Orphan Nuclear Receptor RORalpha Research Articles

Orphan Nuclear Receptor RORalpha Exerts Protective Potential in Acute Kidney Injury via Transcriptional Activation of HIF-1

Orphan Nuclear Receptor RORalpha Exerts Protective Potential in Acute Kidney Injury via Transcriptional Activation of HIF-1

Arachidonic Acid Pathway Members PLA2G7, HPGD, EPHX2, and CYP4F8 Identified as Putative Novel Therapeutic Targets in Prostate Cancer

The arachidonic acid and prostaglandin pathway has been implicated in prostate carcinogenesis, but comprehensive studies of the individual members in this key pathway are lacking. Here, we first conducted a systematic bioinformatic study of the expression of 36 arachidonic acid pathway genes across 9783 human tissue samples. The results showed that the PLA2G7, HPGD, EPHX2, and CYP4F8 genes are highly expressed in prostate cancer. Functional studies using RNA interference in prostate cancer cells indicated that all four genes are also essential for cell growth and survival. Clinical validation confirmed high PLA2G7 expression, especially in ERG oncogene-positive prostate cancers, and its silencing sensitized ERG-positive prostate cancer cells to oxidative stress. HPGD was highly expressed in androgen receptor (AR)-overexpressing advanced tumors, as well as in metastatic prostate cancers. EPHX2 mRNA correlated with AR in primary prostate cancers, and its inhibition in vitro reduced AR signaling and potentiated the effect of antiandrogen flutamide in cultured prostate cancer cells. In summary, we identified four novel putative therapeutic targets with biomarker potential for different subtypes of prostate cancer. In addition, our results indicate that inhibition of these enzymes may be particularly powerful when combined with other treatments, such as androgen deprivation or induction of oxidative stress.

T helper17 Cells Are Sufficient But Not Necessary to Induce Acute Graft-Versus-Host Disease

T helper (Th)1 cells were considered responsible for the induction of graft-versus-host disease (GVHD), but recently the concept has been challenged. Th17 cells play a critical role in mediating autoimmune diseases, but their role in the pathogenesis of GVHD remains unclear. Herein we compare the ability of in vitro generated Th1 and Th17 cells from C57BL/6 mice to induce GVHD in lethally irradiated BALB/c recipients. Allogeneic Th17 cells had superior expansion and infiltration capabilities in GVHD target organs, which correlated with their increased pathogenicity when compared with naïve or Th1 controls. Th17 cells caused no pathology in the syngeneic recipients, indicating that antigen-activation was required for their pathogenicity. Polarized Th17 cells could not maintain their phenotype in vivo as they produced a significant amount of interferon (IFN)-gamma after being transplanted into allogeneic recipients; however, IFN-gamma was not required for Th17 cell-induced GVHD. Further, we evaluated the pathogenesis of Th17 cells in GVHD by using polyclonal nonprimed CD4T cells in a clinically relevant allogeneic bone marrow transplantation (BMT) setting. We found that disruption of Th17-differentiation alone by targeting RORgammat (Th17-specific transcription factor) had no significant effect on GVHD development. We conclude that Th17 cells are sufficient but not necessary to induce GVHD.

Absence of the SRC-2 coactivator results in a glycogenopathy resembling Von Gierke's disease.

Hepatic glucose production is critical for basal brain function and survival when dietary glucose is unavailable. Glucose-6-phosphatase (G6Pase) is an essential, rate-limiting enzyme that serves as a terminal gatekeeper for hepatic glucose release into the plasma. Mutations in G6Pase result in Von Gierke's disease (glycogen storage disease-1a), a potentially fatal genetic disorder. We have identified the transcriptional coactivator SRC-2 as a regulator of fasting hepatic glucose release, a function that SRC-2 performs by controlling the expression of hepatic G6Pase. SRC-2 modulates G6Pase expression directly by acting as a coactivator with the orphan nuclear receptor RORalpha. In addition, SRC-2 ablation, in both a whole-body and liver-specific manner, resulted in a Von Gierke's disease phenotype in mice. Our results position SRC-2 as a critical regulator of mammalian glucose production.

