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Pinocembrin (5,7-dihydroxyflavanone) is one of the primary flavonoids in propolis. Angiotensin II (AngII) is a biologically active peptide that induces vasoconstriction via the activation of the angiotensin type 1 receptor (AT1R). In the present study, we investigated the vasorelaxant effect of pinocembrin on AngII-induced vasoconstriction and the molecular mechanism of action. Pinocembrin was observed to inhibit AngII-induced vasoconstriction in rat aortic rings with either intact or denuded endothelium. In endothelium-denuded tissues, pinocembrin (pD́'2pD2(') 4.28±0.15) counteracted the contractions evoked by cumulative concentrations of AngII. In a docking model, pinocembrin showed effective binding at the active site of AT1R. Pinocembrin was shown to inhibit both AngII-induced Ca(2+) release from internal stores and Ca(2+) influx. Moreover, the increase in the phosphorylation of extracellular signal-regulated kinase (ERK1/2) and myosin light chain 2 (MLC2) induced by AngII was blocked by pinocembrin. These results demonstrate that pinocembrin inhibits AngII-induced rat aortic ring contraction, and these inhibitory effects may be related to the reduction of the AngII-induced increase in [Ca(2+)]i and ERK1/2 activation via blocking AT1R., (Copyright © 2013 Elsevier Inc. All rights reserved.)

The proinflammatory cytokine TNF-alpha plays an important role in stimulating inflammatory responses of vascular smooth muscle cells (VSMCs). The anti-inflammatory function of Sirtuin 1 (SIRT1), a NAD-dependent class III histone/protein deacetylase, has been well documented, but how SIRT1 is regulated under inflammatory conditions is largely unknown. In the present research, we showed that levels of SIRT1 mRNA and protein expression increased in TNF-alpha-treated VSMCs. Overexpression of the p65/RelA subunit of NF-kappaB, a TNF-alpha-activated inflammatory transcription factor, in A7r5 cells, upregulated SIRT1 mRNA and protein expression as well as SIRT1 promoter activity, while knockdown of endogenous p65/RelA expression by RNAi not only led to a decrease in SIRT1's basal protein expression and promoter activity, but almost abolished the TNF-alpha-induced elevation of SIRT1 protein expression and SIRT1 promoter activity. Furthermore, using promoter deletion analysis and chromatin immunoprecipitation assays, we found that p65/RelA bound to the SIRT1 promoter at a consensus NF-kappaB binding site. Our study indicates that p65/RelA mediates the TNF-alpha-induced elevated expression of SIRT1 in VSMCs, shedding new light on the regulation of SIRT1 under inflammatory conditions.

The nuclear location and relocation of genes play crucial regulatory roles in gene expression. SATB1, a MAR-binding protein, has been found to regulate beta-like globin genes through chromatin remodeling. In this study, we generated K562 cells over-expressing wild-type or nuclear matrix targeting sequences (NMTS)-deficient SATB1 and found that like wild-type SATB1, NMTS-deficient SATB1 induces out loop of beta-globin cluster from its chromosome territory (CT), while it is unable to associate the cluster with the nuclear matrix as wild-type SATB1 does and had no regulatory functions to the beta-globin cluster. Besides, our data showed that the transacting factor occupancies and chromatin modifications at beta-globin cluster were differentially affected by wild-type and NMTS-deficient SATB1. These results indicate that SATB1 regulates beta-like globin genes at the nuclear level interlaced with chromatin and DNA level, and emphasize the nuclear matrix binding activity of SATB1 to its regulatory function.

Histone deacetylase (HDAC) inhibitors are one of promising drugs to induce fetal hemoglobin (HbF) for treatment of sickle cell disease (SCD) and beta-thalassemia. The HDAC inhibitor apicidin was recently reported as a powerful inducer of HbF via a mechanism involving p38 signaling. In this study, we further investigated the signaling effects on the transcriptional activation of gamma-globin gene. First, we compared histone 3 (H3) acetylation patterns of approximately 70kb beta-globin loci in K562 erythroid versus HeLa cells upon apicidin treatment by chromatin immunoprecipitation assays. The results showed that the level of H3 acetylation was globally increased from the LCR to the promoter of gamma-globin gene in K562 cells, but not in non-erythroid, HeLa cells. Inhibition of p38 signaling blocks the effects of apicidin-induced gamma-globin expression and H3 acetylation. In parallel, we assessed the recruitment of transcriptional complex to beta-globin locus following apicidin treatment. The binding of GATA-1, Sp1 and RNA polymerase II (pol II) were observed to increase over several regulatory regions of beta-globin locus. Inhibitor study revealed that p38 pathway was not involved in their recruitments by apicidin. Collectively, our results provide a molecular basis to elucidate the underlying mechanisms involving p38 signaling pathway in the inducement of gamma-globin transcriptional expression by apicidin.

The ideal gene-therapy vector for treating genetic disorders should deliver intact therapeutic genes and their essential regulatory elements into the specific "safe genomic site" and realize long-term, self-regulatory expression. For beta-thalassemia gene therapy, viral vectors have been broadly used, but the accompanying insertional mutation and immunogenicity remain problematic. Hence, we aimed to develop new non-viral vectors that are efficient and safe in treating diseases. As previous studies have demonstrated that physiological expression of beta-globin genes requires both a 5' locus control region and 3' specific elements, we constructed a new human chromosome-derived targeting vector to transfer the intact beta-globin gene cluster into K562 cells. The whole beta-globin gene cluster was precisely integrated into the target site and expressed in a self-regulatory pattern. The results proved that the human chromosome-derived vector was specifically targeted to the human genome and this could provide a novel platform for further gene therapy research.