Isolation and Characterization of Human ADAMTS13 Gene Promoter Region - Control of ADAMTS13 Gene Expression by Hepatic Transcription Factors and Inflammatory Cytokines.

Abstract

ADAMTS13 (A Disintegrin And Metalloprotease with ThromboSpondin type 1 repeats-13) controls von Willebrand factor multimer sizes by cleaving the Tyr1605-Met1606 bond in the central A2 domain. Deficiency of plasma ADAMTS13 activity can result in a lethal syndrome, thrombotic thrombocytopenic purpura (TTP). ADAMTS13 is primarily synthesized in hepatic stellate cells (HSCs), endothelial cells and megakaryocytes. We determined the transcription initiation site, the core region for promoter activity, the putative transcription factor binding sites as well as the influence of inflammatory cytokines on ADAMTS13 promoter activity. To explore the transcriptional control of ADAMTS13 gene expression, we constructed reporter genes containing 991 base pairs (bp) of the ADAMTS13 5′ untranslated (UT) region. We showed by deletion mutagenesis and luciferase reporter expression that the proximal-most 197 bp region was required for maximal luciferase activity in transfected cells in the human hepatic stellate cell line (LX-2) and in the human hepatocyte-like cell line (HepG2); the major transcription initiation site determined by 5′ - RACE was found at 77 bp upstream from the translation start site (ATG). However, the minimal sequences that were required for the promoter activity varied depending on the cells, with required sequences of approximately 147 and 127 bp in LX-2 and HepG2 cells, respectively. The proximal ADAMTS13 promoter region is evolutionally conserved between humans, mice and rats. This region is rich in GC content (72%) and contains putative binding sites for the transcription factors heat shock factor-2 (HSF2), FOXa2 [also named hepatocyte nuclear factor 3beta (HNF-3b)] and AP-1. A footprint assay demonstrated that the region between −116 and −126, containing the putative FOXa2 binding site, was largely protected by Dnase I digestion. The luciferase reporter activity was suppressed in cells transfected with the plasmid containing the proximal 314 bp human 5′ UT ADAMTS13 sequence in parallel with the inflammatory cytokines found to be elevated in patients with TTP: IL-4, TNF-alpha and INF-gamma. These inflammatory cytokines inhibited the Adamts13 mRNA and protein expression in rat primary HSCs in culture in a dose dependent manner. Approximately 70%, 71% and 80% of Adamts13 mRNA (by real time RT-PCR) and 77%, 78% and 92% of Adamts13 proteolytic activity (by FRETS-VWF73) were suppressed at 48 hours by IL-4 (10 ng/ml), TNF-alpha (10 ng/ml) and INF-gamma (100 ng/ml), respectively. We conclude that under physiological conditions ADAMTS13 synthesis may be strictly maintained at relatively low levels by binding transcription factors, whereas under pathological conditions inflammatory cytokines, released due to systemic inflammation, may further suppress ADAMTS13 gene expression, which may result in thrombotic complications. However, the mechanism regarding how the inflammatory cytokines negatively regulate ADAMTS13 (or Adamts13) synthesis remains to be determined.