Generation of T Follicular Helper Cells Is Mediated by Interleukin-21 but Independent of T Helper 1, 2, or 17 Cell Lineages

After activation, CD4(+) helper T (Th) cells differentiate into distinct effector subsets. Although chemokine (C-X-C motif) receptor 5-expressing T follicular helper (Tfh) cells are important in humoral immunity, their developmental regulation is unclear. Here we show that Tfh cells had a distinct gene expression profile and developed in vivo independently of the Th1 or Th2 cell lineages. Tfh cell generation was regulated by ICOS ligand (ICOSL) expressed on B cells and was dependent on interleukin-21 (IL-21), IL-6, and signal transducer and activator of transcription 3 (STAT3). However, unlike Th17 cells, differentiation of Tfh cells did not require transforming growth factor beta (TGF-beta) or Th17-specific orphan nuclear receptors RORalpha and RORgamma in vivo. Finally, naive T cells activated in vitro in the presence of IL-21 but not TGF-beta signaling preferentially acquired Tfh gene expression and promoted germinal-center reactions in vivo. This study thus demonstrates that Tfh is a distinct Th cell lineage.

Regulation of T Helper 17 Differentiation by Orphan Nuclear Receptors: It's Not Just RORγt Anymore

T cells that produce IL-17 (T helper 17 cells) are implicated in autoimmune pathogenesis. In this issue of Immunity, Yang et al. (2008) report that the closely related orphan nuclear receptors ROR alpha and ROR gamma t work together to regulate T helper (Th)17 cell differentiation.

Neurodegeneration: A Case of Arrested Development?

The neurodegenerative disease Spinocerebellar ataxia type 1 (SCA1) is a polyglutamine expansion disorder characterized by the death of Purkinje neurons in the brain. In this issue, Serra et al. (2006) implicate the impaired function of the orphan nuclear receptor RORalpha in SCA1 pathogenesis. Their intriguing results suggest that derailing a transcription program during embryonic development may render adult neurons more susceptible to toxic insults.

Activation of the orphan nuclear receptor RORalpha counteracts the proliferative effect of fatty acids on prostate cancer cells: crucial role of 5-lipoxygenase.

The incidence of prostate carcinoma is very low in Eastern countries, such as Japan, suggesting that life style conditions may play a crucial role in the development of this pathology. Dietary omega-6 polyunsaturated fatty acids, such as linoleic (LA) and arachidonic (AA) acids, have been shown to stimulate the proliferation of prostate cancer cells after being converted into 5-HETE by means of the 5-lipoxygenase (5-LOX) pathway. Blockade of 5-LOX activity has been proposed as an attractive target for the prevention of the mitogenic action of dietary fats on prostate cancer. The 5-LOX gene has been shown to carry a response element for the orphan nuclear receptor RORalpha (for its RORalpha1 isoform in particular) in its promoter region. We attempt to clarify whether activation of RORalpha might modulate the expression of 5-LOX, thus interfering with the mitogenic activity of fatty acids in prostate cancer cells. We show that in androgen-independent DU 145 prostate cancer cells, LA, AA and their metabolite 5-HETE exert a strong stimulatory action on cell proliferation. This effect is completely counteracted by the simultaneous treatment of the cells with a non redox inhibitor of 5-LOX activity. We then demonstrate that: i) RORalpha, and specifically its RORalpha1 isoform, is expressed in DU 145 cells; ii) activation of RORalpha, by means of the thiazolidinedione derivative CGP 52608 (the synthetic RORalpha activator), significantly reduces 5-LOX expression, both at mRNA (as evaluated by comparative RT-PCR) and at protein (as investigated by Western blot analysis) level (this was confirmed by the reduced activity of 5-LOX in CGP 52608 treated cells); and iii) the treatment of DU 145 cells with CGP 52608 completely abrogated the proliferative action of both LA and AA. These results have been confirmed in another androgen-independent prostate cancer cell line (PC3). Our data indicate that, by decreasing the expression of 5-LOX, activation of RORalpha might interfere with the mitogenic activity of fatty acids on prostate cancer. We have shown previously that CGP 52608 reduces the proliferation and the metastatic behavior of DU 145 cells. These observations indicate that the orphan nuclear receptor RORalpha might be considered as a molecular target for the development of new chemopreventive or chemotherapeutic strategies for prostate carcinoma.

