HAb18G/CD147 enhances the secretion of matrix metalloproteinases (MMP) via cGMP/NO-sensitive capacitative calcium entry (CCE) and accordingly attenuates adhesion ability of fibroblasts

Abstract

The present study examined the effect of hepatoma-associated antigen HAb18G (homologous to CD147) expression on the NO/cGMP-regulated Ca 2+ mobilization to induce matrix metalloproteinases (MMP) production and attenuate adhesion ability of mouse fibroblast NIH/3T3 cells. HAb18G/CD147 cDNA was transfected into fibroblast 3T3 cells to obtain a cell line stably expressing HAb18G/CD147, t3T3, as demonstrated by immunofluorescence staining and flow cytometry assays. 8-Bromo-cGMP inhibited the thapsigargin-induced Ca 2+ entry in 3T3 cells, whereas an inhibitor of protein kinase G, KT5823 (1 μM), led to an increase in Ca 2+ entry. Expression of HAb18G/CD147 in t3T3 cells decreased the inhibitory response to cGMP. A similar effect on the Ca 2+ entry was observed in 3T3 cells in response to an NO donor, (±)- S-nitroso- N-acetylpenicillamine (SNAP). The inhibitory effect of SNAP on the thapsigargin-induced Ca 2+ entry was also reduced in HAb18G/CD147-expressing t3T3 cells, indicating a role for HAb18G/CD147 in NO/cGMP-regulated Ca 2+ entry. Results of gelatin zymography assays showed that addition of extracellular Ca 2+ induced MMP (MMP-2, MMP-9) release and activation in a dose-dependent manner, and expression of HAb18G/CD147 enhanced the secretion of MMP-2 and MMP-9 in 3T3 cells. 8-Bromo-cGMP and SNAP reduced the production of MMP in 3T3 cells but not in t3T3 with HAb18G/CD147 expression. RT-PCR experiments substantiated that the expression of MMP-2 and MMP-9 mRNA in HAb18G/CD147-expressing t3T3 cell was significantly greater than that in 3T3 cells. Experiments investigating adhesion potentials demonstrated that HAb18G/CD147-expressing t3T3 cells pretreated with Ca 2+ attached to Matrigel-coated culture plates significantly less efficiently than 3T3 cells. The proportion of attached cells could be increased by treatment with 8-bromo-cGMP and SNAP in 3T3 cells, but not in t3T3. These results suggest that HAb18G/CD147 attenuates adhesion potentials in fibroblasts by enhancing the secretion of MMP through NO/cGMP-sensitive capacitative Ca 2+ entry.