X-box binding protein 1 is a novel key regulator of peroxisome proliferator-activated receptor γ2.

X-box binding protein 1 (XBP1), a transcription factor of the unfolded protein response, plays various roles in many biological processes. We examined its pro-adipogenic activity and target genes during adipogenic differentiation in wild-type and genetically modified 3T3-L1 cells. Signalling pathways that contribute to Xbp1 mRNA splicing, and the correlation of the transcriptionally active XBP1 isoform (XBP1s) level with body mass index and the level of peroxisome proliferator-activated receptor γ2 (PPARγ2) in human adipose tissues were also examined. The mRNA and nuclear protein expression levels of XBP1s increased immediately following hormonal induction of adipogenesis, reaching a peak at 6 h. Results from cDNA microarray and gene expression analyses using genetically modified cells indicated that PPARγ2 was a principal target of XBP1s. The XBP1s-specific binding motif, which is distinct from the CCAAT/enhancer-binding protein α binding site, was identified in the PPARγ2 promoter by site-directed mutagenesis. Fetal bovine serum, insulin, 3-isobutyl-1-methylxanthine and dexamethasone contributed independently to Xbp1 mRNA splicing. In human subcutaneous adipose tissues, the levels of both Xbp1s and Pparγ2 mRNA increased proportionally with body mass index, and there was a significant positive correlation between the two genes. These data suggest for the first time that positive regulation of PPARγ2 is a principal mechanism of XBP1s-mediated adipogenesis in 3T3-L1 cells.

Hypoxia-responsive microRNA-101 promotes angiogenesis via heme oxygenase-1/vascular endothelial growth factor axis by targeting cullin 3.

AIMS: Hypoxia induces expression of various genes and microRNAs (miRs) that regulate angiogenesis and vascular function. In this study, we investigated a new functional role of new hypoxia-responsive miR-101 in angiogenesis and its underlying mechanism for regulating heme oxygenase-1 (HO-1) and vascular endothelial growth factor (VEGF) expression. RESULTS: We found that hypoxia induced miR-101, which binds to the 3'untranslated region of cullin 3 (Cul3) and stabilizes nuclear factor erythroid-derived 2-related factor 2 (Nrf2) via inhibition of the proteasomal degradation pathway. miR-101 overexpression promoted Nrf2 nuclear accumulation, which was accompanied with increases in HO-1 induction, VEGF expression, and endothelial nitric oxide synthase (eNOS)-derived nitric oxide (NO) production. The elevated NO-induced S-nitrosylation of Kelch-like ECH-associated protein 1 and subsequent induction of Nrf2-dependent HO-1 lead to further elevation of VEGF production via a positive feedback loop between the Nrf2/HO-1 and VEGF/eNOS axes. Moreover, miR-101 promoted angiogenic signals and angiogenesis both in vitro and in vivo, and these events were attenuated by inhibiting the biological activity of HO-1, VEGF, or eNOS. Moreover, these effects were also observed in aortic rings from HO-1(+/-) and eNOS(-/-) mice. Local overexpression of miR-101 improved therapeutic angiogenesis and perfusion recovery in the ischemic mouse hindlimb, whereas antagomiR-101 diminished regional blood flow. INNOVATION: Hypoxia-responsive miR-101 stimulates angiogenesis by activating the HO-1/VEGF/eNOS axis via Cul3 targeting. Thus, miR-101 is a novel angiomir. CONCLUSION: Our results provide new mechanistic insights into a functional role of miR-101 as a potential therapeutic target in angiogenesis and vascular remodeling.

Functional role of NF-κB in expression of human endothelial nitric oxide synthase.

The transcription factor NF-κB has an essential role in inflammation in endothelial cells. Endothelial nitric oxide synthase (eNOS)-derived nitric oxide (NO) prevents vascular inflammation. However, the molecular mechanism underlying NF-κB-mediated regulation of eNOS expression has not been clearly elucidated. We here found that NF-κB-activating stimuli, such as lipopolysaccharide, tumor necrosis factor-α (TNF-α), and interleukin-1ß, suppressed eNOS mRNA and protein levels by decreasing mRNA stability, without affecting promoter activity. TNF-α-mediated suppression of eNOS expression, mRNA stability, and 3'-untranslated region (3'UTR) activity were inhibited by NF-κB inhibitors and Dicer knockdown, but not by p38 MAPK and MEK inhibitors, suggesting the involvement of NF-κB-responsive miRNAs in eNOS expression. Moreover, TNF-α increased MIR155HG expression and promoter activity as well as miR-155 biogenesis, and these increases were blocked by NF-κB inhibitors. Transfection with antagomiR-155 blocked TNF-α-mediated suppression of eNOS 3'UTR activity, eNOS mRNA and protein levels, and NO and cGMP production. These data provide evidence that NF-κB is a negative regulator of eNOS expression via upregulation of miR-155 under inflammatory conditions. These results suggest that NF-κB is a potential therapeutic target for preventing vascular inflammation and endothelial dysfunction induced by suppression of miR-155-mediated eNOS expression.

