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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

Detection of MMP-28 expression in SW1353 cells using immunofluorescence. SW1353 cells stably transfected with MMP28 constructs were probed for protein expression with an anti-FLAG primary antibody and an Alexa 488-conjugated secondary antibody. Cells were permeabilised (A to D) or non-permeabilised (E to H) to detect cell surface localisation of MMP-28. Wild-type cells treated with an IgG isotype control primary antibody were also included as a control, both permeabilised (I) and non-permeabilised (J). Cells were counterstained with DAPI and viewed under 20× magnification.
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fig4: Detection of MMP-28 expression in SW1353 cells using immunofluorescence. SW1353 cells stably transfected with MMP28 constructs were probed for protein expression with an anti-FLAG primary antibody and an Alexa 488-conjugated secondary antibody. Cells were permeabilised (A to D) or non-permeabilised (E to H) to detect cell surface localisation of MMP-28. Wild-type cells treated with an IgG isotype control primary antibody were also included as a control, both permeabilised (I) and non-permeabilised (J). Cells were counterstained with DAPI and viewed under 20× magnification.

Mentions: Intracellular MMP-28 was detected in permeabilised SW1353 cells stably transfected with MMP28 expression constructs (Fig. 4A–C) but not in empty vector control cells (Fig. 4D). Cells expressing the WT form demonstrated strong perinuclear staining. The EA mutant transfects (Fig. 4C) also demonstrated punctate staining that appeared to localise the periphery of the cells with WT transfects demonstrating this to a lesser extent. In non-permeabilised cells both WT (Fig. 4E) and EA mutant (Fig. 4F) stable transfects showed similar cell surface staining, though the pro-cat form was barely detectable at the cell surface (Fig. 4G). Empty vector control cells showed no cell surface staining for MMP-28 (Fig. 4H). IgG controls were negative for intracellular and cell surface staining of MMP-28 (Fig. 4I and J respectively).


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)

Detection of MMP-28 expression in SW1353 cells using immunofluorescence. SW1353 cells stably transfected with MMP28 constructs were probed for protein expression with an anti-FLAG primary antibody and an Alexa 488-conjugated secondary antibody. Cells were permeabilised (A to D) or non-permeabilised (E to H) to detect cell surface localisation of MMP-28. Wild-type cells treated with an IgG isotype control primary antibody were also included as a control, both permeabilised (I) and non-permeabilised (J). Cells were counterstained with DAPI and viewed under 20× magnification.
© Copyright Policy
Related In: Results  -  Collection

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

fig4: Detection of MMP-28 expression in SW1353 cells using immunofluorescence. SW1353 cells stably transfected with MMP28 constructs were probed for protein expression with an anti-FLAG primary antibody and an Alexa 488-conjugated secondary antibody. Cells were permeabilised (A to D) or non-permeabilised (E to H) to detect cell surface localisation of MMP-28. Wild-type cells treated with an IgG isotype control primary antibody were also included as a control, both permeabilised (I) and non-permeabilised (J). Cells were counterstained with DAPI and viewed under 20× magnification.
Mentions: Intracellular MMP-28 was detected in permeabilised SW1353 cells stably transfected with MMP28 expression constructs (Fig. 4A–C) but not in empty vector control cells (Fig. 4D). Cells expressing the WT form demonstrated strong perinuclear staining. The EA mutant transfects (Fig. 4C) also demonstrated punctate staining that appeared to localise the periphery of the cells with WT transfects demonstrating this to a lesser extent. In non-permeabilised cells both WT (Fig. 4E) and EA mutant (Fig. 4F) stable transfects showed similar cell surface staining, though the pro-cat form was barely detectable at the cell surface (Fig. 4G). Empty vector control cells showed no cell surface staining for MMP-28 (Fig. 4H). IgG controls were negative for intracellular and cell surface staining of MMP-28 (Fig. 4I and J respectively).

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