Metformin Prevents Hyperglycemia-Associated, Oxidative Stress-Induced Vascular Endothelial Dysfunction: Essential Role for the Orphan Nuclear Receptor Human Nuclear Receptor 4A1 (Nur77).

Vascular pathology is increased in diabetes because of reactive-oxygen-species (ROS)-induced endothelial cell damage. We found that in vitro and in a streptozotocin diabetes model in vivo, metformin at diabetes-therapeutic concentrations (1-50 µM) protects tissue-intact and cultured vascular endothelial cells from hyperglycemia/ROS-induced dysfunction typified by reduced agonist-stimulated endothelium-dependent, nitric oxide-mediated vasorelaxation in response to muscarinic or proteinase-activated-receptor 2 agonists. Metformin not only attenuated hyperglycemia-induced ROS production in aorta-derived endothelial cell cultures but also prevented hyperglycemia-induced endothelial mitochondrial dysfunction (reduced oxygen consumption rate). These endothelium-protective effects of metformin were absent in orphan-nuclear-receptor Nr4a1-null murine aorta tissues in accord with our observing a direct metformin-Nr4a1 interaction. Using in silico modeling of metformin-NR4A1 interactions, Nr4a1-mutagenesis, and a transfected human embryonic kidney 293T cell functional assay for metformin-activated Nr4a1, we identified two Nr4a1 prolines, P505/P549 (mouse sequences corresponding to human P501/P546), as key residues for enabling metformin to affect mitochondrial function. Our data indicate a critical role for Nr4a1 in metformin's endothelial-protective effects observed at micromolar concentrations, which activate AMPKinase but do not affect mitochondrial complex-I or complex-III oxygen consumption rates, as does 0.5 mM metformin. Thus, therapeutic metformin concentrations requiring the expression of Nr4a1 protect the vasculature from hyperglycemia-induced dysfunction in addition to metformin's action to enhance insulin action in patients with diabetes. SIGNIFICANCE STATEMENT: Metformin improves diabetic vasodilator function, having cardioprotective effects beyond glycemic control, but its mechanism to do so is unknown. We found that metformin at therapeutic concentrations (1-50µM) prevents hyperglycemia-induced endothelial dysfunction by attenuating reactive oxygen species-induced damage, whereas high metformin (>250 µM) impairs vascular function. However, metformin's action requires the expression of the orphan nuclear receptor NR4A1/Nur77. Our data reveal a novel mechanism whereby metformin preserves diabetic vascular endothelial function, with implications for developing new metformin-related therapeutic agents.

Nr4a1 as a myogenic factor is upregulated in satellite cells/myoblast under proliferation and differentiation state.

Myogenic differentiation is precisely regulated with a cascade of genes and pathways. The previous study has demonstrated the muscle-specific deletion of Nr4a1 impairs muscle growth. However, it is still unclear whether muscular Nr4a1 deletion may directly impact myoblast physiology. Here, the present study delves into the molecular mechanism of Nr4a1 in C2C12. Through the analysis of RNAseq and microarray data, Nr4a1 was identified to highly correlate with the expression of myogenic factors. In C2C12, except confirming the induction of Nr4a1 mRNA and protein levels upon the initiation of differentiation, we observed a novel shuttling phenomenon of Nr4a1 from nucleus to cytoplasm in myoblast with a higher expression of MyoD or differentiated myotubes. Furthermore, Nr4a1 overexpression in C2C12 accelerates myoblasts' differentiation and increases myoblast fusion. In contrast, ablation of Nr4a1 expression in C2C12 inhibits the differentiation and fusion process. Meanwhile, in quiescent satellite cells, Nr4a1 expressed is not detected, while its protein level is highly induced in both BaCl2-induced muscle regeneration followed with satellite cells activation and satellite cells of cultured single myofiber. The mechanism may be through the Nr4a1-mediated expression of myogenic factors, e.g. MyoD and MyoG. In summary, the current investigation demonstrates that Nr4a1 is an essential myogenic factor involved in myoblast differentiation.

Translational regulation of specific mRNAs controls feedback inhibition and survival during macrophage activation.

