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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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IκBα degradation is both [Ca2+]i and p42/p44MAPK activity dependent. (A) IκBα degradation, p42/p44MAPK phosphorylation, and Akt phosphorylation were determined by Western blotting after 10 min of serum stimulation, using antibodies directed against IκBα (1:1,000), phospho-p42/p44 MAPK (Thr185/202/Tyr185/204; 1:5,000), and phospho-Akt (Ser 473; 1:5,000) and actin (1:10,000). Swiss 3T3 fibroblasts were serum starved for 24 h before serum stimulation, and the pharmacological inhibitors were added, where indicated, 20 min before the serum stimulation at the following concentrations: PD98059, 50 μM; BAPTA-AM, 10 μM; wortmannin, 100 nM. (B) The kinetics of p42/p44MAPK and Akt activation were determined by Western blotting using anti-phospho-p42/p44 MAPK and anti-phospho-Akt antibodies. 24-h serum-starved 3T3 cells were stimulated by 10% FCS for indicated time points before cell lysis.
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fig7: IκBα degradation is both [Ca2+]i and p42/p44MAPK activity dependent. (A) IκBα degradation, p42/p44MAPK phosphorylation, and Akt phosphorylation were determined by Western blotting after 10 min of serum stimulation, using antibodies directed against IκBα (1:1,000), phospho-p42/p44 MAPK (Thr185/202/Tyr185/204; 1:5,000), and phospho-Akt (Ser 473; 1:5,000) and actin (1:10,000). Swiss 3T3 fibroblasts were serum starved for 24 h before serum stimulation, and the pharmacological inhibitors were added, where indicated, 20 min before the serum stimulation at the following concentrations: PD98059, 50 μM; BAPTA-AM, 10 μM; wortmannin, 100 nM. (B) The kinetics of p42/p44MAPK and Akt activation were determined by Western blotting using anti-phospho-p42/p44 MAPK and anti-phospho-Akt antibodies. 24-h serum-starved 3T3 cells were stimulated by 10% FCS for indicated time points before cell lysis.

Mentions: Candidate signaling enzymes that might be involved in the serum-induced NF-κB activity were p42/p44MAPK and Akt, which are known to be activated during cell proliferation (Pages et al., 1993; Davies et al., 1999), and have been reported to regulate the IκB/NF-κB signaling pathway (Romashkova and Makarov, 1999; Madrid et al., 2001). Other candidates were investigated including Ca-calmodulin kinases (CaMKs), calcineurin, and calmodulin. Inhibition of these either had no significant effect (cyclosporin A and KN93; Fig. S2, available at http://www.jcb.org/cgi/content/full/jcb.200402136/DC1) or inconclusive effects (calmidazolium; unpublished data). We measured IκBα degradation (by Western blotting) after a 20-min pretreatment with either the calcium chelator BAPTA-AM, the MAPK kinase (MEK) inhibitor PD98059, or the PtdIns3k inhibitor wortmannin. IκBα degradation was completely inhibited by PD98059 (Fig. 7 A). A similar level of inhibition also occurred in the presence of BAPTA-AM. However, wortmannin had no effect on IκBα degradation, suggesting that IκBα degradation was calcium- and MAPK-dependent, but not PtdIns3k dependent. When the inhibitors were applied together, no additive effect was observed. Measurement of IκBα degradation and p65 translocation by confocal microscopy of the fluorescent fusion proteins further revealed that IκBα degradation was delayed and strongly reduced in presence of PD98059 (50% of IκBα remained versus 10% in control cells, 100 min after serum stimulation; Fig. S4, available at http://www.jcb.org/cgi/content/full/jcb.200402136/DC1). The consequent p65 translocation was also abolished in the presence of PD98059 (Fig. S4).


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)

IκBα degradation is both [Ca2+]i and p42/p44MAPK activity dependent. (A) IκBα degradation, p42/p44MAPK phosphorylation, and Akt phosphorylation were determined by Western blotting after 10 min of serum stimulation, using antibodies directed against IκBα (1:1,000), phospho-p42/p44 MAPK (Thr185/202/Tyr185/204; 1:5,000), and phospho-Akt (Ser 473; 1:5,000) and actin (1:10,000). Swiss 3T3 fibroblasts were serum starved for 24 h before serum stimulation, and the pharmacological inhibitors were added, where indicated, 20 min before the serum stimulation at the following concentrations: PD98059, 50 μM; BAPTA-AM, 10 μM; wortmannin, 100 nM. (B) The kinetics of p42/p44MAPK and Akt activation were determined by Western blotting using anti-phospho-p42/p44 MAPK and anti-phospho-Akt antibodies. 24-h serum-starved 3T3 cells were stimulated by 10% FCS for indicated time points before cell lysis.
© Copyright Policy
Related In: Results  -  Collection

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

fig7: IκBα degradation is both [Ca2+]i and p42/p44MAPK activity dependent. (A) IκBα degradation, p42/p44MAPK phosphorylation, and Akt phosphorylation were determined by Western blotting after 10 min of serum stimulation, using antibodies directed against IκBα (1:1,000), phospho-p42/p44 MAPK (Thr185/202/Tyr185/204; 1:5,000), and phospho-Akt (Ser 473; 1:5,000) and actin (1:10,000). Swiss 3T3 fibroblasts were serum starved for 24 h before serum stimulation, and the pharmacological inhibitors were added, where indicated, 20 min before the serum stimulation at the following concentrations: PD98059, 50 μM; BAPTA-AM, 10 μM; wortmannin, 100 nM. (B) The kinetics of p42/p44MAPK and Akt activation were determined by Western blotting using anti-phospho-p42/p44 MAPK and anti-phospho-Akt antibodies. 24-h serum-starved 3T3 cells were stimulated by 10% FCS for indicated time points before cell lysis.
Mentions: Candidate signaling enzymes that might be involved in the serum-induced NF-κB activity were p42/p44MAPK and Akt, which are known to be activated during cell proliferation (Pages et al., 1993; Davies et al., 1999), and have been reported to regulate the IκB/NF-κB signaling pathway (Romashkova and Makarov, 1999; Madrid et al., 2001). Other candidates were investigated including Ca-calmodulin kinases (CaMKs), calcineurin, and calmodulin. Inhibition of these either had no significant effect (cyclosporin A and KN93; Fig. S2, available at http://www.jcb.org/cgi/content/full/jcb.200402136/DC1) or inconclusive effects (calmidazolium; unpublished data). We measured IκBα degradation (by Western blotting) after a 20-min pretreatment with either the calcium chelator BAPTA-AM, the MAPK kinase (MEK) inhibitor PD98059, or the PtdIns3k inhibitor wortmannin. IκBα degradation was completely inhibited by PD98059 (Fig. 7 A). A similar level of inhibition also occurred in the presence of BAPTA-AM. However, wortmannin had no effect on IκBα degradation, suggesting that IκBα degradation was calcium- and MAPK-dependent, but not PtdIns3k dependent. When the inhibitors were applied together, no additive effect was observed. Measurement of IκBα degradation and p65 translocation by confocal microscopy of the fluorescent fusion proteins further revealed that IκBα degradation was delayed and strongly reduced in presence of PD98059 (50% of IκBα remained versus 10% in control cells, 100 min after serum stimulation; Fig. S4, available at http://www.jcb.org/cgi/content/full/jcb.200402136/DC1). The consequent p65 translocation was also abolished in the presence of PD98059 (Fig. S4).

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