CALCIUM IONOPHORES DECREASE PERICELLULAR GELATINOLYTIC ACTIVITY VIA INHIBITION OF 92-KDA GELATINASE EXPRESSION AND DECREASE OF 72-KDA GELATINASE ACTIVATION

To understand the roles of intracellular calcium levels on gelatinase/ type IV collagenase expression, we analyzed the effects of calcium ion ophores on the expression of 92- and 72-kDa gelatinases (MMP-9 and MMP -2) in human fibrosarcoma cells (HT-1080). Calcium ionophores ionomyci n and A23187 reduced the levels of pericellular gelatinolytic activity in both untreated and phorbol 12-myristate 13-acetate (PMA) or tumor necrosis factor-alpha (TNF alpha)-stimulated cells as determined by de gradation of radiolabeled gelatin. Gelatin zymography and immunoblotti ng revealed a dose-dependent decrease in the levels of secreted 92-kDa gelatinase, which was paralleled by a decrease of its mRNA. Treatment of cells with thapsigargin caused similar decreases of 92-kDa gelatin ase mRNA and protein. The decrease of 99-kDa gelatinase expression was due to lower transcription rate as determined by transfection assays with 92-kDa gelatinase/luciferase construct. The expression of 72-kDa gelatinase was only slightly decreased by ionophores. Treatment of HT- 1080 cells with PMA, TNF alpha, or concanavalin A resulted in the conv ersion of 72-kDa gelatinase proenzyme to its presumed 64- and 62-kDa a ctive forms as determined by gelatin zymography and immunoblotting. Si multaneous treatment with the ionophores or thapsigarin resulted in in hibition of PMA-induced gelatinase activation. The expression of membr ane-type matrix metalloproteinase, a potential activator of 72-kDa gel atinase, was not affected by ionophores. The results indicate that cal cium ionophores decrease gelatinolysis by repressing both the expressi on of 92-kDa gelatinase and the activation of the 72-kDa gelatinase.