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.     

Matrix metalloproteinase 2 (gelatinase A) is related to migration of keratinocytes.

The role of matrix metalloproteinases (MMPs) in cell migration was studied by measuring cell growth, migration, and production of MMP-2 and -9 in oral mucosal and skin keratinocytes cultured in the presence of synthetic MMP inhibitors. MMP-2 was the major gelatinolytic MMP produced by these cells while MMP-9 was produced at a low basal level. Inhibitor effects on MMP-9 production were therefore studied in keratinocytes stimulated by tumor necrosis factor alpha (TNFalpha). Tetracycline analogues at concentrations that inhibited the production of MMP-2 but not MMP-9 were able to drastically inhibit migration of both mucosal and skin keratinocytes. Tetracycline analogues also inhibited keratinocyte growth, an effect not found for the other inhibitors tested. Heterocyclic carbonate-derived compounds (LWs) that inhibited MMP-9 but not MMP-2 production had no effect on cell migration. Batimastat, a potent MMP inhibitor, did not have any effect on MMP production or cell growth but did inhibit keratinocyte migration. Tumor growth factor beta (TGFbeta) increased keratinocyte migration as well as both cell-associated and secreted MMP-2 production in wounded cell cultures. The secreted enzyme was partially converted into an active form. In this model batimastat totally blocked TGFbeta-promoted keratinocyte migration. Immunostaining of keratinocytes advancing into the wound revealed that MMP-2 was localized in extracellular matrix contactlike structures against the endogenously produced laminin-5-rich matrix. MMP-9 was localized diffusely along the cell membranes. Using in situ hybridization we observed that in chronically inflamed human gingiva MMP-2 is expressed in epithelium extending into subepithelial connective tissue. These results suggest that MMP-2 plays a specific role in epithelial migration, possibly by detaching the advancing cells from the pericellular matrix or by activating other MMPs.     

Mastitis Whey — a Good Medium for Bacteria?

Growth of mastitis pathogenic bacteria was measured in bovine whey samples by a turbidometric microtechnique. Whey from mastitis cows supported growth as compared with whey prepared from normal milk. Blood proteins leak into milk during mastitis. A study was undertaken to analyze which molecules from blood would promote bacterial growth in whey Fractions containing hemoglobin showed a distinct growth-promoting effect. An inadequate iron supply is one of the restricting growth factors for bacteria in milk. By utilizing heme-compounds the pathogens can by-pass the effect of antimicrobial iron-binding present in milk in the form of lactoferrin.     

Matrix metalloproteinases process the laminin-5 gamma 2-chain and regulate epithelial cell migration

Matrix metalloproteinase (MMP)-2 and membrane type 1-MMP can process the laminin-5 (Ln-5) gamma2-chain, revealing a cryptic site inducing epithelial cell migration. We investigated whether other MMPs process the Ln-5 gamma2-chain and related their ability to induce epithelial cell migration. The N-terminal sequences of the MMP-3, -12, -13, and -20 processed 80kDa Ln-5 gamma2x-chains were identical whereas the N-terminus of the 80kDa(MMP-8) Ln-5 gamma2x-chain was not. MMP-3, -13, -14, and -20 induced MCF-7 cell migration over Ln-5 while MMP-8 was a poor inducer of MCF-7 cell migration. In conclusion, several MMPs can process the Ln-5 gamma2-chain and induce epithelial cell migration.