Limits...
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.

Show MeSH

Related in: MedlinePlus

S100A4 target gene expression is reduced by H-7 and staurosporine. Real-time RT-PCR demonstrating dose dependent inhibition of ephrin-A1 mRNA (A and C) and optineurin mRNA (B and D) expression in II-11b cells stimulated for 2 hours with 2 μM S100A4 in presence of the indicated concentrations of H-7 (A and B) or staurosporine (C and D). Bars represent mean values ± S.E of three independent experiments. St = staurosporine. *, p < 0.05
© Copyright Policy - open-access
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC2902441&req=5

Figure 4: S100A4 target gene expression is reduced by H-7 and staurosporine. Real-time RT-PCR demonstrating dose dependent inhibition of ephrin-A1 mRNA (A and C) and optineurin mRNA (B and D) expression in II-11b cells stimulated for 2 hours with 2 μM S100A4 in presence of the indicated concentrations of H-7 (A and B) or staurosporine (C and D). Bars represent mean values ± S.E of three independent experiments. St = staurosporine. *, p < 0.05

Mentions: We have previously reported that S100A4 stimulates NF-κB activity through the classical activation pathway in the II-11b cell line, by demonstrating increased phosphorylation of IκBα [11]. To investigate the upstream signal transduction mechanisms involved in S100A4-induced NF-κB activation, II-11b cells were treated with inhibitors of various signal transduction pathways: Ser/Thr kinases (H-7 and staurosporine), phospholipase C (U-73122 and compound 48/80), protein tyrosine kinases (AG 18 and genistein), protein kinase C (GF 109203X), G-protein coupled receptors (GDPβ and suramin) and phosphatidylinositol 3-kinases (PI 3-kinases; LY294002). The levels of phosphorylated IκBα were used to measure activity in the NF-κB pathway. The Ser/Thr kinase inhibitors H-7 and staurosporine reduced IκBα phosphorylation levels in a dose dependent manner (Fig. 1A and 1B). A partial inhibitory effect was observed with genistein and compound 48/80 at the highest concentrations, whereas no inhibition was observed with the other signal transduction inhibitors (Fig. 1C-J). Based on these initial experiments, H-7 and staurosporine were chosen for further studies. As previously shown [11], total IκBα expression levels were reduced upon treatment with S100A4 (Fig. 2A and 2B; compare lanes 1 and 2). In S100A4-stimulated cells, increasing concentrations of H-7 or staurosporine resulted in decreased levels of IκBα (Fig. 2A and 2B; compare lanes 4, 6, 8 and 10 with lane 2). IκBα expression was also reduced in cells treated with staurosporine alone. Importantly, H-7 and staurosporine displayed a potent and dose dependent inhibition of S100A4-induced NF-κB activation in a luciferase based activity assay, both using II-11b cells (Fig. 3A and 3B) and the human osteosarcoma cell line KPDX (Fig. 3C). Recently, we demonstrated that S100A4 augmented expression of ephrin-A1 and optineurin in II-11b cells, and that the induction was dependent on NF-κB activity [11]. Addition of H-7 or staurosporine suppressed S100A4-induced expression of these target genes in a dose dependent manner (Fig. 4). Taken together, these findings indicate that Ser/Thr kinases are central players 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)

S100A4 target gene expression is reduced by H-7 and staurosporine. Real-time RT-PCR demonstrating dose dependent inhibition of ephrin-A1 mRNA (A and C) and optineurin mRNA (B and D) expression in II-11b cells stimulated for 2 hours with 2 μM S100A4 in presence of the indicated concentrations of H-7 (A and B) or staurosporine (C and D). Bars represent mean values ± S.E of three independent experiments. St = staurosporine. *, p < 0.05
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 4: S100A4 target gene expression is reduced by H-7 and staurosporine. Real-time RT-PCR demonstrating dose dependent inhibition of ephrin-A1 mRNA (A and C) and optineurin mRNA (B and D) expression in II-11b cells stimulated for 2 hours with 2 μM S100A4 in presence of the indicated concentrations of H-7 (A and B) or staurosporine (C and D). Bars represent mean values ± S.E of three independent experiments. St = staurosporine. *, p < 0.05
Mentions: We have previously reported that S100A4 stimulates NF-κB activity through the classical activation pathway in the II-11b cell line, by demonstrating increased phosphorylation of IκBα [11]. To investigate the upstream signal transduction mechanisms involved in S100A4-induced NF-κB activation, II-11b cells were treated with inhibitors of various signal transduction pathways: Ser/Thr kinases (H-7 and staurosporine), phospholipase C (U-73122 and compound 48/80), protein tyrosine kinases (AG 18 and genistein), protein kinase C (GF 109203X), G-protein coupled receptors (GDPβ and suramin) and phosphatidylinositol 3-kinases (PI 3-kinases; LY294002). The levels of phosphorylated IκBα were used to measure activity in the NF-κB pathway. The Ser/Thr kinase inhibitors H-7 and staurosporine reduced IκBα phosphorylation levels in a dose dependent manner (Fig. 1A and 1B). A partial inhibitory effect was observed with genistein and compound 48/80 at the highest concentrations, whereas no inhibition was observed with the other signal transduction inhibitors (Fig. 1C-J). Based on these initial experiments, H-7 and staurosporine were chosen for further studies. As previously shown [11], total IκBα expression levels were reduced upon treatment with S100A4 (Fig. 2A and 2B; compare lanes 1 and 2). In S100A4-stimulated cells, increasing concentrations of H-7 or staurosporine resulted in decreased levels of IκBα (Fig. 2A and 2B; compare lanes 4, 6, 8 and 10 with lane 2). IκBα expression was also reduced in cells treated with staurosporine alone. Importantly, H-7 and staurosporine displayed a potent and dose dependent inhibition of S100A4-induced NF-κB activation in a luciferase based activity assay, both using II-11b cells (Fig. 3A and 3B) and the human osteosarcoma cell line KPDX (Fig. 3C). Recently, we demonstrated that S100A4 augmented expression of ephrin-A1 and optineurin in II-11b cells, and that the induction was dependent on NF-κB activity [11]. Addition of H-7 or staurosporine suppressed S100A4-induced expression of these target genes in a dose dependent manner (Fig. 4). Taken together, these findings indicate that Ser/Thr kinases are central players 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