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The kinase MSK1 is required for induction of c-fos by lysophosphatidic acid in mouse embryonic stem cells.

Schuck S, Soloaga A, Schratt G, Arthur JS, Nordheim A - BMC Mol. Biol. (2003)

Bottom Line: We propose that MSK1 contributes to ERK-mediated c-fos promoter activation by targeting CRE binding proteins.In addition, they indicate that MSK1 could act through CRE binding proteins to achieve c-fos promoter activation.Thus, they further our understanding of the complex regulation of the model immediate-early gene c-fos.

View Article: PubMed Central - HTML - PubMed

Affiliation: Interfakultäres Institut für Zellbiologie, Abteilung Molekularbiologie, Universität Tübingen, Auf der Morgenstelle 15, 72076 Tübingen, Germany. schuck@mpi-cbg.de

ABSTRACT

Background: The regulation of the immediate-early gene c-fos serves as a paradigm for signal-activated gene induction. Lysophosphatidic acid is a potent serum-borne mitogen able to induce c-fos.

Results: Analysing the signalling events following stimulation of mouse embryonic stem cells with serum and lysophosphatidic acid, we show that the extracellular signal-regulated kinase (ERK) pathway is involved in mediating c-fos induction. We demonstrate that the ERK-activated kinase MSK1 is required for full c-fos promoter activation, as well as for the phosphorylation of cAMP-responsive element (CRE) binding proteins. We propose that MSK1 contributes to ERK-mediated c-fos promoter activation by targeting CRE binding proteins.

Conclusion: These results show that MSK1 is an important ERK-activated mediator of mitogen-stimulated c-fos induction. In addition, they indicate that MSK1 could act through CRE binding proteins to achieve c-fos promoter activation. Thus, they further our understanding of the complex regulation of the model immediate-early gene c-fos.

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Induction of c-fos in WT ES cells. A – Cells were serum-starved for 36 h and incubated with 25 μM PD98059 during the last hour of starvation where indicated. Cells were stimulated with 15% FCS or 20 μM LPA for 30 min, followed by quantitative RT-PCR. Based on relative mRNA levels, fold inductions of c-fos were calculated from three independent experiments (FCS: 82.1 ± 27.6, FCS + PD: 43.8 ± 25.7, LPA: 63.1 ± 43.5, LPA + PD: 39.7 ± 25.2). To eliminate experiment-to-experiment variations in the induction levels and to more clearly show the effect of PD98059, induction without inhibitor was set to 100% for each individual experiment and percent c-fos induction was calculated for the samples treated with inhibitor. B – Cells were serum-starved and treated as in A. Stimulation was done for 10 min and ERK1/ERK2 phosphorylation was analysed as in Figure 1. C – Cells were serum-starved and treated as in A, except that PD98059 was replaced by 5 μM Ro318220. Fold inductions from three independent experiments were FCS: 76.2 ± 42.0, FCS + Ro: 15.7 ± 10.5, LPA: 67.9 ± 20.3, LPA + Ro: 2.8 ± 1.9. Representation of the data as in A.
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Figure 2: Induction of c-fos in WT ES cells. A – Cells were serum-starved for 36 h and incubated with 25 μM PD98059 during the last hour of starvation where indicated. Cells were stimulated with 15% FCS or 20 μM LPA for 30 min, followed by quantitative RT-PCR. Based on relative mRNA levels, fold inductions of c-fos were calculated from three independent experiments (FCS: 82.1 ± 27.6, FCS + PD: 43.8 ± 25.7, LPA: 63.1 ± 43.5, LPA + PD: 39.7 ± 25.2). To eliminate experiment-to-experiment variations in the induction levels and to more clearly show the effect of PD98059, induction without inhibitor was set to 100% for each individual experiment and percent c-fos induction was calculated for the samples treated with inhibitor. B – Cells were serum-starved and treated as in A. Stimulation was done for 10 min and ERK1/ERK2 phosphorylation was analysed as in Figure 1. C – Cells were serum-starved and treated as in A, except that PD98059 was replaced by 5 μM Ro318220. Fold inductions from three independent experiments were FCS: 76.2 ± 42.0, FCS + Ro: 15.7 ± 10.5, LPA: 67.9 ± 20.3, LPA + Ro: 2.8 ± 1.9. Representation of the data as in A.

Mentions: Given its rapid activation, the ERK pathway could be involved in c-fos induction, which causes an increase of c-fos mRNA within 10 min of serum stimulation [24]. To test this, WT ES cells were stimulated with FCS and LPA in the absence or presence of PD98059, an inhibitor of the ERK pathway [25]. Accumulation of c-fos mRNA after 30 min of stimulation was measured by quantitative RT-PCR (Figure 2a). Induction of c-fos by both stimuli was reduced in the presence of PD98059. Figure 2b shows that PD98059 inhibited ERK activation. It should be noted, however, that PD98059 did not completely block ERK activation in this cell type (data not shown), so that our data may in fact underestimate the importance of the ERK pathway. To further corroborate that the ERK pathway mediates c-fos induction by FCS and LPA, Ro318220 was used to inhibit MAPKAP-K1/RSK and MSK1 [15,26], two downstream targets of ERK1/ERK2 (Figure 2c). Induction of c-fos by both stimuli was strongly inhibited by Ro318220. Therefore, the ERK pathway is involved in c-fos induction by FCS and LPA in mouse ES cells. In addition, the inhibitory effect of Ro318220 suggests that signalling through the ERK pathway involves MAPKAP-K1 or MSK1, or both kinases.


