Differential inhibition of endothelial cell proliferation and migration by urokinase subdomains: amino-terminal fragment and kringle domain

Kwang Sei Kim,Young Ae Joe,Soo-Ik Chang,Soo Il Chung,Joo-Young Shin,Yong-Kil Hong,Yoon Lee

doi:10.1038/emm.2003.76

Kwang Sei Kim, Young Ae Joe + Show 5 more

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https://doi.org/10.1038/emm.2003.76

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Abstract

The serine protease urokinase-type plasminogen activator (uPA) is implicated in pericellular proteolysis in a variety of physiological and pathological processes including angiogenesis and tumor metastasis. The kringle domain of uPA (UK1) has proven to be an anti-angiogenic molecule with unknown mechanism and amino terminal fragment of uPA (u-ATF) with additional growth factor-like domain can be used for blocking interaction of uPA and uPA receptor. Here, we compared anti-angiogenic activities of these two molecules in vitro and in vivo. The recombinant u-ATF from E. coli and refolded in vitro was found to bind to uPAR with high affinity, whereas E. coli-derived UK1 showed no binding by Biacore analysis. In contrast to UK1 having potent inhibitory effect, u-ATF exhibited low inhibitory effect on bovine capillary endothelial cell growth (ED(50)>320 nM). Furthermore, u-ATF inhibition of VEGF-induced migration of human umbilical vein endothelial cell was far less sensitive (IC(50) = 600 nM) than those observed with UK1, and angiogenesis inhibition was marginal in chorioallantoic membrane. These results suggest that kringle domain alone is sufficient for potent anti- angiogenic activity and additional growth factor-like domain diverts this molecule in undergoing different mechanism such as inhibition of uPA/uPAR interaction rather than undergoing distinct anti- angiogenic mechanism driven by kringle domain.

Highlights

Urokinase-type plasmingen activator andits receptor areimportant components of cell surface proteolysis used bytumorcells andcapillary endothelial cellsfor basement membraneinvasion and implicatedin the progression, metastasis, and angiogenesisofnumeroustumors(deVriesetal,1996; Andreasen et al, 1997; Reuninget al., 1998). uPA is secreted from cells as a single-chain proenzyme thatiscleaved by plasminintoitstwo-chain active form (Petersenet al., 1988)
Boththe single and two-chain forms can bind to u-PA receptor
Single-chainuPA(scu-PA)isa multidomainprotein composed of a carboxyl-terminal protease domain, termedlow molecular weightscu-PA(LMW-scu-PA) andanamino-terminalfragment(u-ATF), whichcan befurthersubdividedintoagrowthfactor-likedomain and a kringle domain (Figure 1A).Thegeneralfeatureofkringlestructureiscomposed of 78-80 amino acidsinter-connected by a triple disulfide-linkedloop

Summary

Introduction

Urokinase-type plasmingen activator (uPA) andits receptor areimportant components of cell surface proteolysis used bytumorcells andcapillary endothelial cellsfor basement membraneinvasion and implicatedin the progression, metastasis, and angiogenesisofnumeroustumors(deVriesetal,,1996; Andreasen et al, 1997; Reuninget al., 1998). uPA is secreted from cells as a single-chain proenzyme thatiscleaved by plasminintoitstwo-chain active form (Petersenet al., 1988). Urokinase-type plasmingen activator (uPA) andits receptor areimportant components of cell surface proteolysis used bytumorcells andcapillary endothelial cellsfor basement membraneinvasion and implicatedin the progression, metastasis, and angiogenesisofnumeroustumors(deVriesetal,,1996; Andreasen et al, 1997; Reuninget al., 1998). UPA is secreted from cells as a single-chain proenzyme thatiscleaved by plasminintoitstwo-chain active form (Petersenet al., 1988). Boththe single and two-chain forms can bind to u-PA receptor (uPAR). The two chain form consists of A (aa 158) and B (aa 159-411) polypeptide chains linked by disulfide bonds. The high level expression of uPA and uPA receptor (uPAR)arecorrelatedwithapoorprognosisinanumber of malignancies,including breast, gastric, brain andlungcancer(Sappinoetal.,1987;Yamamotoet al., 1994b; Foekens et al, 1995; Park et al, 1997) and is often localized to the leading, invasive edge of a tumor(Estreicheretal.,1990;Yamamotoetal.,1994a). Blockage of the interaction of uPA and its receptor hasresultedininhibition ofthe migration and spreadingof malignanttumorcells(Crowleyetal.,1993; Mohanam et al, 2002)

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