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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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Related in: MedlinePlus

The serum-induced [Ca2+]i peak is essential for NF-κB–dependent signaling. (A) Starved cells were loaded with both 0.6 μM Diazo-2 and 0.6 μM Fluo-4 for 20 min at 37°C. For uncaging Diazo-2, 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–D) Swiss 3T3 cells were plated on a marked dish and transfected with 0.5 μg p65-dsRed and 1.5 μg of the reporter NF-κB–luc. After 24 h of serum starvation, the cells were loaded with both Fluo-4 and Diazo-2 as previously described. After one wash, the cells were stimulated with 10% FCS. 1 s later the cells were either illuminated for 10 s (open squares) or untreated (ct, filled squares). (B) Confocal microscopy was used as previously described to measure calcium content every second for 2 min. (C) p65 localization was assessed every 2 min for 90 min as already described. (D) An intensified CCD camera (VIM; Hamamatsu Corporation) was used to assess the luminescence from fields of individual control or photoactivated cells as previously described. 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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fig5: The serum-induced [Ca2+]i peak is essential for NF-κB–dependent signaling. (A) Starved cells were loaded with both 0.6 μM Diazo-2 and 0.6 μM Fluo-4 for 20 min at 37°C. For uncaging Diazo-2, 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–D) Swiss 3T3 cells were plated on a marked dish and transfected with 0.5 μg p65-dsRed and 1.5 μg of the reporter NF-κB–luc. After 24 h of serum starvation, the cells were loaded with both Fluo-4 and Diazo-2 as previously described. After one wash, the cells were stimulated with 10% FCS. 1 s later the cells were either illuminated for 10 s (open squares) or untreated (ct, filled squares). (B) Confocal microscopy was used as previously described to measure calcium content every second for 2 min. (C) p65 localization was assessed every 2 min for 90 min as already described. (D) An intensified CCD camera (VIM; Hamamatsu Corporation) was used to assess the luminescence from fields of individual control or photoactivated cells as previously described. 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: BAPTA can inhibit many calcium-dependent events in cells, leading to nonspecific toxicity after 4 h. To be able to study the long-term role of the serum-induced [Ca2+]i increase, we developed a noninvasive method to abolish the calcium increase, based on photoactivation of the caged calcium scavenger Diazo-2, which displays a low affinity for Ca2+ before photolysis. This affinity is increased 30-fold after illumination at 360 nm. Diazo-2 was loaded into 3T3 cells that were then subjected to a brief (10 s) period of photoactivation. This photoactivation alone induced a transient 20% decrease in [Ca2+]i levels (Fig. 5 A). No significant decrease in [Ca2+]i occurred in cells that were loaded with Diazo-2 but were not illuminated, nor in cells that were illuminated but not loaded with Diazo-2 (unpublished data). Experiments where illumination was performed immediately after serum induction showed that the photolysis of Diazo-2 inhibited the serum-induced calcium peak (Fig. 5 B), but had no effect on subsequent intracellular calcium levels. Photoactivation of Diazo-2 after serum stimulation also blocked detectable p65 translocation into the nucleus (Fig. 5 C) as well as NF-κB–dependent gene transcription from the NF-κB consensus and CD1 promoters (threefold decrease; Fig. 5 D). For all these experiments, the control cells shown were the nonilluminated fields from the same illuminated experiment. Additional controls (analyzing the effects of serum stimulation on illuminated cells that were not previously loaded with Diazo-2) were also performed to ensure that the lack of translocation and transcription was not due to illumination, UV-toxicity, or free radical production. These control experiments gave identical results to the nonilluminated cells with respect to p65 translocation and reporter luminescence (unpublished data). Additionally, we also analyzed the effects of serum on cells that were loaded with Diazo-2 and subjected to photoactivation 5 min before serum stimulation (Fig. S1, available at http://www.jcb.org/cgi/content/full/jcb.200402136/DC1). In this important control experiment, the calcium response of the cells to the serum stimulation was identical to the usual response that was obtained in the absence of previous uncaging. Furthermore, the NF-κB translocation and NF-κB–Luc responses to serum were unaffected by the uncaging procedure (Fig. S1).


