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

Grotterød I, Maelandsmo GM, Boye K - BMC Cancer (2010)

Bottom Line: 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.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Tumor Biology, Institute for Cancer Research, The Norwegian Radium Hospital, Oslo University Hospital, Montebello, 0310 Oslo, Norway.

ABSTRACT

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.

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RAGE-independent NF-κB activation upon S100A4-stimulation of II-11b cells. A. Western blot showing reduced RAGE protein expression by siRNA transfection. 48 hours after transfection the cells were stimulated with S100A4 for one hour and cell lysates subjected to immunoblotting using anti-phospho-IκBα. The results shown are representative of three independent experiments. siNC = siRNA negative control. B. Western blot showing RAGE expression in a panel of cell lines. NF-κB activation refers to the levels of S100A4-induced NF-κB activation demonstrated in a previous study [11], except for osteoblasts, where it refers to IκBα phosphorylation levels shown in Fig. 7C. C. Western blot showing IκBα phosphorylation in human osteoblasts after S100A4 stimulation for the indicated time periods. α-tubulin was used as loading control.
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Figure 7: RAGE-independent NF-κB activation upon S100A4-stimulation of II-11b cells. A. Western blot showing reduced RAGE protein expression by siRNA transfection. 48 hours after transfection the cells were stimulated with S100A4 for one hour and cell lysates subjected to immunoblotting using anti-phospho-IκBα. The results shown are representative of three independent experiments. siNC = siRNA negative control. B. Western blot showing RAGE expression in a panel of cell lines. NF-κB activation refers to the levels of S100A4-induced NF-κB activation demonstrated in a previous study [11], except for osteoblasts, where it refers to IκBα phosphorylation levels shown in Fig. 7C. C. Western blot showing IκBα phosphorylation in human osteoblasts after S100A4 stimulation for the indicated time periods. α-tubulin was used as loading control.

Mentions: RAGE has been suggested as receptor for several S100 proteins. In an attempt to investigate the possible role of RAGE in S100A4-induced NF-κB signaling, siRNA molecules targeting RAGE mRNA were utilized. Fig. 7A shows that S100A4 induces phosphorylation of IκBα to the same extent even with RAGE expression levels substantially reduced by siRNA transfection. Furthermore, RAGE expression in a panel of cell lines previously analyzed for NF-κB activation [11] was investigated, and no association between RAGE levels and S100A4-induced NF-κB activation was observed (Fig. 7B). Finally, S100A4-mediated phosphorylation of IκBα was detected in human osteoblasts expressing low levels of RAGE (Fig. 7C). Altogether, these results indicate that RAGE is not involved in S100A4-induced NF-κB activation.


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

Grotterød I, Maelandsmo GM, Boye K - BMC Cancer (2010)

RAGE-independent NF-κB activation upon S100A4-stimulation of II-11b cells. A. Western blot showing reduced RAGE protein expression by siRNA transfection. 48 hours after transfection the cells were stimulated with S100A4 for one hour and cell lysates subjected to immunoblotting using anti-phospho-IκBα. The results shown are representative of three independent experiments. siNC = siRNA negative control. B. Western blot showing RAGE expression in a panel of cell lines. NF-κB activation refers to the levels of S100A4-induced NF-κB activation demonstrated in a previous study [11], except for osteoblasts, where it refers to IκBα phosphorylation levels shown in Fig. 7C. C. Western blot showing IκBα phosphorylation in human osteoblasts after S100A4 stimulation for the indicated time periods. α-tubulin was used as loading control.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2902441&req=5

Figure 7: RAGE-independent NF-κB activation upon S100A4-stimulation of II-11b cells. A. Western blot showing reduced RAGE protein expression by siRNA transfection. 48 hours after transfection the cells were stimulated with S100A4 for one hour and cell lysates subjected to immunoblotting using anti-phospho-IκBα. The results shown are representative of three independent experiments. siNC = siRNA negative control. B. Western blot showing RAGE expression in a panel of cell lines. NF-κB activation refers to the levels of S100A4-induced NF-κB activation demonstrated in a previous study [11], except for osteoblasts, where it refers to IκBα phosphorylation levels shown in Fig. 7C. C. Western blot showing IκBα phosphorylation in human osteoblasts after S100A4 stimulation for the indicated time periods. α-tubulin was used as loading control.
Mentions: RAGE has been suggested as receptor for several S100 proteins. In an attempt to investigate the possible role of RAGE in S100A4-induced NF-κB signaling, siRNA molecules targeting RAGE mRNA were utilized. Fig. 7A shows that S100A4 induces phosphorylation of IκBα to the same extent even with RAGE expression levels substantially reduced by siRNA transfection. Furthermore, RAGE expression in a panel of cell lines previously analyzed for NF-κB activation [11] was investigated, and no association between RAGE levels and S100A4-induced NF-κB activation was observed (Fig. 7B). Finally, S100A4-mediated phosphorylation of IκBα was detected in human osteoblasts expressing low levels of RAGE (Fig. 7C). Altogether, these results indicate that RAGE is not involved in S100A4-induced NF-κB activation.

Bottom Line: 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.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Tumor Biology, Institute for Cancer Research, The Norwegian Radium Hospital, Oslo University Hospital, Montebello, 0310 Oslo, Norway.

ABSTRACT

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.

Show MeSH
Related in: MedlinePlus