Regulation of thrombomodulin by tumor necrosis factor-alpha: comparison of transcriptional and posttranscriptional mechanisms

The procoagulant properties of cultured vascular endothelial cells are enhanced in response to inflammatory cytokines such as tumor necrosis factor-alpha (TNF). A major component of this response is a reduction in expression of thrombomodulin, a cell surface cofactor for the activation of protein C. Regulation of thrombomodulin expression by TNF has been reported to occur through multiple mechanisms. To determine the relative roles of transcriptional and posttranscriptional regulation, the effect of TNF on the turnover of thrombomodulin protein and mRNA was examined in human and bovine endothelial cells. Quantitative nuclease S1 protection assays showed a 70% to 90% reduction in thrombomodulin mRNA within 4 hours of the addition of 1.0 nmol/L TNF to the culture medium. The decrease in thrombomodulin mRNA resulted from inhibition of transcription, followed by rapid degradation of thrombomodulin transcripts (t1/2 less than or equal to 3 hours). In pulse-chase incubations, thrombomodulin synthesis decreased parallel with mRNA, but the rate of degradation of radiolabeled thrombomodulin was not significantly altered by TNF. Human thrombomodulin was degraded with a t1/2 of 8.2 +/- 2.4 hours (SD) or 7.5 +/- 1.3 hours (SD) in the absence or presence of TNF, respectively. We conclude that TNF acts primarily to inhibit thrombomodulin transcription. The subsequent decrease in activity results from the inherent instability of thrombomodulin mRNA and protein in these cells, and not from the regulation of thrombomodulin degradation.