For a rapid induction and efficient resolution of the inflammatory response, gene expression in cells of the immune system is tightly regulated at the transcriptional and post-transcriptional level. The control of mRNA translation has emerged as an important determinant of protein levels, yet its role in macrophage activation is not well understood. We systematically analyzed the contribution of translational regulation to the early phase of the macrophage response by polysome fractionation from mouse macrophages stimulated with lipopolysaccharide (LPS). Individual mRNAs whose translation is specifically regulated during macrophage activation were identified by microarray analysis. Stimulation with LPS for 1 h caused translational activation of many feedback inhibitors of the inflammatory response including NF-κB inhibitors (Nfkbid, Nfkbiz, Nr4a1, Ier3), a p38 MAPK antagonist (Dusp1) and post-transcriptional suppressors of cytokine expression (Zfp36 and Zc3h12a). Our analysis showed that their translation is repressed in resting and de-repressed in activated macrophages. Quantification of mRNA levels at a high temporal resolution by RNASeq allowed us to define groups with different expression patterns. Thereby, we were able to distinguish mRNAs whose translation is actively regulated from mRNAs whose polysomal shifts are due to changes in mRNA levels. Active up-regulation of translation was associated with a higher content in AU-rich elements (AREs). For one example, Ier3 mRNA, we show that repression in resting cells as well as de-repression after stimulation depends on the ARE. Bone-marrow derived macrophages from Ier3 knockout mice showed reduced survival upon activation, indicating that IER3 induction protects macrophages from LPS-induced cell death. Taken together, our analysis reveals that translational control during macrophage activation is important for cellular survival as well as the expression of anti-inflammatory feedback inhibitors that promote the resolution of inflammation.

High expression of orphan nuclear receptor NR4A1 in a subset of ovarian tumors with worse outcome.

OBJECTIVE: Nuclear receptors (NRs) play a vital role in the development and progression of several cancers including breast and prostate. Using TCGA data, we sought to identify critical nuclear receptors in high grade serous ovarian cancers (HGSOC) and to confirm these findings using in vitro approaches. METHODS: In silico analysis of TCGA data was performed to identify relevant NRs in HGSOC. Ovarian cancer cell lines were screened for NR expression and functional studies were performed to determine the significance of these NRs in ovarian cancers. NR expression was analyzed in ovarian cancer tissue samples using immunohistochemistry to identify correlations with histology and stage of disease. RESULTS: The NR4A family of NRs was identified as a potential driver of ovarian cancer pathogenesis. Overexpression of NR4A1 in particular correlated with worse progression free survival. Endogenous expression of NR4A1 in normal ovarian samples was relatively high compared to that of other tissue types, suggesting a unique role for this orphan receptor in the ovary. Expression of NR4A1 in HGSOC cell lines as well as in patient samples was variable. NR4A1 primarily localized to the nucleus in normal ovarian tissue while co-localization within the cytoplasm and nucleus was noted in ovarian cancer cell lines and patient tissues. CONCLUSIONS: NR4A1 is highly expressed in a subset of HGSOC samples from patients that have a worse progression free survival. Studies to target NR4A1 for therapeutic intervention should include HGSOC.

Soluble CD163 masks fibronectin-binding protein A-mediated inflammatory activation of Staphylococcus aureus infected monocytes.

Binding to fibronectin (FN) is a crucial pathogenic factor of Staphylococcus aureus mediated by fibronectin-binding protein A (FnBP-A) and extracellular adherence protein (Eap). Recently, we have shown that binding of soluble CD163 (sCD163) to FN linked to these molecules exhibits anti-microbial effects by enhancing phagocytosis and killing activity of S. aureus-infected monocytes. However, it remained unclear whether sCD163 also influences the monocytic activation status. Using genetically modified staphylococcal strains we now identified FnBP-A, but not Eap, as activator of the inflammatory response of monocytes to infection. FnBP-A-mediated inflammatory activation was masked by sCD163 binding to S. aureus promoting efficient pathogen elimination. Thus, sCD163 protects monocytes from overwhelming activation upon staphylococcal infection by dampening the secretion of pro-inflammatory cytokines TNFα, IL-1ß, IL-6 and IL-8 and DAMP molecule MRP8/14. Moreover, sCD163 limited expression of pro-apoptotic transcription factor NR4A1 induced during S. aureus infection and inhibited induction of chemokine CXCL2promoting survival of staphylococci in vivo. sCD163-mediated effects were not due to general immunosuppression since MAP kinase activation and ROS production were unaltered during infection of monocytes with sCD163-bound bacteria. Thus, sCD163 promotes a specific defence of the immune system against FnBP-A-mediated inflammatory activation enabling successful pathogen elimination, tissue recovery and resolution of inflammation.

