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 respons es by upregulating various proinflammatory gene products including cytokine s and adhesion molecules. A highly potent mediator of this process is tumor necrosis factor-alpha (TNF), In the present study, the suppression subtrac tive hybridization (SSH) method was employed to identify rarely transcribed TNF-inducible genes in human umbilical arterial endothelial cells. Followi ng 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, inte rleukin-8 and I kappa B alpha, an inhibitor of the transcription factor nuc lear factor-kappa B. Interestingly, also syntenin, a PDZ motif-containing p rotein which binds to the cytoplasmic domain of syndecans, was identified b y SSH, Time course studies using RT-PCR analysis confirmed that all genes w ere differentially expressed and rapidly induced by TNF, Our data reveal th at SSH is a powerful technique of high sensitivity for the detection of dif ferential gene expression in primary arterial endothelial cells. (C) 2000 F ederation of European Biochemical Societies.