title: Differential effect of tumor necrosis factor-alpha on thrombomodulin gene expression by human monocytoid (THP-1) cell versus endothelial cells.
abstract: Human mononuclear phagocytes (MO) express a functional form of thrombomodulin (TM), the anticoagulant molecule typically considered purely in the context of regulation of conversion of protein C (PC) to activated PC (aPC) by thrombin-bound TM at the endothelial cell surface. We have been interested in the anti-inflammatory actions of aPC, including its ability to suppress MO production of multiple pro-inflammatory cytokines, such as tumor necrosis factor-alpha (TNF-alpha) and interleukin-1 (IL-1), leading us to consider whether MO surface expression of TM and resultant local aPC generation, might contribute to autoregulation of MO activation at sites of inflammation involving thrombin and fibrin formation. Since TNF-alpha and IL-1 are known to downregulate endothelial expression of TM, this study investigated the effects of TNF-alpha on production of TM by the monocytic leukemic cell line, THP-1. THP-1 cells display many monocyte-like properties, providing a convenient source for biochemical and molecular studies. Western blotting of lysates of THP-1 cells versus cultured human umbilical vein endothelial cells showed that after 24 h of stimulation, TNF-alpha decreased TM protein expression in endothelial but not THP-1 cells and comparable responses were noted by flow cytometry. Subsequent Northern blot analysis showed that at 24 h, TNF-alpha diminished TM steady state mRNA in endothelial but not THP-1 cells, although Northern analysis of the kinetics of TM steady state mRNA did show a rapid and transient modulation by TNF-alpha at 2 h of stimulation, which was confirmed by nuclear run-on analysis of the effect of TNF-alpha on TM gene transcription rates in THP-1 cells, analysis of protein expression by flow cytometry and Western blotting showed similar effects. In contrast to the divergent effects of TNF-alpha on THP-1 vs endothelial cells, agonists such as cyclic adenosine monophosphate (c-AMP) and phorbol ester (PMA) had comparable effects on THP-1 and endothelial cells, resulting in parallel increases or decreases in TM mRNA and protein expression, respectively. Hence, there is a 'split' in the nature of endothelial vs THP-1 cellular responses to TNF-alpha as compared to non-inflammatory stimuli, suggesting cell-specific differences in regulation of the TM promoter. We conclude that in contrast to its effects on TM expression by endothelial cells, exposure of THP-1 cells to TNF-alpha causes a rapid and transient decrease in TM mRNA production which is followed by sustained and high level expression, supporting the concept that MO expression of TM may contribute to regulation of MO activation and cytokine production at inflammatory sites.
authors.affiliation: Center for Immunobiology, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, MA 02215, USA.
authors.forename: S T
authors.initials: ST
authors.lastname: Grey
authors.forename: V
authors.initials: V
authors.lastname: Csizmadia
authors.forename: W W
authors.initials: WW
authors.lastname: Hancock
mesh_terms: D002460:Cell Line; D004730:Endothelium, Vascular / Q000166:cytology / Q000187:drug effects* / Q000378:metabolism; D005786:Gene Expression Regulation / Q000187:drug effects*; D006801:Humans; D009000:Monocytes / Q000187:drug effects* / Q000378:metabolism; D018180:Thrombomodulin / Q000235:genetics*; D014409:Tumor Necrosis Factor-alpha / Q000494:pharmacology*
chemical_list: D018180:Thrombomodulin; D014409:Tumor Necrosis Factor-alpha