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Cleavage of E-Cadherin by Matrix Metalloproteinase-7 Promotes Cellular Proliferation in Nontransformed Cell Lines via Activation of RhoA.

Lynch CC, Vargo-Gogola T, Matrisian LM, Fingleton B - J Oncol (2010)

Bottom Line: Previously, we demonstrated that MMP-7, a protease implicated in mammary and intestinal tumor growth, can process the adherens junction component E-cadherin.This observation leads us to test whether MMP-7 processing of E-cadherin could directly impact cell proliferation in nontransformed epithelial cell lines (MDCK and C57MG).Our goal was to investigate the possibility that MMP-7 produced by cancer cells may have effects on adjacent normal epithelium.

View Article: PubMed Central - PubMed

Affiliation: Department of Cancer Biology, Vanderbilt University School of Medicine, 734 PRB 2220 Pierce Ave, Nashville, TN 37232, USA.

ABSTRACT
Perturbations in cell-cell contact machinery occur frequently in epithelial cancers and result in increased cancer cell migration and invasion. Previously, we demonstrated that MMP-7, a protease implicated in mammary and intestinal tumor growth, can process the adherens junction component E-cadherin. This observation leads us to test whether MMP-7 processing of E-cadherin could directly impact cell proliferation in nontransformed epithelial cell lines (MDCK and C57MG). Our goal was to investigate the possibility that MMP-7 produced by cancer cells may have effects on adjacent normal epithelium. Here, we show that MMP-7 processing of E-cadherin mediates, (1) loss of cell-cell contact, (2) increased cell migration, (3) a loss of epithelial cell polarization and (4) increased cell proliferation via RhoA activation. These data demonstrate that MMP-7 promotes epithelial cell proliferation via the processing of E-cadherin and provide insights into the molecular mechanisms that govern epithelial cell growth.

No MeSH data available.


Related in: MedlinePlus

MMP-7 cleavage of E-cadherin does not affect p27kip-1 levels but enhances RhoA activity. (a) Change in p27kip-1 levels in polarized MDCK cells over time (hours, h) in the absence (−) or presence (+) of exogenous MMP-7 (100 ng/mL) was assessed by immunoblot analysis of the cell lysates (upper panel).  p27kip-1 levels (arrow) in cell extracts of confluent vector control and MMP-7 expressing cell lines were also examined (lower panel). (b) Change in confluent-polarized MDCK RhoA activity in response to the addition of exogenous MMP-7 (100 ng/mL) over time (minutes, min) was examined using a Rhotekin pull down assay as described in the materials and methods followed by immunoblot analysis for active RhoA. Direct immunoblot analysis for total RhoA served as a control for loading. Arrow and arrowhead indicate active and total RhoA, respectively. LPA was used as a positive control for RhoA activity. The level of active RhoA in confluent vector control and MMP-7 expressing cell lines was also determined. (c) RhoA activity (arrow) in lysates obtained from confluent empty vector control and MMP-7 expressing cell lines treated either with IgG control or with E-cadherin blocking (α-E-cad) antibodies in addition to exogenous MMP-7 (100 ng/mL) for 1 hour was examined using the rhotekin pull down assay followed by immunoblot analysis of RhoA (upper panel). Total RhoA (arrowhead) and actin (open arrow head) levels were examined by direct immunoblot analysis of the cell lysates and served as a loading control. (d) Analysis of cyclin D1 (arrowhead) levels in whole cell lysates derived from preconfluent and confluent empty vector control and MMP-7 expressing cell lines. Actin was used as a loading control (open arrowhead).
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fig6: MMP-7 cleavage of E-cadherin does not affect p27kip-1 levels but enhances RhoA activity. (a) Change in p27kip-1 levels in polarized MDCK cells over time (hours, h) in the absence (−) or presence (+) of exogenous MMP-7 (100 ng/mL) was assessed by immunoblot analysis of the cell lysates (upper panel). p27kip-1 levels (arrow) in cell extracts of confluent vector control and MMP-7 expressing cell lines were also examined (lower panel). (b) Change in confluent-polarized MDCK RhoA activity in response to the addition of exogenous MMP-7 (100 ng/mL) over time (minutes, min) was examined using a Rhotekin pull down assay as described in the materials and methods followed by immunoblot analysis for active RhoA. Direct immunoblot analysis for total RhoA served as a control for loading. Arrow and arrowhead indicate active and total RhoA, respectively. LPA was used as a positive control for RhoA activity. The level of active RhoA in confluent vector control and MMP-7 expressing cell lines was also determined. (c) RhoA activity (arrow) in lysates obtained from confluent empty vector control and MMP-7 expressing cell lines treated either with IgG control or with E-cadherin blocking (α-E-cad) antibodies in addition to exogenous MMP-7 (100 ng/mL) for 1 hour was examined using the rhotekin pull down assay followed by immunoblot analysis of RhoA (upper panel). Total RhoA (arrowhead) and actin (open arrow head) levels were examined by direct immunoblot analysis of the cell lysates and served as a loading control. (d) Analysis of cyclin D1 (arrowhead) levels in whole cell lysates derived from preconfluent and confluent empty vector control and MMP-7 expressing cell lines. Actin was used as a loading control (open arrowhead).

