X-box binding protein l splicing attenuates brain microvascular endothelial cell damage induced by oxygen-glucose deprivation through the activation of phosphoinositide 3-kinase/protein kinase B,extracellular signal-regulated kinases,and hypoxia-inducible factor-1α/vascular endothelial growth factor signaling pathways

Angiogenesis is positively correlated with the survival rate of stroke patients. Therefore, studying factors that initiate and promote angiogenesis after ischemic stroke is crucial for finding novel and effective treatment targets that improve the prognosis of stroke. X-box binding protein l splicing (XBP1s) plays a positive regulatory role in cell proliferation and angiogenesis. However, the role and mechanism of XBP1s on the proliferation of brain microvascular endothelial cells (BMECs) and angiogenesis after cerebral ischemia remains unclear. In the current study, we investigated the role XBP1s plays in BMEC proliferation and angiogenesis following cerebral ischemia. In this study, the roles of XBP1s on cell survival, apoptosis, cycle migration, and angiogenesis were determined in oxygen-glucose deprivation (OGD) treated BMECs. The expression of XBP1s in BMECs, which were exposed to OGD at 0, 2, 4, and 6 hr, increased in a time-dependent manner. The overexpression of XBP1s promoted cell survival, cell cycle, migration, and angiogenesis of BMECs, and inhibited the apoptosis in OGD-treated BMECs. In addition, the overexpression of XBP1s promoted the expression of cyclin D1, matrix metalloproteinase (MMP-2), and MMP-9, but inhibited cleaved Caspase-3 and cleaved Caspase-9 expression in OGD-treated BMECs. The overexpression of XBP1s also promoted the expression of hypoxia-inducible factor 1-alpha, vascular endothelial growth factor, phosphatidylinositol-4,5-bisphosphate 3-kinase, p-AKT, p-mTOR, p-GSK3β, and p-extracellular signal-regulated kinase1/2 in OGD-treated BMECs. The effect of XBP1s silencing was opposite to that of XBP1s overexpression. In conclusion, using an in vitro OGD model, we demonstrated that XBP1s may be a promising target for ischemic stroke therapy to maintain BMECs survival and induce angiogenesis.     

miR-221 is required for endothelial tip cell behaviors during vascular development

Angiogenesis requires coordination of distinct cell behaviors between tip and stalk cells. Although this process is governed by regulatory interactions between the vascular endothelial growth factor (Vegf) and Notch signaling pathways, little is known about the potential role of microRNAs. Through deep sequencing and functional screening in zebrafish, we find that miR-221 is essential for angiogenesis. miR-221 knockdown phenocopied defects associated with loss of the tip cell-expressed Flt4 receptor. Furthermore, miR-221 was required for tip cell proliferation and migration, as well as tip cell potential in mosaic blood vessels. miR-221 knockdown also prevented "hyper-angiogenesis" defects associated with Notch deficiency and miR-221 expression was inhibited by Notch signaling. Finally, miR-221 promoted tip cell behavior through repression of two targets: cyclin dependent kinase inhibitor 1b (cdkn1b) and phosphoinositide-3-kinase regulatory subunit 1 (pik3r1). These results identify miR-221 as an important regulatory node through which tip cell migration and proliferation are controlled during angiogenesis.     

Histone deacetylases in Trypanosoma brucei: two are essential and another is required for normal cell cycle progression

