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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 stimulation promotes p65 translocation into the nucleus as well as IκBα degradation. (A) p65-dsRed (red staining) and IκBα-EGFP (green staining) were cotransfected into Swiss 3T3 fibroblasts. 24 h after serum starvation, the cells were stimulated with 10% FCS. Confocal images were collected every 2 min from transfected living cells cultured in a humidified CO2 incubator (5% CO2, 37°C) (Video 1, available at http://www.jcb.org/cgi/content/full/jcb.200402136/DC1). (B) Mean fluorescence intensities were measured for each time point in both the nucleus and cytoplasm for p65-dsRed, and the results are shown as a ratio. The IκBα-EGFP mean fluorescence intensities were measured at each time point in the cytoplasm alone and the results presented as the relative intensity to the starting fluorescence levels. Experiments were performed at least four times, with four fields. In each field there were typically 3–4 transfected cells. (C) Endogenous p65 levels in the nucleus were assessed after 10% FCS stimulation for the indicated time by Western blotting nuclear extracts using an anti-p65 antibody (1:1,000). Endogenous IκBα was assessed in whole-cell extracts using an anti-IκBα antibody (1:1,000).
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fig3: Serum stimulation promotes p65 translocation into the nucleus as well as IκBα degradation. (A) p65-dsRed (red staining) and IκBα-EGFP (green staining) were cotransfected into Swiss 3T3 fibroblasts. 24 h after serum starvation, the cells were stimulated with 10% FCS. Confocal images were collected every 2 min from transfected living cells cultured in a humidified CO2 incubator (5% CO2, 37°C) (Video 1, available at http://www.jcb.org/cgi/content/full/jcb.200402136/DC1). (B) Mean fluorescence intensities were measured for each time point in both the nucleus and cytoplasm for p65-dsRed, and the results are shown as a ratio. The IκBα-EGFP mean fluorescence intensities were measured at each time point in the cytoplasm alone and the results presented as the relative intensity to the starting fluorescence levels. Experiments were performed at least four times, with four fields. In each field there were typically 3–4 transfected cells. (C) Endogenous p65 levels in the nucleus were assessed after 10% FCS stimulation for the indicated time by Western blotting nuclear extracts using an anti-p65 antibody (1:1,000). Endogenous IκBα was assessed in whole-cell extracts using an anti-IκBα antibody (1:1,000).

Mentions: To investigate the mechanism by which serum stimulation activates the NF-κB signaling pathway, we made use of fluorescent fusion constructs expressing the p65 Rel/NF-κB protein and the inhibitor IκBα (Nelson et al., 2002a). We monitored p65-dsRed translocation into the nucleus and IκBα-EGFP degradation in living cells by using time-lapse confocal microscopy at 2-min intervals. p65-dsRed was found to translocate into the nucleus and reached a peak nuclear concentration 25 min after serum stimulation (Fig. 3, A and B; Video 1, available at http://www.jcb.org/cgi/content/full/jcb.200402136/DC1). IκBα was found to be completely degraded after 15 min. The same timing of movement and degradation was also observed in experiments to study the endogenous p65 and IκBα proteins using Western blotting (Fig. 3 C) on nuclear extracts (for p65) and whole-cell extracts (for IκBα). Therefore, these data suggested that the fluorescent fusion proteins faithfully reported the dynamics of the endogenous p65 and IκBα response to serum in 3T3 cells.


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 stimulation promotes p65 translocation into the nucleus as well as IκBα degradation. (A) p65-dsRed (red staining) and IκBα-EGFP (green staining) were cotransfected into Swiss 3T3 fibroblasts. 24 h after serum starvation, the cells were stimulated with 10% FCS. Confocal images were collected every 2 min from transfected living cells cultured in a humidified CO2 incubator (5% CO2, 37°C) (Video 1, available at http://www.jcb.org/cgi/content/full/jcb.200402136/DC1). (B) Mean fluorescence intensities were measured for each time point in both the nucleus and cytoplasm for p65-dsRed, and the results are shown as a ratio. The IκBα-EGFP mean fluorescence intensities were measured at each time point in the cytoplasm alone and the results presented as the relative intensity to the starting fluorescence levels. Experiments were performed at least four times, with four fields. In each field there were typically 3–4 transfected cells. (C) Endogenous p65 levels in the nucleus were assessed after 10% FCS stimulation for the indicated time by Western blotting nuclear extracts using an anti-p65 antibody (1:1,000). Endogenous IκBα was assessed in whole-cell extracts using an anti-IκBα antibody (1:1,000).
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Related In: Results  -  Collection

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fig3: Serum stimulation promotes p65 translocation into the nucleus as well as IκBα degradation. (A) p65-dsRed (red staining) and IκBα-EGFP (green staining) were cotransfected into Swiss 3T3 fibroblasts. 24 h after serum starvation, the cells were stimulated with 10% FCS. Confocal images were collected every 2 min from transfected living cells cultured in a humidified CO2 incubator (5% CO2, 37°C) (Video 1, available at http://www.jcb.org/cgi/content/full/jcb.200402136/DC1). (B) Mean fluorescence intensities were measured for each time point in both the nucleus and cytoplasm for p65-dsRed, and the results are shown as a ratio. The IκBα-EGFP mean fluorescence intensities were measured at each time point in the cytoplasm alone and the results presented as the relative intensity to the starting fluorescence levels. Experiments were performed at least four times, with four fields. In each field there were typically 3–4 transfected cells. (C) Endogenous p65 levels in the nucleus were assessed after 10% FCS stimulation for the indicated time by Western blotting nuclear extracts using an anti-p65 antibody (1:1,000). Endogenous IκBα was assessed in whole-cell extracts using an anti-IκBα antibody (1:1,000).
Mentions: To investigate the mechanism by which serum stimulation activates the NF-κB signaling pathway, we made use of fluorescent fusion constructs expressing the p65 Rel/NF-κB protein and the inhibitor IκBα (Nelson et al., 2002a). We monitored p65-dsRed translocation into the nucleus and IκBα-EGFP degradation in living cells by using time-lapse confocal microscopy at 2-min intervals. p65-dsRed was found to translocate into the nucleus and reached a peak nuclear concentration 25 min after serum stimulation (Fig. 3, A and B; Video 1, available at http://www.jcb.org/cgi/content/full/jcb.200402136/DC1). IκBα was found to be completely degraded after 15 min. The same timing of movement and degradation was also observed in experiments to study the endogenous p65 and IκBα proteins using Western blotting (Fig. 3 C) on nuclear extracts (for p65) and whole-cell extracts (for IκBα). Therefore, these data suggested that the fluorescent fusion proteins faithfully reported the dynamics of the endogenous p65 and IκBα response to serum in 3T3 cells.

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