Amelioration of palmitate-induced metabolic dysfunction in L6 muscle cells expressing low levels of receptor-interacting protein 140.

We have shown that reduced expression of receptor-interacting protein 140 (RIP140) alters the regulation of fatty-acid (FA) oxidation in muscle. To determine whether a high level of FA availability alters the effects of RIP140 on metabolic regulation, L6 myotubes were transfected with or without RNA interference oligonucleotide sequences to reduce RIP140 expression, and then incubated with high levels of palmitic acid, with or without insulin. High levels of palmitate reduced basal (53%-58%) and insulin-treated (24%-44%) FA uptake and oxidation, and increased basal glucose uptake (88%). In cells incubated with high levels of palmitate, low RIP140 increased basal FA uptake and insulin-treated FA oxidation and glucose uptake, and decreased basal glucose uptake and insulin-treated FA uptake. Under basal conditions, low RIP140 increased the mRNA content of FAT/CD36 (159%) and COX4 (61%), as well as the protein content of Nur77 (68%), whereas the mRNA expression of FGF21 (50%) was decreased, as was the protein content of CPT1b (35%) and FGF21 (44%). Under insulin-treated conditions, low RIP140 expression increased the mRNA content of MCAD (84%) and Nur77 (84%), as well as the protein content of Nur77 (23%). Thus, a low level of RIP140 restores the rates of FA uptake in the basal state, in part via a reduction in upstream insulin signaling. Our data also indicate that the protein expression of Nur77 may be modulated by RIP140 when muscle cells are metabolically challenged by high levels of palmitate.

Orphan nuclear receptor Nur77 promotes colorectal cancer invasion and metastasis by regulating MMP-9 and E-cadherin.

Nur77, an orphan member of the nuclear receptor superfamily, has been implicated in tumorigenesis. However, its contributions to colorectal cancer (CRC) invasion and metastasis are largely under characterized. Here, we present the first evidence that the invasion and metastasis of CRC is regulated by Nur77. High expression of Nur77 was observed in clinical CRC tissues, and this elevated expression was significantly associated with advanced tumor, lymph nodes, distant metastasis stage (P = 0.003), lymph node metastasis (P = 0.001) and poor survival (P = 0.03). Overexpression of Nur77 in CRC cells enhanced cell invasion in vitro, whereas knockdown of Nur77 diminished cell invasion and metastasis both in vitro and in vivo. In studying the possible mechanism by which overexpression of Nur77 contributes to CRC invasion and metastasis, we observed that the nuclear protein Nur77 promoted the expression of matrix metalloproteinase (MMP)-9, a novel downstream target of Nur77, and subsequently decreased the expression of E-cadherin. Examination of clinical samples further showed that Nur77 expression is positively correlated with MMP-9, whereas negatively correlated with E-cadherin. Interestingly, Nur77-mediated CRC invasion via MMP-9 and E-cadherin could be mimicked by some metastasis-inducible factors including hypoxia and prostaglandin E2. Collectively, our study demonstrated that Nur77 could promote the invasion and metastasis of CRC cells through regulation of MMP-9/E-cadherin signaling. These observations provide a possible new strategy for potentially treating or preventing the metastasis of CRC through targeting of Nur77.

Class IIa HDACs repressive activities on MEF2-depedent transcription are associated with poor prognosis of ER⁺ breast tumors.

