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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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NF-κB dependent transcription is essential for cell cycle progression. (A) Swiss 3T3 fibroblasts were serum starved for 24 h. They were then stimulated with 10% FCS for 18 h in the absence or presence of an NF-κB inhibitor (5 μM Bay117082). Cell cycle stage was determined by flow cytometry analysis of propidium iodide–stained cells. (B) Swiss 3T3 cells were transfected with the indicated reporter vectors (NF-κB luc and −1745 CD1-luc). 24 h after serum starvation, firefly luciferin (Biosynth) was added to the medium (0.5-mM final concentration). 2 h later, serum stimulation was performed and luminescence imaging was carried out using a Hamamatsu 4880-65 liquid nitrogen–cooled CCD camera. Images were acquired using 30-min integration times. (C) RT-PCR analysis was performed using primers specific to CD1 or cyclophilin A (control), with total RNA prepared from cells stimulated with 10% FCS at indicated time points. (D) RT-PCR analysis, with total RNA prepared from nonstimulated starved cells (ct) or cells stimulated 8 h with 10% FCS in the presence or absence of Bay117082.
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fig1: NF-κB dependent transcription is essential for cell cycle progression. (A) Swiss 3T3 fibroblasts were serum starved for 24 h. They were then stimulated with 10% FCS for 18 h in the absence or presence of an NF-κB inhibitor (5 μM Bay117082). Cell cycle stage was determined by flow cytometry analysis of propidium iodide–stained cells. (B) Swiss 3T3 cells were transfected with the indicated reporter vectors (NF-κB luc and −1745 CD1-luc). 24 h after serum starvation, firefly luciferin (Biosynth) was added to the medium (0.5-mM final concentration). 2 h later, serum stimulation was performed and luminescence imaging was carried out using a Hamamatsu 4880-65 liquid nitrogen–cooled CCD camera. Images were acquired using 30-min integration times. (C) RT-PCR analysis was performed using primers specific to CD1 or cyclophilin A (control), with total RNA prepared from cells stimulated with 10% FCS at indicated time points. (D) RT-PCR analysis, with total RNA prepared from nonstimulated starved cells (ct) or cells stimulated 8 h with 10% FCS in the presence or absence of Bay117082.

Mentions: We investigated whether NF-κB regulates cell cycle entry and CD1 transcription in Swiss 3T3 cells. Serum starvation induced significant cell cycle arrest in 3T3 cells. 84% of cells were in G0/G1 phase and 11% in S-phase 24 h after serum starvation (Fig. 1 A). 18 h after serum stimulation, 44% of the cells were in S-phase. This indicated that, as expected, serum stimulation induced cell cycle progression in 3T3 cells. In the presence of an NF-κB inhibitor, Bay117082, only 23% of cells were found to be in S-phase 18 h after serum stimulation. Therefore, these data provided preliminary evidence that NF-κB activation is a key step for cell cycle progression in 3T3 fibroblasts.


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)

NF-κB dependent transcription is essential for cell cycle progression. (A) Swiss 3T3 fibroblasts were serum starved for 24 h. They were then stimulated with 10% FCS for 18 h in the absence or presence of an NF-κB inhibitor (5 μM Bay117082). Cell cycle stage was determined by flow cytometry analysis of propidium iodide–stained cells. (B) Swiss 3T3 cells were transfected with the indicated reporter vectors (NF-κB luc and −1745 CD1-luc). 24 h after serum starvation, firefly luciferin (Biosynth) was added to the medium (0.5-mM final concentration). 2 h later, serum stimulation was performed and luminescence imaging was carried out using a Hamamatsu 4880-65 liquid nitrogen–cooled CCD camera. Images were acquired using 30-min integration times. (C) RT-PCR analysis was performed using primers specific to CD1 or cyclophilin A (control), with total RNA prepared from cells stimulated with 10% FCS at indicated time points. (D) RT-PCR analysis, with total RNA prepared from nonstimulated starved cells (ct) or cells stimulated 8 h with 10% FCS in the presence or absence of Bay117082.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2172420&req=5

fig1: NF-κB dependent transcription is essential for cell cycle progression. (A) Swiss 3T3 fibroblasts were serum starved for 24 h. They were then stimulated with 10% FCS for 18 h in the absence or presence of an NF-κB inhibitor (5 μM Bay117082). Cell cycle stage was determined by flow cytometry analysis of propidium iodide–stained cells. (B) Swiss 3T3 cells were transfected with the indicated reporter vectors (NF-κB luc and −1745 CD1-luc). 24 h after serum starvation, firefly luciferin (Biosynth) was added to the medium (0.5-mM final concentration). 2 h later, serum stimulation was performed and luminescence imaging was carried out using a Hamamatsu 4880-65 liquid nitrogen–cooled CCD camera. Images were acquired using 30-min integration times. (C) RT-PCR analysis was performed using primers specific to CD1 or cyclophilin A (control), with total RNA prepared from cells stimulated with 10% FCS at indicated time points. (D) RT-PCR analysis, with total RNA prepared from nonstimulated starved cells (ct) or cells stimulated 8 h with 10% FCS in the presence or absence of Bay117082.
Mentions: We investigated whether NF-κB regulates cell cycle entry and CD1 transcription in Swiss 3T3 cells. Serum starvation induced significant cell cycle arrest in 3T3 cells. 84% of cells were in G0/G1 phase and 11% in S-phase 24 h after serum starvation (Fig. 1 A). 18 h after serum stimulation, 44% of the cells were in S-phase. This indicated that, as expected, serum stimulation induced cell cycle progression in 3T3 cells. In the presence of an NF-κB inhibitor, Bay117082, only 23% of cells were found to be in S-phase 18 h after serum stimulation. Therefore, these data provided preliminary evidence that NF-κB activation is a key step for cell cycle progression in 3T3 fibroblasts.

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