title: Two-dimensional zymography differentiates gelatinase isoforms in stimulated microglial cells and in brain tissues of acute brain injuries.
abstract: Excessive activation of gelatinases (MMP-2/-9) is a key cause of detrimental outcomes in neurodegenerative diseases. A single-dimension zymography has been widely used to determine gelatinase expression and activity, but this method is inadequate in resolving complex enzyme isoforms, because gelatinase expression and activity could be modified at transcriptional and posttranslational levels. In this study, we investigated gelatinase isoforms under in vitro and in vivo conditions using two-dimensional (2D) gelatin zymography electrophoresis, a protocol allowing separation of proteins based on isoelectric points (pI) and molecular weights. We observed organomercuric chemical 4-aminophenylmercuric acetate-induced activation of MMP-2 isoforms with variant pI values in the conditioned medium of human fibrosarcoma HT1080 cells. Studies with murine BV-2 microglial cells indicated a series of proform MMP-9 spots separated by variant pI values due to stimulation with lipopolysaccharide (LPS). The MMP-9 pI values were shifted after treatment with alkaline phosphatase, suggesting presence of phosphorylated isoforms due to the proinflammatory stimulation. Similar MMP-9 isoforms with variant pI values in the same molecular weight were also found in mouse brains after ischemic and traumatic brain injuries. In contrast, there was no detectable pI differentiation of MMP-9 in the brains of chronic Zucker obese rats. These results demonstrated effective use of 2D zymography to separate modified MMP isoforms with variant pI values and to detect posttranslational modifications under different pathological conditions.
authors: Department of Pathology and Anatomical Sciences, University of Missouri School of Medicine, Columbia, Missouri, United States of America; Center for Translational Neuroscience, University of Missouri School of Medicine, Columbia, Missouri, United States of America; Interdisciplinary Neuroscience Program, University of Missouri, Columbia, Missouri, United States of America.
authors: Shanyan
authors: S
authors: Chen
authors: Department of Pathology and Anatomical Sciences, University of Missouri School of Medicine, Columbia, Missouri, United States of America; Center for Translational Neuroscience, University of Missouri School of Medicine, Columbia, Missouri, United States of America.
authors: Fanjun
authors: F
authors: Meng
authors: Department of Pathology and Anatomical Sciences, University of Missouri School of Medicine, Columbia, Missouri, United States of America; Center for Translational Neuroscience, University of Missouri School of Medicine, Columbia, Missouri, United States of America.
authors: Zhenzhou
authors: Z
authors: Chen
authors: Department of Pathology and Anatomical Sciences, University of Missouri School of Medicine, Columbia, Missouri, United States of America; Center for Translational Neuroscience, University of Missouri School of Medicine, Columbia, Missouri, United States of America; MS in Pathology program, University of Missouri Graduate School, Columbia, Missouri, United States of America.
authors: Brittany N
authors: BN
authors: Tomlinson
authors: Department of Pathology and Anatomical Sciences, University of Missouri School of Medicine, Columbia, Missouri, United States of America; Center for Translational Neuroscience, University of Missouri School of Medicine, Columbia, Missouri, United States of America.
authors: Jennifer M
authors: JM
authors: Wesley
authors: Department of Pathology and Anatomical Sciences, University of Missouri School of Medicine, Columbia, Missouri, United States of America; Center for Translational Neuroscience, University of Missouri School of Medicine, Columbia, Missouri, United States of America; Department of Biochemistry, University of Missouri School of Medicine, Columbia, Missouri, United States of America.
authors: Grace Y
authors: GY
authors: Sun
authors: Department of Internal Medicine Diabetes and Cardiovascular Center, University of Missouri School of Medicine, Columbia, Missouri, United States of America; Harry S. Truman Memorial Veterans' Hospital, Columbia, Missouri, United States of America.
authors: Adam T
authors: AT
authors: Whaley-Connell
authors: Department of Internal Medicine Diabetes and Cardiovascular Center, University of Missouri School of Medicine, Columbia, Missouri, United States of America; Harry S. Truman Memorial Veterans' Hospital, Columbia, Missouri, United States of America.
authors: James R
authors: JR
authors: Sowers
authors: Department of Pathology and Anatomical Sciences, University of Missouri School of Medicine, Columbia, Missouri, United States of America; Center for Translational Neuroscience, University of Missouri School of Medicine, Columbia, Missouri, United States of America; Harry S. Truman Memorial Veterans' Hospital, Columbia, Missouri, United States of America.
authors: Jiankun
authors: J
authors: Cui
authors: Department of Pathology and Anatomical Sciences, University of Missouri School of Medicine, Columbia, Missouri, United States of America; Center for Translational Neuroscience, University of Missouri School of Medicine, Columbia, Missouri, United States of America; Harry S. Truman Memorial Veterans' Hospital, Columbia, Missouri, United States of America.
authors: Zezong
authors: Z
authors: Gu
mesh_terms: D000818:Animals; D001921:Brain / Q000378:metabolism / Q000473:pathology; D001930:Brain Injuries / Q000175:diagnosis / Q000201:enzymology*; D002545:Brain Ischemia / Q000378:metabolism / Q000473:pathology; D002460:Cell Line; D017077:Culture Media, Conditioned / Q000378:metabolism; D004195:Disease Models, Animal; D018093:Gelatinases / Q000378:metabolism*; D006801:Humans; D007527:Isoenzymes; D008297:Male; D020780:Matrix Metalloproteinase 9 / Q000378:metabolism; D051379:Mice; D017628:Microglia / Q000378:metabolism*; D020078:Neurogenic Inflammation / Q000378:metabolism; D051381:Rats
chemical_list: D017077:Culture Media, Conditioned; D007527:Isoenzymes; D018093:Gelatinases; D020780:Matrix Metalloproteinase 9