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Calcium-dependent regulation of the cell cycle via a novel MAPK--NF-kappaB pathway in Swiss 3T3 cells.

Sée V, Rajala NK, Spiller DG, White MR - J. Cell Biol. (2004)

Bottom Line: Nuclear factor kappa B (NF-kappaB) has been implicated in the regulation of cell proliferation and transformation.We further showed that the serum-induced mitogen-activated protein kinase (MAPK) phosphorylation is calcium dependent.These data suggest that a serum-dependent calcium signal regulates the cell cycle via a MAPK--NF-kappaB pathway in Swiss 3T3 cells.

View Article: PubMed Central - PubMed

Affiliation: Centre for Cell Imaging, School of Biological Sciences, University of Liverpool, Liverpool L69 7ZB, England, UK.

ABSTRACT
Nuclear factor kappa B (NF-kappaB) has been implicated in the regulation of cell proliferation and transformation. We investigated the role of the serum-induced intracellular calcium increase in the NF-kappaB--dependent cell cycle progression in Swiss 3T3 fibroblasts. Noninvasive photoactivation of a calcium chelator (Diazo-2) was used to specifically disrupt the transient rise in calcium induced by serum stimulation of starved Swiss 3T3 cells. The serum-induced intracellular calcium peak was essential for subsequent NF-kappaB activation (measured by real-time imaging of the dynamic p65 and IkappaBalpha fluorescent fusion proteins), cyclin D1 (CD1) promoter-directed transcription (measured by real-time luminescence imaging of CD1 promoter-directed firefly luciferase activity), and progression to cell division. We further showed that the serum-induced mitogen-activated protein kinase (MAPK) phosphorylation is calcium dependent. Inhibition of the MAPK- but not the PtdIns3K-dependent pathway inhibited NF-kappaB signaling, and further, CD1 transcription and cell cycle progression. These data suggest that a serum-dependent calcium signal regulates the cell cycle via a MAPK--NF-kappaB pathway in Swiss 3T3 cells.

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p42/p44MAPK activation is determinant for CD1 transcription and further cell cycle progression. (A) Swiss 3T3 cells were transfected with the indicated reporter vectors (NF-κB luc and −1745 CD1-luc). 24 h after serum starvation, cells were stimulated or not (ct) with 10% FCS in the absence or presence of MAPK inhibitor (PD98059, 50 μM; 20-min preincubation) or Akt inhibitor (100 nM wortmannin, 20-min preincubation) as indicated. 6 h after serum stimulation, in vitro luciferase activity was measured. Histograms are means ± SEM of triplicate values. Each experiment was performed three times. (B) Swiss 3T3 fibroblasts were serum starved for 24 h. 24 h after serum starvation, the cells were stimulated with 10% FCS for 18 h in the absence (ct) or presence of PD98059 (50 μM, 20-min preincubation). Cell cycle stage was determined by flow cytometric DNA histogram analysis of propidium iodide–stained cells.
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fig8: p42/p44MAPK activation is determinant for CD1 transcription and further cell cycle progression. (A) Swiss 3T3 cells were transfected with the indicated reporter vectors (NF-κB luc and −1745 CD1-luc). 24 h after serum starvation, cells were stimulated or not (ct) with 10% FCS in the absence or presence of MAPK inhibitor (PD98059, 50 μM; 20-min preincubation) or Akt inhibitor (100 nM wortmannin, 20-min preincubation) as indicated. 6 h after serum stimulation, in vitro luciferase activity was measured. Histograms are means ± SEM of triplicate values. Each experiment was performed three times. (B) Swiss 3T3 fibroblasts were serum starved for 24 h. 24 h after serum starvation, the cells were stimulated with 10% FCS for 18 h in the absence (ct) or presence of PD98059 (50 μM, 20-min preincubation). Cell cycle stage was determined by flow cytometric DNA histogram analysis of propidium iodide–stained cells.

Mentions: The key role of p42/p44MAPK in NF-κB-dependent cell cycle progression was further investigated by studying its role in gene transcription and cell cycle progression. We tested the effects of both PD98059 and wortmannin on consensus NF-κB and CD1 promoter activity. PD98059 was found to block the serum-induced NF-κB–Luc and CD1-Luc reporter activity (Fig. 8 A), whereas the PtdIns3k inhibitor wortmannin gave rise to only weak inhibition of gene expression. The effect on cell cycle progression was further investigated by flow cytometer analysis. In the presence of the MEK inhibitor PD98059, 22% of cells were found to be in S-phase after 18 h serum stimulation, compared to the control without inhibitor, where 46% of cells were in S-phase (Fig. 8 B). These data support the concept that p42/p44MAPK is a key regulator of NF-κB–dependent gene transcription (specifically CD1 transcription) and subsequent cell cycle progression.


