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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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Serum-induced p65 translocation into the nucleus is [Ca2+]i dependent. (A) Starved cells were loaded with 1 μM Fluo-4-AM for 20 min at 37°C and intracellular calcium changes were recorded using laser scanning confocal microscopy (488-nm excitation, 505–550-nm emission) in response to serum. Images were taken every second for 2 min, and the increase of fluorescence was calculated taking level in the control unstimulated cells as 100%. (B) Swiss 3T3 cells were transfected with p65-dsRed. Confocal images were obtained and analyzed as described above. 24 h after serum starvation, the cells were either stimulated with serum alone (filled squares) or pretreated for 10 min with 10 μM BAPTA-AM (open circles). Experiments were performed at least four times, with four fields. In each field there were typically 3–4 transfected cells.
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fig4: Serum-induced p65 translocation into the nucleus is [Ca2+]i dependent. (A) Starved cells were loaded with 1 μM Fluo-4-AM for 20 min at 37°C and intracellular calcium changes were recorded using laser scanning confocal microscopy (488-nm excitation, 505–550-nm emission) in response to serum. Images were taken every second for 2 min, and the increase of fluorescence was calculated taking level in the control unstimulated cells as 100%. (B) Swiss 3T3 cells were transfected with p65-dsRed. Confocal images were obtained and analyzed as described above. 24 h after serum starvation, the cells were either stimulated with serum alone (filled squares) or pretreated for 10 min with 10 μM BAPTA-AM (open circles). Experiments were performed at least four times, with four fields. In each field there were typically 3–4 transfected cells.

Mentions: Calcium is a major second messenger that is activated during the cell cycle and by growth factor stimulation (Means, 1994; Santella, 1998; Ichikawa and Kiyohara, 2001). We investigated how serum stimulation modulates [Ca2+]i by loading the cell-permeant fluorescent dye Fluo-4-AM. Serum induced an immediate and significant peak in [Ca2+]i (Fig. 4 A). The transient increase in [Ca2+]i started 1 s after serum stimulation and lasted for 30 s. No further oscillations of [Ca2+]i were observed (over 5 min; unpublished data). We used a calcium chelator (BAPTA-AM) to investigate the effect of ablation of the serum-induced calcium signal on p65-dsRed translocation. 10 μM BAPTA-AM was loaded into the cells for 20 min and the cells were then stimulated with 10% serum. Treatment with the calcium chelator led to a drastic inhibition in the level of p65 translocation in comparison to control cells (Fig. 4 B). The ratio of the nuclear/cytoplasmic fluorescence intensities reached a maximum of 1.6 in control conditions versus 0.4 in presence of BAPTA.


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)

Serum-induced p65 translocation into the nucleus is [Ca2+]i dependent. (A) Starved cells were loaded with 1 μM Fluo-4-AM for 20 min at 37°C and intracellular calcium changes were recorded using laser scanning confocal microscopy (488-nm excitation, 505–550-nm emission) in response to serum. Images were taken every second for 2 min, and the increase of fluorescence was calculated taking level in the control unstimulated cells as 100%. (B) Swiss 3T3 cells were transfected with p65-dsRed. Confocal images were obtained and analyzed as described above. 24 h after serum starvation, the cells were either stimulated with serum alone (filled squares) or pretreated for 10 min with 10 μM BAPTA-AM (open circles). Experiments were performed at least four times, with four fields. In each field there were typically 3–4 transfected cells.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2172420&req=5

fig4: Serum-induced p65 translocation into the nucleus is [Ca2+]i dependent. (A) Starved cells were loaded with 1 μM Fluo-4-AM for 20 min at 37°C and intracellular calcium changes were recorded using laser scanning confocal microscopy (488-nm excitation, 505–550-nm emission) in response to serum. Images were taken every second for 2 min, and the increase of fluorescence was calculated taking level in the control unstimulated cells as 100%. (B) Swiss 3T3 cells were transfected with p65-dsRed. Confocal images were obtained and analyzed as described above. 24 h after serum starvation, the cells were either stimulated with serum alone (filled squares) or pretreated for 10 min with 10 μM BAPTA-AM (open circles). Experiments were performed at least four times, with four fields. In each field there were typically 3–4 transfected cells.
Mentions: Calcium is a major second messenger that is activated during the cell cycle and by growth factor stimulation (Means, 1994; Santella, 1998; Ichikawa and Kiyohara, 2001). We investigated how serum stimulation modulates [Ca2+]i by loading the cell-permeant fluorescent dye Fluo-4-AM. Serum induced an immediate and significant peak in [Ca2+]i (Fig. 4 A). The transient increase in [Ca2+]i started 1 s after serum stimulation and lasted for 30 s. No further oscillations of [Ca2+]i were observed (over 5 min; unpublished data). We used a calcium chelator (BAPTA-AM) to investigate the effect of ablation of the serum-induced calcium signal on p65-dsRed translocation. 10 μM BAPTA-AM was loaded into the cells for 20 min and the cells were then stimulated with 10% serum. Treatment with the calcium chelator led to a drastic inhibition in the level of p65 translocation in comparison to control cells (Fig. 4 B). The ratio of the nuclear/cytoplasmic fluorescence intensities reached a maximum of 1.6 in control conditions versus 0.4 in presence of BAPTA.

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