Mentions: Our data suggest that MMP-7 processing of E-cadherin mediated the proliferation of nontransformed epithelial cells. Next, we examined the precise molecular mechanism through which E-cadherin mediated this effect. Previous reports have identified that E-cadherin can regulate cell cycle progression via negative regulation of the cell cycle inhibitor, p27kip1 [28]. However, analysis of lysates derived from control and MMP-7-treated MDCK cells and from the empty vector control and MMP-7 expressing mammary epithelial cells revealed no major differences in the levels of p27kip1 (Figure 6(a)). In separate experiments, no changes in cytosolic versus nuclear p27kip-1 were observed (data not shown). Several studies have tied RhoA activity status to E-cadherin function [29–35], which prompted us to analyze the activity of RhoA in both the MDCK cells after treatment with exogenous MMP-7, and in the C57MG control and MMP-7-expressing clones. We detected heightened RhoA activity (normalized to total RhoA) compared to controls in both cases (Figure 6(b)). We then used the C57MG clones to test whether the enhanced RhoA activity was downstream of E-cadherin. In the C57MG empty vector control cell line, blocking E-cadherin homotypic binding via the addition of blocking E-cadherin antibodies significantly enhanced RhoA activation in comparison to IgG-treated controls (Figure 6(c)). An identical result was achieved when control cells were treated with recombinant active MMP-7. As expected, in cells already expressing MMP-7 in which E-cadherin is already perturbed, neither blocking antibody nor addition of exogenous MMP-7 had any effect on RhoA activity (Figure 6(c)). Thus, perturbation of E-cadherin by either antibody blockade or MMP-7-mediated processing results in the activation of RhoA.


Cleavage of E-Cadherin by Matrix Metalloproteinase-7 Promotes Cellular Proliferation in Nontransformed Cell Lines via Activation of RhoA.

Lynch CC, Vargo-Gogola T, Matrisian LM, Fingleton B - J Oncol (2010)

MMP-7 cleavage of E-cadherin does not affect p27kip-1 levels but enhances RhoA activity. (a) Change in p27kip-1 levels in polarized MDCK cells over time (hours, h) in the absence (−) or presence (+) of exogenous MMP-7 (100 ng/mL) was assessed by immunoblot analysis of the cell lysates (upper panel).  p27kip-1 levels (arrow) in cell extracts of confluent vector control and MMP-7 expressing cell lines were also examined (lower panel). (b) Change in confluent-polarized MDCK RhoA activity in response to the addition of exogenous MMP-7 (100 ng/mL) over time (minutes, min) was examined using a Rhotekin pull down assay as described in the materials and methods followed by immunoblot analysis for active RhoA. Direct immunoblot analysis for total RhoA served as a control for loading. Arrow and arrowhead indicate active and total RhoA, respectively. LPA was used as a positive control for RhoA activity. The level of active RhoA in confluent vector control and MMP-7 expressing cell lines was also determined. (c) RhoA activity (arrow) in lysates obtained from confluent empty vector control and MMP-7 expressing cell lines treated either with IgG control or with E-cadherin blocking (α-E-cad) antibodies in addition to exogenous MMP-7 (100 ng/mL) for 1 hour was examined using the rhotekin pull down assay followed by immunoblot analysis of RhoA (upper panel). Total RhoA (arrowhead) and actin (open arrow head) levels were examined by direct immunoblot analysis of the cell lysates and served as a loading control. (d) Analysis of cyclin D1 (arrowhead) levels in whole cell lysates derived from preconfluent and confluent empty vector control and MMP-7 expressing cell lines. Actin was used as a loading control (open arrowhead).
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Related In: Results  -  Collection

