Anti-angiogenic activity of the recombinant kringle domain of urokinase and its specific entry into endothelial cells.
Urokinase plasminogen activator (uPA) belongs to a family of proteins that contains kringle domain and plays an important role in inflammation, tissue remodeling, angiogenesis, and tumor metastasis by pericellular plasminogen activation. Kringle domains of plasminogen have been shown to demonstrate anti-angiogenic and anti-tumor activities. Here, we report our investigation of the kringle domain of uPA for anti-angiogenic activity and a possible cellular mechanism of action. The recombinant kringle domain of uPA (Asp(45)-Lys(135)) (UK1) inhibited endothelial cell proliferation stimulated by basic fibroblast growth factor, vascular endothelial growth factor (VEGF), or epidermal growth factor. It also inhibited migration of endothelial cells induced by VEGF or uPA, and in vivo angiogenesis on the chick chorioallantoic membrane. It did not block plasminogen activation by activated uPA in clot lysis and chromogenic substrate assays. Neither binding of UK1 to immobilized uPA receptor nor competitive inhibition of uPA binding were confirmed by real-time interaction analysis. However, internalization of UK1 followed by translocation from cytosol to nucleus was determined to be specific to endothelial cells. It also elicited a transient increase of Ca(2+) flux of more than 2-fold within 2 min of exposure in an endothelial cell-specific manner. These results suggest that the kringle domain of uPA exhibits anti-angiogenic activity and that its anti-angiogenic activity may occur through a different mechanism from inhibition of uPA-uPA receptor interaction or uPA proteolytic activity and may be associated with endothelial-cell specific internalization not mediated by the uPA receptor.
Kim
Kwang Sei
KS
Cancer Research Institute, Catholic Research Institutes of Medical Sciences, The Catholic University of Korea, Seoul 137-701, Korea.
Hong
Yong-Kil
YK
Joe
Young Ae
YA
Lee
Yoon
Y
Shin
Joo-Young
JY
Park
Hyo-Eun
HE
Lee
Il-Ha
IH
Lee
Soo-Young
SY
Kang
Dong-Ku
DK
Chang
Soo-Ik
SI
Chung
Soo Il
SI
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D016466:CHO Cells; D002118:Calcium / Q000378:metabolism; D006224:Cricetinae; D017931:DNA Primers; D016228:Endothelial Growth Factors / Q000037:antagonists & inhibitors; D004730:Endothelium, Vascular / Q000166:cytology / Q000378:metabolism*; D006801:Humans; D036341:Intercellular Signaling Peptides and Proteins; D018082:Kringles; D008222:Lymphokines / Q000037:antagonists & inhibitors; D008969:Molecular Sequence Data; D010958:Plasminogen / Q000378:metabolism; D011994:Recombinant Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D014568:Urokinase-Type Plasminogen Activator / Q000737:chemistry / Q000302:isolation & purification / Q000502:physiology*; D042461:Vascular Endothelial Growth Factor A; D042442:Vascular Endothelial Growth Factors
D017931:DNA Primers; D016228:Endothelial Growth Factors; D036341:Intercellular Signaling Peptides and Proteins; D008222:Lymphokines; D011994:Recombinant Proteins; D042461:Vascular Endothelial Growth Factor A; D042442:Vascular Endothelial Growth Factors; D010958:Plasminogen; D014568:Urokinase-Type Plasminogen Activator; D002118:Calcium