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Ribosomal protein S6 kinase (RSK)-2 as a central effector molecule in RON receptor tyrosine kinase mediated epithelial to mesenchymal transition induced by macrophage-stimulating protein.

Ma Q, Guin S, Padhye SS, Zhou YQ, Zhang RW, Wang MH - Mol. Cancer (2011)

Bottom Line: These effects relied on RON and Erk1/2 phosphorylation, which is significantly potentiated by transforming growth factor (TGF)-β1, an EMT-inducing cytokine.Specific RSK inhibitor SL0101 completely prevented MSP-induced RSK phosphorylation, which results in inhibition of MSP-induced spindle-like morphology and suppression of cell migration associated with EMT.Moreover, specific siRNA-mediated silencing of RSK2 but not RSK1 in L3.6pl pancreatic cancer cells significantly inhibited MSP-induced EMT-like phenotype and cell migration.

View Article: PubMed Central - HTML - PubMed

Affiliation: Division of Cancer Biology at State Key Laboratory for Diagnosis & Treatment of Infectious Diseases, First Affiliated Hospital, Zhejiang University College of Medicine, Hangzhou 310003, China.

ABSTRACT

Background: Epithelial to mesenchymal transition (EMT) occurs during cancer cell invasion and malignant metastasis. Features of EMT include spindle-like cell morphology, loss of epithelial cellular markers and gain of mesenchymal phenotype. Activation of the RON receptor tyrosine kinase by macrophage-stimulating protein (MSP) has been implicated in cellular EMT program; however, the major signaling determinant(s) responsible for MSP-induced EMT is unknown.

Results: The study presented here demonstrates that RSK2, a downstream signaling protein of the Ras-Erk1/2 pathway, is the principal molecule that links MSP-activated RON signaling to complete EMT. Using MDCK cells expressing RON as a model, a spindle-shape based screen was conducted, which identifies RSK2 among various intracellular proteins as a potential signaling molecule responsible for MSP-induced EMT. MSP stimulation dissociated RSK2 with Erk1/2 and promoted RSK2 nuclear translocation. MSP strongly induced RSK2 phosphorylation in a dose-dependent manner. These effects relied on RON and Erk1/2 phosphorylation, which is significantly potentiated by transforming growth factor (TGF)-β1, an EMT-inducing cytokine. Specific RSK inhibitor SL0101 completely prevented MSP-induced RSK phosphorylation, which results in inhibition of MSP-induced spindle-like morphology and suppression of cell migration associated with EMT. In HT-29 cancer cells that barely express RSK2, forced RSK2 expression results in EMT-like phenotype upon MSP stimulation. Moreover, specific siRNA-mediated silencing of RSK2 but not RSK1 in L3.6pl pancreatic cancer cells significantly inhibited MSP-induced EMT-like phenotype and cell migration.

Conclusions: MSP-induced RSK2 activation is a critical determinant linking RON signaling to cellular EMT program. Inhibition of RSK2 activity may provide a therapeutic opportunity for blocking RON-mediated cancer cell migration and subsequent invasion.

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Related in: MedlinePlus

Effect of MSP on RSK2 phosphorylation and its nuclear translocation: M-RON cells (3 × 106 cells per dish) in DMEM with 1% FBS were stimulated with MSP (2 nM), TGF-β1 (5 ng/ml), or MSP plus TGF-β1 for various times. Cellular proteins (50 μg/sample) from cell lysates were subjected to Western blot analysis using antibodies specific to regular or phospho-RSK (Ser380) and Erk1/2. A) MSP induced RSK2 phosphorylation and nuclear translocation. B) Effect of MSP and TGF-β1 on RSK2 phosphorylation and nuclear translocation. C) Immunofluorescent analysis of MSP or MSP plus TGF-β1-induced RSK2 nuclear translocation. M-RON cells were stimulated with MSP or MSP plus TGF-β1v for various times. Cells were fixed with cold acetone, blocked with 1% BSA, followed by incubation with antibody specific to RSK2. FITC coupled rabbit anti-mouse IgG was used as the detecting antibody. Immunofluorescence was observed by Olympus BT71 microscope equipped with fluorescent apparatus as previously described [45]. Scale bars represent 5 μm.
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Figure 3: Effect of MSP on RSK2 phosphorylation and its nuclear translocation: M-RON cells (3 × 106 cells per dish) in DMEM with 1% FBS were stimulated with MSP (2 nM), TGF-β1 (5 ng/ml), or MSP plus TGF-β1 for various times. Cellular proteins (50 μg/sample) from cell lysates were subjected to Western blot analysis using antibodies specific to regular or phospho-RSK (Ser380) and Erk1/2. A) MSP induced RSK2 phosphorylation and nuclear translocation. B) Effect of MSP and TGF-β1 on RSK2 phosphorylation and nuclear translocation. C) Immunofluorescent analysis of MSP or MSP plus TGF-β1-induced RSK2 nuclear translocation. M-RON cells were stimulated with MSP or MSP plus TGF-β1v for various times. Cells were fixed with cold acetone, blocked with 1% BSA, followed by incubation with antibody specific to RSK2. FITC coupled rabbit anti-mouse IgG was used as the detecting antibody. Immunofluorescence was observed by Olympus BT71 microscope equipped with fluorescent apparatus as previously described [45]. Scale bars represent 5 μm.

