Bjørnland K, Winberg J O, Odegaard O T, Hovig E, Loennechen T, Aasen A O, Fodstad O, Maelandsmo G M
Institute for Surgical Research, The National Hospital, Rikshospitalet, Oslo, Norway.
Cancer Res. 1999 Sep 15;59(18):4702-8.
The biological function of the metastasis-associated gene S100A4 is not fully understood, although there is evidence indicating interactions between the gene product and the cytoskeleton. We have examined whether an association could exist between S100A4 and the regulation of matrix metalloproteinases (MMPs) and their endogenous inhibitors (TIMPs). For these studies, three clones of a highly metastatic human osteosarcoma cell line (OHS) transfected with a hammerhead ribozyme directed against the S100A4 gene transcript were used. The clones demonstrated different expression levels of S100A4 and also different metastatic capacity. In the clone with the most prominent down-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 patterns of MMPs and TIMPs at the protein level. In the clones with an intermediate expression of S100A4, reduced expression of MT1-MMP and TIMP-1 was detected, whereas the expression of MMP-2 was at the same level as in the control 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-regulation of S100A4. The transwell chamber assay demonstrated that the capacity of the ribozyme-transfected cells to cross uncoated filters was reduced, relative to control cells, according to the reduction in the S100A4 expression level. The clone with the lowest reduction in S100A4 did not demonstrate different motility compared with control cells, whereas transfectants with only 5% S100A4 mRNA showed a 50% reduction in motility. Interestingly, this trend was even more striking when the capacity to cross Matrigel-coated filters was analyzed, as all the clones demonstrated between 40 and 75% reduced invasion. It is concluded that S100A4 may exert its effect on metastasis formation not only by stimulating the motility of tumor cells but also by affecting their invasive properties through influencing the expression of MMPs and their endogenous inhibitors.
转移相关基因S100A4的生物学功能尚未完全明确，尽管有证据表明该基因产物与细胞骨架之间存在相互作用。我们研究了S100A4与基质金属蛋白酶（MMPs）及其内源性抑制剂（TIMPs）的调节之间是否存在关联。在这些研究中，使用了针对S100A4基因转录本的锤头状核酶转染的高转移性人骨肉瘤细胞系（OHS）的三个克隆。这些克隆表现出不同的S100A4表达水平以及不同的转移能力。在S100A4下调最显著的克隆中，指数生长期培养物中MMP2、膜型（MT）1-MMP和TIMP-1的mRNA水平显著降低。蛋白质印迹、明胶酶谱分析和酶联免疫吸附测定显示，在蛋白质水平上，MMPs和TIMPs具有相似的表达模式。在S100A4表达中等的克隆中，检测到MT1-MMP和TIMP-1的表达降低，而MMP-2的表达与对照细胞处于同一水平。与其他因子不同，TIMP-2在所有克隆中均上调，与核酶诱导的S100A4下调程度无关。Transwell小室测定表明，相对于对照细胞，核酶转染细胞穿越未包被滤膜的能力根据S100A4表达水平的降低而降低。S100A4降低程度最低的克隆与对照细胞相比，未表现出不同的运动能力，而S100A4 mRNA仅为5%的转染细胞运动能力降低了50%。有趣的是，当分析穿越基质胶包被滤膜的能力时，这种趋势更加明显，因为所有克隆的侵袭能力均降低了40%至75%。得出的结论是，S100A4可能不仅通过刺激肿瘤细胞的运动能力，还通过影响MMPs及其内源性抑制剂的表达来影响其侵袭特性，从而对转移形成产生影响。
