S100A4 involvement in metastasis: Deregulation of matrix metalloproteinases and tissue inhibitors of matrix metalloproteinases in osteosarcoma cells transfected with an anti-S100A4 ribozyme

The biological function of the metastasis-associated gene S100A4 is not ful ly understood, although there is evidence indicating interactions between t he gene product and the cytoskeleton. We have examined whether an associati on could exist between S100A4 and the regulation of matrix metalloproteinas es (MMPs) and their endogenous inhibitors (TIMPs). For these studies, three clones of a highly metastatic human osteosarcoma cell line (OHS) transfect ed with a hammerhead ribozyme directed against the S100A4 gene transcript w ere used. The clones demonstrated different expression levels of S100A4 and also different metastatic capacity. In the clone with the most prominent d own-regulation of S100A4, the mRNA levels of MMP2, membrane type (MT) 1-MMP , and TIMP-1 were significantly reduced in exponentially growing cultures. Western blots, gelatin zymography, and ELISA showed similar expression patt erns of MMPs and TIMPs at the protein level. In the clones with an intermed iate expression of S100A4, reduced expression of MT1-MMP and TIMP-1 was det ected, whereas the expression of MMP-2 was at the same level as in the cont rol cells. In contrast to the other factors, TIMP-2 was up-regulated in all of the clones independent of the extent of ribozyme-induced down-regulatio n of S100A4. The transwell chamber assay demonstrated that the capacity of the ribozyme-transfected cells to cross uncoated filters was reduced, relat ive to control cells, according to the reduction in the S100A4 expression l evel. The clone with the lowest reduction in S100A4 did not demonstrate dif ferent motility compared with control cells, whereas transfectants with onl y 5% S100A4 mRNA shelved a 50% reduction in motility. Interestingly, this t rend was even more striking when the capacity to cross Matrigel-coated filt ers was analyzed, as all the clones demonstrated between 40 and 75% reduced invasion, It is concluded that S100A4 may exert its effect on metastasis f ormation not only by stimulating the motility of tumor cells but also by af fecting their invasive properties through influencing the expression of MMP s and their endogenous inhibitors.