Evaluation of matrix metalloproteinase-2 (mmp-2) activity with film in situ zymography for improved cytological diagnosis of Breast tumors

Fujiwara, Akira; Shibata, Eri; Terashima, Hitomi; Shishido, Akemi; Nishiki, Junko; Yoshida, Kanji; Miyauchi, Keisuke; Madachi, Ayako; Matsuura, Nariaki

doi:10.2325/jbcs.13.272

Cited by 8 publications

(10 citation statements)

References 18 publications

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“…In particular, we showed that silencing MMP2 expression all but eliminated gelatinase activity in AF cells and profoundly compromised their ability to remodel collagen matrices. We also demonstrated that, similar to tumor invadopodia [20-23], MMP-2 preferentially functions in a localized region surrounding AF cells, rather than being secreted and acting at a distance. Contrary to our expectations, the impaired ability of AF cells to degrade and remodel their local microstructure did not translate to the macroscale characterization of the collagen-gel mechanical properties.…”

Section: Discussionsupporting

confidence: 52%

MMP-2 mediates local degradation and remodeling of collagen by annulus fibrosus cells of the intervertebral disc

et al. 2013

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IntroductionDegeneration of the intervertebral disc (IVD) is characterized by marked degradation and restructuring of the annulus fibrosus (AF). Although several matrix metalloproteinases (MMPs) have been found to be more prevalent in degenerate discs, their coordination and function within the context of the disease process are still not well understood. In this study, we sought to determine whether MMP-2 is associated with degenerative changes in the AF and to identify the manner by which AF cells use MMP-2.MethodsTwo established animal models of disc degeneration, static compression and transannular needle puncture of rodent caudal discs, were examined for MMP-2 immunopositivity. With lentiviral transduction of an shRNA expression cassette, we screened and identified an effective shRNA sequence for generating stable RNA interference to silence MMP-2 expression in primary rat AF cells. Gelatin films were used to compare gelatinase activity and spatial patterns of degradation between transduced cells, and both noninfected and nonsense shRNA controls. The functional significance of MMP-2 was determined by assessing the ability for cells to remodel collagen gels.ResultsBoth static compression and 18-g annular puncture of rodent caudal discs stimulated an increase in MMP-2 activity with concurrent lamellar disorganization in the AF, whereas 22-g and 26-g needle injuries did not. To investigate the functional role of MMP-2, we established lentivirus-mediated RNAi to induce stable knockdown of transcript levels by as much as 88%, and protein levels by as much as 95% over a 10-day period. Culturing transduced cells on gelatin films confirmed that MMP-2 is the primary functional gelatinase in AF cells, and that MMP-2 is used locally in regions immediately around AF cells. In collagen gels, transduced cells demonstrated an inability to remodel collagen matrices.ConclusionsOur study indicates that increases in MMP-2 observed in human degenerate discs are mirrored in experimentally induced degenerative changes in rodent animal models. AF cells appear to use MMP-2 in a very directed fashion for local matrix degradation and collagen remodeling. This suggests that MMP-2 may have a functionally significant role in the etiology of degenerative disc disease and could be a potential therapeutic target.

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Section: Discussionsupporting

confidence: 52%

MMP-2 mediates local degradation and remodeling of collagen by annulus fibrosus cells of the intervertebral disc

et al. 2013

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“…To detect the localization of gelatinolytic MMP activity in skeletal muscle tissues, we performed film in situ zymography (FIZ) as previously described [42-48]. 6 μm-thick cryosections from frozen samples were placed on a polyethylene terephthalate base film coated with 7 μm thickness cross-linked gelatin with (FIZ-GI, Wako Pure Chemical, Osaka, Japan) or without (FIZ-GN, Wako Pure Chemical) 1,10-phenanthroline (inhibitor of MMPs, but not of other proteinases such as trypsin).…”

Section: Methodsmentioning

confidence: 99%

Activation and localization of matrix metalloproteinase-2 and -9 in the skeletal muscle of the muscular dystrophy dog (CXMDJ)

Fukushima

Nakamura

Ueda

et al. 2007

BMC Musculoskelet Disord

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Background: Matrix metalloproteinases (MMPs) are key regulatory molecules in the formation, remodeling and degradation of all extracellular matrix (ECM) components in both physiological and pathological processes in various tissues. The aim of this study was to examine the involvement of gelatinase MMP family members, MMP-2 and MMP-9, in dystrophin-deficient skeletal muscle. Towards this aim, we made use of the canine X-linked muscular dystrophy in Japan (CXMD J ) model, a suitable animal model for Duchenne muscular dystrophy.

