Figure 6.

MMP-7 cleavage of E-cadherin does not affect p27^kip-1 levels but enhances RhoA activity. (a) Change in p27^kip-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). p27^kip-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).