MEF2s transcription factors and class IIa HDACs compose a fundamental axis for several differentiation pathways. Functional relationships between this axis and cancer are largely unexplored. We have found that class IIa HDACs are heterogeneously expressed and display redundant activities in breast cancer cells. Applying gene set enrichment analysis to compare the expression profile of a list of putative MEF2 target genes, we have discovered a correlation between the down-regulation of the MEF2 signature and the aggressiveness of ER(+) breast tumors. Kaplan-Meier analysis in ER(+) breast tumors evidenced an association between increased class IIa HDACs expression and reduced survival. The important role of the MEF2-HDAC axis in ER(+) breast cancer was confirmed in cultured cells. MCF7 ER(+) cells were susceptible to silencing of class IIa HDACs in terms of both MEF2-dependent transcription and apoptosis. Conversely, in ER(-) MDA-MB-231 cells, the repressive influence of class IIa HDACs was dispensable. Similarly, a class IIa HDAC-specific inhibitor preferentially promoted the up-regulation of several MEF2 target genes and apoptosis in ER(+) cell lines. The prosurvival function of class IIa HDACs could be explained by the repression of NR4A1/Nur77, a proapoptotic MEF2 target. In summary, our studies underscore a contribution of class IIa HDACs to aggressiveness of ER(+) tumors.-Clocchiatti, A., Di Giorgio, E., Ingrao, S., Meyer-Almes, F.-J., Tripodo, C., Brancolini, C. Class IIa HDACs repressive activities on MEF2-depedent transcription are associated with poor prognosis of ER(+) breast tumors.

Crosstalk between lysine-specific demethylase 1 (LSD1) and histone deacetylases mediates antineoplastic efficacy of HDAC inhibitors in human breast cancer cells.

Our previous studies demonstrated that lysine-specific demethylase 1 (LSD1) and histone deacetylases (HDACs) closely interact in controlling growth of breast cancer cells. However, the underlying mechanisms are largely unknown. In this study, we showed that knockdown of LSD1 expression (LSD1-KD) by RNAi decreased mRNA levels of HDAC isozymes in triple-negative breast cancer (TNBC) cells. Small interfering RNA (siRNA)-mediated depletion of HDAC5 expression induced the most significant accumulation of H3K4me2, a specific substrate of LSD1. Combined treatment with LSD1 inhibitor, pargyline, and HDAC inhibitor, SAHA (Vorinostat), led to superior growth inhibition and apoptotic death in TNBC cells, but exhibited additive or antagonistic effect on growth inhibition in non-TNBC counterparts or non-tumorigenic breast cells. Additionally, LSD1-KD enhanced SAHA-induced reexpression of a subset of aberrantly silenced genes, such as NR4A1, PCDH1, RGS16, BIK, and E-cadherin whose reexpression may be tumor suppressive. Genome-wide microarray study in MDA-MB-231 cells identified a group of tumor suppressor genes whose expression was induced by SAHA and significantly enhanced by LSD1-KD. We also showed that concurrent depletion of RGS16 by siRNA reduced overall cytotoxicity of SAHA and blocked the reexpression of E-cadherin, CDKN1C and ING1 in LSD1-deficient MDA-MB-231 cells. Furthermore, cotreatment with RGS16 siRNA reversed the downregulation of nuclear factor-kappaB expression induced by combined inhibition of LSD1 and HDACs, suggesting a crucial role of RGS16 in controlling key pathways of cell death in response to combination therapy. Taken together, these results provide novel mechanistic insight into the breast cancer subtype-dependent role of LSD1 in mediating HDAC activity and therapeutic efficacy of HDAC inhibitor.

Exercise training enhances white adipose tissue metabolism in rats selectively bred for low- or high-endurance running capacity.

Impaired visceral white adipose tissue (WAT) metabolism has been implicated in the pathogenesis of several lifestyle-related disease states, with diminished expression of several WAT mitochondrial genes reported in both insulin-resistant humans and rodents. We have used rat models selectively bred for low- (LCR) or high-intrinsic running capacity (HCR) that present simultaneously with divergent metabolic phenotypes to test the hypothesis that oxidative enzyme expression is reduced in epididymal WAT from LCR animals. Based on this assumption, we further hypothesized that short-term exercise training (6 wk of treadmill running) would ameliorate this deficit. Approximately 22-wk-old rats (generation 22) were studied. In untrained rats, the abundance of mitochondrial respiratory complexes I-V, citrate synthase (CS), and PGC-1 was similar for both phenotypes, although CS activity was greater than 50% in HCR (P = 0.09). Exercise training increased CS activity in both phenotypes but did not alter mitochondrial protein content. Training increased the expression and phosphorylation of proteins with roles in ß-adrenergic signaling, including ß3-adrenergic receptor (16% increase in LCR; P < 0.05), NOR1 (24% decrease in LCR, 21% decrease in HCR; P < 0.05), phospho-ATGL (25% increase in HCR; P < 0.05), perilipin (25% increase in HCR; P < 0.05), CGI-58 (15% increase in LCR; P < 0.05), and GLUT4 (16% increase in HCR; P < 0.0001). A training effect was also observed for phospho-p38 MAPK (12% decrease in LCR, 20% decrease in HCR; P < 0.05) and phospho-JNK (29% increase in LCR, 20% increase in HCR; P < 0.05). We conclude that in the LCR-HCR model system, mitochondrial protein expression in WAT is not affected by intrinsic running capacity or exercise training. However, training does induce alterations in the activity and expression of several proteins that are essential to the intracellular regulation of WAT metabolism.

