title: Identification of syntenin and other TNF-inducible genes in human umbilical arterial endothelial cells by suppression subtractive hybridization.
abstract: 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-alpha (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 IkappaBalpha, an inhibitor of the transcription factor nuclear factor-kappaB. 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.
authors.affiliation: Medizinische Poliklinik, University of Bonn, Wilhelmstr. 35-37, D-53111, Bonn, Germany.
authors.forename: S
authors.initials: S
authors.lastname: Stier
authors.forename: G
authors.initials: G
authors.lastname: Totzke
authors.forename: E
authors.initials: E
authors.lastname: Grünewald
authors.forename: T
authors.initials: T
authors.lastname: Neuhaus
authors.forename: S
authors.initials: S
authors.lastname: Fronhoffs
authors.forename: A
authors.initials: A
authors.lastname: Sachinidis
authors.forename: H
authors.initials: H
authors.lastname: Vetter
authors.forename: K
authors.initials: K
authors.lastname: Schulze-Osthoff
authors.forename: Y
authors.initials: Y
authors.lastname: Ko
mesh_terms: D002352:Carrier Proteins / Q000235:genetics*; D002469:Cell Separation; D018076:DNA, Complementary / Q000378:metabolism*; D004730:Endothelium, Vascular / Q000166:cytology / Q000187:drug effects / Q000378:metabolism*; D006801:Humans; D047908:Intracellular Signaling Peptides and Proteins; D008565:Membrane Proteins; D009693:Nucleic Acid Hybridization / Q000379:methods*; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods; D053676:Syntenins; D014409:Tumor Necrosis Factor-alpha / Q000494:pharmacology*; D014469:Umbilical Arteries
chemical_list: D002352:Carrier Proteins; D018076:DNA, Complementary; D047908:Intracellular Signaling Peptides and Proteins; D008565:Membrane Proteins; C109621:SDCBP protein, human; D053676:Syntenins; D014409:Tumor Necrosis Factor-alpha