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)

The serum-induced [Ca2+]i peak is essential for NF-κB–dependent signaling. (A) Starved cells were loaded with both 0.6 μM Diazo-2 and 0.6 μM Fluo-4 for 20 min at 37°C. For uncaging Diazo-2, 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–D) Swiss 3T3 cells were plated on a marked dish and transfected with 0.5 μg p65-dsRed and 1.5 μg of the reporter NF-κB–luc. After 24 h of serum starvation, the cells were loaded with both Fluo-4 and Diazo-2 as previously described. After one wash, the cells were stimulated with 10% FCS. 1 s later the cells were either illuminated for 10 s (open squares) or untreated (ct, filled squares). (B) Confocal microscopy was used as previously described to measure calcium content every second for 2 min. (C) p65 localization was assessed every 2 min for 90 min as already described. (D) An intensified CCD camera (VIM; Hamamatsu Corporation) was used to assess the luminescence from fields of individual control or photoactivated cells as previously described. 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

Show All Figures
getmorefigures.php?uid=PMC2172420&req=5

fig5: The serum-induced [Ca2+]i peak is essential for NF-κB–dependent signaling. (A) Starved cells were loaded with both 0.6 μM Diazo-2 and 0.6 μM Fluo-4 for 20 min at 37°C. For uncaging Diazo-2, 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–D) Swiss 3T3 cells were plated on a marked dish and transfected with 0.5 μg p65-dsRed and 1.5 μg of the reporter NF-κB–luc. After 24 h of serum starvation, the cells were loaded with both Fluo-4 and Diazo-2 as previously described. After one wash, the cells were stimulated with 10% FCS. 1 s later the cells were either illuminated for 10 s (open squares) or untreated (ct, filled squares). (B) Confocal microscopy was used as previously described to measure calcium content every second for 2 min. (C) p65 localization was assessed every 2 min for 90 min as already described. (D) An intensified CCD camera (VIM; Hamamatsu Corporation) was used to assess the luminescence from fields of individual control or photoactivated cells as previously described. 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: BAPTA can inhibit many calcium-dependent events in cells, leading to nonspecific toxicity after 4 h. To be able to study the long-term role of the serum-induced [Ca2+]i increase, we developed a noninvasive method to abolish the calcium increase, based on photoactivation of the caged calcium scavenger Diazo-2, which displays a low affinity for Ca2+ before photolysis. This affinity is increased 30-fold after illumination at 360 nm. Diazo-2 was loaded into 3T3 cells that were then subjected to a brief (10 s) period of photoactivation. This photoactivation alone induced a transient 20% decrease in [Ca2+]i levels (Fig. 5 A). No significant decrease in [Ca2+]i occurred in cells that were loaded with Diazo-2 but were not illuminated, nor in cells that were illuminated but not loaded with Diazo-2 (unpublished data). Experiments where illumination was performed immediately after serum induction showed that the photolysis of Diazo-2 inhibited the serum-induced calcium peak (Fig. 5 B), but had no effect on subsequent intracellular calcium levels. Photoactivation of Diazo-2 after serum stimulation also blocked detectable p65 translocation into the nucleus (Fig. 5 C) as well as NF-κB–dependent gene transcription from the NF-κB consensus and CD1 promoters (threefold decrease; Fig. 5 D). For all these experiments, the control cells shown were the nonilluminated fields from the same illuminated experiment. Additional controls (analyzing the effects of serum stimulation on illuminated cells that were not previously loaded with Diazo-2) were also performed to ensure that the lack of translocation and transcription was not due to illumination, UV-toxicity, or free radical production. These control experiments gave identical results to the nonilluminated cells with respect to p65 translocation and reporter luminescence (unpublished data). Additionally, we also analyzed the effects of serum on cells that were loaded with Diazo-2 and subjected to photoactivation 5 min before serum stimulation (Fig. S1, available at http://www.jcb.org/cgi/content/full/jcb.200402136/DC1). In this important control experiment, the calcium response of the cells to the serum stimulation was identical to the usual response that was obtained in the absence of previous uncaging. Furthermore, the NF-κB translocation and NF-κB–Luc responses to serum were unaffected by the uncaging procedure (Fig. S1).

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