Extracellular Cleavage of the Vascular Endothelial Growth Factor 189-Amino Acid Form by Urokinase Is Required for Its Mitogenic Effect

Abstract

Alternative splicing of vascular endothelial growth factor (VEGF) mRNA results in three distinct molecular forms of 121 or 165 (V165) amino acids that are released in the conditioned medium of cultured cells and one longer isoform of 189 amino acids (V189) that remains cell-associated. V189 has been expressed in wild type CHO-K1 cells and in glycosaminoglycan-deficient pgsA-745 Chinese hamster ovary (CHO) mutant cells. It could be released from CHO-K1 cell membranes by heparin or a synthetic peptide designed on the sequence encoded by exon 6 but was freely released from CHO mutant cells. In both cases, the immunoreactive V189 was mainly released as a 40-kDa cleaved form, provided that the serine protease urokinase, but not plasmin, was active. Recombinant V189 was purified from insect cells infected with a recombinant baculovirus as a nonmitogenic 50-kDa precursor that binds to the receptor Flt-1 but not to Flk-1. It could be matured by urokinase as a 38-kDa fragment able to bind to Flk-1 and to trigger cell proliferation. V165 and V189, however, could be cleaved by plasmin as 34-kDa fragments that exhibit a decreased mitogenic activity. These findings indicate that the carboxyl-terminal domain of V189 masks its binding domain to Flk-1.

Cell Release of Bioactive Fibroblast Growth Factor 2 by Exon 6-encoded Sequence of Vascular Endothelial Growth Factor

“…Cell culture trays were from Costar. Recombinant human V165 and V189 were synthesized in Sf9 cells infected with a recombinant baculovirus containing the V165 or V189 cDNA and purified by cation exchange and heparin affinity chromatography (18). Two molecular species were purified from V189-infected Sf9 cells: the 50-kDa native V189 and the 38-kDa matured V189.…”

“…The binding domains of VEGF to Flt-1 and KDR/Flk-1 have been identified as the sequences 63-67 and 82-86, respectively (17). We recently demonstrated that native V189 binds to Flt-1 but requires a cleavage by urokinase located within the exon 6-encoded sequence to bind to KDR/Flk-1 and to exert a mitogenic effect on vascular endothelial cells (18). However the mitogenic effect of the shorter form generated by plasmin cleavage between Arg 110 and Ala 111 was reduced 10 -100-fold as compared with that of urokinase-matured V189 (18) or V165 (19).…”

“…We recently demonstrated that native V189 binds to Flt-1 but requires a cleavage by urokinase located within the exon 6-encoded sequence to bind to KDR/Flk-1 and to exert a mitogenic effect on vascular endothelial cells (18). However the mitogenic effect of the shorter form generated by plasmin cleavage between Arg 110 and Ala 111 was reduced 10 -100-fold as compared with that of urokinase-matured V189 (18) or V165 (19). Because these results suggested that exon 6 acted as a mitogenic enhancer, we then compared the physiological roles exerted by V165, V189, and a synthetic peptide designed on the sequence encoded by exon 6 (Ex6P).…”

Adenovirus-mediated delivery of a uPA/uPAR antagonist suppresses angiogenesis-dependent tumor growth and dissemination in mice

“…The inhibition of angiogenesis that occurred in AdmATFtreated mice might also have resulted from a concurrent action of ATF in hampering the bioavailability of angiogenic factors. [21][22][23][24][25] Because the dialogue between cancer and endothelial cells is of a 'symbiotic' nature, 2 ATF therapy may be particularly relevant because of its potential to target both the tumoral and endothelial compartments.…”

“…Finally, uPA and plasmin activate morphogenic or angiogenic factors such as VEGF, HGF, FGFs and TGF␤, further supporting cell-associated uPA in controlling cell migration and angiogenesis. [21][22][23][24][25] The relevance of targeting uPA and uPAR for cancer therapy is supported by epidemiological studies in which high level expression of uPA and uPAR are correlated with a poor prognosis in a number of malignancies, including ovarian, breast, gastric, brain and lung cancer. [26][27][28][29][30][31] Clinical observations also indicate the presence of enhanced uPA activity at the invasive edge of the tumors.…”