Abstract 5687: Identification of HGF as a Novel Vascular ADAM Metalloprotease Substrate by Phage Display

Activation of a metalloprotease, ADAM17, and subsequent EGF receptor transactivation via proteolytic cleavage of its pro-ligand has been implicated in vascular remodeling induced by angiotensin II (AngII). The known substrates of ADAM17 include proHB-EGF and proTNF-alpha. However, whether other unidentified ADAM17 substrates are involved in AngII-induced vascular remodeling remains unanswered. To search for such substrates, we made a rat aortic cDNA phage library that has a hexa-histidine tag. After Ni-NTA agarose binding, the library phage was incubated with recombinant ADAM17 and only the cleaved phages were rescued and amplified (bio-panning). Upon repeating the panning 3 times, we obtained about 4000 phage colonies which potentially displayed ADAM17 substrates. From 128 colonies, we identified 71 candidate proteins in 88 readable sequences. Among these proteins, eleven proteins were known as ADAM17 substrates including (amphiregulin, Notch etc.) while six proteins were other protease substrates indicating strong reliability and provability of the technique. Among the novel substrate candidates, we focused on hepatocyte growth factor (HGF), a multifunctional growth factor with a proteolytic mechanism of activation. Upon transfection with a shedding assay vector encoding proHGF with its N terminus fused with ALP, the ALP activity in the condition medium was significantly reduced in ADAM17-null MEF cells compared to the control MEF cells. Moreover, the reduction was rescued with adenovirus vector encoding ADAM17. In rat VSMCs transfected with the assay vector, AngII markedly enhanced the ALP activity in the conditioned medium that was completely blocked with a MMP/ADAM inhibitor, BiPS. Also, AngII stimulated secretion of HGF-alpha in VSMCs. Recombinant HGF markedly stimulated ERK1/2 activity via the Met receptor in VSMCs. It also stimulated proliferation of VSMCs. In conclusion, we have established a novel phage display system for identification of a protease substrate with high probability. HGF was identified as a novel ADAM17 substrate potentially involved in vascular remodeling induced by AngII. This research has received full or partial funding support from the American Heart Association, National Center.