Mentions: To further determine the effect of MSP on RSK2, we studied RSK2 nuclear translocation in comparison with Erk1/2 activation. Cells were stimulated by MSP or MSP plus TGF-β1 for various times and cytoplasmic and nuclear proteins were prepared. RSK2 was mainly detected in cytoplasmic fraction in non-stimulated M-RON cells. A small amount of RSK2 was also present in nuclear proteins (Figure 3A). This pattern was similar to that of Erk1/2, in which Erk1/2 in both cytoplasmic and nuclear fractions was observed. Upon MSP stimulation, the amounts of RSK in nuclear fraction were dramatically increased in a time-dependent manner. Phosphorylation was observed not only in cytosolic but also in nuclear RSK2. Again, a similar pattern was documented for Erk1/2, in which phosphorylated Erk1/2 was detected in nuclear proteins. Results in Figure 3B demonstrated that MSP in combination with TGF-β1 induced RSK2 nuclear translocation and phosphorylation. This effect was accompanied by Erk1/2 phosphorylation. A major difference was that the time course for both RSK2 and Erk1/2 phosphorylation lasted longer in MSP and TGF-β1 co-stimulated cells than in cell treated with MSP alone.


Ribosomal protein S6 kinase (RSK)-2 as a central effector molecule in RON receptor tyrosine kinase mediated epithelial to mesenchymal transition induced by macrophage-stimulating protein.

Ma Q, Guin S, Padhye SS, Zhou YQ, Zhang RW, Wang MH - Mol. Cancer (2011)

Effect of MSP on RSK2 phosphorylation and its nuclear translocation: M-RON cells (3 × 106 cells per dish) in DMEM with 1% FBS were stimulated with MSP (2 nM), TGF-β1 (5 ng/ml), or MSP plus TGF-β1 for various times. Cellular proteins (50 μg/sample) from cell lysates were subjected to Western blot analysis using antibodies specific to regular or phospho-RSK (Ser380) and Erk1/2. A) MSP induced RSK2 phosphorylation and nuclear translocation. B) Effect of MSP and TGF-β1 on RSK2 phosphorylation and nuclear translocation. C) Immunofluorescent analysis of MSP or MSP plus TGF-β1-induced RSK2 nuclear translocation. M-RON cells were stimulated with MSP or MSP plus TGF-β1v for various times. Cells were fixed with cold acetone, blocked with 1% BSA, followed by incubation with antibody specific to RSK2. FITC coupled rabbit anti-mouse IgG was used as the detecting antibody. Immunofluorescence was observed by Olympus BT71 microscope equipped with fluorescent apparatus as previously described [45]. Scale bars represent 5 μm.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3117816&req=5

