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Background: MT1-MMP is a cell-surface enzyme whose regulation of pro-MMP-2 and ERK activation position it as a key facilitator of ECM remodelling and cell migration. These processes are modulated by endogenous MMP inhibitors, such as RECK, a GPI-anchored protein which has been shown to inhibit both MT1-MMP and MMP-2 activity. Our previous studies have revealed a link between MT1-MMP levels, and pro-MMP-2 and ERK activation in mammalian cells, as well as MT1-MMP and RECK co-localization in Xenopus embryos. We here investigated how modulation of RECK would impact MT1-MMP and MMP-2 levels, as well as ERK signalling in Xenopus A6 cells.
Results: We used a Morpholino approach to knockdown RECK, plasmid transfection to overexpress RECK, and PI-PLC treatment to shed RECK from the cell surface of Xenopus A6 cells. RECK reduction did not alter pERK or MT1-MMP levels, nor MMP-2 activity as measured by zymography; thus RECK-knockdown cells maintained the ability to remodel the ECM. RECK overexpression and PI-PLC treatment both increased ECM remodelling potential through increased MT1-MMP protein and relative MMP-2 activation levels.
Conclusions: RECK changes that reduce the ability of the cell to remodel the ECM (overexpression and cell surface shedding) are compensated for by increases in MT1-MMP, and MMP-2 levels as seen by zymography.
Fig. 1. Treatment of A6 cells with RECK MO resulted in decreased RECK protein levels. Immunoblot analysis was used to confirm reduction of RECK protein following treatment of A6 cells with RECK MO using Endo-porter delivery reagent. Protein was extracted from cells 48 h following treatment with increasing concentrations of RECK MO. a Knockdown of RECK protein occurred in a dose-dependent manner. β-actin is used as a loading control. b Quantification of protein levels in a were graphed and data is based on 3 biological replicates (mean ± SD) and normalized to control (set to 100%). Data is analyzed via t test; *, p ≤ 0.05; ***, p ≤ 0.001; ****, p ≤ 0.0001
Fig. 2. RECK reduction did not alter MT1-MMP or pERK protein levels nor MMP-2 activity levels. a Densitometry quantification of MT1-MMP immunoblotted protein normalized to β-actin. MT1-MMP protein levels did not significantly change in RECK knockdown cells compared to control (set to 1). β-actin is used as a loading control. b Densitometry quantification of pERK normalized to total ERK levels. pERK protein levels did not significantly change in RECK reduced cells compared to control (set to 1). c Gelatin zymography was used to measure protein activity of secreted pro-, intermediate, and active MMP-2 following RECK knockdown in A6 cells. Data is presented as the ratio between active and total (pro-, intermediate, and active) MMP-2 levels between control and RECK reduced cells. Active/total MMP-2 levels did not significantly change in the media of RECK reduced cells compared to control (set to 1). Secreted MMP-9 protein could not be detected at the expected size of 92 kDa (shown by the arrow). Graphed data is based on 3 biological replicates (mean ± SD). Data is analyzed via t-test; ns, p > 0.05
Fig. 3. Transfection of full-length HA-tagged RECK constructs in A6 cells resulted in increased RECK levels. a Immunoblot analysis was used to confirm overexpression of RECK protein following transfection of RECK cDNA constructs. Mock-transfected and GFP-transfected A6 cells were used as controls. Protein was extracted from cells 24 h following transfection. β-actin is used as a loading control. Left panel demonstrates increased levels of RECK protein compared to control using a RECK antibody, while right panel confirms the presence of HA-tagged proteins only in RECK-transfected A6 cells using an HA antibody. b Graphed data is based on 3 biological replicates (mean ± SD) and normalized to control (set to 100%). Data is analyzed via t-test; ***, p ≤ 0.001
Fig. 4. RECK overexpression increased MT1-MMP protein and MMP-2 activity levels and decreased pERK protein levels. a Densitometry quantification of immunoblots of MT1-MMP protein normalized to β-actin. MT1-MMP protein levels significantly increased in RECK-overexpressing cells compared to control (set to 1). b Densitometry quantification of pERK normalized to total ERK levels. pERK protein levels significantly decreased in RECK-overexpressing cells compared to control (set to 1). c Gelatin zymography was used to measure protein levels of secreted pro-, intermediate, and active MMP-2 following RECK overexpression in A6 cells. Data is presented as the ratio between active and total (pro-, intermediate, and active) MMP-2 levels between control and RECK-overexpressing cells. Active/total MMP-2 levels significantly increased in the media of RECK-overexpressing cells compared to control (set to 1). Secreted MMP-9 protein could not be detected at the expected size of 92 kDa (shown by the arrow). Graphed data is based on 3 biological replicates (mean ± SD). Data is analyzed via t-test; *, p ≤ 0.05; **, p ≤ 0.01
Fig. 5. Solubilization of RECK proteins following PI-PLC treatment. a Twenty-four hours following transfection of RECK constructs, control and transfected A6 cells were treated with PI-PLC for 24 h and cell lysate collected and analyzed by Western blot. b Densitometry quantification demonstrates that the level of RECK proteins bound to the cell is significantly reduced following PI-PLC treatment in both mock-transfected and RECK-transfected cells. β-actin is used as a loading control. Graphed data is based on 3 biological replicates (mean ± SD). Data is analyzed via t-test; *, p ≤ 0.05; **, p ≤ 0.01
Fig. 6. PI-PLC treatment caused an increase in MT1-MMP protein and MMP-2 activity levels. a Densitometry quantification of MT1-MMP protein level normalized to β-actin in control A6 cells treated with PI-PLC. MT1-MMP protein levels significantly increased in PI-PLC-treated cells compared to untreated cells (set to 1). b Densitometry quantification of pERK normalized to total ERK levels. pERK protein levels did not significantly change following PI-PLC treatment. c Gelatin zymography was used to measure protein levels of secreted pro-, intermediate, and active MMP-2 following PI-PLC treatment in A6 cells. Data is presented as the ratio between active and total (pro-, intermediate, and active) MMP-2 levels between untreated cells and PI-PLC treated cells. Active/total MMP-2 levels significantly increased in the media of PI-PLC-treated cells compared to untreated cells (set to 1). Secreted MMP-9 protein could not be detected at the expected size of 92 kDa (shown by the arrow). Graphed data is based on 3 biological replicates (mean ± SD). Data is analyzed via t-test; ns, p > 0.05; *, p ≤ 0.05; **, p ≤ 0.01
Fig. 7. Effect of RECK knockdown, RECK overexpression, and PI-PLC treatment on mRNA levels. The levels of MMP-2, MMP-9, MT1-MMP, and TIMP-2 mRNA were measured following RECK knockdown, overexpression, and PI-PLC treatment in A6 cells using real-time qPCR. a Following RECK reduction in cells, MMP-2, -9, and MT1-MMP levels did not change significantly compared to control; however, TIMP-2 levels decreased significantly. b Following RECK overexpression, MMP-2 and TIMP-2 mRNA levels did not change significantly, however, MMP-9 and MT1-MMP mRNA levels increased significantly compared to control. c Treatment of cells with PI-PLC did not change MMP-2, -9, MT1-MMP, or TIMP-2 mRNA levels. Changes in gene expression were measured relative to EF1α and normalized to control cells (set to 1). Results are based on 3 biological replicates (mean ± SEM; technical replicates, N = 9). Data is analyzed via t-test; *, p ≤ 0.05; **, p ≤ 0.01
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