Role of the orphan nuclear receptor RORα in the control of the metastatic behavior of androgen-independent prostate cancer cells

Orphan nuclear receptors constitute a subgroup of the superfamily of steroid/thyroid/retinoid receptors for which no endogenous ligand has been identified. The orphan nuclear receptor ROR alpha has been shown to be involved in the control of cell growth and differentiation. We have previously shown that, in DU 145 androgen-independent prostate cancer cells, ROR alpha activation brings about a significant decrease of cell proliferation and affects cell cycle progression through the modulation of cell cycle-related genes. The experiments here described have been performed to clarify whether ROR alpha might also be involved in the control of the metastatic behavior of DU 145 cells. We have shown that the thiazolidinedione derivative CGP 52608, the specific ROR alpha ligand and activator, reduces the ability of DU 145 cells to invade a reconstituted basement membrane (Matrigel). CGP 52608 also significantly decreased the capacity of prostate cancer cells to migrate towards a chemotactic stimulus (fibronectin), when plated in the upper compartment of a Boyden's chamber. Moreover, ROR alpha activation resulted in a decreased expression of alpha v beta 3 integrin and an increased level of expression of beta 4 integrin subunit. These findings indicate that the activation of the orphan nuclear receptor ROR alpha reduces the invasive and migratory capacities of androgen-independent prostate cancer cells, at least partially, by affecting integrin expression.

Cyclical regulation of GnRH gene expression in GT1-7 GnRH-secreting neurons by melatonin.

The pineal hormone melatonin plays an important role in the neuroendocrine control of reproductive physiology, but its effects on hypothalamic GnRH neurons are not yet known. We have found that GT1-7 GnRH-secreting neurons express membrane-bound G protein-coupled melatonin receptors, mt1 (Mel-1a) and MT2 (Mel-1b) as well as the orphan nuclear receptors ROR alpha and RZR beta. Melatonin (1 nM) significantly downregulates GnRH mRNA levels in a 24-h cyclical manner, an effect that is specifically inhibited by the melatonin receptor antagonist luzindole (10 microM). Repression of GnRH gene expression by melatonin appears to occur at the transcriptional level and can be mapped to the GnRH neuron-specific enhancer located within the 5' regulatory region of the GnRH gene. Using transient transfection of GT1-7 cells, downregulation of GnRH gene expression by melatonin was further localized to five specific regions within the GnRH enhancer including -1827/-1819, -1780/-1772, -1746/-1738, -1736/-1728, and -1697/-1689. Interestingly, the region located at -1736/-1728 includes sequences that correspond to two direct repeats of hexameric consensus binding sites for members of the ROR/RZR orphan nuclear receptor family. To begin to dissect the mechanisms involved in the 24-h cyclical regulation of GnRH transcription, we have found that melatonin (10 nM) induces rapid internalization of membrane-bound mt1 receptors through a beta-arrestin 1-mediated mechanism. These results provide the first evidence that melatonin may mediate its neuroendocrine control on reproductive physiology through direct actions on the GnRH neurons of the hypothalamus, both at the level of GnRH gene expression and through the regulation of G protein-coupled melatonin receptors.

Activation of the orphan nuclear receptor RORalpha induces growth arrest in androgen-independent DU 145 prostate cancer cells.

RORalpha is a transcription factor which belongs to the family of orphan nuclear receptors. The regulatory functions of this receptor are still poorly understood. However, response elements for RORalpha are present on the promoter of cell cycle-related genes suggesting that it might be involved in the control of cell proliferation. In this study, we investigated the expression and the possible function of RORalpha in a human androgen-independent prostate cancer cell line (DU 145). The thiazolidinedione-derivative CGP 52608 has been utilized as the specific ligand and activator of RORalpha. The effects of CGP 52608 on DU 145 cell proliferation and cell cycle distribution were analyzed by hemocytometer and by FACS analysis, respectively. The expression of RORalpha as well as the effects of RORalpha activation on the expression of cell cycle-related genes were evaluated by RT-PCR. To clarify whether RORalpha activation might affect the proliferation of prostate cancer cells also in vivo, nude mice bearing DU 145 tumor xenografts were treated with CGP 52608 at different doses and the growth of the tumors was followed by caliper measurement. RORalpha is expressed in DU 145 cells and the treatment of the cells with the thiazolidinedione-derivative CGP 52608 brought about a dose-dependent and significant decrease of cell proliferation. Ligand-induced activation of RORalpha affected cell cycle distribution, inducing an accumulation in the G(0)/G(1) phase and a decrease in the S phase. This effect was accompanied by an increased expression of the cyclin-dependent kinase inhibitor p21(WAF1/CIP1) and a decreased expression of cyclin A. The growth of DU 145 tumors in nude mice was significantly reduced by treatment with CGP 52608. These data indicate that, in androgen-independent DU 145 prostate cancer cells, activation of the orphan nuclear receptor RORalpha inhibits cell growth, both in vitro and in vivo. RORalpha also induces cell cycle arrest, possibly through the modulation of the expression of cell cycle-related genes.