Figure 3: Effect of MSP on RSK2 phosphorylation and its nuclear translocation: M-RON cells (3 × 106 cells per dish) in DMEM with 1% FBS were stimulated with MSP (2 nM), TGF-β1 (5 ng/ml), or MSP plus TGF-β1 for various times. Cellular proteins (50 μg/sample) from cell lysates were subjected to Western blot analysis using antibodies specific to regular or phospho-RSK (Ser380) and Erk1/2. A) MSP induced RSK2 phosphorylation and nuclear translocation. B) Effect of MSP and TGF-β1 on RSK2 phosphorylation and nuclear translocation. C) Immunofluorescent analysis of MSP or MSP plus TGF-β1-induced RSK2 nuclear translocation. M-RON cells were stimulated with MSP or MSP plus TGF-β1v for various times. Cells were fixed with cold acetone, blocked with 1% BSA, followed by incubation with antibody specific to RSK2. FITC coupled rabbit anti-mouse IgG was used as the detecting antibody. Immunofluorescence was observed by Olympus BT71 microscope equipped with fluorescent apparatus as previously described [45]. Scale bars represent 5 μm.
Mentions: To further determine the effect of MSP on RSK2, we studied RSK2 nuclear translocation in comparison with Erk1/2 activation. Cells were stimulated by MSP or MSP plus TGF-β1 for various times and cytoplasmic and nuclear proteins were prepared. RSK2 was mainly detected in cytoplasmic fraction in non-stimulated M-RON cells. A small amount of RSK2 was also present in nuclear proteins (Figure 3A). This pattern was similar to that of Erk1/2, in which Erk1/2 in both cytoplasmic and nuclear fractions was observed. Upon MSP stimulation, the amounts of RSK in nuclear fraction were dramatically increased in a time-dependent manner. Phosphorylation was observed not only in cytosolic but also in nuclear RSK2. Again, a similar pattern was documented for Erk1/2, in which phosphorylated Erk1/2 was detected in nuclear proteins. Results in Figure 3B demonstrated that MSP in combination with TGF-β1 induced RSK2 nuclear translocation and phosphorylation. This effect was accompanied by Erk1/2 phosphorylation. A major difference was that the time course for both RSK2 and Erk1/2 phosphorylation lasted longer in MSP and TGF-β1 co-stimulated cells than in cell treated with MSP alone.

Bottom Line: These effects relied on RON and Erk1/2 phosphorylation, which is significantly potentiated by transforming growth factor (TGF)-β1, an EMT-inducing cytokine.Specific RSK inhibitor SL0101 completely prevented MSP-induced RSK phosphorylation, which results in inhibition of MSP-induced spindle-like morphology and suppression of cell migration associated with EMT.Moreover, specific siRNA-mediated silencing of RSK2 but not RSK1 in L3.6pl pancreatic cancer cells significantly inhibited MSP-induced EMT-like phenotype and cell migration.

View Article: PubMed Central - HTML - PubMed

Affiliation: Division of Cancer Biology at State Key Laboratory for Diagnosis & Treatment of Infectious Diseases, First Affiliated Hospital, Zhejiang University College of Medicine, Hangzhou 310003, China.

ABSTRACT

Background: Epithelial to mesenchymal transition (EMT) occurs during cancer cell invasion and malignant metastasis. Features of EMT include spindle-like cell morphology, loss of epithelial cellular markers and gain of mesenchymal phenotype. Activation of the RON receptor tyrosine kinase by macrophage-stimulating protein (MSP) has been implicated in cellular EMT program; however, the major signaling determinant(s) responsible for MSP-induced EMT is unknown.

Results: The study presented here demonstrates that RSK2, a downstream signaling protein of the Ras-Erk1/2 pathway, is the principal molecule that links MSP-activated RON signaling to complete EMT. Using MDCK cells expressing RON as a model, a spindle-shape based screen was conducted, which identifies RSK2 among various intracellular proteins as a potential signaling molecule responsible for MSP-induced EMT. MSP stimulation dissociated RSK2 with Erk1/2 and promoted RSK2 nuclear translocation. MSP strongly induced RSK2 phosphorylation in a dose-dependent manner. These effects relied on RON and Erk1/2 phosphorylation, which is significantly potentiated by transforming growth factor (TGF)-β1, an EMT-inducing cytokine. Specific RSK inhibitor SL0101 completely prevented MSP-induced RSK phosphorylation, which results in inhibition of MSP-induced spindle-like morphology and suppression of cell migration associated with EMT. In HT-29 cancer cells that barely express RSK2, forced RSK2 expression results in EMT-like phenotype upon MSP stimulation. Moreover, specific siRNA-mediated silencing of RSK2 but not RSK1 in L3.6pl pancreatic cancer cells significantly inhibited MSP-induced EMT-like phenotype and cell migration.

Conclusions: MSP-induced RSK2 activation is a critical determinant linking RON signaling to cellular EMT program. Inhibition of RSK2 activity may provide a therapeutic opportunity for blocking RON-mediated cancer cell migration and subsequent invasion.

Show MeSH
Related in: MedlinePlus