X-box binding protein 1 enhances adipogenic differentiation of 3T3-L1 cells through the downregulation of Wnt10b expression.

Differentiation of preadipocytes into adipocytes is controlled by various transcription factors. Recently, the pro-adipogenic function of XBP1, a transcription factor upregulated by endoplasmic reticulum stress, has been reported. In this study, we demonstrated that XBP1 suppresses the expression of Wnt10b, an anti-adipogenic Wnt, during the differentiation of 3T3-L1 preadipocytes. The expression pattern of XBP1 was reciprocal to that of Wnt10b during the early stage of adipogenesis. The intracellular protein levels of ß-catenin were negatively regulated by XBP1. Direct binding of XBP1 to the Wnt10b promoter and the subsequent decrease of the ß-catenin signalling pathway represent a novel adipogenic differentiation mechanism.

Inhibition of mouse brown adipocyte differentiation by second-generation antipsychotics.

Brown adipose tissue is specialized to burn lipids for thermogenesis and energy expenditure. Second-generation antipsychotics (SGA) are the most commonly used drugs for schizophrenia with several advantages over first-line drugs, however, it can cause clinically-significant weight gain. To reveal the involvement of brown adipocytes in SGA-induced weight gain, we compared the effect of clozapine, quetiapine, and ziprasidone, SGA with different propensities to induce weight gain, on the differentiation and the expression of brown fat-specific markers, lipogenic genes and adipokines in a mouse brown preadipocyte cell line. On Oil Red-O staining, the differentiation was inhibited almost completely by clozapine (40 µM) and partially by quetiapine (30 µM). Clozapine significantly down-regulated the brown adipogenesis markers PRDM16, C/EBPß, PPARγ2, UCP-1, PGC-1α, and Cidea in dose- and time-dependent manners, whereas quetiapine suppressed PRDM16, PPARγ 2, and UCP-1 much weakly than clozapine. Clozapine also significantly inhibited the mRNA expressions of lipogenic genes ACC, SCD1, GLUT4, aP2, and CD36 as well as adipokines such as resistin, leptin, and adiponectin. In contrast, quetiapine suppressed only resistin and leptin but not those of lipogenic genes and adiponectin. Ziprasidone (10 µM) did not alter the differentiation as well as the gene expression patterns. Our results suggest for the first time that the inhibition of brown adipogenesis may be a possible mechanism to explain weight gain induced by clozapine and quetiapine.

Purple corn anthocyanins inhibit diabetes-associated glomerular monocyte activation and macrophage infiltration.

Diabetic nephropathy (DN) is one of the major diabetic complications and the leading cause of end-stage renal disease. In early DN, renal injury and macrophage accumulation take place in the pathological environment of glomerular vessels adjacent to renal mesangial cells expressing proinflammatory mediators. Purple corn utilized as a daily food is rich in anthocyanins exerting disease-preventive activities as a functional food. This study elucidated whether anthocyanin-rich purple corn extract (PCA) could suppress monocyte activation and macrophage infiltration. In the in vitro study, human endothelial cells and THP-1 monocytes were cultured in conditioned media of human mesangial cells exposed to 33 mM glucose (HG-HRMC). PCA decreased the HG-HRMC-conditioned, media-induced expression of endothelial vascular cell adhesion molecule-1, E-selectin, and monocyte integrins-ß1 and -ß2 through blocking the mesangial Tyk2 pathway. In the in vivo animal study, db/db mice were treated with 10 mg/kg PCA daily for 8 wk. PCA attenuated CXCR2 induction and the activation of Tyk2 and STAT1/3 in db/db mice. Periodic acid-Schiff staining showed that PCA alleviated mesangial expansion-elicited renal injury in diabetic kidneys. In glomeruli, PCA attenuated the induction of intracellular cell adhesion molecule-1 and CD11b. PCA diminished monocyte chemoattractant protein-1 expression and macrophage inflammatory protein 2 transcription in the diabetic kidney, inhibiting the induction of the macrophage markers CD68 and F4/80. These results demonstrate that PCA antagonized the infiltration and accumulation of macrophages in diabetic kidneys through disturbing the mesangial IL-8-Tyk-STAT signaling pathway. Therefore, PCA may be a potential renoprotective agent treating diabetes-associated glomerulosclerosis.

