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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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A photoactivated induced [Ca2+]i peak is not sufficient to promote p65 translocation. (A) Starved cells were loaded with both 0.6 μM NP-EGTA and 0.6 μM Fluo-4 for 20 min at 37°C. For uncaging of NP-EGTA, cells were illuminated for 10 s with a Micro-point photoactivation system (Photonic Instruments). Intracellular calcium content was monitored every second by confocal microscopy (488-nm excitation, 505–550-nm emission) of Fluo-4 fluorescence. (B) Subsequently, p65 localization was assessed every 2 min for 90 min as already described. The control (ct) is nonilluminated cells stimulated with serum. Experiments were performed at least four times, with four fields. In each field there were typically 3–4 transfected cells. The horizontal line in A and B marks the period of photoactivation.
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fig6: A photoactivated induced [Ca2+]i peak is not sufficient to promote p65 translocation. (A) Starved cells were loaded with both 0.6 μM NP-EGTA and 0.6 μM Fluo-4 for 20 min at 37°C. For uncaging of NP-EGTA, cells were illuminated for 10 s with a Micro-point photoactivation system (Photonic Instruments). Intracellular calcium content was monitored every second by confocal microscopy (488-nm excitation, 505–550-nm emission) of Fluo-4 fluorescence. (B) Subsequently, p65 localization was assessed every 2 min for 90 min as already described. The control (ct) is nonilluminated cells stimulated with serum. Experiments were performed at least four times, with four fields. In each field there were typically 3–4 transfected cells. The horizontal line in A and B marks the period of photoactivation.

Mentions: To further characterize the relationship between the transient [Ca2+]i increase and subsequent p65 activation, we performed the same experiment using flash photolysis with a caged calcium donor. Photoactivation of NP-EGTA induced a 22% transient increase above the resting level in [Ca2+]i (Fig. 6 A). This increase was smaller, but of the same magnitude to that obtained with serum stimulation (22% with illumination compared to 60% above the resting level with serum). The increase in [Ca2+]i after illumination lasted for a similar duration to that obtained with serum stimulation (∼20 s). Despite the similar dynamics in [Ca2+]i levels (compared to serum stimulation), the illumination of NP-EGTA was unable to induce any detectable p65 translocation (Fig. 6 B). Together, these data (Fig. 5 and Fig. 6) suggest that p65 translocation and downstream regulation of gene transcription is dependent on a transient increase of [Ca2+]i. However, that increase alone, without the presence of other serum-dependent signals, was not sufficient to activate p65. Therefore, we next set out to investigate which other factor(s) upstream of IκBα degradation were activated by growth factors and were dependent on calcium signaling.


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)

A photoactivated induced [Ca2+]i peak is not sufficient to promote p65 translocation. (A) Starved cells were loaded with both 0.6 μM NP-EGTA and 0.6 μM Fluo-4 for 20 min at 37°C. For uncaging of NP-EGTA, cells were illuminated for 10 s with a Micro-point photoactivation system (Photonic Instruments). Intracellular calcium content was monitored every second by confocal microscopy (488-nm excitation, 505–550-nm emission) of Fluo-4 fluorescence. (B) Subsequently, p65 localization was assessed every 2 min for 90 min as already described. The control (ct) is nonilluminated cells stimulated with serum. Experiments were performed at least four times, with four fields. In each field there were typically 3–4 transfected cells. The horizontal line in A and B marks the period of photoactivation.
© Copyright Policy
Related In: Results  -  Collection

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fig6: A photoactivated induced [Ca2+]i peak is not sufficient to promote p65 translocation. (A) Starved cells were loaded with both 0.6 μM NP-EGTA and 0.6 μM Fluo-4 for 20 min at 37°C. For uncaging of NP-EGTA, cells were illuminated for 10 s with a Micro-point photoactivation system (Photonic Instruments). Intracellular calcium content was monitored every second by confocal microscopy (488-nm excitation, 505–550-nm emission) of Fluo-4 fluorescence. (B) Subsequently, p65 localization was assessed every 2 min for 90 min as already described. The control (ct) is nonilluminated cells stimulated with serum. Experiments were performed at least four times, with four fields. In each field there were typically 3–4 transfected cells. The horizontal line in A and B marks the period of photoactivation.
Mentions: To further characterize the relationship between the transient [Ca2+]i increase and subsequent p65 activation, we performed the same experiment using flash photolysis with a caged calcium donor. Photoactivation of NP-EGTA induced a 22% transient increase above the resting level in [Ca2+]i (Fig. 6 A). This increase was smaller, but of the same magnitude to that obtained with serum stimulation (22% with illumination compared to 60% above the resting level with serum). The increase in [Ca2+]i after illumination lasted for a similar duration to that obtained with serum stimulation (∼20 s). Despite the similar dynamics in [Ca2+]i levels (compared to serum stimulation), the illumination of NP-EGTA was unable to induce any detectable p65 translocation (Fig. 6 B). Together, these data (Fig. 5 and Fig. 6) suggest that p65 translocation and downstream regulation of gene transcription is dependent on a transient increase of [Ca2+]i. However, that increase alone, without the presence of other serum-dependent signals, was not sufficient to activate p65. Therefore, we next set out to investigate which other factor(s) upstream of IκBα degradation were activated by growth factors and were dependent on calcium signaling.

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