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“…The human fibrosarcoma cell line HT1080 is known to secrete relatively high amounts of proMMP-2 [25]. Treatment of the cells with (GPO) 10 almost doubled the invasive capacity of the tumour cells, indicating an enhanced activation of endogenous proMMP-2 by (GPO) 10 in the supernatant.…”

Section: Discussionmentioning

confidence: 99%

The synthetic hydroxyproline-containing collagen analogue (Gly-Pro-Hyp)₁₀promotes enzymatic activity of matrixmetalloproteinase-2in vitro

Freise

Ruehl²,

Erben³

et al. 2012

European Journal of Microbiology and Immunology

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Diseases such as liver fibrosis and intestinal inflammation are characterized by accumulated components of the extracellular matrix (ECM). Given that fibrillar collagen structures were shown to serve as storage site for inactive proforms of matrixmetalloproteinases (MMPs), modulating this MMP-collagen interaction might offer a rational interventional (therapeutic) approach to enhance degradation of accumulated ECM. The synthetic triple helical collagen analogue (Gly-Pro-Hyp) 10 -(GPO) 10 -was shown to trigger release and enzymatic activation of collagen sequestered proMMP-2. In the presented study, we, for the first time, investigated how MMP-(GPO) 10 interaction impacts cellular responses in vitro. We found that recombinant proMMP-2 induced proliferation of hepatic stellate cells (HSC), which was enhanced after addition of (GPO) 10 reaching comparable levels following incubation with fully activated MMP-2. In addition, (GPO) 10 induced HSC migration similar to the platelet-derived growth factor subunit-B. Further, the MMP-2-dependent invasion of HT1080 fibrosarcoma cells through an ECM membrane was enhanced after addition of (GPO) 10 . Since cellular proliferation and migration concomitant with matrix degradation is stimulated, we conclude that the MMP-(GPO) 10 interaction also functions in a physiological environment. Thus, a potential therapeutic effect of (GPO) 10 should be further tested in animal models for MMP-associated diseases such as colitis or fibrosis.

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Evaluation of matrix metalloproteinase-2 (mmp-2) activity with film in situ zymography for improved cytological diagnosis of Breast tumors

Abstract: The present study demonstrated that measurement of gelatinolytic activity by FIZ analysis of aspiration cytology samples may be useful for improving the cytological diagnosis of breast tumors.

Cited by 8 publications

References 18 publications

MMP-2 mediates local degradation and remodeling of collagen by annulus fibrosus cells of the intervertebral disc

MMP-2 mediates local degradation and remodeling of collagen by annulus fibrosus cells of the intervertebral disc

Activation and localization of matrix metalloproteinase-2 and -9 in the skeletal muscle of the muscular dystrophy dog (CXMDJ)

The synthetic hydroxyproline-containing collagen analogue (Gly-Pro-Hyp)₁₀promotes enzymatic activity of matrixmetalloproteinase-2in vitro

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Evaluation of matrix metalloproteinase-2 (mmp-2) activity with film in situ zymography for improved cytological diagnosis of Breast tumors

Abstract: The present study demonstrated that measurement of gelatinolytic activity by FIZ analysis of aspiration cytology samples may be useful for improving the cytological diagnosis of breast tumors.

Cited by 8 publications

References 18 publications

MMP-2 mediates local degradation and remodeling of collagen by annulus fibrosus cells of the intervertebral disc

MMP-2 mediates local degradation and remodeling of collagen by annulus fibrosus cells of the intervertebral disc

Activation and localization of matrix metalloproteinase-2 and -9 in the skeletal muscle of the muscular dystrophy dog (CXMDJ)

The synthetic hydroxyproline-containing collagen analogue (Gly-Pro-Hyp)10promotes enzymatic activity of matrixmetalloproteinase-2in vitro

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The synthetic hydroxyproline-containing collagen analogue (Gly-Pro-Hyp)₁₀promotes enzymatic activity of matrixmetalloproteinase-2in vitro