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Expression and function of matrix metalloproteinase (MMP)-28.

Rodgers UR, Kevorkian L, Surridge AK, Waters JG, Swingler TE, Culley K, Illman S, Lohi J, Parker AE, Clark IM - Matrix Biol. (2009)

Bottom Line: Adhesion to type II collagen and fibronectin was increased, and migration across the former was decreased.These data demonstrate that expression of MMP28 alters cell phenotype towards a more adhesive, less migratory behaviour.Further, MMP-28 activity may reside predominantly in the extracellular matrix, and we are currently searching for substrates in this compartment.

View Article: PubMed Central - PubMed

Affiliation: Biomedical Research Centre, School of Biological Sciences, University of East Anglia, Norwich, UK.

ABSTRACT
Matrix metalloproteinase-28 (MMP-28, epilysin) is highly expressed in the skin by keratinocytes, the developing and regenerating nervous system and a number of other normal human tissues. In epithelial cells, over-expression of MMP-28 mediates irreversible epithelial to mesenchymal transition concomitant with loss of E-cadherin from the cell surface and an increase in active transforming growth factor beta. We recently reported the expression of MMP-28 in both cartilage and synovium where expression is increased in patients with osteoarthritis. In human chondrosarcoma cells MMP-28 was activated by proprotein convertases and the active form of the enzyme preferentially associated with the extracellular matrix in a C-terminal independent manner. over-expression of MMP-28 in chondrosarcoma cells led to altered cell morphology with increased organisation of actin. Adhesion to type II collagen and fibronectin was increased, and migration across the former was decreased. MMP-28 was localised to the cell surface, at least transiently, in a C-terminal dependent manner. Heparin prevented both extracellular matrix association and cell surface binding of MMP-28 suggesting that both are via heparan sulphate proteoglycans. Over-expression of activatable MMP-28, but not catalytically inactive EA mutant increased the expression and activity of MMP-2, and all forms of MMP-28 tested increased expression of MMP19 and TIMP3 mRNA. These data demonstrate that expression of MMP28 alters cell phenotype towards a more adhesive, less migratory behaviour. Further, MMP-28 activity may reside predominantly in the extracellular matrix, and we are currently searching for substrates in this compartment.

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Furin activity is required for processing of proMMP-28. HeLa cells were transiently transfected with the wild-type MMP28 gene or the pcDNA4FLAG vector as a negative control. Cells were treated with 100 μM of the furin inhibitor Decanoyl-Arg-Val-Lys-Arg-chloromethyl ketone prior to transfection. The same dose was repeated 24 h after transfection. Untreated cells were included as controls. Protein expression in the extracellular matrix was detected by western blotting, using an anti-FLAG antibody.
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fig2: Furin activity is required for processing of proMMP-28. HeLa cells were transiently transfected with the wild-type MMP28 gene or the pcDNA4FLAG vector as a negative control. Cells were treated with 100 μM of the furin inhibitor Decanoyl-Arg-Val-Lys-Arg-chloromethyl ketone prior to transfection. The same dose was repeated 24 h after transfection. Untreated cells were included as controls. Protein expression in the extracellular matrix was detected by western blotting, using an anti-FLAG antibody.

Mentions: In order to ascertain the mechanism of processing, the effect of a proprotein convertase inhibitor, Decanoyl Arg-Val-Lys-Arg Chloromethylketone, was tested. As the active (processed) form of MMP-28 appeared to be largely associated with the ECM, samples from the ECM were assessed. Samples treated with the inhibitor demonstrated only the presence of the pro form of MMP-28 (Fig. 2), confirming that MMP-28 processing is mediated by a furin-like proprotein convertase. The anti-FLAG antibody also detected a high molecular weight band present in both vector only and MMP-28 transfected cells.


Expression and function of matrix metalloproteinase (MMP)-28.

Rodgers UR, Kevorkian L, Surridge AK, Waters JG, Swingler TE, Culley K, Illman S, Lohi J, Parker AE, Clark IM - Matrix Biol. (2009)

Furin activity is required for processing of proMMP-28. HeLa cells were transiently transfected with the wild-type MMP28 gene or the pcDNA4FLAG vector as a negative control. Cells were treated with 100 μM of the furin inhibitor Decanoyl-Arg-Val-Lys-Arg-chloromethyl ketone prior to transfection. The same dose was repeated 24 h after transfection. Untreated cells were included as controls. Protein expression in the extracellular matrix was detected by western blotting, using an anti-FLAG antibody.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2724077&req=5

fig2: Furin activity is required for processing of proMMP-28. HeLa cells were transiently transfected with the wild-type MMP28 gene or the pcDNA4FLAG vector as a negative control. Cells were treated with 100 μM of the furin inhibitor Decanoyl-Arg-Val-Lys-Arg-chloromethyl ketone prior to transfection. The same dose was repeated 24 h after transfection. Untreated cells were included as controls. Protein expression in the extracellular matrix was detected by western blotting, using an anti-FLAG antibody.
Mentions: In order to ascertain the mechanism of processing, the effect of a proprotein convertase inhibitor, Decanoyl Arg-Val-Lys-Arg Chloromethylketone, was tested. As the active (processed) form of MMP-28 appeared to be largely associated with the ECM, samples from the ECM were assessed. Samples treated with the inhibitor demonstrated only the presence of the pro form of MMP-28 (Fig. 2), confirming that MMP-28 processing is mediated by a furin-like proprotein convertase. The anti-FLAG antibody also detected a high molecular weight band present in both vector only and MMP-28 transfected cells.

Bottom Line: Adhesion to type II collagen and fibronectin was increased, and migration across the former was decreased.These data demonstrate that expression of MMP28 alters cell phenotype towards a more adhesive, less migratory behaviour.Further, MMP-28 activity may reside predominantly in the extracellular matrix, and we are currently searching for substrates in this compartment.

View Article: PubMed Central - PubMed

Affiliation: Biomedical Research Centre, School of Biological Sciences, University of East Anglia, Norwich, UK.

ABSTRACT
Matrix metalloproteinase-28 (MMP-28, epilysin) is highly expressed in the skin by keratinocytes, the developing and regenerating nervous system and a number of other normal human tissues. In epithelial cells, over-expression of MMP-28 mediates irreversible epithelial to mesenchymal transition concomitant with loss of E-cadherin from the cell surface and an increase in active transforming growth factor beta. We recently reported the expression of MMP-28 in both cartilage and synovium where expression is increased in patients with osteoarthritis. In human chondrosarcoma cells MMP-28 was activated by proprotein convertases and the active form of the enzyme preferentially associated with the extracellular matrix in a C-terminal independent manner. over-expression of MMP-28 in chondrosarcoma cells led to altered cell morphology with increased organisation of actin. Adhesion to type II collagen and fibronectin was increased, and migration across the former was decreased. MMP-28 was localised to the cell surface, at least transiently, in a C-terminal dependent manner. Heparin prevented both extracellular matrix association and cell surface binding of MMP-28 suggesting that both are via heparan sulphate proteoglycans. Over-expression of activatable MMP-28, but not catalytically inactive EA mutant increased the expression and activity of MMP-2, and all forms of MMP-28 tested increased expression of MMP19 and TIMP3 mRNA. These data demonstrate that expression of MMP28 alters cell phenotype towards a more adhesive, less migratory behaviour. Further, MMP-28 activity may reside predominantly in the extracellular matrix, and we are currently searching for substrates in this compartment.

Show MeSH
Related in: MedlinePlus