Oncostatic activity of a thiazolidinedione derivative on human androgen-dependent prostate cancer cells.

Thiazolidinedione derivatives with potent antiarthritic activity, such as CGP 52608, have been suggested to exert their biological effects through the activation of the orphan nuclear receptor RORalpha. Since response elements for this receptor are present in the promoter region of cell cycle-related genes (i.e., p21(WAF1/CIP1) and cyclin A), we reasoned that CGP 52608 might affect cell proliferation, cell cycle progression and the expression of cell cycle-related genes. This hypothesis has been verified in the human androgen-dependent prostate cancer cell line LNCaP. We found that the treatment of LNCaP cells with CGP 52608 brings about a significant and dose-dependent decrease of cell proliferation. Thiazolidinedione affected cell cycle distribution, inducing an accumulation of the cells in the G0/G1 phase and a decrease in the S phase. This effect was accompanied by an increased expression of the cyclin-dependent kinase inhibitor p21(WAF1/CIP1) and a decreased expression of cyclin A. These data indicate that, in human androgen-dependent LNCaP prostate cancer cells, the thiazolidinedione derivative CGP 52608 exerts a strong cytostatic activity, by reducing cell proliferation and by affecting cell cycle distribution through the modulation of the expression of cell cycle-related genes. These biological actions of CGP 52608 might be mediated by the activation of the orphan nuclear RORalpha receptor, which is expressed in LNCaP cells.

Coactivators for the orphan nuclear receptor RORalpha.

A mutation in the nuclear orphan receptor RORalpha results in a severe impairment of cerebellar development by unknown mechanisms. We have shown previously that RORalpha contains a strong constitutive activation domain in its C terminus. We therefore searched for mammalian RORalpha coactivators using the minimal activation domain as bait in a two-hybrid screen. Several known and putative coactivators were isolated, including glucocorticoid receptor-interacting protein-1 (GRIP-1) and peroxisome proliferator-activated receptor (PPAR)-binding protein (PBP/TRAP220/DRIP205). These interactions were confirmed in vitro and require the intact activation domain of RORalpha although different requirements for interaction with GRIP-1 and PBP were detected. Even in the absence of exogenous ligand, RORalpha interacts with a complex or complexes of endogenous proteins, similar to those that bind to ligand-occupied thyroid hormone and vitamin D receptors. Both PBP and GRIP-1 were shown to be present in these complexes. Thus we have identified several potential RORalpha coactivators that, in contrast to the interactions with hormone receptors, interact with RORalpha in yeast, in bacterial extracts, and in mammalian cells in vivo and in vitro in the absence of exogenous ligand. GRIP-1 functioned as a coactivator for the RORalpha both in yeast and in mammalian cells. Thus, GRIP-1 is the first proven coactivator for RORalpha.

ROR alpha gene expression in the perinatal rat cerebellum: ontogeny and thyroid hormone regulation.

