MicroRNA‐27a‐3p suppression of peroxisome proliferator‐activated receptor‐γ contributes to cognitive impairments resulting from sevoflurane treatment

Sevoflurane is the most widely used anaesthetic administered by inhalation. Exposure to sevoflurane in neonatal mice can induce learning deficits and abnormal social behaviours. MicroRNA (miR)‐27a‐3p, a short, non‐coding RNA that functions as a tumour suppressor, is up‐regulated after inhalation of anaesthetic, and peroxisome proliferator‐activated receptor γ (PPAR‐γ) is one of its target genes. The objective of this study was to investigate how the miR‐27a‐3p–PPAR‐γ interaction affects sevoflurane‐induced neurotoxicity. A luciferase reporter assay was employed to identify the interaction between miR‐27a‐3p and PPAR‐γ. Primary hippocampal neuron cultures prepared from embryonic day 0 C57BL/6 mice were treated with miR‐27a‐3p inhibitor or a PPAR‐γ agonist to determine the effect of miR‐27a‐3p and PPAR‐γ on sevoflurane‐induced cellular damage. Cellular damage was assessed by a flow cytometry assay to detect apoptotic cells, immunofluorescence to detect reactive oxygen species, western blotting to detect NADPH oxidase 1/4 and ELISA to measure inflammatory cytokine levels. In vivo experiments were performed using a sevoflurane‐induced anaesthetic mouse model to analyse the effects of miR‐27a‐3p on neurotoxicity by measuring the number of apoptotic neurons using the Terminal‐deoxynucleoitidyl Transferase Mediated Nick End Labeling (TUNEL) method and learning and memory function by employing the Morris water maze test. Our results revealed that PPAR‐γ expression was down‐regulated by miR‐27a‐3p following sevoflurane treatment in hippocampal neurons. Down‐regulation of miR‐27a‐3p expression decreased sevoflurane‐induced hippocampal neuron apoptosis by decreasing inflammation and oxidative stress‐related protein expression through the up‐regulation of PPAR‐γ. In vivo tests further confirmed that inhibition of miR‐27a‐3p expression attenuated sevoflurane‐induced neuronal apoptosis and learning and memory impairment. Our findings suggest that down‐regulation of miR‐27a‐3p expression ameliorated sevoflurane‐induced neurotoxicity and learning and memory impairment through the PPAR‐γ signalling pathway. MicroRNA‐27a‐3p may, therefore, be a potential therapeutic target for preventing or treating sevoflurane‐induced neurotoxicity.

     

Functional characterization of ecdysone receptor gene switches in mammalian cells

Regulated expression of transgene is essential in basic research as well as for many therapeutic applications. The main purpose of the present study is to understand the functioning of the ecdysone receptor (EcR)-based gene switch in mammalian cells and to develop improved versions of EcR gene switches. We utilized EcR mutants to develop new EcR gene switches that showed higher ligand sensitivity and higher magnitude of induction of reporter gene expression in the presence of ligand. We also developed monopartite versions of EcR gene switches with reduced size of the components that are accommodated into viral vectors. Ligand binding assays revealed that EcR alone could not bind to the nonsteroidal ligand, RH-2485. The EcR's heterodimeric partner, ultraspiracle, is required for efficient binding of EcR to the ligand. The essential role of retinoid X receptor (RXR) or its insect homolog, ultraspiracle, in EcR function is shown by RXR knockdown experiments using RNAi. Chromatin immunoprecipitation assays demonstrated that VP16 (activation domain, AD):GAL4(DNA binding domain, DBD):EcR(ligand binding domain, LBD) or GAL4(DBD):EcR(LBD) fusion proteins can bind to GAL4 response elements in the absence of ligand. The VP16(AD) fusion protein of a chimera between human and locust RXR could heterodimerize with GAL4(DBD):EcR(LBD) in the absence of ligand but the VP16(AD) fusion protein of Homo sapiens RXR requires ligand for its heterodimerization with GAL4(DBD):EcR(LBD).     

