Identification of syntenin and other TNF-inducible genes in human umbilical arterial endothelial cells by suppression subtractive hybridization.
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.
Stier
S
S
Medizinische Poliklinik, University of Bonn, Wilhelmstr. 35-37, D-53111, Bonn, Germany.
Totzke
G
G
Grünewald
E
E
Neuhaus
T
T
Fronhoffs
S
S
Sachinidis
A
A
Vetter
H
H
Schulze-Osthoff
K
K
Ko
Y
Y
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
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