Biochimica et Biophysica Acta - Gene Structure and Expression

Sphingosine kinase 1 (SphK1) is a lipid kinase implicated in mitogenic signaling pathways in vascular smooth muscle cells. We demonstrate that human coronary artery smooth muscle (HCASM) cells require SphK1 for growth and that SphK1 mRNA and protein levels are elevated in PDGF stimulated HCASM cells. To determine the mechanism of PDGF-induced SphK1 expression, we used pharmacological inhibitors of the PI3K/AKT/mTOR signaling pathway. Wortmannin, SH-5, and rapamycin significantly blocked PDGF-stimulated induction of SphK1 mRNA and protein expression, indicating a regulatory role of the PI3K/AKT/mTOR pathway in SphK1 expression. To determine which isoform of AKT regulates SphK1 mRNA and protein levels, siRNAs specific for AKT1, AKT2, and AKT3 were used. We show that AKT2 siRNA significantly blocked PDGF-stimulated increases in SphK1 mRNA and protein expression levels as well as SphK1 enzymatic activity levels. In contrast, AKT1 or AKT3 siRNA did not have an effect. Together, these results demonstrate that the PI3K/AKT/mTOR signaling pathway is involved in regulation of SphK1, with AKT2 playing a key role in PDGF-induced SphK1 expression in HCASM cells.

Jacquelyn M. Francy

Arpita Nag

Elizabeth J. Conroy

Jeremy A. Hengst

Jong K. Yun

Biochimica et Biophysica Acta (BBA) - Gene Structure and Expression

Sphingosine kinase 1 expression is regulated by signaling through PI3K, AKT2, and mTOR in human coronary artery smooth muscle cells

鞘氨醇激酶1表达受PI3K、AKT2和mTOR信号通路调控于人冠状动脉平滑肌细胞中

E2F and FOXO transcription factors both play a role in neuronal apoptosis. In addition, both E2F-induced apoptosis and FOXO function are inhibited by the kinase Akt. We therefore tested whether FOXO is downstream of E2F-1 during neuronal apoptosis. We found that expression of endogenous FOXO1 and FOXO3a is induced by E2F-1. The presence of putative E2F binding sites in the promoters of both genes suggested that FOXO genes are direct targets of E2F-1. Indeed, a 4-hydroxytamoxifen activated E2F-1-ER fusion protein induced FOXO expression in the presence of cycloheximide. Moreover, E2F-1 activated the FOXO1 promoter in transient reporter assays, and E2F-1-ER as well as endogenous E2F bound to the FOXO1 promoter in vivo. Yet, E2F-1-mediated apoptosis of differentiated PC12 cells after withdrawal of NGF was not accompanied by changes in FOXO expression, indicating that no transcriptional induction of FOXO occurs during E2F-1-dependent neuronal apoptosis. In summary, our data identify E2F-1 as a first transcription factor regulating FOXO expression, providing a link between E2F and FOXO proteins in the control of cell fate.

Katrin Nowak

Katrin Killmer

Christine Gessner

Werner Lutz

E2F-1 regulates expression of FOXO1 and FOXO3a

E2F-1 调控 FOXO1 和 FOXO3a 的表达

Plant Ku genes were identified very recently in Arabidopsis thaliana, and their roles in repair of double-stranded break DNA and maintenance of telomere integrity were scrutinized. In this study, the cDNAs encoding Ku70 (VrKu70) and Ku80 (VrKu80) were isolated from mung bean (Vigna radiata L.) hypocotyls. Both genes were expressed widely among different tissues of mung bean with the highest levels in hypocotyls and leaves. The VrKu gene expression was stimulated by exogenous auxins in a concentration- and time-dependent manner. The stimulation could be abolished by auxin transport inhibitors, N-(1-naphthyl) phthalamic acid and 2,3,5-triiodobenzoic acid implicating that exogenous auxins triggered the effects following their uptake by the cells. Further analysis using specific inhibitors of auxin signaling showed that the stimulation of VrKu expression by 2,4-dichlorophenoxyacetic acid (2,4-D) was suppressed by intracellular Ca2+ chelators, calmodulin antagonists, and calcium/calmodulin dependent protein kinase inhibitors, suggesting the involvement of calmodulin in the signaling pathway. On the other hand, exogenous indole-3-acetic acid (IAA) and α-naphthalene acetic acid (NAA) stimulated VrKu expression through the mitogen-activated protein kinase/extracellular signal-regulated kinase pathway. Altogether, it is thus proposed that 2,4-D and IAA (or NAA) regulate the expression of VrKu through two distinct pathways.