Activation of RXR–PPAR heterodimers by organotin environmental endocrine disruptors

The nuclear receptor retinoid X receptor‐α (RXR‐α)–peroxisome proliferator‐activated receptor‐γ (PPAR‐γ) heterodimer was recently reported to have a crucial function in mediating the deleterious effects of organotin compounds, which are ubiquitous environmental contaminants. However, because organotins are unrelated to known RXR‐α and PPAR‐γ ligands, the mechanism by which these compounds bind to and activate the RXR‐α–PPAR‐γ heterodimer at nanomolar concentrations has remained elusive. Here, we show that tributyltin (TBT) activates all three RXR–PPAR‐α, ‐γ, ‐δ heterodimers, primarily through its interaction with RXR. In addition, the 1.9 Å resolution structure of the RXR‐α ligand‐binding domain in complex with TBT shows a covalent bond between the tin atom and residue Cys 432 of helix H11. This interaction largely accounts for the high binding affinity of TBT, which only partly occupies the RXR‐α ligand‐binding pocket. Our data allow an understanding of the binding and activation properties of the various organotins and suggest a mechanism by which these tin compounds could affect other nuclear receptor signalling pathways.     

Differential responses of PPARalpha, PPARdelta, and PPARgamma reporter cell lines to selective PPAR synthetic ligands

To characterize the specificity of synthetic compounds for peroxisome proliferator-activated receptors (PPARs), three stable cell lines expressing the ligand binding domain (LBD) of human PPARalpha, PPARdelta, or PPARgamma fused to the yeast GAL4 DNA binding domain (DBD) were developed. These reporter cell lines were generated by a two-step transfection procedure. First, a stable cell line, HG5LN, expressing the reporter gene was developed. These cells were then transfected with the different receptor genes. With the help of the three PPAR reporter cell lines, we assessed the selectivity and activity of PPAR agonists GW7647, WY-14-643, L-165041, GW501516, BRL49653, ciglitazone, and pioglitazone. GW7647, L-165041, and BRL49653 were the most potent and selective agonists for hPPARalpha, hPPARdelta, and hPPARgamma, respectively. Two PPAR antagonists, GW9662 and BADGE, were also tested. GW9662 was a selective PPARgamma antagonist, whereas BADGE was a low-affinity PPAR ligand. Furthermore, GW9662 was a full antagonist on PPARgamma and PPARdelta, whereas it showed partial agonism on PPARalpha. We conclude that our stable models allow specific and sensitive measurement of PPAR ligand activities and are a high-throughput, cell-based screening tool for identifying and characterizing PPAR ligands.     

MeCP2‐421‐mediated RPE epithelial‐mesenchymal transition and its relevance to the pathogenesis of proliferative vitreoretinopathy

Proliferative vitreoretinopathy (PVR) is a blinding eye disease. Epithelial‐mesenchymal transition (EMT) of RPE cells plays an important role in the pathogenesis of PVR. In the current study, we sought to investigate the role of the methyl‐CpG‐binding protein 2 (MeCP2), especially P‐MeCP2‐421 in the pathogenesis of PVR. The expressions of P‐MeCP2‐421, P‐MeCP2‐80, PPAR‐γ and the double labelling of P‐MeCP2‐421 with α‐SMA, cytokeratin, TGF‐β and PPAR‐γ in human PVR membranes were analysed by immunohistochemistry. The effect of knocking down MeCP2 using siRNA on the expressions of α‐SMA, phospho‐Smad2/3, collagen I, fibronectin and PPAR‐γ; the expression of α‐SMA stimulated by recombinant MeCP2 in ARPE‐19; and the effect of TGF‐β and 5‐AZA treatment on PPAR‐γ expression were analysed by Western blot. Chromatin immunoprecipitation was used to determine the binding of MeCP2 to TGF‐β. Our results showed that P‐MeCP2‐421 was highly expressed in PVR membranes and was double labelled with α‐SMA, cytokeratin and TGF‐β, knocking down MeCP2 inhibited the activation of Smad2/3 and the expression of collagen I and fibronectin induced by TGF‐β. TGF‐β inhibited the expression of PPAR‐γ, silence of MeCP2 by siRNA or using MeCP2 inhibitor (5‐AZA) increased the expression of PPAR‐γ. α‐SMA was up‐regulated by the treatment of recombinant MeCP2. Importantly, we found that MeCP2 bound to TGF‐β as demonstrated by Chip assay. The results suggest that MeCP2 especially P‐MeCP2‐421 may play a significant role in the pathogenesis of PVR and targeting MeCP2 may be a potential therapeutic approach for the treatment of PVR.     

