Identification of syntenin and other TNF‐inducible genes in human umbilical arterial endothelial cells by suppression subtractive hybridization

FEBS Letters

Schulze-Osthoff, Klaus¹  Neuhaus, Thomas²  Totzke, Gudrun²  Grünewald, Elisabeth²  Vetter, Hans²  Stier, Sebastian²  Ko, Yon²  Sachinidis, Agapios²  Fronhoffs, Stefan² 

[1] Department of Immunology and Cell Biology, University of Münster, Münster, Germany;Medizinische Poliklinik, University of Bonn, Wilhelmstr. 35-37, D-53111 Bonn, Germany

关键词: Adhesion molecule;    Endothelial cell;    Syntenin;    Nuclear factor-κB;    Suppression subtractive hybridization;    Tumor necrosis factor;    HUAEC;    human umbilical arterial endothelial cells;    IL;    interleukin;    MCP-1;    monocyte chemoattractant protein-1;    NF-κB;    nuclear factor-κB;    IκB;    inhibitor of NF-κB;    PCR;    polymerase chain reaction;    RT;    reverse transcription;    SSH;    suppression subtractive hybridization;    TNF;    tumor necrosis factor-α;    VCAM-1;    vascular cell adhesion molecule-1;   

DOI  :  10.1016/S0014-5793(00)01177-7

学科分类：生物化学/生物物理

来源: John Wiley & Sons Ltd.

【 摘 要 】

Endothelial cells play an important regulatory role in inflammatory responses by upregulating various proinflammatory gene products including cytokines and adhesion molecules. A highly potent mediator of this process is tumor necrosis factor-α (TNF). In the present study, the suppression subtractive hybridization (SSH) method was employed to identify rarely transcribed TNF-inducible genes in human umbilical arterial endothelial cells. Following mRNA isolation of non-stimulated and TNF-stimulated cells, cDNAs of both populations were prepared and subtracted by suppression PCR. Sequencing of the enriched cDNAs identified 12 genes differentially expressed including vascular cell adhesion molecule-1, monocyte chemoattractant protein-1, interleukin-8 and IκBα, an inhibitor of the transcription factor nuclear factor-κB. Interestingly, also syntenin, a PDZ motif-containing protein which binds to the cytoplasmic domain of syndecans, was identified by SSH. Time course studies using RT-PCR analysis confirmed that all genes were differentially expressed and rapidly induced by TNF. Our data reveal that SSH is a powerful technique of high sensitivity for the detection of differential gene expression in primary arterial endothelial cells.

【 授权许可】

Unknown