal corticotrophs but not in pituitary Cushing tumors. COUP-TFI expression in ACTH-secreting tumor cells blocked retinoic acid action. Retinoic acid also inhibited cell proliferation and, after prolonged treatment, increased caspase-3 activity and induced cell death in ACTH-secreting cells. In adrenal cortex cells, retinoic acid inhibited corticosterone production and cell proliferation. The antiproliferative action and the inhibition of ACTH and corticosterone produced by retinoic acid were confirmed in vivo in experimental ACTH-secreting tumors in nude mice. Thus, we conclude that the effects of retinoic acid combine in vivo to reverse the endocrine alterations and symptoms observed in experimental Cushing syndrome.

Involvement of the nuclear orphan receptor NURR1 in the regulation of corticotropin‐releasing hormone expression and actions in human inflammatory arthritis

Involvement of the nuclear orphan receptor NURR1 in the regulation of corticotropin‐releasing hormone expression and actions in human inflammatory arthritis

Regulation of the Nuclear Orphan Receptor Nur77 in Bone by Parathyroid Hormone

Osteoblasts function under the control of several hormones and growth factors. Among them, parathyroid hormone (PTH) and steroid hormones have significant effects on bone metabolism. We show that PTH induced the expression of Nur77, a member of the NGFI-B subfamily of nuclear orphan receptors in bone. PTH rapidly and transiently induced Nur77 mRNA in primary mouse osteoblasts that peaked at 1 h and at 10 nM of hormone. Cycloheximide did not affect the induction of Nur77 mRNA, suggesting that protein synthesis is not required for the PTH effect. PTH also induced Nur77 mRNA in calvariae cultures. Finally Nur77 protein expression was induced in nuclear protein extracts of cells treated with PTH. NGFI-B nuclear receptors have been implicated in retinoic acid, vitamin D, and thyroid hormone signaling. We propose that induction of NGFI-B nuclear orphan receptors represents a potential cross-talk mechanism between PTH and steroid hormone signaling to regulate bone metabolism.

Parathyroid hormone induces expression of the nuclear orphan receptor Nurr1 in bone cells.

Following PTH treatment, immediate changes in osteoblast gene expression involve induction of primary response genes. Primary gene products subsequently mediate the osteoblast response to PTH. Using representational difference analysis (RDA) to isolate primary genes induced by PTH in osteoblasts, we identified Nurr1, a member of the NGFI-B nuclear orphan receptor subfamily. Nurr1 binds DNA as a monomer but also heterodimerizes with the 9-cis retinoic acid receptor (RXR). Nurr1's importance in retinoic acid, vitamin D, and thyroid hormone signaling has been hypothesized. Nurr1 messenger RNA (mRNA) levels were maximal at 1 h and at 10 nM of PTH in primary mouse osteoblasts (MOB). Activation of the PKA and PKC pathways by 10 microM forskolin and 1 microM PMA, respectively, induced Nurr1 mRNA levels. However, inhibition of the PKA but not the PKC pathway significantly inhibited the PTH induction of Nurr1. Moreover, PTH(3-34) at 1-100 nM did not induce Nurr1 mRNA levels. Thus, PTH induction of Nurr1 in primary mouse osteoblasts is mediated primarily through the cAMP/PKA pathway. PTH also stimulated Nurr1 protein in MOB cells and Nurr1 mRNA in calvarial organ cultures. Nurr1 induction represents a potential cross-talk mechanism between PTH and steroid hormone signaling at the transcription factor level.

Parathyroid Hormone Induces Expression of the Nuclear Orphan Receptor Nurr1 in Bone Cells

Parathyroid Hormone Induces Expression of the Nuclear Orphan Receptor Nurr1 in Bone Cells

Involvement of the nuclear orphan receptor NURR1 in the regulation of corticotropin-releasing hormone expression and actions in human inflammatory arthritis.