Gliotoxin is a potent NOTCH2 transactivation inhibitor and efficiently induces apoptosis in chronic lymphocytic leukaemia (CLL) cells.

Chronic lymphocytic leukaemia (CLL) cells express constitutively activated NOTCH2 in a protein kinase C (PKC)- dependent manner. The transcriptional activity of NOTCH2 correlates not only with the expression of its target gene FCER2 (CD23) but is also functionally linked with CLL cell viability. In the majority of CLL cases, DNA-bound NOTCH2 complexes are less sensitive to the γ-secretase inhibitor (GSI) DAPT. Therefore, we searched for compounds that interfere with NOTCH2 signalling at the transcription factor level. Using electrophoretic mobility shift assays (EMSA), we identified the Aspergillum-derived secondary metabolite gliotoxin as a potent NOTCH2 transactivation inhibitor. Gliotoxin completely blocked the formation of DNA-bound NOTCH2 complexes in CLL cells independent of their sensitivity to DAPT. The inhibition of NOTCH2 signalling by gliotoxin was associated with down regulation of CD23 (FCER) expression and induction of apoptosis. Short time exposure of CLL cells indicated that the early apoptotic effect of gliotoxin is independent of proteasome regulated nuclear factor κB activity, and is associated with up regulation of NOTCH3 and NR4A1 expression. Gliotoxin could overcome the supportive effect of primary bone marrow stromal cells in an ex vivo CLL microenvironment model. In conclusion, we identified gliotoxin as a potent NOTCH2 inhibitor with a promising therapeutic potential in CLL.

Enrichment of Nur77 mediated by retinoic acid receptor ß leads to apoptosis of human hepatocellular carcinoma cells induced by fenretinide and histone deacetylase inhibitors.

UNLABELLED: The synthetic retinoid fenretinide is one of the most promising clinically tested retinoids. Previously, we have shown that fenretinide induces apoptosis of Huh7 cells, but HepG2 cells are relatively resistant to fenretinide-induced apoptosis. This study examines the interactive role of fenretinide and histone deacetylase inhibitors (HDACi) in inducing apoptosis of human hepatocellular carcinoma (HCC) cells and the underlying mechanism. Trichostatin A and scriptaid can either enhance fenretinide-induced apoptosis in the fenretinide sensitive HCC cells (Huh7 and Hep3B) or sensitize the fenretinide resistant cells (HepG2) to become sensitive to the apoptotic effect of fenretinide in a cancer cell-specific manner. The sensitivity of cells to fenretinide-induced apoptosis was not associated with reactive oxygen species production nor with antioxidant gene expression. However, the level of retinoic acid receptor ß (RARß) and Nur77 (NR4A1) was important for inducing apoptosis. Upon fenretinide and HDACi treatment, the expression of RARß and Nur77 were induced and colocalized in the cytosol. The induction of Nur77 protein level, but not the messenger RNA level, was RARß-dependent. In addition, RARß interacted with Nur77. Nur77 was essential for fenretinide-induced and HDACi-induced apoptosis of Huh7 cells. Induction of the expression, the interaction, and the nuclear export of RARß and Nur77 mediate fenretinide-induced and HDACi-induced apoptosis. CONCLUSION: Our findings suggest that targeting Nur77 and RARß simultaneously provides an effective way to induce HCC cell death.

