Abstract 5898: ADAMTS-1 is an Endothelial Cell-specific Hypoxia-inducible Gene
Background: We have previously reported that ADAMTS1 (A disintegrin and metalloprotease with thrombospondin motifs), a member of the metalloproteinase family, is induced in the infarcted heart in rat model. In this study, we examined whether hypoxia induces ADAMTS1 gene expression, and investigated its molecular mechanism.
Methods: Endothelial cells (Human umbilical vein endothelial cells (HUVEC), human microvascular endothelial cells (HMEC) and human pulmonary artery endothelial cells (HPAEC)) and non-endothelial cells (human skin fibroblasts, human retinal pigment epithelial cells (ARPE)) were cultured under normoxia (20% O2) or hypoxia (1% O2)(control, 1hr, 3hr, 6hr and 24hr, respectively). We examined ADAMTS1 mRNA expression level by quantitative real-time RT-PCR and Western blot analysis. We studied the impact of RNA synthesis inhibitor (actinomycin D) and protein synthesis inhibitor (cycloheximide) on ADAMTS1 transcription and then LY294002 (phosphatidylinositol 3-kinase (PI3K) inhibitor) and SB203580 (p38-MAPK inhibitor) were used for signaling mechanism regulating ADAMTS1 induction. Finally, we performed ChIP assay to determine the binding of Hypoxia-inducible factor-1 (HIF-1) to the ADAMTS1 promoter.
Results: ADAMTS1 mRNA expression increased approximately 4-fold by hypoxia (p<0.005). CoCl2, chemically mimicked hypoxia, also increased ADAMTS1 mRNA level in HUVEC. Hypoxic induction of ADAMTS1 was observed in endothelial cells (HUVEC, HMEC and HPAEC) and ADAMTS1 induction by hypoxia was not observed in non-endothelial cells. Actinomycin D blocked the increase of ADAMTS-1 mRNA expression by hypoxia, while cycloheximide did not affect, indicating that this increase is due to the stabilized transcriptional factor by hypoxia. LY294002 and SB203580 dose-dependently inhibited the increase of ADAMTS1 mRNA expression in hypoxia, while only LY294002 ameliorated VEGF mRNA induction. ChIP assay demonstrated that there are at least two HIF-1 binding sites in the ADAMTS1 promoter region under hypoxia.
Conclusion: Our data indicated that ADAMTS1 is a unique endothelial cell-specific hypoxia-inducible gene and its molecular mechanism is distinctly different from that of VEGF in hypoxia.