Fig. 5. KE increased proMMP-2 and proMT1-MMP expression and activation in HMECs. (A) HMECs were cultured as monolayers and incubated in the presence of various concentrations of KE for 24 hours. ProMMP-2 and MMP-2 in conditioned media (a) and in cell extracts (b) were assessed by gelatin zymography analysis. Arrows indicate the positions of proMMP-2 and MMP-2. (c) TIMPs in conditioned medium of HMECs were analyzed by reverse zymography analysis. Positions of TIMP-1 and TIMP-2 are shown. (d) Representative western blot of MT1-MMP from cell extracts of HMECs cultured in the absence or presence of KE. Rabbit polyclonal antibodies were used to reveal MT1-MMP (hinge region). Molecular masses of the bands are indicated, the 66-kDa species corresponds to proMT1-MMP, the 60 and 55-kDa species to MT1-MMP and the band at 44 kDa corresponds to a species processed further (Ellerbroek et al., 1999). (B) Semi-quantitative RT-PCR analysis of proMMP-2 and proMT1-MMP was performed in HMECs incubated for various periods in the absence (white column) or presence of 10 µg/ml KE (black column) and with various concentrations of KE for 24 hours. The time- and concentration-dependent histograms, each based on four experiments, display the mean±s.d. of proMMP-2 and proMT1-MMP values that have been normalized to the levels of GAPDH mRNA and expressed relative to control cell value (time 0 and not KE treated). NS, not significant; ^*P<0.05; ^**P<0.01; P<0.001.