Identification of annexin A1 as a novel substrate for E6AP‐mediated ubiquitylation

E6‐associated protein (E6AP) is a cellular ubiquitin protein ligase that mediates ubiquitylation and degradation of p53 in conjunction with the high‐risk human papillomavirus E6 proteins. However, the physiological functions of E6AP are poorly understood. To identify a novel biological function of E6AP, we screened for binding partners of E6AP using GST pull‐down and mass spectrometry. Here we identified annexin A1, a member of the annexin superfamily, as an E6AP‐binding protein. Ectopic expression of E6AP enhanced the degradation of annexin A1 in vivo. RNAi‐mediated downregulation of endogenous E6AP increased the levels of endogenous annexin A1 protein. E6AP interacted with annexin A1 and induced its ubiquitylation in a Ca²⁺‐dependent manner. GST pull‐down assay revealed that the annexin repeat domain III of annexin A1 is important for the E6AP binding. Taken together, our data suggest that annexin A1 is a novel substrate for E6AP‐mediated ubiquitylation. Our findings raise the possibility that E6AP may play a role in controlling the diverse functions of annexin A1 through the ubiquitin‐proteasome pathway. J. Cell. Biochem. 106: 1123–1135, 2009. © 2009 Wiley‐Liss, Inc.     

Ginsenoside Rb1 ameliorates CKD‐associated vascular calcification by inhibiting the Wnt/β‐catenin pathway

Vascular calcification (VC) is a pathological process underpinning major cardiovascular conditions and has attracted public attention due to its high morbidity and mortality. Chronic kidney disease (CKD) is a common disease related to VC. Ginsenoside Rb1 (Rb1) has been reported to protect the cardiovascular system against vascular diseases, yet its role in VC and the underlying mechanisms remain unclear. In this study, we established a CKD‐associated VC rat model and a β‐glycerophosphate (β‐GP)‐induced vascular smooth muscle cell (VSMC) calcification model to investigate the effects of Rb1 on VC. Our results demonstrated that Rb1 ameliorated calcium deposition and VSMC osteogenic transdifferentiation both in vivo and in vitro. Rb1 treatment inhibited the Wnt/β‐catenin pathway by activating peroxisome proliferator‐activated receptor‐γ (PPAR‐γ), and confocal microscopy was used to show that Rb1 inhibited β‐catenin nuclear translocation in VSMCs. Furthermore, SKL2001, an agonist of the Wnt/β‐catenin pathway, compromised the vascular protective effect of Rb1. GW9662, a PPAR‐γ antagonist, reversed Rb1's inhibitory effect on β‐catenin. These results indicate that Rb1 exerted anticalcific properties through PPAR‐γ/Wnt/β‐catenin axis, which provides new insights into the potential theraputics of VC.     