Reversible protein acetylation is established as a modification of major regulatory significance. In particular, histone acetylation regulates access to genetic information in eukaryotes. For example, class I and class II histone deacetylases are regulatory components of corepressor complexes involved in cell cycle progression and differentiation. Here, we have investigated the function of such enzymes in Trypanosoma brucei, mono-flagellated parasitic protozoa that branched very early from the eukaryotic lineage. Four T. brucei genes encoding histone deacetylase orthologues have been identified, cloned and characterized. The predicted deacetylases, DAC1-4 are approximately 43, 61, 75 and 64 kDa respectively. They share significant similarity with mammalian and yeast class I (DAC1 and DAC2) and class II (DAC3 and DAC4) histone deacetylases, and all except DAC2 have the critical residues predicted to be required for deacetylase activity. In gene targeting experiments, DAC1 and DAC3 appear to be essential whereas DAC2 and DAC4 are not required for viability. Of the two mutant cell types, the dac4 mutant displays a delay in the G2/M phase of the cell cycle. Our results provide genetic validation of DAC1 and DAC3 as potential chemotherapy targets and demonstrate that T. brucei expresses at least three probable histone deacetylases with distinct function.     

转录因子X-box结合蛋白1相互作用蛋白的筛选和鉴定

X-box 结合蛋白 1 是一种重要的转录因子,参与体内多项信号转导过程. 为进一步研究 XBP1 的生物学功能,运用酵母双杂交技术在肝细胞文库中筛选 XBP1 的结合蛋白. 首先运用 PCR 技术扩增获得 XBP1 的编码序列,克隆至 pGEM-T 载体,经测序鉴定后,亚克隆至诱饵载体 pGBKT7 中,转化酵母 AH109(a type). 免疫印迹检测诱饵质粒 pGBKT7-XBP1 在AH109 酵母中的表达之后,含有诱饵质粒的酵母 AH109 与含有肝细胞 cDNA 文库质粒 pACT2 的酵母 Y187(αtype)配合,配合后的二倍体酵母生长在含有 X-α-gal 的营养缺陷型培养基上 (SD/-Trp-Leu-His-Ade) 进行选择和筛选,经测序和序列比对确定阳性克隆的开放读码框 ORF,得到 7 种不同的蛋白质. 为了进一步验证这些筛选蛋白质与 XBP1 的相互作用,克隆其中一种蛋白质 MT1E,并运用 GST pulldown 和免疫共沉淀技术成功检测了 MT1E 和 XBP1 的相互作用(体外 / 体内),结果提示,MT1E 可能是 XBP1 的一个新的调节蛋白. 通过酵母双杂交技术筛选得到的 7 种蛋白质分别与肝细胞基础代谢、蛋白质的合成与运输、细胞的增殖与凋亡密切相关. 上述结果有助于揭示 XBP1 的生物学功能,为进一步探讨 XBP1 的表达和调控机制提供新线索.     

转录因子XBP1S基因表达谱差异分析

目的 应用基因表达谱芯片技术了解XBP1S在肝细胞中可能上调或下调的基因,了解其可能的调节功能线索.方法 构建pcDNA3.1（-）-XBP1S真核表达载体,转染HepG2细胞,同时以空载体pcDNA3.1（-）处理相同细胞系作为对照.48 h后制备细胞裂解液,提取mRNA,应用基因表达谱芯片技术对差异表达mRNA进行检测和分析.结果 构建的表达载体经过限制性内切酶分析和DNA序列测定,证实准确无误,提取高质量的总mRNA并进行逆转录成为cDNA,进行基因表达谱芯片技术分析.经过差异基因表达谱的筛选,发现HepG2细胞转染XBP1S以后,有38个基因表达水平显著上调,30个基因表达水平显著下调.结论 成功构建XBP1S的真核表达载体pcDNA3.1（-）-XBP1S,运用基因表达谱芯片技术成功筛选了XBP1S转染细胞后的差异表达基因,这些差异表达基因包括细胞周期、蛋白质的翻译合成及运输、能量代谢、体内免疫调节、细胞凋亡及细胞内的信号转导等方面起重要作用及肿瘤发生相关的基因,为进一步阐明XBP1S可能存在的调控机制及XBP1S蛋白可能的生物学功能提供理论依据.     

