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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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Effect of over-expression of MMP28 on expression of other MMP and TIMP family members. Expression of all of the members of the MMP and TIMP family, including MMP28, was measured using qRT-PCR and normalised to 18S rRNA in two clones each of cells stably transfected with MMP28 cDNA constructs (vector only (VO), wild-type (WT), EA mutant (EA) and pro-cat (PC)). Expression of each of the MMP28 constructs was verified (A) with MMP28 undetectable in the vector-only transfects. The expression of three genes altered in response to MMP28 over-expression: MMP2 (B), MMP19 (C) and TIMP3 (D). Data are plotted as mean +/− s.e.m. (n = 6). Statistical significance indicated by: ⁎⁎⁎⁎, p < 0.0001; ⁎⁎⁎, p < 0.001: ⁎⁎, p < 0.01 and ⁎, p < 0.05.
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fig8: Effect of over-expression of MMP28 on expression of other MMP and TIMP family members. Expression of all of the members of the MMP and TIMP family, including MMP28, was measured using qRT-PCR and normalised to 18S rRNA in two clones each of cells stably transfected with MMP28 cDNA constructs (vector only (VO), wild-type (WT), EA mutant (EA) and pro-cat (PC)). Expression of each of the MMP28 constructs was verified (A) with MMP28 undetectable in the vector-only transfects. The expression of three genes altered in response to MMP28 over-expression: MMP2 (B), MMP19 (C) and TIMP3 (D). Data are plotted as mean +/− s.e.m. (n = 6). Statistical significance indicated by: ⁎⁎⁎⁎, p < 0.0001; ⁎⁎⁎, p < 0.001: ⁎⁎, p < 0.01 and ⁎, p < 0.05.

Mentions: SW1353 cells were stably transfected with the wild-type MMP28/pcDNA4-FLAG construct (WT), an E241A (EA) mutant form in which the glutamate residue in the catalytic domain was substituted for an alanine, or a truncated form, in which the hemopexin (C-terminal) domain is absent (pro-cat). Clonal cell lines were established for each construct as well as for an empty vector control. All established lines were screened for MMP-28 mRNA expression by qRT-PCR analysis (see Fig. 8A for example). Two of these clonal lines were selected for each construct and control, and analysed for MMP-28 protein expression. Full-length, wild-type MMP-28 (WT) was found (though in variable amounts in different experiments) in the conditioned medium, cell lysates and ECM (Fig. 3A). Both the pro and active (processed) forms were detected in each fraction, at molecular sizes of approximately 58 kDa and 50 kDa respectively. However, it appeared that the pro form was predominant in the cell lysates, whereas in the conditioned medium and especially the ECM, the active form was predominant. The C-terminal domain was also detected, at approximately 33 kDa. The expression of the EA mutant followed a slightly different pattern to that of WT (Fig. 3B). The EA mutant was barely detected in the conditioned medium, and instead appeared to be largely associated with the cell lysate and the ECM. Also, only the pro form of the EA mutant was detected in the cell lysates, in striking contrast to the ECM, which shows the presence of both the pro and (active) processed forms. This eliminates intramolecular autocatalytic activity of MMP-28 as a means of processing, as the EA mutant is catalytically inactive. MMP28 expression in the SW1353 cell line or vector-only transfects was undetectable by qRT-PCR, thus intermolecular autocatalysis is also unlikely. The pro-cat form of MMP-28 was detected as two smaller bands in the ECM (Fig. 3C), at approximately 37 kDa and 28 kDa. These are the pro and active (processed) forms respectively. The smaller form corresponds to the catalytic domain alone. Only the pro form was detected in the cell lysates. Neither the pro or active forms were detected in the conditioned medium.


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)

Effect of over-expression of MMP28 on expression of other MMP and TIMP family members. Expression of all of the members of the MMP and TIMP family, including MMP28, was measured using qRT-PCR and normalised to 18S rRNA in two clones each of cells stably transfected with MMP28 cDNA constructs (vector only (VO), wild-type (WT), EA mutant (EA) and pro-cat (PC)). Expression of each of the MMP28 constructs was verified (A) with MMP28 undetectable in the vector-only transfects. The expression of three genes altered in response to MMP28 over-expression: MMP2 (B), MMP19 (C) and TIMP3 (D). Data are plotted as mean +/− s.e.m. (n = 6). Statistical significance indicated by: ⁎⁎⁎⁎, p < 0.0001; ⁎⁎⁎, p < 0.001: ⁎⁎, p < 0.01 and ⁎, p < 0.05.
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Related In: Results  -  Collection

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

fig8: Effect of over-expression of MMP28 on expression of other MMP and TIMP family members. Expression of all of the members of the MMP and TIMP family, including MMP28, was measured using qRT-PCR and normalised to 18S rRNA in two clones each of cells stably transfected with MMP28 cDNA constructs (vector only (VO), wild-type (WT), EA mutant (EA) and pro-cat (PC)). Expression of each of the MMP28 constructs was verified (A) with MMP28 undetectable in the vector-only transfects. The expression of three genes altered in response to MMP28 over-expression: MMP2 (B), MMP19 (C) and TIMP3 (D). Data are plotted as mean +/− s.e.m. (n = 6). Statistical significance indicated by: ⁎⁎⁎⁎, p < 0.0001; ⁎⁎⁎, p < 0.001: ⁎⁎, p < 0.01 and ⁎, p < 0.05.
Mentions: SW1353 cells were stably transfected with the wild-type MMP28/pcDNA4-FLAG construct (WT), an E241A (EA) mutant form in which the glutamate residue in the catalytic domain was substituted for an alanine, or a truncated form, in which the hemopexin (C-terminal) domain is absent (pro-cat). Clonal cell lines were established for each construct as well as for an empty vector control. All established lines were screened for MMP-28 mRNA expression by qRT-PCR analysis (see Fig. 8A for example). Two of these clonal lines were selected for each construct and control, and analysed for MMP-28 protein expression. Full-length, wild-type MMP-28 (WT) was found (though in variable amounts in different experiments) in the conditioned medium, cell lysates and ECM (Fig. 3A). Both the pro and active (processed) forms were detected in each fraction, at molecular sizes of approximately 58 kDa and 50 kDa respectively. However, it appeared that the pro form was predominant in the cell lysates, whereas in the conditioned medium and especially the ECM, the active form was predominant. The C-terminal domain was also detected, at approximately 33 kDa. The expression of the EA mutant followed a slightly different pattern to that of WT (Fig. 3B). The EA mutant was barely detected in the conditioned medium, and instead appeared to be largely associated with the cell lysate and the ECM. Also, only the pro form of the EA mutant was detected in the cell lysates, in striking contrast to the ECM, which shows the presence of both the pro and (active) processed forms. This eliminates intramolecular autocatalytic activity of MMP-28 as a means of processing, as the EA mutant is catalytically inactive. MMP28 expression in the SW1353 cell line or vector-only transfects was undetectable by qRT-PCR, thus intermolecular autocatalysis is also unlikely. The pro-cat form of MMP-28 was detected as two smaller bands in the ECM (Fig. 3C), at approximately 37 kDa and 28 kDa. These are the pro and active (processed) forms respectively. The smaller form corresponds to the catalytic domain alone. Only the pro form was detected in the cell lysates. Neither the pro or active forms were detected in the conditioned medium.

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