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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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Related in: MedlinePlus

Expression of MMP-28 in transiently transfected HeLa cells. HeLa cells were transiently transfected with the wild-type (WT) MMP28 cDNA, previously cloned into a modified pcDNA4FLAG vector. Vector-only (VO) transfects were included as a negative control. Protein expression in conditioned medium (A), cell lysate (B) and extracellular matrix (C) was detected by western blotting, using an anti-FLAG antibody. The pro and active forms of MMP-28 are indicated. CTD = C-terminal domain.
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fig1: Expression of MMP-28 in transiently transfected HeLa cells. HeLa cells were transiently transfected with the wild-type (WT) MMP28 cDNA, previously cloned into a modified pcDNA4FLAG vector. Vector-only (VO) transfects were included as a negative control. Protein expression in conditioned medium (A), cell lysate (B) and extracellular matrix (C) was detected by western blotting, using an anti-FLAG antibody. The pro and active forms of MMP-28 are indicated. CTD = C-terminal domain.

Mentions: HeLa cells were transiently transfected with MMP28/pcDNA4-FLAG construct (WT) or empty vector and protein expression assessed by western blot. Two bands were detected in the conditioned medium of WT transient transfects, at approximately 60 kDa and 30 kDa (Fig. 1A). These bands were independently confirmed to be the pro form and C-terminal domain respectively, using domain-specific antibodies (see Experimental procedures, data not shown). Predicted molecular weights for the unmodified polypeptides of these two forms are 56.5 kDa and 26.7 kDa respectively. An approximate 60 kDa band was also detected in cell lysates (Fig. 1B), again verified as the pro form with a domain-specific antibody (data not shown). In the ECM, two bands of approximately 60 kDa and 50 kDa were detected (Fig. 1C) and were independently confirmed to be the pro and active forms of MMP-28 (data not shown); in this study, the term ‘active’ refers to MMP-28 following removal of the pro domain. Predicted molecular weight for the active form is 44.9 kDa.


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)

Expression of MMP-28 in transiently transfected HeLa cells. HeLa cells were transiently transfected with the wild-type (WT) MMP28 cDNA, previously cloned into a modified pcDNA4FLAG vector. Vector-only (VO) transfects were included as a negative control. Protein expression in conditioned medium (A), cell lysate (B) and extracellular matrix (C) was detected by western blotting, using an anti-FLAG antibody. The pro and active forms of MMP-28 are indicated. CTD = C-terminal domain.
© Copyright Policy
Related In: Results  -  Collection

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

fig1: Expression of MMP-28 in transiently transfected HeLa cells. HeLa cells were transiently transfected with the wild-type (WT) MMP28 cDNA, previously cloned into a modified pcDNA4FLAG vector. Vector-only (VO) transfects were included as a negative control. Protein expression in conditioned medium (A), cell lysate (B) and extracellular matrix (C) was detected by western blotting, using an anti-FLAG antibody. The pro and active forms of MMP-28 are indicated. CTD = C-terminal domain.
Mentions: HeLa cells were transiently transfected with MMP28/pcDNA4-FLAG construct (WT) or empty vector and protein expression assessed by western blot. Two bands were detected in the conditioned medium of WT transient transfects, at approximately 60 kDa and 30 kDa (Fig. 1A). These bands were independently confirmed to be the pro form and C-terminal domain respectively, using domain-specific antibodies (see Experimental procedures, data not shown). Predicted molecular weights for the unmodified polypeptides of these two forms are 56.5 kDa and 26.7 kDa respectively. An approximate 60 kDa band was also detected in cell lysates (Fig. 1B), again verified as the pro form with a domain-specific antibody (data not shown). In the ECM, two bands of approximately 60 kDa and 50 kDa were detected (Fig. 1C) and were independently confirmed to be the pro and active forms of MMP-28 (data not shown); in this study, the term ‘active’ refers to MMP-28 following removal of the pro domain. Predicted molecular weight for the active form is 44.9 kDa.

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