To examine the regulation and mode of action of peripheral corticotropin-releasing hormone (CRH) in human inflammatory arthritis. CRH messenger RNA (mRNA) levels were measured in normal and inflamed synovial tissue and in primary synoviocytes prior to and following cytokine stimulation. Primary synoviocytes were transiently transfected with CRH promoter/reporter constructs, and promoter activity in response to cytokines was assessed. Immunohistochemical staining established CRH receptor expression, and Northern blot analysis confirmed that the nuclear transcription factors NUR77 and NURR1 contributed to synovial CRH receptor-mediated signaling. Primary synoviocytes were treated with pro- and antiinflammatory mediators, and the time course of NURR1 and NUR77 modulation was examined. Nuclear extracts were analyzed by electrophoretic mobility shift assay to determine NURR1 binding to the CRH promoter/enhancer. CRH mRNA was up-regulated in the synovial tissue in rheumatoid arthritis (RA), psoriatic arthritis (PsA), and sarcoid arthritis, but not in normal synovium. Inflammatory cytokines, such as interleukin-1beta and tumor necrosis factor alpha, enhanced the transcriptional activity of the human CRH promoter and increased levels of CRH mRNA in primary synoviocytes. Synovial CRH functioned in a paracrine manner to induce NURR1 and NUR77. NURR1 was abundantly expressed in the inflammatory cells of both RA and PsA synovium. NURR1 and NUR77 were differentially regulated, and NURR1 was the major cytokine-regulated member of the NURR subfamily as well as the mediator of cytokine- and CRH-dependent inflammatory responses in synovium. Furthermore, glucocorticoids dramatically suppressed cytokine- and CRH-induced synovial NURR1 mRNA. These data demonstrate the involvement of the transcription factor NURR1 in the regulation of CRH expression and activity in human inflammatory arthritis.

Modulation of retinoic acid sensitivity in lung cancer cells through dynamic balance of orphan receptors nur77 and COUP-TF and their heterodimerization.

The diverse function of retinoic acid (RA) is mediated by its nuclear receptors, the retinoic acid receptors (RARs) and retinoid X receptors (RXRs). However, the RA response is often lost in cancer cells that express the receptors. Previously, it was demonstrated that the RA response is regulated by the COUP-TF orphan receptors. Here, we present evidence that nur77, another orphan receptor whose expression is highly induced by phorbol esters and growth factors, is involved in modulation of the RA response. Expression of nur77 enhances ligand-independent transactivation of RA response elements (RAREs) and desensitizes their RA responsiveness. Conversely, expression of COUP-TF sensitizes RA responsiveness of RAREs by repressing their basal transactivation activity. Unlike the effect of COUP-TFs, the function of nur77 does not require direct binding of nur77 to the RAREs, but is through interaction between nur77 and COUP-TFs. The interaction occurs in solution and results in inhibition of COUP-TF RARE binding and transcriptional activity. Unlike other nuclear receptors, a large portion of the carboxy-terminal end of nur77 is not required for its interaction with COUP-TF. In human lung cancer cell lines, COUP-TF is highly expressed in RA-sensitive cell lines while nur77 expression is associated with RA resistance. Stable expression of COUP-TF in nur77-positive, RA-resistant lung cancer cells enhances the inducibility of RARbeta gene expression and growth inhibition by RA. These observations demonstrate that a dynamic equilibrium between orphan receptors nur77 and COUP-TF, through their heterodimerization that regulates COUP-TF RARE binding, is critical for RA responsiveness of human lung cancer cells.

Functional domains and phosphorylation of the orphan receptor Nur77.

Nur77 represents a unique class within the steroid receptor superfamily since its synthesis is tightly regulated by extracellular signals and it is capable of potent transactivation activity in the absence of an exogenously added ligand. In this study, we sought to dissect the functional domains regulating the activities of Nur77 by deletion mapping. We demonstrate that whereas the transactivation activity of Nur77 resides in the amino-terminal domain, the carboxy-terminal domain regulates this activity. A short deletion from the carboxy terminus eliminates transactivation activity while a further deletion restores the activity. Deletion of the domain immediately carboxyl to the zinc fingers motif eliminates both DNA binding activity and nuclear localization, thus abolishing transactivation. Nur77 is posttranslationally modified predominantly by phosphorylation, which occurs primarily at the N-terminal domain. The growth-related kinase pp90rsk, but neither the pp44mapk nor the pp70s6k, can phosphorylate recombinant Nur77 in vitro. Furthermore, we have identified a site within the region required for sequence-specific DNA binding, Ser-354, that is phosphorylated by pp90rsk in vitro; this site is also phosphorylated in vivo. The possibility that phosphorylation might affect DNA binding is discussed.

Functional domains and phosphorylation of the orphan receptor Nur77

Functional domains and phosphorylation of the orphan receptor Nur77