Venlafaxine-induced adrenergic signaling stimulates Leydig cells steroidogenesis via Nur77 overexpression: A possible role of EGF.

Venlafaxine, a norepinephrine and serotonin reuptake inhibitor, impairs rat sperm parameters, spermatogenesis and causes high intratesticular estrogen and testosterone levels, indicating that Leydig cells (LCs) may be a venlafaxine target. We evaluated the effect of venlafaxine treatment on rat LCs, focusing on adrenergic signaling, EGF immunoexpression and steroidogenesis. Germ cells mitotic/meiotic activity and UCHL1 levels were also evaluated in the seminiferous epithelium. Eighteen adult male rats received 30 mg/kg of venlafaxine (n = 9) or distilled water (n = 9). The seminiferous tubules, epithelium and LCs nuclear areas were measured, and the immunoexpression of Ki-67, UCHL1, StAR, EGF, c-Kit and 17ß-HSD was evaluated. UCHL1, StAR and EGF protein levels and Adra1a, Nur77 and Ndrg2 expression were analyzed. Malondialdehyde (MDA) and nitrite testicular levels, and serum estrogen and testosterone levels were measured. Venlafaxine induced LCs hypertrophy and Ndrg2 upregulation in parallel to increased number of Ki-67, c-Kit- and 17ß-HSD-positive interstitial cells, indicating that this antidepressant stimulates LCs lineage proliferation and differentiation. Upregulation of Adra1a and Nur77 could explain the high levels of StAR and testosterone levels, as well as aromatization. Enhanced EGF immunoexpression in LCs suggests that this growth fact is involved in adrenergically-induced steroidogenesis, likely via upregulation of Nur77. Slight tubular atrophy and weak Ki-67 immunoexpression in germ cells, in association with high UCHL1 levels, indicate that spermatogenesis is likely impaired by this enzyme under supraphysiological estrogen levels. These data corroborate the unchanged MDA and nitrite levels. Therefore, venlafaxine stimulates LCs steroidogenesis via adrenergic signaling, and EGF may be involved in this process.

Nuclear Receptor Nr4a1 Regulates Striatal Striosome Development and Dopamine D1 Receptor Signaling.

The GABAergic medium-size spiny neuron (MSN), the striatal output neuron, may be classified into striosome, also known as patch, and matrix, based on neurochemical differences between the two compartments. At this time, little is known regarding the regulation of the development of the two compartments. Nr4a1, primarily described as a nuclear receptor/immediate early gene involved in the homeostasis of the dopaminergic system, is a striosomal marker. Using Nr4a1-overexpressing and Nr4a1-null mice, we sought to determine whether Nr4a1 is necessary and/or sufficient for striosome development. We report that in vivo and in vitro, Nr4a1 and Oprm1 mRNA levels are correlated. In the absence of Nr4a, there is a decrease in the percentage of striatal surface area occupied by striosomes. Alterations in Nr4a1 expression leads to dysregulation of multiple mRNAs of members of the dopamine receptor D1 signal transduction system. Constitutive overexpression of Nr4a1 decreases both the induction of phosphorylation of ERK after a single cocaine exposure and locomotor sensitization following chronic cocaine exposure. Nr4a1 overexpression increases MSN excitability but reduces MSN long-term potentiation. In the resting state, type 5 adenylyl cyclase (AC5) activity is normal, but the ability of AC5 to be activated by Drd1 G-protein-coupled receptor inputs is decreased. Our results support a role for Nr4a1 in determination of striatal patch/matrix structure and in regulation of dopaminoceptive neuronal function.

Androgen upregulates NR4A1 via the TFAP2A and ETS signaling networks.

