Biochemical and Biophysical Research Communications
Urokinase directly activates matrix metalloproteinases-9: A potential role in glioblastoma invasion
Section snippets
Materials and methods
Biochemical cleavage assay of MMP-9 by uPA. Purified recombinant uPA (0.02 μM, a gift from Drs. Jack Henkin and Andrew Mazar of Abbott Laboratories, Abbott Park, IL) was incubated with purified pro-MMP-9 (pMMP-9, 0.2 μM), purified MMP-9/TIMP-1 (0.2 μM), and purified MMP-9–lipocalin complex (0.2 μM), MMP-9 monomer (mMMP-9, 0.2 μM) in the presence or absence of purified recombinant N-terminal domain of TIMP-1 (N-TIMP-1, 0.2 μM) [12] in glycine buffer (0.1 M glycine, pH 8.0) at 37 °C for 24 h. To compare
uPA directly cleaves latent MMP-9
Our biochemical assays revealed that purified 92 kDa pro-MMP-9 was cleaved directly by uPA, generating two new bands (86 and 80 kDa) showed gelatinolytic activity (Fig. 1). A specific uPA inhibitor B428 (7.5 μM) completely blocked the cleavage of MMP-9 by uPA (Fig. 1). Furthermore, uPA-mediated cleavage of pro-MMP-9 was time-dependent. These results suggest that pro-MMP-9 can be cleaved by uPA directly and generated two new bands with gelatinolytic activity in vitro.
To identify the cleavage sites
Discussion
Our current results demonstrate that uPA directly cleaved latent MMP-9 to generate two new bands (86 kDa and 80 kDa) that showed gelatinolytic activity (Fig. 1B). The 86 kDa band was the product of MMP-9 cleaved by uPA at the C-terminus, while the 80 kDa band was cleaved at both N-terminus (at Lys65-Ser66 site) and C-terminus. Although we do not know exactly the cleavage sites of 86 kDa and 80 kDa bands at C-terminus from the mass spectrum analysis, the detected C-terminus fragments of 86 kDa and 80
Acknowledgments
We gratefully acknowledge Dr. Galina Kuznetsov at Eisai Research Institute for providing the uPA inhibitor (B428). This work was supported by the Farrow Fellowship and the UVA Cancer Center Support Grant, P30 CA44570 (to Y.G.Z.), NIH Grants NS35122 and CA90851 (to I.M.H.) and AR40994 (to K.B.).
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