We have discovered a role for coactivators binding to the AF-2 surface of the vitamin D receptor (VDR) in its negative effects on gene transcription. We tested nine amino acid residues (Ser(235), Ile(242), Lys(246), Asp(253), Ile(260), Leu(263), Leu(417), Leu(419), and Glu(420)) in human VDR which, based on homology to the human thyroid hormone receptor, would be predicted to lie in or near the coactivator-binding site. Mutation of six of these residues in VDR resulted in loss of both the activation (assessed with a transfected DR3 TK luciferase reporter) and inhibition (assessed with an hANPCAT reporter) functions of the receptor when tested in cultured neonatal rat atrial myocytes and HeLa cells. Collectively, these mutations also suppressed association of VDR with the coactivators GRIP1 and steroid receptor coactivator 1 in vitro but had little or no effect on ligand binding, heterodimerization with the retinoid X receptor, or association with a VDR-specific DNA recognition element. Co-transfection with GRIP1 or steroid receptor coactivator 1 amplified both the positive and negative responses to wild type VDR but had little or no effect on the functionally impaired mutants described above. The interaction between VDR and GRIP1 proved to be heavily dependent upon the integrity of nuclear box III in the latter protein. Mutations in this region of GRIP1 impaired its ability to associate with VDR in vitro and to amplify VDR activity in intact cells. These studies establish a role for coactivators recruited to the same receptor surface in both the activating and inhibitory activity of the liganded receptor.

Abstract-The type B natriuretic peptide receptor (NPR-B) is the cognate receptor for the C-type natriuretic peptide and, as such, is responsible for signaling growth-suppressant activity in vascular smooth muscle cells. Here we report the isolation and characterization of the human (h) NPR-B gene promoter. Using 5Ј rapid amplification of cDNA ends analysis, we have identified the 5Ј terminus of the hNPR-B gene transcript Ϸ732 base pairs upstream from the presumed translation start site of the protein. We generated a series of 5Ј deletion mutants linked to a luciferase reporter and introduced these constructs into rat aortic smooth muscle cells or neonatal rat cardiac fibroblasts. Maximal expression was seen with a construct harboring 441 base pairs of 5Ј flanking sequence. Site-directed mutagenesis of the proximal promoter revealed a series of GC-rich sequences, 5 of which contributed modestly (Ϸ25%) to basal hNPR-B promoter activity. Mutation of a sixth GC-rich sequence led to a Ͼ90% reduction in promoter activity. This sequence was shown to associate with Sp1 and Sp3 in vitro. The same mutation that resulted in loss of functional activity also resulted in loss of binding activity in vitro. Overexpression of Sp1 or Sp3 in Drosophila Schneider cells resulted in an increase in hNPR-B promoter activity that was completely nullified with the Sp1 binding site mutation described above. These studies provide the first description and characterization of the NPR-B gene promoter and suggest that this promoter's activity is dominated by a single cluster of Sp1-binding elements in the proximal 5Ј flanking sequence of the gene.

Natural phytochemicals are attracting increasing interest as anticancer agents. The aim of this study is to evaluate the therapeutic potential of geraniin, a major ellagitannin extracted from Geranium sibiricum L., in human glioma. Human U87 and LN229 glioma cells were treated with different concentrations of geraniin, and cell viability, apoptosis, and gene expression were assessed. The involvement of STAT3 signaling in the action of geraniin was examined. We found that geraniin treatment for 48 h significantly (P \ 0.05) impaired the phosphorylation of STAT3 and reduced the expression of downstream target genes Bcl-xL, Mcl-1, Bcl-2, and cyclin D1. Exposure to geraniin led to a concentration-dependent decline in cell viability and increase in apoptosis in glioma cells, but had no significant impact on the viability of normal human astrocytes. Measurement of caspase-3 activity showed that geraniin-treated U87 and LN229 cells showed a 1.8-2.5-fold higher caspase-3 activity than control cells. Overexpression of constitutively active STAT3 significantly (P \ 0.05) reversed geraniin-mediated growth suppression and apoptosis, which was accompanied by restoration of Bcl-xL, Mcl-1, Bcl-2, and cyclin D1 expression. In an xenograft tumor mouse model, geraniin treatment significantly retarded tumor growth and induced apoptosis. Western blot analysis confirmed the suppression of STAT3 phosphorylation in glioma

microRNAs (miRNAs) play important roles in the pathogenesis of atherosclerosis. A previous study has reported that miR-497 is elevated in advanced atherosclerotic lesions in an apoE-deficient (apoE-/-) mouse model. The purpose of this study is to test whether miR-497 can modulate macrophage foam cell formation, an initiating event in atherosclerosis. We found that miR-497 was upregulated in THP-1 macrophages after treatment with oxidized low-density lipoprotein (oxLDL). Enforced expression of miR-497 promoted lipid accumulation and decreased cholesterol efflux in oxLDL-exposed THP-1 macrophages. In contrast, downregulation of miR-497 suppressed oxLDL-induced lipid accumulation in THP-1 macrophages. Apelin was identified to be a downstream target of miR-497. Overexpression of miR-497 significantly reduced the expression of apelin in THP-1 macrophages. Interestingly, delivery of a miR-497-resistant variant of apelin significantly inhibited lipid accumulation and enhanced cholesterol efflux in miR-497-overexpressing THP-1 macrophages in response to oxLDL. In addition, miR-497 expression was increased and negatively correlated with apelin protein expression in human atherosclerotic lesions. In conclusion, miR-497 contributes to oxLDL-induced lipid deposition in macrophages largely via targeting of apelin and thus represents a potential therapeutic target for atherosclerosis.

Hepatocellular carcinoma (HCC) is one of the most common cancers and a leading cause of cancer-related deaths worldwide. Early diagnosis of HCC remains a great challenge in clinic. Novel and effective biomarkers are in urgent need in early diagnosis of HCC.Serum levels of neurotrophin-receptor-interacting melanoma antigen-encoding gene homolog (NRAGE) were measured for 107 patients with HCC, 98 patients with benign liver diseases, and 89 healthy controls using quantitative real-time polymerase chain reaction. Receiver operating characteristic curve was applied to evaluate the diagnostic capacity of serum NRAGE in HCC.NRAGE expression was significantly higher in patients with HCC than in controls (all, P < .05). Moreover, its expression was tightly correlated with TNM stage (P = .004). NRAGE could distinguish patients with HCC from healthy controls with the area under the curve (AUC) of 0.874, yielding a sensitivity of 81.3% and a specificity of 78.7%. Additionally, in differentiation between benign liver diseases and HCC, the AUC value of NRAGE was 0.726, with a sensitivity of 63.6% and a specificity of 73.5%. Meanwhile, alpha-fetoprotein also could distinguish patients with HCC from benign liver disease cases, with an AUC of 0.677, a sensitivity of 64.4%, and a specificity of 60.2%.NRAGE could be a potential biomarker for HCC early diagnosis.