Calcium-dependent NF- B activity during cell cycle | Sée et al. 669 Figure 8. p42/p44MAPK activation is determinant for CD1 transcription and further cell cycle progression. (A) Swiss 3T3 cells were transfected with the indi- cated reporter vectors (NF- B luc and 1745 CD1-luc). 24 h after serum star- vation, cells were stimulated or not (ct) with 10% FCS in the absence or presence of MAPK inhibitor (PD98059, 50 M; 20-min preincubation) or Akt inhibitor (100 nM wortmannin, 20-min pre- incubation) as indicated. 6 h after serum stimulation, in vitro luciferase activity was measured. Histograms are means SEM of triplicate values. Each experi- ment was performed three times. (B) Swiss 3T3 fibroblasts were serum starved for 24 h. 24 h after serum starvation, the cells were stimulated with 10% FCS for 18 h in the absence (ct) or presence of PD98059 (50 M, 20-min preincubation). Cell cycle stage was determined by flow cytometric DNA histogram analysis of propidium iodide–stained cells. scription (Fig. 8 A). Ryan et al. (2000) showed that p53- induced NF- B activation can be mediated by MEK1 and pp90rsk, suggesting that MAPK signaling can directly regu- late NF- B. Madrid et al. (2001) also reported regulation of IKK through p38 after IL-1 stimulation. However, it is un- likely that p38 activity has this role in serum stimulation be- cause p38 is known to be an inhibitor of CD1 and cell cycle progression (Lavoie et al., 1996). Therefore, we concentrated on p42/p44MAPK, which has been widely reported to be criti- cal for cell cycle commitment. Our results suggest that p42/ p44MAPK activity is an early event (2 min) after serum stimu- lation and that it is critical for IKK phosphorylation (Fig. S4), I B phosphorylation (Fig. S4), and degradation (Fig. 7A; Fig. S4), and for NF- B activation (Fig. S4) and CD1 transcription (Fig. 8 A). These observations establish for the first time a clear link between mitogenic MAPK signaling, NF- B, CD1 transcription, and cell cycle commitment. We investigated whether there was a link between PtdIns3K/Akt, the NF- B pathway, and cell cycle commit- ment in fibroblasts stimulated by serum. A significant num- ber of previous papers have reported a link between Akt and NF- B (Ozes et al., 1999; Madrid et al., 2001; Meng et al., 2002). However, there remains considerable controversy about the position of Akt in the pathway and whether it lies upstream or downstream of the NF- B activity. Some reports have indicated that Akt has a functional role upstream of NF- B and induces its activity upon TNF stimulation (Ozes et al., 1999) or PDGF stimulation (Romashkova and Makarov, 1999). Our results do not agree with these previous reports because wortmannin does not inhibit I B degradation (even though Western blots confirm effective Akt inhibition by this agent; see Fig. 7 A). A further paper by Meng et al. (2002) reported that Akt could be a downstream target of NF- B when the NF- B pathway was activated by TNF and lipopolysaccharides in NIH 3T3 cells. The determina- tion of the exact position of Akt in this pathway was beyond the objectives of this work, yet our results support a parallel or a downstream role for Akt because the first time point of de- tectable Akt activation was at 10 min; the same time at which the I B degradation was observed. Moreover, Akt was found to be dependent on MAPK activity, but MAPK activ- ity was independent of Akt activation (Fig. 7). We further in- vestigated the role of Akt and MAPK on NF- B–dependent transcription and CD1 promoter activity. MAPK inhibition strongly blocked CD1 and consensus NF- B promoter activ- ity, strengthening the case for an essential upstream role for MAPK signaling (Fig. 8 A). In a significant addition to previ- ous reports (Lavoie et al., 1996), we demonstrated for the first time that this MAPK-dependent CD1 promoter activity oc- curs through the NF- B pathway. The absence of any effect of wortmannin on CD1 promoter activity conflicts with a pa- per by Albanese et al. (2003), who reported an inhibitory ef- fect of another PtdIns3K inhibitor, LY294002, on CD1 tran- scription and expression in mouse embryonic fibroblasts. We also showed the critical role of calcium signaling in p65 translocation to the nucleus (Fig. 4). Other reports (Takuwa et al., 1995; Ichikawa and Kiyohara, 2001) have demonstrated that the increase in calcium is dependent on both calcium influx through plasma membrane channels and on calcium release from internal stores. In order to investi-