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.

Resveratrol Suppresses Matrix Metalloproteinase-2 Activation Induced by Lipopolysaccharide in Mouse Osteoblasts via Interactions with AMP-Activated Protein Kinase and Suppressor of Cytokine Signaling 1

“…The gelatinolytic activities of MC3T3-E1 cell supernatants were determined via gelatin zymography assay (Xinfan Technology, Shanghai, China) according to the manufacturer’s instructions. HT1080 (human fibrosarcoma) cell media conditioned for 24 h was used as a positive control, as it is enriched with gelatinases [ 47 ]. Supernatants were collected from culcured cells and examined with 8% polyactylamide gels containing 0.1% gelatin.…”

Substrate-zymography: a still worthwhile method for gelatinases analysis in biological samples

“…For example, Chen et al, by using 2D gelatin zymography were able to detect organomercuric chemical 4-aminophenylmercuricacetate-induced activation of MMP-2 isoforms with variant pI values in the conditioned medium of human fibrosarcoma cells and also several MMP-9 pro-forms, with different pI values, in murine LPS-stimulated BV-2 cells [40].…”

Gelatinase activity imaged by activatable cell-penetrating peptides in cell-based and in vivo models of stroke

“…Nevertheless, we observed that MMP-9 was the predominant gelatinase under our experimental conditions. 8,20,21,32 It should be noted, however, that our present ACPPs do not discriminate between MMP-2 and MMP-9, although we are actively developing more selective ACPPs.…”