Hyperandrogenism is one of the clinical and biochemical characteristics of polycystic ovary syndrome (PCOS). Our previous studies confirmed that nuclear receptor subfamily 4 group A member 1 (NR4A1), as a differentially expressed gene in the ovaries of PCOS patients, was upregulated by increased androgen. However, the potential mechanism of NR4A1 upregulation remains unknown. To elucidate the molecular mechanisms involved in NR4A1 regulation, we cloned and characterized the promoter regions of the NR4A1 gene using a series of truncated promoter plasmids in luciferase reporter assays. We identified two unique core promoters of NR4A1 located within the +1055/+1251 and +3183/+3233 regions relative to the transcription start site. TFAP2A downregulated NR4A1 expression, while five ETS transcription factors, ETS1, ELK1, ERG, FLI1 and SPI1, could upregulate NR4A1 promoter activity in HeLa cells. Of these transcription factors, ETS1 and ELK1 were the most effective ones. Moreover, all six transcription factors were confirmed to interact directly with the NR4A1 promoter. In conclusion, this study presents the first description that TFAP2A and ETS family signaling networks are involved in the androgen-mediated transcriptional regulation of NR4A1, which contributes to the understanding of the molecular mechanisms involved in the TFAP2A-NR4A1 and ETS-NR4A1 signaling networks in PCOS.

Targeting nuclear receptor NR4A1-dependent adipocyte progenitor quiescence promotes metabolic adaptation to obesity.

Adipocyte turnover in adulthood is low, suggesting that the cellular source of new adipocytes, the adipocyte progenitor (AP), resides in a state of relative quiescence. Yet the core transcriptional regulatory circuitry (CRC) responsible for establishing a quiescent state and the physiological significance of AP quiescence are incompletely understood. Here, we integrate transcriptomic data with maps of accessible chromatin in primary APs, implicating the orphan nuclear receptor NR4A1 in AP cell-state regulation. NR4A1 gain and loss of function in APs ex vivo decreased and enhanced adipogenesis, respectively. Adipose tissue of Nr4a1-/- mice demonstrated higher proliferative and adipogenic capacity compared with that of WT mice. Transplantation of Nr4a1-/- APs into the subcutaneous adipose tissue of WT obese recipients improved metrics of glucose homeostasis relative to administration of WT APs. Collectively, these data identify NR4A1 as a previously unrecognized constitutive regulator of AP quiescence and suggest that augmentation of adipose tissue plasticity may attenuate negative metabolic sequelae of obesity.

Injectable Supramolecular Hydrogels as Delivery Agents of Bcl-2 Conversion Gene for the Effective Shrinkage of Therapeutic Resistance Tumors.

Injectable hydrogels to deliver therapeutic genes in a minimally invasive manner and to achieve long term sustained release at tumor sites to minimize side effects are attractive for cancer therapy and precision medicine, but its rational design remains a challenge. In this report, an injectable supramolecular hydrogel system is designed based on the polypesudorotaxane formation between α-cyclodextrin (α-CD) and cationic methoxy-poly(ethylene glycol)-b-poly(ε-caprolactone)-b-poly(ethylene imine) (MPEG-PCL-PEI) copolymer, with the ability to form polyplexes with anionic plasmid DNA for effective sustained gene delivery. To be mentioned, the MPEG-PCL-PEI copolymers show similar pDNA binding ability, better gene transfection efficiency, lower cytotoxicity than nonviral gene transfection gold standard PEI (25 kDa), due to the formation of micelles and more stable polyplexes. More importantly, this MPEG-PCL-PEI/α-CD/pDNA supramolecular hydrogel shows a sustained release of pDNA in form of polyplex for up to 7 d. By taking these advantages, this supramolecular hydrogel system is applied as an injectable carrier for sustained Bcl-2 conversion gene release, in an in vivo rodent model of therapeutic resistant hepatocarcinoma with high expression of antiapoptotic Bcl-2 protein. This work represents the first time that injectable MPEG-PCL-PEI/α-CD supramolecular hydrogels possess good controllable release effect of Bcl-2 conversion genes in the form of polyplex to effectively inhibit in vivo tumor growth and this "enemy to friend" strategy will benefit various applications, including on-demand gene delivery and personalized medicine.

Orphan Nuclear Receptor Nur77 Inhibits Angiotensin II-Induced Vascular Remodeling via Downregulation of ß-Catenin.

