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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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The induction of CD1 promoter activity by serum is NF-κB dependent. (A) Cells were transfected with the CD1 reporter vector. After a period of 24 h of serum starvation, cells were stimulated or not (ct) with 10% FCS in the absence (ct) or presence of an NF-κB inhibitor (5 μM Bay117082; 20 min preincubation), or were cotransfected with a truncated active mutant of IκBα. (B) Cells were cotransfected with the CD1 reporter vector and either a control empty vector, or a vector expressing either transdominant-negative IKKα or IKKβ. (C) Schematic diagrams of the several CD1-luciferase reporters used. (D) Cells were transfected with the indicated reporters. (A, B, and D) Results are expressed as fold activation relative to the level measured in nonstimulated starved cells. Cells were assayed for luciferase activity 6 h after serum stimulation. Histograms are means ± SEM of triplicate values. Each experiment was performed three times. **, (P < 0.05) indicates statistically significant difference (two-tailed t test).
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fig2: The induction of CD1 promoter activity by serum is NF-κB dependent. (A) Cells were transfected with the CD1 reporter vector. After a period of 24 h of serum starvation, cells were stimulated or not (ct) with 10% FCS in the absence (ct) or presence of an NF-κB inhibitor (5 μM Bay117082; 20 min preincubation), or were cotransfected with a truncated active mutant of IκBα. (B) Cells were cotransfected with the CD1 reporter vector and either a control empty vector, or a vector expressing either transdominant-negative IKKα or IKKβ. (C) Schematic diagrams of the several CD1-luciferase reporters used. (D) Cells were transfected with the indicated reporters. (A, B, and D) Results are expressed as fold activation relative to the level measured in nonstimulated starved cells. Cells were assayed for luciferase activity 6 h after serum stimulation. Histograms are means ± SEM of triplicate values. Each experiment was performed three times. **, (P < 0.05) indicates statistically significant difference (two-tailed t test).

Mentions: To assess the role of NF-κB–dependent transcription in cell cycle progression, we transfected a reporter vector, which contained five copies of the NF-κB consensus binding site, in front of the firefly luciferase reporter gene. Then, we used noninvasive luminescence imaging to measure the time course of luciferase gene expression levels. We observed a peak of luminescence activity 7 h after serum stimulation (Fig. 1 B). When the same experiment was performed using the CD1 promoter (which contains three NF-κB binding sites; Hinz et al., 1999) upstream of the luciferase reporter gene, the luminescence activity reached a maximum at 3 h after serum stimulation (Fig. 1 B). A second peak of luminescence was also observed 20 h after serum stimulation, corresponding to the timing of cell division and the beginning of a new cell cycle. Analysis of endogenous CD1 mRNA levels by RT-PCR showed an accumulation of CD1 mRNA after 4 h, which reached a peak 8 h after serum stimulation (Fig. 1 C). This increase in CD1 mRNA was inhibited by the NF-κB inhibitor Bay117082 (Fig. 1 D). The serum-induced activation of CD1 promoter activity (at 6 h) was also inhibited by treatment with Bay117082 (Fig. 2 A). In order to provide further evidence for the direct role of NF-κB in serum-induced CD1 promoter activity, we studied the effect on coexpression with the CD1 promoter of either truncated IκBα (Fig. 2 A) or transdominant-negative IKKα or IKKβ (Fig. 2 B). Cotransfection with each of these expression vectors significantly repressed CD1 luciferase reporter activation, with the repression by the transdominant IKKβ expression being particularly strong.


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 induction of CD1 promoter activity by serum is NF-κB dependent. (A) Cells were transfected with the CD1 reporter vector. After a period of 24 h of serum starvation, cells were stimulated or not (ct) with 10% FCS in the absence (ct) or presence of an NF-κB inhibitor (5 μM Bay117082; 20 min preincubation), or were cotransfected with a truncated active mutant of IκBα. (B) Cells were cotransfected with the CD1 reporter vector and either a control empty vector, or a vector expressing either transdominant-negative IKKα or IKKβ. (C) Schematic diagrams of the several CD1-luciferase reporters used. (D) Cells were transfected with the indicated reporters. (A, B, and D) Results are expressed as fold activation relative to the level measured in nonstimulated starved cells. Cells were assayed for luciferase activity 6 h after serum stimulation. Histograms are means ± SEM of triplicate values. Each experiment was performed three times. **, (P < 0.05) indicates statistically significant difference (two-tailed t test).
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2172420&req=5

fig2: The induction of CD1 promoter activity by serum is NF-κB dependent. (A) Cells were transfected with the CD1 reporter vector. After a period of 24 h of serum starvation, cells were stimulated or not (ct) with 10% FCS in the absence (ct) or presence of an NF-κB inhibitor (5 μM Bay117082; 20 min preincubation), or were cotransfected with a truncated active mutant of IκBα. (B) Cells were cotransfected with the CD1 reporter vector and either a control empty vector, or a vector expressing either transdominant-negative IKKα or IKKβ. (C) Schematic diagrams of the several CD1-luciferase reporters used. (D) Cells were transfected with the indicated reporters. (A, B, and D) Results are expressed as fold activation relative to the level measured in nonstimulated starved cells. Cells were assayed for luciferase activity 6 h after serum stimulation. Histograms are means ± SEM of triplicate values. Each experiment was performed three times. **, (P < 0.05) indicates statistically significant difference (two-tailed t test).
Mentions: To assess the role of NF-κB–dependent transcription in cell cycle progression, we transfected a reporter vector, which contained five copies of the NF-κB consensus binding site, in front of the firefly luciferase reporter gene. Then, we used noninvasive luminescence imaging to measure the time course of luciferase gene expression levels. We observed a peak of luminescence activity 7 h after serum stimulation (Fig. 1 B). When the same experiment was performed using the CD1 promoter (which contains three NF-κB binding sites; Hinz et al., 1999) upstream of the luciferase reporter gene, the luminescence activity reached a maximum at 3 h after serum stimulation (Fig. 1 B). A second peak of luminescence was also observed 20 h after serum stimulation, corresponding to the timing of cell division and the beginning of a new cell cycle. Analysis of endogenous CD1 mRNA levels by RT-PCR showed an accumulation of CD1 mRNA after 4 h, which reached a peak 8 h after serum stimulation (Fig. 1 C). This increase in CD1 mRNA was inhibited by the NF-κB inhibitor Bay117082 (Fig. 1 D). The serum-induced activation of CD1 promoter activity (at 6 h) was also inhibited by treatment with Bay117082 (Fig. 2 A). In order to provide further evidence for the direct role of NF-κB in serum-induced CD1 promoter activity, we studied the effect on coexpression with the CD1 promoter of either truncated IκBα (Fig. 2 A) or transdominant-negative IKKα or IKKβ (Fig. 2 B). Cotransfection with each of these expression vectors significantly repressed CD1 luciferase reporter activation, with the repression by the transdominant IKKβ expression being particularly strong.

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