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Zymographic techniques for the analysis of matrix metalloproteinases and their inhibitors
 
Patricia A.M. Snoek-van Beurden Johannes W. Von den Hoff
University Medical Centre Nijmegen, Nijmegen, The Netherlands
BioTechniques, Vol. 38, No. 1, January 2005, pp. 73–83
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Abstract

The balance between matrix metalloproteinases (MMPs) and their inhibitors, the tissue inhibitors of metalloproteinases (TIMPs), is largely responsible for the remodeling of tissues. Deregulation of this balance is a characteristic of extensive tissue degradation in certain degenerative diseases. To analyze the role of MMPs and TIMPs in tissue remodeling under normal and pathological conditions, it is important to have reliable detection methods. This review will focus on zymographical techniques for the analysis of MMPs and TIMPs. MMPs can be analyzed with several zymographical techniques, but substrate zymography is the most commonly used. This technique identifies MMPs by the degradation of their preferential substrate and by their molecular weight. Several substrates that can be used for zymography are described. Reverse zymography, which detects TIMPs by their ability to inhibit MMPs, is also discussed. Finally, in situ zymography is described, which is used to localize MMPs in tissue sections. Common problems encountered during sample preparation, zymography itself, and the data analysis are discussed. Hints are given to improve the sensitivity and accuracy of zymographical methods. In conclusion, zymography is a valuable tool for research purposes and for the development of new diagnostic techniques and therapies for pathological conditions such as rheumatoid and osteoarthritis, and tumor progression.

Introduction

Matrix metalloproteinases (MMPs) are a family of calcium-dependent, zinc-containing endopeptidases that are structurally and functionally related (1). They are secreted in an inactive (latent) form, which is called a zymogen or a pro-MMP. These latent MMPs require an activation step before they are able to cleave extracellular matrix (ECM) components (1). The activity of MMPs is regulated by several types of inhibitors, of which the tissue inhibitors of metalloproteinases (TIMPs) are the most important (2). The TIMPs are also secreted proteins, but they may be located at the cell surface in association with membrane-bound MMPs (3). The balance between MMPs and TIMPs is largely responsible for the control of degradation of ECM proteins (4). MMPs are involved in the remodeling of tissues during embryonic development, cell migration, wound healing, and tooth development (5,6,7,8). However, a deregulation of the balance between MMPs and TIMPs is a characteristic of diverse pathological conditions, such as rheumatoid and osteoarthritis, cancer progression, and acute and chronic cardiovascular diseases (3,9,10). To analyze the role of MMPs and TIMPs in tissue remodeling under normal and pathological conditions, it is important to have reliable detection methods. This review will briefly describe all known MMPs, their activation, and their role in tissue remodeling and pathology. It will focus on zymographical techniques for the analysis of MMPs and TIMPs.

The MMP Family

The family of human MMPs consists of 23 different forms that are divided into six groups (11,12,13,14). In order to classify the MMPs, knowledge of their characteristics is essential. It has been shown that each MMP consists of a specific domain sequence with several domain motifs. This sequence includes the signal peptide, the propeptide domain, the catalytic domain, and the C-terminal hemopexin-like domain, which are present in almost all MMPs (15). However, several MMPs have additional domains such as a transmembrane or a cytoplasmic domain (15,16). The organization of the MMP domains, together with their substrate specificity and sequence similarity, define the MMP classification. Six groups can be distinguished ((Table 1); subgroups 1–6). (1.) The collagenase group includes MMP-1, MMP-8, and MMP-13. These are generally able to cleave the interstitial collagens I, II, and III. Collagenases are also able to digest certain other ECM and non-ECM proteins (8,12,17). (2.) The gelatinase group, which consists of MMP-2 and MMP-9, mainly digests gelatin, the denatured form of collagen (8,12). (3.) The stromelysins, MMP-3 and MMP-10, digest ECM components such as collagen IV and fibronectin. MMP-11 is also called stromelysin-3, but its sequence and substrate specificity are different from that of MMP-3 and MMP-10. Therefore, MMP-11 is usually placed in the heterogeneous subgroup (see subgroup 6) (8,12,17). (4.) The matrilysins, MMP-7 and MMP-26, which are categorized differently among the MMP subgroups by several authors (4,12,18,19). Both matrilysins digest several ECM components, such as fibronectin and gelatin (20). They lack the C-terminal hemopexin-like domain present in all other MMPs and are therefore also called the minimal-domain MMPs (16,21). (5.) The membrane-type matrix metalloproteinases (MT-MMP), of which six forms are known, can digest a number of ECM proteins such as gelatin, fibronectin, and laminin (22). Moreover, most MT-MMPs can activate pro-MMP-2 (8,12). (6.) The remaining MMPs are gathered in a more heterogeneous subgroup because of their different substrate specificity, amino acid sequence, or domain organization. This group includes MMP-12, MMP-19, MMP-20, MMP-21, MMP-23, MMP-27, and MMP-28 (8,12), which cleave substrates such as elastin and aggrecan (23,24). The classification presented here is a general one, and several other classifications are used (4,8,12,17). The main difference between these classifications is the assignment of the matrilysins, which are sometimes designated separately or to the stromelysins or the heterogeneous subgroup (4,8,12,17).

Table 1. Members of the MMP Family


MMPs are categorized according to the organization of their peptide domains, their substrate specificity, and their sequence similarity (8,2,17,22,23,24,85,86,87). MMP, matrix metalloproteinase; MT-MMP, membrane-type matrix metallopro-teinase.

In healthy tissues, some MMPs such as MMP-7, MMP-19, MMP-24, MMP-25, and MMP-26 are expressed at low levels (25). Many of the other MMPs, such as MMP-1, MMP-3, MMP-9, MMP-10, MMP-11, and MMP-13 are absent or only marginally expressed in normal, healthy, resting tissues. During repair or remodeling processes and in diseased or inflamed tissues, MMP expression is often increased (25,26).

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