In vivo localization of gelatinases (MMP-2 and -9) by in situ zymography with a selective gelatinase inhibitor.
In situ zymography provides a tool to localize proteolytic activity in tissues in vivo. However, it has been difficult to discriminate between the proteases responsible for the detected activity. We used a selective tissue-permeable gelatinase inhibitor, the CTTHWGFTLC-peptide (CTT) in inflamed human gingiva. The CTT-peptide was evidenced to home, target to, and selectively inhibit the areas of gelatinolytic activity in inflamed human gingiva expressing MMP-2 and -9. Gelatinolytic activity, MMP-9 immunoreactivity, and mRNA expression as well as CD-45-positive inflammatory cells colocalized well in the inflamed human gingival connective tissue. Gelatinolytic activity corresponding to MMP-2 colocalized with laminin-5 gamma2-chain immunoreactivity and was detected in the close vicinity of the sulcular basement membrane region. Furthermore, the CTT-peptide inhibited beta-caseinolysis by human MMP-2 and MMP-9 as well as laminin-5 gamma2-chain degradation by MMP-2 in vitro. Thus, the CTT-peptide may prove to be a useful tool (i) to discriminate between gelatinolytic proteases detected by in situ zymography and (ii) to preventMMP-2-dependent induction of epithelial cell migration and gelatinase-dependent tissue destruction in inflammatory and malignant diseases.
Pirilä
E
E
Department of Clinical Veterinary Sciences, University of Helsinki, Finland. pirila@hammas.helsinki.fi
Maisi
P
P
Salo
T
T
Koivunen
E
E
Sorsa
T
T
D015815:Cell Adhesion Molecules / Q000737:chemistry; D004305:Dose-Response Relationship, Drug; D004791:Enzyme Inhibitors / Q000494:pharmacology*; D004847:Epithelial Cells / Q000201:enzymology; D005881:Gingiva / Q000201:enzymology; D006801:Humans; D007150:Immunohistochemistry; D017403:In Situ Hybridization / Q000379:methods; D017493:Leukocyte Common Antigens / Q000096:biosynthesis; D020778:Matrix Metalloproteinase 2 / Q000096:biosynthesis*; D020780:Matrix Metalloproteinase 9 / Q000096:biosynthesis*; D010456:Peptides, Cyclic / Q000494:pharmacology*; D011485:Protein Binding; D011994:Recombinant Proteins / Q000378:metabolism; D014665:Vasodilator Agents / Q000494:pharmacology; D000097794:Kalinin
D015815:Cell Adhesion Molecules; D004791:Enzyme Inhibitors; D017493:Leukocyte Common Antigens; D020778:Matrix Metalloproteinase 2; D020780:Matrix Metalloproteinase 9; D010456:Peptides, Cyclic; D011994:Recombinant Proteins; D014665:Vasodilator Agents; D000097794:Kalinin; C415426:CTTHWGFTLC peptide