662 The Journal of Cell Biology | Volume 166, Number 5, 2004 NF- B pathway by [Ca2 ]i has been studied in several mod- els, including T-cell stimulation (Truneh et al., 1985), B lymphocytes (Dolmetsch et al., 1998), and cerebellar gran- ule neurons (Lilienbaum and Israel, 2003). In the absence of growth factors, 3T3 fibroblasts enter the G0 quiescent state. The addition of growth factors initiates cells to enter the G1 phase of the cell cycle, the synthesis of DNA (S phase) and cell division (M phase) (Lau and Nathans, 1985; Bravo, 1990). During the G0–G1 transi- tion, the expression of several transcription factors regulates cell growth and DNA synthesis. In most cell types, the mito- genic signal is relayed by the activation of the ubiquitously expressed p42/p44MAPK (ERK1/ERK2) (Chen et al., 1992; Lenormand et al., 1993). Previous works have shown that sustained activation of p42/p44MAPK is required for fibro- blasts to pass the restriction point in G1 and enter S-phase (Pages et al., 1993; Brondello et al., 1995). The p42/ p44MAPK activity has been reported to be dependent on cal- cium concentration in a swine carotid artery model (Katoch et al., 1999). This might suggest that p42/p44MAPK could have a role in cell cycle regulation downstream from the se- rum-induced calcium increase. The activation of p42/ p44MAPK results in the activation of a range of transcription factors, and CD1 expression is positively regulated by this p42/p44MAPK signal (Lavoie et al., 1996). A link between intracellular calcium increase, mitogenic activation, and NF- B pathway activation has not previ- ously been established. In the present work, we have applied flash photolysis of caged calcium chelator in living cells to study the precise role of calcium in CD1 gene transcription and subsequent cell proliferation after serum stimulation of serum-starved Swiss 3T3 fibroblasts. We have investigated the relationship between growth factor activation, [Ca2 ]i increase, MAPK and NF- B activation, cell cycle gene tran- scription, and cell cycle progression. We report that serum stimulation of starved 3T3 fibroblasts induces a calcium- dependent increase in p42/p44MAPK phosphorylation and NF- B signaling, which in turn appears to activate CD1 gene transcription and cell cycle progression. Results Serum stimulation activates the NF- B pathway and subsequent CD1 transcription We investigated whether NF- B regulates cell cycle entry and CD1 transcription in Swiss 3T3 cells. Serum starvation induced significant cell cycle arrest in 3T3 cells. 84% of cells were in G0/G1 phase and 11% in S-phase 24 h after serum starvation (Fig. 1 A). 18 h after serum stimulation, 44% of the cells were in S-phase. This indicated that, as expected, serum stimulation induced cell cycle progression in 3T3 cells. In the presence of an NF- B inhibitor, Bay117082, only 23% of cells were found to be in S-phase 18 h after se- rum stimulation. Therefore, these data provided preliminary evidence that NF- B activation is a key step for cell cycle progression in 3T3 fibroblasts. To assess the role of NF- B–dependent transcription in cell cycle progression, we transfected a reporter vector, which con- tained five copies of the NF- B 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 lu- minescence activity 7 h after serum stimulation (Fig. 1 B). When the same experiment was performed using the CD1 promoter (which contains three NF- B binding sites; Hinz et al., 1999) upstream of the luciferase reporter gene, the lumi- nescence activity reached a maximum at 3 h after serum stim- ulation (Fig. 1 B). A second peak of luminescence was also ob- served 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 in- crease in CD1 mRNA was inhibited by the NF- B 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- B in serum-induced CD1 promoter activity, we studied the effect on coexpression with the CD1 promoter of either truncated I B (Fig. 2 A) or transdomi- nant-negative IKK or IKK (Fig. 2 B). Cotransfection with each of these expression vectors significantly repressed CD1 luciferase reporter activation, with the repression by the trans- dominant IKK expression being particularly strong. In order to confirm the crucial role of the NF- B pathway for serum induction of CD1 gene expression, we investigated deletion mutants of the CD1 promoter (Fig. 2, C and D). The 1745 CD1 promoter gave 1.9-fold 0.14 (SEM) in- duction in response to serum. A 963 CD1 promoter, which retained all three NF- B sites, gave twofold 0.13 (SEM) in- duction. A 66 CD1 promoter, which had two NF- B sites deleted, gave a significantly reduced serum response (1.4-fold 0.09; SEM). Most significantly, mutation of the remaining NF- B site in the 66 construct further reduced the serum response (1.14-fold 0.09; SEM). To investigate the mechanism by which serum stimulation activates the NF- B signaling pathway, we made use of fluorescent fusion constructs expressing the p65 Rel/NF- B protein and the inhibitor I B (Nelson et al., 2002a). We monitored p65-dsRed translocation into the nucleus and I B -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). I B was found to be completely de- graded after 15 min. The same timing of movement and deg- radation was also observed in experiments to study the endog- enous p65 and I B proteins using Western blotting (Fig. 3 C) on nuclear extracts (for p65) and whole-cell extracts (for I B ). Therefore, these data suggested that the fluorescent fusion proteins faithfully reported the dynamics of the en- dogenous p65 and I B response to serum in 3T3 cells. Intracellular calcium rise is required, but not sufficient for serum-induced NF- B activity Calcium is a major second messenger that is activated during the cell cycle and by growth factor stimulation (Means, 1994; Santella, 1998; Ichikawa and Kiyohara, 2001). We in- vestigated how serum stimulation modulates [Ca2 ]i by