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fig6: MMP-7 cleavage of E-cadherin does not affect p27kip-1 levels but enhances RhoA activity. (a) Change in p27kip-1 levels in polarized MDCK cells over time (hours, h) in the absence (−) or presence (+) of exogenous MMP-7 (100 ng/mL) was assessed by immunoblot analysis of the cell lysates (upper panel). p27kip-1 levels (arrow) in cell extracts of confluent vector control and MMP-7 expressing cell lines were also examined (lower panel). (b) Change in confluent-polarized MDCK RhoA activity in response to the addition of exogenous MMP-7 (100 ng/mL) over time (minutes, min) was examined using a Rhotekin pull down assay as described in the materials and methods followed by immunoblot analysis for active RhoA. Direct immunoblot analysis for total RhoA served as a control for loading. Arrow and arrowhead indicate active and total RhoA, respectively. LPA was used as a positive control for RhoA activity. The level of active RhoA in confluent vector control and MMP-7 expressing cell lines was also determined. (c) RhoA activity (arrow) in lysates obtained from confluent empty vector control and MMP-7 expressing cell lines treated either with IgG control or with E-cadherin blocking (α-E-cad) antibodies in addition to exogenous MMP-7 (100 ng/mL) for 1 hour was examined using the rhotekin pull down assay followed by immunoblot analysis of RhoA (upper panel). Total RhoA (arrowhead) and actin (open arrow head) levels were examined by direct immunoblot analysis of the cell lysates and served as a loading control. (d) Analysis of cyclin D1 (arrowhead) levels in whole cell lysates derived from preconfluent and confluent empty vector control and MMP-7 expressing cell lines. Actin was used as a loading control (open arrowhead).
Mentions: Our data suggest that MMP-7 processing of E-cadherin mediated the proliferation of nontransformed epithelial cells. Next, we examined the precise molecular mechanism through which E-cadherin mediated this effect. Previous reports have identified that E-cadherin can regulate cell cycle progression via negative regulation of the cell cycle inhibitor, p27kip1 [28]. However, analysis of lysates derived from control and MMP-7-treated MDCK cells and from the empty vector control and MMP-7 expressing mammary epithelial cells revealed no major differences in the levels of p27kip1 (Figure 6(a)). In separate experiments, no changes in cytosolic versus nuclear p27kip-1 were observed (data not shown). Several studies have tied RhoA activity status to E-cadherin function [29–35], which prompted us to analyze the activity of RhoA in both the MDCK cells after treatment with exogenous MMP-7, and in the C57MG control and MMP-7-expressing clones. We detected heightened RhoA activity (normalized to total RhoA) compared to controls in both cases (Figure 6(b)). We then used the C57MG clones to test whether the enhanced RhoA activity was downstream of E-cadherin. In the C57MG empty vector control cell line, blocking E-cadherin homotypic binding via the addition of blocking E-cadherin antibodies significantly enhanced RhoA activation in comparison to IgG-treated controls (Figure 6(c)). An identical result was achieved when control cells were treated with recombinant active MMP-7. As expected, in cells already expressing MMP-7 in which E-cadherin is already perturbed, neither blocking antibody nor addition of exogenous MMP-7 had any effect on RhoA activity (Figure 6(c)). Thus, perturbation of E-cadherin by either antibody blockade or MMP-7-mediated processing results in the activation of RhoA.

Bottom Line: Previously, we demonstrated that MMP-7, a protease implicated in mammary and intestinal tumor growth, can process the adherens junction component E-cadherin.This observation leads us to test whether MMP-7 processing of E-cadherin could directly impact cell proliferation in nontransformed epithelial cell lines (MDCK and C57MG).Our goal was to investigate the possibility that MMP-7 produced by cancer cells may have effects on adjacent normal epithelium.

View Article: PubMed Central - PubMed

Affiliation: Department of Cancer Biology, Vanderbilt University School of Medicine, 734 PRB 2220 Pierce Ave, Nashville, TN 37232, USA.

ABSTRACT
Perturbations in cell-cell contact machinery occur frequently in epithelial cancers and result in increased cancer cell migration and invasion. Previously, we demonstrated that MMP-7, a protease implicated in mammary and intestinal tumor growth, can process the adherens junction component E-cadherin. This observation leads us to test whether MMP-7 processing of E-cadherin could directly impact cell proliferation in nontransformed epithelial cell lines (MDCK and C57MG). Our goal was to investigate the possibility that MMP-7 produced by cancer cells may have effects on adjacent normal epithelium. Here, we show that MMP-7 processing of E-cadherin mediates, (1) loss of cell-cell contact, (2) increased cell migration, (3) a loss of epithelial cell polarization and (4) increased cell proliferation via RhoA activation. These data demonstrate that MMP-7 promotes epithelial cell proliferation via the processing of E-cadherin and provide insights into the molecular mechanisms that govern epithelial cell growth.

No MeSH data available.


Related in: MedlinePlus