666 The Journal of Cell Biology | Volume 166, Number 5, 2004 Figure 4. Serum-induced p65 translocation into the nucleus is [Ca2 ]i dependent. (A) Starved cells were loaded with 1 M Fluo- 4-AM for 20 min at 37 C and intracellular calcium changes were recorded using laser scanning confocal microscopy (488-nm excita- tion, 505–550-nm emission) in response to serum. Images were taken every second for 2 min, and the increase of fluorescence was calcu- lated taking level in the control unstimulated cells as 100%. (B) Swiss 3T3 cells were transfected with p65-dsRed. Confocal images were obtained and analyzed as described above. 24 h after serum starvation, the cells were either stimulated with serum alone (filled squares) or pretreated for 10 min with 10 M BAPTA-AM (open circles). Experiments were performed at least four times, with four fields. In each field there were typically 3–4 transfected cells. KN93; Fig. S2, available at http://www.jcb.org/cgi/content/ full/jcb.200402136/DC1) or inconclusive effects (calmida- zolium; unpublished data). We measured I B degradation (by Western blotting) after a 20-min pretreatment with ei- ther the calcium chelator BAPTA-AM, the MAPK kinase (MEK) inhibitor PD98059, or the PtdIns3k inhibitor wort- mannin. I B degradation was completely inhibited by PD98059 (Fig. 7 A). A similar level of inhibition also oc- curred in the presence of BAPTA-AM. However, wortman- nin had no effect on I B degradation, suggesting that I B degradation was calcium- and MAPK-dependent, but not PtdIns3k dependent. When the inhibitors were applied together, no additive effect was observed. Measurement of I B degradation and p65 translocation by confocal mi- croscopy of the fluorescent fusion proteins further revealed that I B degradation was delayed and strongly reduced in presence of PD98059 (50% of I B 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). We further investigated both MAPK and Akt activity using antibodies directed against phosphorylated p42/p44MAPK (Thr185/202/Tyr185/204) and phospho-Akt (Ser 473). These enzymes were found to be activated by a 10-min se- rum stimulation. p42/p44MAPK activity was inhibited (as expected) by PD98059, and more significantly was also in- hibited by BAPTA-AM. However, p42/p44MAPK activity was not sensitive to wortmannin. Akt activity was inhibited by all three inhibitors tested (Fig. 7 A). These data support the hypothesis that I B degradation is dependent on in- creases in p42/p44MAPK activity and that this activity is it- self dependent on [Ca2 ]i. However, I B degradation was not dependent on PtdIns3k activity, which therefore ap- peared to act downstream from the [Ca2 ]i increase and MAPK activation. Next, we investigated the order and timing of MAPK and Akt activation after serum stimulation (by Western blotting; Fig. 7 B). The phosphorylation of p42/p44MAPK was detected as early as 2 min and was complete after 5 min of serum stimulation. In contrast, Akt was weakly phosphorylated after 5 min, and reached a maximum after 10 min. These data can be compared to the previous obser- vations that I B was degraded after 10 min (Fig. 3 B) and that p42/p44MAPK activity was sensitive to [Ca2 ] , i which reached a peak much earlier (1 min after stimula- tion; Fig. 4 A). Therefore, they suggest an upstream role for MAPK in I B degradation. The key role of p42/p44MAPK in NF- B-dependent cell cy- cle progression was further investigated by studying its role in gene transcription and cell cycle progression. We tested the effects of both PD98059 and wortmannin on consensus NF- B and CD1 promoter activity. PD98059 was found to block the serum-induced NF- B–Luc and CD1-Luc re- porter activity (Fig. 8 A), whereas the PtdIns3k inhibitor wortmannin gave rise to only weak inhibition of gene expres- sion. The effect on cell cycle progression was further investi- gated by flow cytometer analysis. In the presence of the MEK inhibitor PD98059, 22% of cells were found to be in S-phase after 18 h serum stimulation, compared to the con- trol without inhibitor, where 46% of cells were in S-phase (Fig. 8 B). These data support the concept that p42/p44MAPK is a key regulator of NF- B–dependent gene transcription (specifically CD1 transcription) and subsequent cell cycle progression. Discussion Rel/NF- B proteins have been shown to participate in cellu- lar growth control and neoplasia (for review see Gilmore, 1999), and many tumor cells display constitutively high lev- els of nuclear NF- B activity (Barth et al., 1998; Brownell et al., 1988). The aim of the present work was to identify the mechanism by which the NF- B pathway is activated dur- ing cell cycle commitment. First, we demonstrated the es- sential role of NF- B in Swiss 3T3 cell cycle progression by inhibiting the NF- B pathway with a pharmacological in- hibitor that blocked the cell cycle in G1 phase. This is in line with one previous report (Hinz et al., 1999) where the cell