The kinase MSK1 is required for induction of c-fos by lysophosphatidic acid in mouse embryonic stem cells.

Schuck S, Soloaga A, Schratt G, Arthur JS, Nordheim A - BMC Mol. Biol. (2003)

Induction of c-fos in WT ES cells. A – Cells were serum-starved for 36 h and incubated with 25 μM PD98059 during the last hour of starvation where indicated. Cells were stimulated with 15% FCS or 20 μM LPA for 30 min, followed by quantitative RT-PCR. Based on relative mRNA levels, fold inductions of c-fos were calculated from three independent experiments (FCS: 82.1 ± 27.6, FCS + PD: 43.8 ± 25.7, LPA: 63.1 ± 43.5, LPA + PD: 39.7 ± 25.2). To eliminate experiment-to-experiment variations in the induction levels and to more clearly show the effect of PD98059, induction without inhibitor was set to 100% for each individual experiment and percent c-fos induction was calculated for the samples treated with inhibitor. B – Cells were serum-starved and treated as in A. Stimulation was done for 10 min and ERK1/ERK2 phosphorylation was analysed as in Figure 1. C – Cells were serum-starved and treated as in A, except that PD98059 was replaced by 5 μM Ro318220. Fold inductions from three independent experiments were FCS: 76.2 ± 42.0, FCS + Ro: 15.7 ± 10.5, LPA: 67.9 ± 20.3, LPA + Ro: 2.8 ± 1.9. Representation of the data as in A.
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Figure 2: Induction of c-fos in WT ES cells. A – Cells were serum-starved for 36 h and incubated with 25 μM PD98059 during the last hour of starvation where indicated. Cells were stimulated with 15% FCS or 20 μM LPA for 30 min, followed by quantitative RT-PCR. Based on relative mRNA levels, fold inductions of c-fos were calculated from three independent experiments (FCS: 82.1 ± 27.6, FCS + PD: 43.8 ± 25.7, LPA: 63.1 ± 43.5, LPA + PD: 39.7 ± 25.2). To eliminate experiment-to-experiment variations in the induction levels and to more clearly show the effect of PD98059, induction without inhibitor was set to 100% for each individual experiment and percent c-fos induction was calculated for the samples treated with inhibitor. B – Cells were serum-starved and treated as in A. Stimulation was done for 10 min and ERK1/ERK2 phosphorylation was analysed as in Figure 1. C – Cells were serum-starved and treated as in A, except that PD98059 was replaced by 5 μM Ro318220. Fold inductions from three independent experiments were FCS: 76.2 ± 42.0, FCS + Ro: 15.7 ± 10.5, LPA: 67.9 ± 20.3, LPA + Ro: 2.8 ± 1.9. Representation of the data as in A.
Mentions: Given its rapid activation, the ERK pathway could be involved in c-fos induction, which causes an increase of c-fos mRNA within 10 min of serum stimulation [24]. To test this, WT ES cells were stimulated with FCS and LPA in the absence or presence of PD98059, an inhibitor of the ERK pathway [25]. Accumulation of c-fos mRNA after 30 min of stimulation was measured by quantitative RT-PCR (Figure 2a). Induction of c-fos by both stimuli was reduced in the presence of PD98059. Figure 2b shows that PD98059 inhibited ERK activation. It should be noted, however, that PD98059 did not completely block ERK activation in this cell type (data not shown), so that our data may in fact underestimate the importance of the ERK pathway. To further corroborate that the ERK pathway mediates c-fos induction by FCS and LPA, Ro318220 was used to inhibit MAPKAP-K1/RSK and MSK1 [15,26], two downstream targets of ERK1/ERK2 (Figure 2c). Induction of c-fos by both stimuli was strongly inhibited by Ro318220. Therefore, the ERK pathway is involved in c-fos induction by FCS and LPA in mouse ES cells. In addition, the inhibitory effect of Ro318220 suggests that signalling through the ERK pathway involves MAPKAP-K1 or MSK1, or both kinases.

Bottom Line: We propose that MSK1 contributes to ERK-mediated c-fos promoter activation by targeting CRE binding proteins.In addition, they indicate that MSK1 could act through CRE binding proteins to achieve c-fos promoter activation.Thus, they further our understanding of the complex regulation of the model immediate-early gene c-fos.

View Article: PubMed Central - HTML - PubMed

Affiliation: Interfakultäres Institut für Zellbiologie, Abteilung Molekularbiologie, Universität Tübingen, Auf der Morgenstelle 15, 72076 Tübingen, Germany. schuck@mpi-cbg.de

ABSTRACT

Background: The regulation of the immediate-early gene c-fos serves as a paradigm for signal-activated gene induction. Lysophosphatidic acid is a potent serum-borne mitogen able to induce c-fos.

Results: Analysing the signalling events following stimulation of mouse embryonic stem cells with serum and lysophosphatidic acid, we show that the extracellular signal-regulated kinase (ERK) pathway is involved in mediating c-fos induction. We demonstrate that the ERK-activated kinase MSK1 is required for full c-fos promoter activation, as well as for the phosphorylation of cAMP-responsive element (CRE) binding proteins. We propose that MSK1 contributes to ERK-mediated c-fos promoter activation by targeting CRE binding proteins.

Conclusion: These results show that MSK1 is an important ERK-activated mediator of mitogen-stimulated c-fos induction. In addition, they indicate that MSK1 could act through CRE binding proteins to achieve c-fos promoter activation. Thus, they further our understanding of the complex regulation of the model immediate-early gene c-fos.

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