Pei-Feng Liu

Wen-Chi Chang

Yung-Kai Wang

Suresh-Babu Munisamy

Shen-Hsing Hsu

Hwan-You Chang

Shu-Hsing Wu

Rong-Long Pan

Differential regulation of Ku gene expression in etiolated mung bean hypocotyls by auxins

赤霉素对黄化绿豆下胚轴中Ku基因表达的差异调控

Agrobacterium tumefaciens transfers DNA (T-DNA) to plant cells, where it integrates into the plant genome. Little is known about how T-DNA chooses sites within the plant chromosome for integration. Previous studies indicated that T-DNA preferentially integrates into transcriptionally active regions of the genome, especially in 5′-promoter regions. This would make sense, considering that chromatin structure surrounding active promoters may be more "open" and accessible to foreign DNA. However, recent results suggest that this seemingly non-random pattern of integration may be an artifact of selection bias, and that T-DNA may integrate more randomly than previously thought. In this chapter, I discuss the history of these observations and the role chromatin proteins may play in T-DNA integration and transgene expression. Understanding how chromatin conformation may influence T-DNA integration will be important in developing strategies for reproducible and stable transgene expression, and for gene targeting.

Stanton B. Gelvin

Sang-Ic Kim

Effect of chromatin upon Agrobacterium T-DNA integration and transgene expression

染色质对根癌农杆菌T-DNA整合及转基因表达的影响

Ribosomal protein S12 is a critical component of the decoding center of the 30S ribosomal subunit and is involved in both tRNA selection and the response to streptomycin. We have investigated the interplay between S12 and some of the surrounding 16S rRNA residues by examining the phenotypes of double-mutant ribosomes in strains of Escherichia coli carrying deletions in all chromosomal rrn operons and expressing total rRNA from a single plasmid-borne rrn operon. We show that the combination of S12 and otherwise benign mutations at positions C1409–G1491 in 16S rRNA severely compromises cell growth while the level and range of aminoglycoside resistances conferred by the G1491U/C substitutions is markedly increased by a mutant S12 protein. The G1491U/C mutations in addition confer resistance to the unrelated antibiotic, capreomycin. S12 also interacts with the 912 region of 16S rRNA. Genetic selection of suppressors of streptomycin dependence caused by mutations at proline 90 in S12 yielded a C912U substitution in 16S rRNA. The C912U mutation on its own confers resistance to streptomycin and restricts miscoding, properties that distinguish it from a majority of the previously described error-promoting ram mutants that also reverse streptomycin dependence.

Anton Vila-Sanjurjo

Ying Lu

Jamie L. Aragonez

Rebekah E. Starkweather

Manoj Sasikumar

Michael O'Connor

Modulation of 16S rRNA function by ribosomal protein S12

16S rRNA功能受核糖体蛋白S12的调控

Takashi Toyono

Yuji Seta

Shinji Kataoka

Kuniaki Toyoshima

CCAAT/Enhancer-binding protein β regulates expression of human T1R3 taste receptor gene in the bile duct carcinoma cell line, HuCCT1

CCAAT/增强子结合蛋白β调控人T1R3味觉受体基因在胆管癌细胞系HuCCT1中的表达

A rice diterpene cyclase, OsDTC2, functions as a stemar-13-ene synthase that converts syn-copalyl diphosphate into stemar-13-ene, a putative diterpene hydrocarbon precursor of the phytoalexin oryzalexin S. The transcriptional expression of OsDTC2 is induced by treatment of suspension-cultured rice cells with a chitin oligosaccharide elicitor. To investigate the molecular mechanisms of the elicitor signaling pathway that leads to OsDTC2 expression, we carried out deletion and mutation analysis of the region -1939 bp upstream of the transcription start site of OsDTC2 in rice cells using dual luciferase assays. The region between -1709 and -1450 bp was found to contain six W-box motifs, which are putative recognition sites for WRKY transcription factors, as cis elements involved in elicitor-responsiveness and/or basic promoter activity of OsDTC2.