Monoubiquitylation of GGA3 by hVPS18 regulates its ubiquitin-binding ability

GGAs (Golgi-localizing, gamma-adaptin ear domain homology, ADP-ribosylation factor (ARF)-binding proteins), constitute a family of monomeric adaptor proteins and are associated with protein trafficking from the trans-Golgi network to endosomes. Here, we show that GGA3 is monoubiquitylated by a RING-H2 type-ubiquitin ligase hVPS18 (human homologue of vacuolar protein sorting 18). By in vitro ubiquitylation assays, we have identified lysine 258 in the GAT domain as a major ubiquitylation site that resides adjacent to the ubiquitin-binding site. The ubiquitylation is abolished by a mutation in either the GAT domain or ubiquitin that disrupts the GAT-ubiquitin interaction, indicating that the ubiquitin binding is a prerequisite for the ubiquitylation. Furthermore, the GAT domain ubiquitylated by hVPS18 no longer binds to ubiquitin, indicating that ubiquitylation negatively regulates the ubiquitin-binding ability of the GAT domain. These results suggest that the ubiquitin binding and ubiquitylation of GGA3-GAT domain are mutually inseparable through a ubiquitin ligase activity of hVPS18.     

Hesperidin regresses cardiac hypertrophy by virtue of PPAR‐γ agonistic,anti‐inflammatory,antiapoptotic, and antioxidant properties

Hesperidin (HES), a flavanone glycoside, predominant in citrus fruits, has an agonistic activity on peroxisome proliferator‐activated receptor gamma (PPAR‐γ). PPAR‐γ is an inhibitor of cardiac hypertrophy (CH) signaling pathways. In this study, we investigated the cardioprotective effect of HES in isoproterenol (ISO)‐induced CH through PPAR‐γ agonistic activity. For this, male albino Wistar rats were divided into six groups (n = 6), that is, normal, ISO‐control, HES treatment group (200 mg kg^?1; p.o.), HES per se (200 mg kg^?1; p.o.), enalapril treatment group (30 mg kg^?1; p.o.), and combination group (HES 200 mg kg^?1; p.o.+enalapril 30 mg kg^?1; p.o.). ISO (3 mg kg^?1; s.c.) was administered to all groups except normal and per se to induce CH. HES or enalapril treatment of 28 days significantly attenuated pathological changes, improved cardiac hemodynamics, suppressed oxidative stress, and apoptosis along with an increased PPAR‐γ expression. The combination of enalapril with HES exhibited an effect similar to that of HES or enalapril alone on all the aforementioned parameters. Therefore, HES may be further evaluated as a promising molecule for the alleviation of CH.     

A COP1‐PIF‐HEC regulatory module fine‐tunes photomorphogenesis in Arabidopsis

Light responses mediated by the photoreceptors play crucial roles in regulating different aspects of plant growth and development. An E3 ubiquitin ligase complex CONSTITUTIVELY PHOTOMORPHOGENIC1 (COP1)1/SUPPRESSOR OF PHYA (SPA), one of the central repressors of photomorphogenesis, is critical for maintaining skotomorphogenesis. It targets several positive regulators of photomorphogenesis for degradation in darkness. Recently, we revealed that basic helix‐loop‐helix factors, HECATEs (HECs), function as positive regulators of photomorphogenesis by directly interacting and antagonizing the activity of another group of repressors called PHYTOCHROME‐INTERACTING FACTORs (PIFs). It was also shown that HECs are partially degraded in the dark through the ubiquitin/26S proteasome pathway. However, the underlying mechanism of HEC degradation in the dark is still unclear. Here, we show that HECs also interact with both COP1 and SPA proteins in darkness, and that HEC2 is directly targeted by COP1 for degradation via the ubiquitin/26S proteasome pathway. Moreover, COP1‐mediated polyubiquitylation and degradation of HEC2 are enhanced by PIF1. Therefore, the ubiquitylation and subsequent degradation of HECs are significantly reduced in both cop1 and pif mutants. Consistent with this, the hec mutants partially suppress photomorphogenic phenotypes of both cop1 and pifQ mutants. Collectively, our work reveals that the COP1/SPA‐mediated ubiquitylation and degradation of HECs contributes to the coordination of skoto/photomorphogenic development in plants.     