Deficiency of thyroid hormone (TH) during the perinatal period results in severe neurological abnormalities in rodent cerebellar development. However, the molecular mechanisms of TH action in the developing cerebellum are not fully understood. Of note, a mutant mouse, staggerer, in which the orphan nuclear hormone receptor ROR alpha gene is disrupted, exhibits cerebellar abnormalities similar to those seen in the hypothyroid animals, despite normal thyroid function. We, therefore, speculated that TH (tetraiodo-L-thyronine; T4) may regulate ROR alpha gene expression, which then may regulate genes essential for normal brain development. To test this hypothesis, we studied the changes in ROR alpha gene expression in perinatal hypothyroid rat cerebellum and the effect of TH replacement using Northern blot analysis, ribonuclease protection assay and in situ hybridization histochemistry. During cerebellar development, an approximately 3-fold increase in the cerebellar content of ROR alpha messenger RNA (mRNA) was seen in both propylthiouracil-treated, and propylthiouracil-treated and T4-replaced animals. However, the increase was accelerated when T4 was injected, although the ROR alpha mRNA content was identical, with or without T4, by 30 days after birth (P30). In contrast, T4 treatment suppressed the TH receptor alpha1 and c-erbA alpha2 mRNA content by P30; retinoic acid X receptor-beta mRNA content was not influenced by thyroid status. A significant hybridization signal for ROR alpha mRNA was seen only over Purkinje cells in the cerebellar cortex by in situ hybridization histochemistry. These results indicate that TH alters the timing of expression of the ROR alpha gene in the Purkinje cells of the cerebellar cortex, which may, in turn, influence Purkinje cell differentiation.

Transcriptional activation and repression by RORalpha, an orphan nuclear receptor required for cerebellar development.

Mutation of the orphan nuclear receptor RORalpha results in a severe impairment of cerebellar development by unknown mechanisms. We have found that RORalpha activates transcription from only a subset of sites to which it binds strongly as a monomer. RORalpha also selectively binds as a homodimer to a direct repeat of this monomer site with a 2-bp spacing between the AGGTCA sequences (Rev-DR2 site) and is a much more potent transcriptional activator on this site than on monomer sites or other direct repeats. To better understand the transcriptional regulatory functions of RORalpha, we fused its C terminus to a heterologous DNA-binding domain. Mutational analysis revealed that RORalpha contains both transcriptional activation and transcriptional repression domains, with the repression domain being more active in some cell types. The abilities of RORalpha polypeptides to repress transcription correlate with their abilities to interact with the nuclear receptor corepressors N-CoR and SMRT in vitro. However, the AF2 region of RORalpha inhibits corepressor interaction on DNA, consistent with the lack of repression by the full-length receptor. Thus, transcriptional regulation by RORalpha is complex and likely to be regulated in a cell type- and target gene-specific manner.

Transcriptional regulation of a Purkinje cell-specific gene through a functional interaction between ROR alpha and RAR.

The orphan nuclear receptor ROR alpha is highly expressed in the Purkinje cells of the cerebellum during the postnatal development of brain. A recent observation has been made that the ROR alpha gene is disrupted in staggerer mice-which show a cell-autonomous defect in the development of the Purkinje cells. In order to understand the functions of ROR alpha in cerebellar development, I attempted to identify its target genes. Transient expression study demonstrated that transcription of the Purkinje cell protein-2 (Pcp-2) gene is activated by ROR alpha, which binds as a monomer to a single half-site motif (RORE) within the promoter region. Its transcription was also activated by retinoic acid receptor (RAR) which binds as a heterodimer with RXR to a retinoic acid responsive element (RARE) in the downstream region. Interestingly, the ROR alpha-mediated transcription is further activated synergistically by RAR. That the Pcp-2 gene is a target of ROR alpha, and is suggested that its transcription is also regulated by RAR.

Orphan nuclear receptor ROR alpha gene: isoform-specific spatiotemporal expression during postnatal development of brain.

We analyzed expression of mouse orphan nuclear receptor ROR alpha during postnatal development of rodent brain. Using a riboprobe corresponding to the 3'-end of mROR alpha cDNA a peak of ROR alpha expression was observed at postnatal 16 day (P16) in the Purkinje cells of cerebellum, neurons of the thalamus and the olfactory bulb. The hippocampus was also shown to express ROR alpha with an earlier peak at P7. Expression in cell types other than the Purkinje cells appeared transient. On the other hand, when a probe to the 5'-end of mROR alpha cDNA was used, we observed patterns of ROR alpha expression that are different from those observed with the 3'-probe. No specific transcripts of ROR alpha were detected with the 5'-probe in the Purkinje cells until P16. Additionally, the relative level of the hybridization signals with the 5'-probe and the 3'-probe were different among the various brain regions. Together with the previous findings that ROR alpha comprises at least four isoforms which differ from one another in their N-terminal regions, these observations suggest that the spatiotemporal expression of ROR alpha is under isoform-specific regulation. The timing of its expression suggests that ROR alpha may be involved in regulation of postnatal maturation of specific classes of neurons.