Assembly and function of AsGlu2 fibrillar multimer of oat beta-glucosidase.

Oat beta-glucosidase in plastid exists as a long fibrillar structure of AsGlu1 homomultimer (type I) and heteromultimer of AsGlu1 and AsGlu2 (type II). In spite of the high amino acid sequence homology of AsGlu1 and AsGlu2, AsGlu1 assembles into the fibrillar multimers but AsGlu2 forms a dimer when expressed in E. coli. A swapping analysis of AsGlu2 cDNA with AsGlu1 cDNA indicated that the C-terminal segment of AsGlu1 was critical for the fibrillar multimerization. A single substitution of glutamic acid-495 of AsGlu2 in the C-terminal region with lysine, an AsGlu1 counterpart amino acid for the glutamic acid-495, assembled the AsGlu2 into fibrillar homomultimers. The mutant AsGlu2 homomultimer was highly stable and had relatively faster electric mobility in native gel than the AsGlu1 homomultimer. Multimerization increased enzyme affinity to substrates.

Structural insight into bioremediation of triphenylmethane dyes by Citrobacter sp. triphenylmethane reductase.

Triphenylmethane dyes are aromatic xenobiotic compounds that are widely considered to be one of the main culprits of environmental pollution. Triphenylmethane reductase (TMR) from Citrobacter sp. strain KCTC 18061P was initially isolated and biochemically characterized as an enzyme that catalyzes the reduction of triphenylmethane dyes. Information from the primary amino acid sequence suggests that TMR is a dinucleotide-binding motif-containing enzyme; however, no other functional clues can be derived from sequence analysis. We present the crystal structure of TMR in complex with NADP+ at 2.0-angstroms resolution. Despite limited sequence similarity, the enzyme shows remarkable structural similarity to short-chain dehydrogenase/reductase (SDR) family proteins. Functional assignments revealed that TMR has features of both classic and extended SDR family members and does not contain a conserved active site. Thus, it constitutes a novel class of SDR family proteins. On the basis of simulated molecular docking using the substrate malachite green and the TMR/NADP+ crystal structure, together with site-directed mutagenesis, we have elucidated a potential molecular mechanism for triphenylmethane dye reduction.

Expression and characterization of the integral membrane domain of bacterial histidine kinase SCO3062 for structural studies.

Bacterial histidine kinases play an important role in the response to external stimuli. Structural studies of the histidine kinase transmembrane domain are challenging due to difficulties in protein expression and sample preparation. After carrying out expression screening of a series of histidine kinases, we investigated sample preparation methods for obtaining high quality samples of the periplasmic and transmembrane domain (PTD) of the bacterial histidine kinase SCO3062. Various sample conditions were tested for their ability to give homogeneous NMR spectra of the SCO3062 PTD with well-resolved resonances. Circular dichroism and 3D (15)N-edited NOESY spectrum results demonstrate that the SCO3062 PTD is predominantly alpha-helical. This method should be applicable to the NMR analysis of other transmembrane proteins.

Characterization of a CK II protein kinase from etiolated oat seedlings.

Protein kinases play a central role in controlling the cellular metabolism of living organisms. A protein kinase was purified from etiolated oat seedlings by several steps of ion-exchange and affinity chromatographies. The kinase was a 150-kDa tetrameric protein and composed of three subunits of 34, 37, and 40 kDa proteins. The 34 and 40 kDa proteins had ATP binding sites, suggesting that they are catalytic subunits and that the 37-kDa protein is a regulatory subunit. In the in vitro phosphorylation of a crude oat cell extract, it intensively phosphorylated a serine residue of a 110-kDa protein. The 110-kDa protein was tentatively identified as a DNA topoisomerase I, based on an amino acid sequence homology. Phosphorylation of the 110-kDa protein by the kinase required ATP or GTP as a phosphoryl group donor. The kinase activity was inhibited by 50% at a concentration of 0.05 microg/ml heparin. These results, therefore, indicate that the purified kinase is a CK II protein kinase and may be involved in the regulation of DNA topoisomerase I activity.