Apparent suppression of MMP-9 activity by GD1a as determined by gelatin zymography

• Published in: Biochemical and biophysical research communications 2006-10, Vol.349 (1), p.426-431
• Main Authors: Hu, Dan, Tan, Xuan, Sato, Toshinori, Yamagata, Sadako, Yamagata, Tatsuya
• Format: Article
• Language: English
• Subjects: Animals; Cattle; Cell Line, Tumor; Culture Media, Conditioned - metabolism; Culture Media, Conditioned - pharmacology; Ganglioside; Gangliosides - metabolism; GD1a; Gelatin - chemistry; Gene Expression Regulation, Enzymologic; Gene Expression Regulation, Neoplastic; Matrix Metalloproteinase 2 - metabolism; Matrix Metalloproteinase 9 - metabolism; Matrix Metalloproteinase Inhibitors; Metastasis; Mice; Mice, Inbred BALB C; MMP-2; MMP-9; p38 Mitogen-Activated Protein Kinases - metabolism; Time Factors; Zymography
• ISSN: 0006-291X; 1090-2104
• DOI: 10.1016/j.bbrc.2006.08.062

Gelatin zymography is widely used to detect and evaluate matrix metalloproteinase-9 (MMP-9) activity. MMP-9 transcription was previously shown to be negatively regulated by ganglioside GD1a [D. Hu, Z. Man, T. Xuan, P. Wang, T. Takaku, S. Hyuga, X.S. Yao, T. Sato, S. Yamagata, T. Yamagata, Ganglioside GD1a regulation of matrix metalloproteinase-9 (MMP-9) expression in mouse FBJ cell Lines: GD1a suppression of MMP-9 expression stimulated by PI3K-Akt and p38 though not by the Erk signaling pathway, 2006, submitted for publication.]. Zymography of MMP-9 of FBJ-M5 cells preincubated with GD1a indicated a greater decrease in activity than expected from mRNA suppression. Incubation of conditioned medium containing MMP-9 with GD1a caused MMP-9 activity to decrease. Examination was thus made to confirm that MMP-9 activity is actually suppressed and/or MMP-9 protein undergoes degradation by GD1a. GD1a was found to have no effect on MMP-9 activity and Western blots indicated GD1a not to diminish MMP-9 during electrophoresis under reducing conditions. GD1a appeared to mediate the binding of a portion of MMP-9 with certain molecules, with consequently greater molecular mass on the gel, to cause decrease in the activity of MMP-9 at the site where it would normally appear. Caution should be used in doing gelatin zymography since molecules other than GD1a may similarly work, causing decrease in MMP-9 activity in zymography.