Signal transduction mechanisms involved in S100A4-induced activation of the transcription factor NF-kappaB.
BACKGROUND
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.


METHODS
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.


RESULTS
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.


CONCLUSIONS
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.
Grotterød
Ida
I
Department of Tumor Biology, Institute for Cancer Research, The Norwegian Radium Hospital, Oslo University Hospital, Montebello, 0310 Oslo, Norway.
Maelandsmo
Gunhild M
GM
Boye
Kjetil
K
D000818:Animals; D015153:Blotting, Western; D045744:Cell Line, Tumor; D004305:Dose-Response Relationship, Drug; D005786:Gene Expression Regulation; D006801:Humans; D051550:I-kappa B Kinase / Q000378:metabolism; D020672:I-kappa B Proteins / Q000378:metabolism; D048728:MAP Kinase Kinase Kinase 1 / Q000235:genetics / Q000378:metabolism; D051379:Mice; D000072000:NF-KappaB Inhibitor alpha; D016328:NF-kappa B / Q000235:genetics / Q000378:metabolism*; D012516:Osteosarcoma / Q000235:genetics / Q000378:metabolism*; D010766:Phosphorylation; D047428:Protein Kinase Inhibitors / Q000494:pharmacology; D017346:Protein Serine-Threonine Kinases / Q000235:genetics / Q000378:metabolism; D051057:Proto-Oncogene Proteins c-akt / Q000378:metabolism; D034622:RNA Interference; D012333:RNA, Messenger / Q000378:metabolism; D000067759:Receptor for Advanced Glycation End Products; D011971:Receptors, Immunologic / Q000235:genetics / Q000378:metabolism; D011994:Recombinant Proteins / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D000071999:S100 Calcium-Binding Protein A4; D009418:S100 Proteins / Q000378:metabolism*; D015398:Signal Transduction / Q000187:drug effects; D014162:Transfection; D000097782:NF-kappaB-Inducing Kinase
D051550:I-kappa B Kinase; D020672:I-kappa B Proteins; D048728:MAP Kinase Kinase Kinase 1; D000072000:NF-KappaB Inhibitor alpha; D016328:NF-kappa B; D047428:Protein Kinase Inhibitors; D017346:Protein Serine-Threonine Kinases; D051057:Proto-Oncogene Proteins c-akt; D012333:RNA, Messenger; D000067759:Receptor for Advanced Glycation End Products; D011971:Receptors, Immunologic; D011994:Recombinant Proteins; D000071999:S100 Calcium-Binding Protein A4; D009418:S100 Proteins; D000097782:NF-kappaB-Inducing Kinase; C000606204:NFKBIA protein, human; C000606205:Nfkbia protein, mouse; C470944:S100a4 protein, mouse; C496559:CHUK protein, human; C496560:IKBKB protein, human; C482576:MAP3K1 protein, human