The metastasis-promoting protein S100A4 activates the transcription factor NF-kappaB through the classical NF-kappaB activation pathway. The upstream signal transduction mechanisms leading to increased NF-kappaB activity are, however, incompletely characterized.

The human osteosarcoma cell line II-11b was stimulated with recombinant S100A4 in the presence or absence of inhibitors of common signal transduction pathways, and NF-kappaB activity was examined using a luciferase-based reporter assay and phosphorylation of IkappaBalpha. mRNA expression was analyzed by real-time RT-PCR, protein expression was examined by Western blotting and IKK activity was measured using an in vitro kinase assay. The role of upstream kinases and the cell surface receptor RAGE was investigated by overexpression of dominant negative proteins and by siRNA transfection.

The Ser/Thr kinase inhibitors H-7 and staurosporine inhibited S100A4-induced IkappaBalpha phosphorylation and subsequent NF-kappaB activation. The protein tyrosine kinase inhibitor genistein and the phospholipase C inhibitor compound 48/80 had a partial inhibitory effect on IkappaBalpha phosphorylation, whereas inhibitors of protein kinase C, G-protein coupled receptors and PI 3-kinases had no effect on the level of phosphorylation. Interestingly, S100A4 treatment induced activating phosphorylations of IKKalpha/beta, but neither H-7 nor staurosporine was able to significantly inhibit IKK activation. Dominant negative MEKK1 or NIK did not inhibit S100A4-induced NF-kappaB activity, and S100A4 stimulation did not influence AKT phosphorylation. Furthermore, diminished expression of the putative S100 protein receptor RAGE did not affect the observed phosphorylation of IkappaBalpha.

S100A4 activates NF-kappaB by inducing phosphorylation of IKKalpha/beta, leading to increased IkappaBalpha phosphorylation. The Ser/Thr kinase inhibitors H-7 and staurosporine attenuated S100A4-induced NF-kappaB activation and inhibited IKK-mediated phosphorylation of IkappaBalpha. S100A4-induced NF-kappaB activation was independent of the putative S100 protein receptor RAGE and the Ser/Thr kinases MEKK1, NIK and AKT. These findings lead to increased understanding of S100A4 signaling, which may contribute to the identification of novel targets for anti-metastatic therapy.

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