Role of phospholipase Cε in physiological phosphoinositide signaling networks

Receptor-initiated phospholipase C activation and generation of IP(3) and DAG are important common triggers for a diversity of signal transduction processes in many cell types. Contributing to this diversity is the existence and differential cellular and subcellular distribution of distinct phospholipase C isoforms with distinct regulatory properties. The recently identified PLCε enzyme is an isoform that is uniquely regulated by multiple upstream signals including ras-family GTP binding proteins as well as heterotrimeric G-proteins. In this review we will consider the well documented biochemical regulation of this isoform in the context of cell and whole animal physiology and in the context of other G protein-regulated PLC isoforms. These studies together reveal a surprisingly wide range of unexpected functions for PLCε in cellular signaling, physiology and disease.     

Deregulation of XBP1 expression contributes to myocardial vascular endothelial growth factor‐A expression and angiogenesis during cardiac hypertrophy in vivo

Endoplasmic reticulum (ER) stress has been reported to be involved in many cardiovascular diseases such as atherosclerosis, diabetes, myocardial ischemia, and hypertension that ultimately result in heart failure. XBP1 is a key ER stress signal transducer and an important pro‐survival factor of the unfolded protein response (UPR) in mammalian cells. The aim of this study was to establish a role for XBP1 in the deregulation of pro‐angiogenic factor VEGF expression and potential regulatory mechanisms in hypertrophic and failing heart. Western blots showed that myocardial XBP1s protein was significantly increased in both isoproterenol (ISO)‐induced and pressure‐overload‐induced hypertrophic and failing heart compared to normal control. Furthermore, XBP1 silencing exacerbates ISO‐induced cardiac dysfunction along with a reduction of myocardial capillary density and cardiac expression of pro‐angiogenic factor VEGF‐A in vivo. Consistently, experiments in cultured cardiomyocytes H9c2 (2‐1) cells showed that UPR‐induced VEGF‐A upregulation was determined by XBP1 expression level. Importantly, VEGF‐A expression was increased in failing human heart tissue and blood samples and was correlated with the levels of XBP1. These results suggest that XBP1 regulates VEGF‐mediated cardiac angiogenesis, which contributes to the progression of adaptive hypertrophy, and might provide novel targets for prevention and treatment of heart failure.     

Scalloped and strawberry notch are target genes of Notch signaling in the context of wing margin formation in Drosophila.

The Notch pathway regulates the differentiation of many cell types throughout development of higher metazoa. Different cellular responses are elicited through specific activation of distinct Notch target genes. In the Drosophila wing, for example, the cut gene is activated by Notch signaling along the dorso-ventral boundary but, as we show here, not in other cell types. We identify additional regulatory components, scalloped and strawberry notch, that are targets of the Notch pathway specifically within the wing anlagen. As suggested by physical interactions, these proteins could be co-factors of the cut trans-regulator Vestigial. Additional regulatory input comes from the Wingless pathway. Our data support a model, whereby context specific involvement of distinct co-regulators modulates Notch target gene activation.     

Evidence for Multiple, Local Functions of Ciliary Neurotrophic Factor (CNTF) in Retinal Development: Expression of CNTF and Its Receptor and In Vitro Effects on Target Cells

Abstract: There is increasing, although largely indirect, evidence that neurotrophic factors not only function as target-derived survival factors for projection neurons, but also act locally to regulate developmental processes. We studied the expression of ciliary neurotrophic factor (CNTF) and the CNTF-specific ligand-binding α-subunit of the CNTF receptor complex (CNTFRα) in the rat retina, a well-defined CNS model system, and CNTF effects on cultured retinal neurons. Both CNTF and CNTFRα (mRNA and protein) are expressed during phases of retinal neurogenesis and differentiation. Retina-specific Müller glia are immunocytochemically identified as the site of CNTF production and CNTFRα-expressing, distinct neuronal cell types as potential CNTF targets. Biological effects on corresponding neurons in culture further support the conclusion that locally supplied CNTF plays a regulatory role in the development of various retinal cell types including ganglion cells and interneurons.