Binding of Gamma-glutamyltransferase to toll-like receptor-4 allows tissue factor activation in monocytes

BACKGROUND: Gamma-glutamyltransferase (GGT) is well known to play a key role in the antioxidant processes, however, it also exerts pro-oxidant effects by activating Nf-kB, a redoxsensitive transcription factor key in the induction of Tissue Factor (TF) gene expression, the principal initiator of the clotting cascade. GGT may potentially modulate TF expression, an assumption verified by previous studies. Quite importantly, TF expression in response to GGT stimulation was independent of its enzymatic properties since those experiments were conducted by using human recombinant (hr)GGT, a protein enzymatically inert. Thus, GGT may act through a cytokine-like mechanism. AIMS: To assess whether GGT-induced TF stimulation is a consequence of binding to Toll-Like Receptor (TLR)-4 and activation of Nf-kB, as suggested by results recently obtained in different experimental contexts. METHODS: PBMCs obtained from healthy donors and THP-1 cells, a human monocytic cell line, were incubated with hrGG. LPS-Rs, CLI-095 and BAY-11-7082 were used to block TLR-4 receptors, TLR4 signaling and NF-kB respectively. TF pro-coagulant activity (PCA) was evaluated and results were expressed in pg/ml. HEK-Blue cells, an engineered HEK-293 cell line designed for studying the stimulation of TLR4 by monitoring the activation of NF-κB and AP-1, are used to assed hrGGT as TLR-4 agonist. RESULTS: hrGGT increased TF expression in both PBMCs and THP-1 cells. The GGT-induced TF stimulation was antagonized by TLR-4 antagonists, both extracellular (LPS-Rs) and intracellular (CLI-095 and BAY-11-7082). HEK Blue cells confirmed that the lack of TLR-4 receptor abrogates GGT-induced activation of NfkB. CONCLUSIONS Our data identify for the first time the possible role of TLR-4 as the receptor of GGT in monocytes. The GGT-TLR-4 link may provide a mechanistic explanation to the consistent association between circulating GGT levels and increased risk of acute thrombotic events as well as to the involvement of GGT in the morbid evolution of the silent atherosclerotic plaque in which GGT colocalizes with monocytes and foam cells, the prime sources of TF within the plaque.