Angiotensin II (Ang II) is the predominant effector peptide of the renin-angiotensin system. Ang II contributes to vascular remodeling in many cardiovascular diseases (eg, hypertension, atherosclerosis, restenosis, and aneurysm). Orphan nuclear receptor Nur77 has a crucial role in the functional regulation of vascular cells. The objective of this study was to define the specific role of Nur77 in Ang II-induced vascular remodeling. Nur77 expression was initially found to be elevated in medial vascular smooth muscle cells (VSMCs) of thoracic aortas from mice continuously infused with Ang II for 2 weeks using a subcutaneous osmotic minipump. Cellular studies revealed that Nur77 expression was upregulated by Ang II via the MAPK/PKA-CREB signaling pathway. Ang II-induced proliferation, migration, and phenotypic switching were significantly enhanced in VSMCs isolated from Nur77(-/-) mice compared with wild-type VSMCs. Consistent with the role in VSMCs, we found that compared with wild-type mice, Nur77(-/-) mice had elevated aortic medial areas and luminal diameters, more severe elastin disruption and collagen deposition, increased VSMC proliferation and matrix metalloproteinase production, and decreased VSMC-specific genes SM-22α and α-actin expression, after 2 weeks of exogenous Ang II administration. The results of additional experiments suggested that Nur77 suppressed Ang II-induced ß-catenin signaling pathway activation by promoting ß-catenin degradation and inhibiting its transcriptional activity. Our findings indicated that Nur77 is a critical negative regulator of Ang II-induced VSMC proliferation, migration, and phenotypic switching via the downregulation of ß-catenin activity. Nur77 may reduce Ang II-induced vascular remodeling involved in many cardiovascular diseases.

Dopamine dynamics and cocaine sensitivity differ between striosome and matrix compartments of the striatum.

The striatum is typically classified according to its major output pathways, which consist of dopamine D1 and D2 receptor-expressing neurons. The striatum is also divided into striosome and matrix compartments, based on the differential expression of a number of proteins, including the mu opioid receptor, dopamine transporter (DAT), and Nr4a1 (nuclear receptor subfamily 4, group A, member 1). Numerous functional differences between the striosome and matrix compartments are implicated in dopamine-related neurological disorders including Parkinson's disease and addiction. Using Nr4a1-eGFP mice, we provide evidence that electrically evoked dopamine release differs between the striosome and matrix compartments in a regionally-distinct manner. We further demonstrate that this difference is not due to differences in inhibition of dopamine release by dopamine autoreceptors or nicotinic acetylcholine receptors. Furthermore, cocaine enhanced extracellular dopamine in striosomes to a greater degree than in the matrix and concomitantly inhibited dopamine uptake in the matrix to a greater degree than in striosomes. Importantly, these compartment differences in cocaine sensitivity were limited to the dorsal striatum. These findings demonstrate a level of exquisite microanatomical regulation of dopamine by the DAT in striosomes relative to the matrix.

Honokiol sensitizes breast cancer cells to TNF-α induction of apoptosis by inhibiting Nur77 expression.

BACKGROUND AND PURPOSE: The orphan nuclear receptor Nur77 is implicated in the survival and apoptosis of cancer cells. The purpose of this study was to determine whether and how Nur77 serves to mediate the effect of the inflammatory cytokine TNF-α in cancer cells and to identify and characterize new agents targeting Nur77 for cancer therapy. EXPERIMENTAL APPROACH: The effects of TNF-α on the expression and function of Nur77 were studied using in vitro and in vivo models. Nur77 expression was evaluated in tumour tissues from breast cancer patients. The anticancer effects of honokiol and its mechanism of action were assessed by in vitro, cell-based and animal studies. KEY RESULTS: TNF-α rapidly and potently induced the expression of Nur77 in breast cancer cells through activation of IκB kinase and JNK. Knocking down Nur77 resulted in TNF-α-dependent apoptosis, while ectopic Nur77 expression in MCF-7 cells promoted their growth in animals. Levels of Nur77 were higher in tumour tissues than the corresponding tissues surrounding the tumour in about 50% breast cancer patients studied. Our in vitro and animal studies also identified honokiol as an effective sensitizer of TNF-α-induced apoptosis by inhibiting TNF-α-induced Nur77 mRNA expression, which could be attributed to its interference of TNFR1's interaction with receptor-interacting protein 1 (RIPK1). CONCLUSIONS AND IMPLICATIONS: TNF-α-induced Nur77 serves as a survival factor to attenuate the death effect of TNF-α in cancer cells. With its proven human safety profile, honokiol represents a promising agent that warrants further clinical development.