Calcium-dependent regulation of the cell cycle via a novel MAPK--NF-kappaB pathway in Swiss 3T3 cells.

Sée V, Rajala NK, Spiller DG, White MR - J. Cell Biol. (2004)

p42/p44MAPK activation is determinant for CD1 transcription and further cell cycle progression. (A) Swiss 3T3 cells were transfected with the indicated reporter vectors (NF-κB luc and −1745 CD1-luc). 24 h after serum starvation, cells were stimulated or not (ct) with 10% FCS in the absence or presence of MAPK inhibitor (PD98059, 50 μM; 20-min preincubation) or Akt inhibitor (100 nM wortmannin, 20-min preincubation) as indicated. 6 h after serum stimulation, in vitro luciferase activity was measured. Histograms are means ± SEM of triplicate values. Each experiment was performed three times. (B) Swiss 3T3 fibroblasts were serum starved for 24 h. 24 h after serum starvation, the cells were stimulated with 10% FCS for 18 h in the absence (ct) or presence of PD98059 (50 μM, 20-min preincubation). Cell cycle stage was determined by flow cytometric DNA histogram analysis of propidium iodide–stained cells.
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Related In: Results  -  Collection

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fig8: p42/p44MAPK activation is determinant for CD1 transcription and further cell cycle progression. (A) Swiss 3T3 cells were transfected with the indicated reporter vectors (NF-κB luc and −1745 CD1-luc). 24 h after serum starvation, cells were stimulated or not (ct) with 10% FCS in the absence or presence of MAPK inhibitor (PD98059, 50 μM; 20-min preincubation) or Akt inhibitor (100 nM wortmannin, 20-min preincubation) as indicated. 6 h after serum stimulation, in vitro luciferase activity was measured. Histograms are means ± SEM of triplicate values. Each experiment was performed three times. (B) Swiss 3T3 fibroblasts were serum starved for 24 h. 24 h after serum starvation, the cells were stimulated with 10% FCS for 18 h in the absence (ct) or presence of PD98059 (50 μM, 20-min preincubation). Cell cycle stage was determined by flow cytometric DNA histogram analysis of propidium iodide–stained cells.
Mentions: The key role of p42/p44MAPK in NF-κB-dependent cell cycle progression was further investigated by studying its role in gene transcription and cell cycle progression. We tested the effects of both PD98059 and wortmannin on consensus NF-κB and CD1 promoter activity. PD98059 was found to block the serum-induced NF-κB–Luc and CD1-Luc reporter activity (Fig. 8 A), whereas the PtdIns3k inhibitor wortmannin gave rise to only weak inhibition of gene expression. The effect on cell cycle progression was further investigated by flow cytometer analysis. In the presence of the MEK inhibitor PD98059, 22% of cells were found to be in S-phase after 18 h serum stimulation, compared to the control without inhibitor, where 46% of cells were in S-phase (Fig. 8 B). These data support the concept that p42/p44MAPK is a key regulator of NF-κB–dependent gene transcription (specifically CD1 transcription) and subsequent cell cycle progression.

Bottom Line: Nuclear factor kappa B (NF-kappaB) has been implicated in the regulation of cell proliferation and transformation.We further showed that the serum-induced mitogen-activated protein kinase (MAPK) phosphorylation is calcium dependent.These data suggest that a serum-dependent calcium signal regulates the cell cycle via a MAPK--NF-kappaB pathway in Swiss 3T3 cells.

View Article: PubMed Central - PubMed

Affiliation: Centre for Cell Imaging, School of Biological Sciences, University of Liverpool, Liverpool L69 7ZB, England, UK.

ABSTRACT
Nuclear factor kappa B (NF-kappaB) has been implicated in the regulation of cell proliferation and transformation. We investigated the role of the serum-induced intracellular calcium increase in the NF-kappaB--dependent cell cycle progression in Swiss 3T3 fibroblasts. Noninvasive photoactivation of a calcium chelator (Diazo-2) was used to specifically disrupt the transient rise in calcium induced by serum stimulation of starved Swiss 3T3 cells. The serum-induced intracellular calcium peak was essential for subsequent NF-kappaB activation (measured by real-time imaging of the dynamic p65 and IkappaBalpha fluorescent fusion proteins), cyclin D1 (CD1) promoter-directed transcription (measured by real-time luminescence imaging of CD1 promoter-directed firefly luciferase activity), and progression to cell division. We further showed that the serum-induced mitogen-activated protein kinase (MAPK) phosphorylation is calcium dependent. Inhibition of the MAPK- but not the PtdIns3K-dependent pathway inhibited NF-kappaB signaling, and further, CD1 transcription and cell cycle progression. These data suggest that a serum-dependent calcium signal regulates the cell cycle via a MAPK--NF-kappaB pathway in Swiss 3T3 cells.

Show MeSH
Related in: MedlinePlus