Tadahiro Nemoto

Atsushi Okada

Kazunori Okada

Naoto Shibuya

Tomonobu Toyomasu

Hideaki Nojiri

Hisakazu Yamane

Promoter analysis of the rice stemar-13-ene synthase gene OsDTC2, which is involved in the biosynthesis of the phytoalexin oryzalexin S

水稻倍半萜烯合酶基因OsDTC2启动子分析及其在植物抗毒素稻瘟菌素S生物合成中的作用

We have learned a great deal over the last several years about the molecular mechanisms that govern cell growth, cell division and cell death. Normal cells pass through cell cycle (growth) and divide in response to mitogenic signals that are transduced through their cognate cell surface receptors to the nucleus. Despite the fact that cellular growth and division are mechanistically distinct steps, they are usually coordinately regulated, which is critical for normal cellular proliferation. The precise mechanistic basis for this coordinated regulation is unclear. TFII-I is a unique, signal-induced multifunctional transcription factor that is activated upon a variety of signaling pathways and appears to participate in distinct phases of cell growth. For instance, TFII-I is required for growth factor-induced transcriptional activation of the c-fos gene, which is essential for cell cycle entry. Two alternatively spliced isoforms of TFII-I exhibit opposing but necessary functions for mitogen-induced transcriptional activation of c-fos. Besides transcriptional activation of the c-fos proto-oncogene and eventual entry into cell cycle, TFII-I also appears to have a role in later phases of the cell cycle and cell division. Here we discuss how a multitude of signaling inputs target TFII-I isoforms, which may exert their functions in distinct phases of the cell cycle and play a key role in the coordinated regulation of cellular proliferation.

Ananda L. Roy

Signal-induced functions of the transcription factor TFII-I

转录因子TFII-I的信号诱导功能

Ribonuclease P (RNase P) complexed with an external guide sequence (EGS) represents a novel nucleic acid-based gene interference approach to modulate gene expression. This enzyme is a ribonucleoprotein complex for tRNA processing. In Escherichia coli, RNase P contains a catalytic RNA subunit (M1 ribozyme) and a protein subunit (C5 cofactor). EGSs, which are RNAs derived from natural tRNAs, bind to a target mRNA and render the mRNA susceptible to hydrolysis by RNase P and M1 ribozyme. When covalently linked with a guide sequence, M1 can be engineered into a sequence-specific endonuclease, M1GS ribozyme, which cleaves any target RNAs that base pair with the guide sequence. Studies have demonstrated efficient cleavage of mRNAs by M1GS and RNase P complexed with EGSs in vitro. Moreover, highly active M1GS and EGSs were successfully engineered using in vitro selection procedures. EGSs and M1GS ribozymes are effective in blocking gene expression in both bacteria and human cells, and exhibit promising activity for antimicrobial, antiviral, and anticancer applications. In this review, we highlight some recent results using the RNase P-based technology, and offer new insights into the future of using EGS and M1GS RNA as tools for basic research and as gene-targeting agents for clinical applications.

Kihoon Kim

Fenyong Liu

Inhibition of gene expression in human cells using RNase P-derived ribozymes and external guide sequences

使用RNase P衍生的核酶和外部引导序列抑制人类细胞中的基因表达

The Publisher regrets that this article is an accidental duplication of an article that has already been published in Biochem. Biophys. Acta, doi:10.1016/j.bbagrm.2007.09.001. The duplicate article has therefore been withdrawn.

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