Effects of the new C1q/TNF-related protein (CTRP-3) "cartonectin" on the adipocytic secretion of adipokines

Background: Cartonectin (collagenous repeat‐containing sequence of 26‐kDa protein; CORS‐26) was described as a new adipokine of the C1q/TNF molecular superfamily C1q/TNF‐related protein‐3 (CTRP‐3), secreted by the adipocytes of mice and humans. The receptor and function of cartonectin are unknown and the recombinant protein is not commercially available. Objective: To investigate the effects of recombinant cartonectin on the secretion of adipokines such as adiponectin, leptin, and resistin from adipocytes of human and murine origin. The effect of the BMI of the adipocyte donor was also investigated. Methods and Procedures: Human adipocytes from pooled lean and preobese healthy individuals and murine 3T3‐L1 adipocytes were used for stimulation experiments. Recombinant cartonectin was expressed in insect H5 cells. Adipokine secretion was measured using enzyme‐linked immunosorbent assay. In addition, western blot analysis and luciferase reporter gene assays were employed. Results: Cartonectin (1, 10, 50, and 250 ng/ml) in higher doses stimulates the secretion of adiponectin and resistin from murine adipocytes. This effect is not caused by an induction of peroxisome proliferator‐activated receptor‐γ (PPAR‐γ) protein expression, as confirmed by western blot analysis. Also, luciferase reporter gene assay revealed that cartonectin failed to induce luciferase activity at the peroxisome proliferator‐activated receptor responsive element site containing the adiponectin/luciferase promoter fragment. Human adipocytes from lean individuals secrete higher amounts of adiponectin and leptin when compared with adipocytes of individuals with a preobesity BMI (25–30 kg/m²). Cartonectin failed to stimulate adiponectin or leptin secretion from human adipocytes, irrespective of the BMI value. Discussion: Cartonectin is a new adipokine that differentially regulates the secretion of classical adipokines, with marked differences between the human and the murine systems. These effects are species‐dependent, while basal adipokine secretion is influenced by the BMI.     

IL‐12 stimulates the osteoclast inhibitory peptide‐1 (OIP‐1/hSca) gene expression in CD4+ T cells

Immune cell products such as interferon (IFN)‐γ and interleukin (IL)‐12 are potent inhibitors of osteoclast formation. We previously characterized the human osteoclast inhibitory peptide‐1 (OIP‐1/hSca), a Ly‐6 gene family member and showed IFN‐γ modulation of OIP‐1 expression in bone marrow cells. Whether, IL‐12 regulates OIP‐1 expression in the bone microenvironment is unclear. Real‐time PCR analysis revealed that IL‐12 treatment significantly enhanced OIP‐1 mRNA expression in human bone marrow mononuclear cells. Because IL‐12 induces IFN‐γ production by T cells, we tested whether IFN‐γ participates in IL‐12 stimulation of OIP‐1 gene expression in these cells. IL‐12 treatment in the presence of IFN‐γ neutralizing antibody significantly increased OIP‐1 mRNA expression, suggesting that IL‐12 directly regulates OIP‐1 gene expression. Interestingly, real‐time PCR analysis demonstrated that IL‐12 induces OIP‐1 expression (3.2‐fold) in CD4+ T cells; however, there was no significant change in CD8+ T cells. Also, IL‐12 (10 ng/ml) treatment of Jurkat cells transfected with OIP‐1 gene (?1 to ?1,988 bp) promoter‐luciferase reporter plasmid demonstrated a 5‐fold and 2.7‐fold increase in OIP‐1 gene promoter activity in the presence and absence of antibody against IFN‐γ, respectively. We showed that STAT‐1,3 inhibitors treatment significantly decreased IL‐12 stimulated OIP‐1 promoter activity. Chromatin immunoprecipitation (ChIP) assay confirmed STAT‐3, but not STAT‐1 binding to the OIP‐1 gene promoter in response to IL‐12 stimulation. These results suggest that IL‐12 stimulates the OIP‐1 gene expression through STAT‐3 activation in CD4+ T cells. J. Cell. Biochem. 107: 104–111, 2009. © 2009 Wiley‐Liss, Inc.