JCB Article Calcium-dependent regulation of the cell cycle via a novel MAPK–NF- B pathway in Swiss 3T3 cells Violaine Sée, Nina K.M. Rajala, David G. Spiller, and Michael R.H. White Centre for Cell Imaging, School of Biological Sciences, University of Liverpool, Liverpool L69 7ZB, England, UK y g o l o i B l l e C f o l a n r u o J e h T Nin the regulation of cell proliferation and transfor- uclear factor kappa B (NF- B) has been implicated mation. We investigated the role of the serum-induced intracellular calcium increase in the NF- B–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 sub- sequent NF- B activation (measured by real-time imaging of the dynamic p65 and I B ﬂuorescent fusion proteins), Introduction The nuclear factor kappa B (NF- B)/Rel family of transcrip- tion factors consists of five members (p50, p52, p65/RelA, c-Rel, and RelB) that form a variety of homo- or hetero- dimeric complexes (Sen and Baltimore, 1986). NF- B is activated by its release from cytoplasmic I B proteins and subsequently translocates into the nucleus (Baeuerle and Baltimore, 1996; Baldwin, 1996). Activation is triggered by signal-induced phosphorylation of I Bs, which targets the inhibitors for rapid degradation by the proteasome (Thanos and Maniatis, 1995). Several papers have suggested a role for the NF- B and I B gene products in cell proliferation, transformation, and tumor development (Rayet and Gelinas, 1999; Chapman et al., 2002). Cell cycle activation is in part coordinated by D-type cyclins, which are rate limiting and essential for the progression through the G1 phase of the cell cycle (Coqueret, 2002). Cyclin D1 (CD1) is expressed in most mammalian cells and varies in abundance during the cell cycle, peaking in mid-G1. The level of CD1 protein is largely controlled by the rate of CD1 gene transcription, which is regulated by multiple transcription factors. The CD1 promoter region includes The online version of this article includes supplemental material. Address correspondence to M.R.H. White, Centre for Cell Imaging, School of Biological Sciences, University of Liverpool, Liverpool L69 7ZB, England, UK. Tel.: (44) 151-795-4424. Fax: (44) 151-795-4404. email: mwhite@liv.ac.uk Key words: calcium; NF- B; MAPK; cyclin D1; ﬂash photolysis © The Rockefeller University Press, 0021-9525/2004/08/661/12 $8.00 The Journal of Cell Biology, Volume 166, Number 5, August 30, 2004 661–672 http://www.jcb.org/cgi/doi/10.1083/jcb.200402136 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- B signaling, and further, CD1 tran- scription and cell cycle progression. These data suggest that a serum-dependent calcium signal regulates the cell cycle via a MAPK–NF- B pathway in Swiss 3T3 cells. binding sites for NF- B (Guttridge et al., 1999; Hinz et al., 1999; Joyce et al., 1999), E2F/DP, sp-1/sp-3 (Watanabe et al., 1998), AP-1 (Lee et al., 1999), STAT (Matsumura et al., 1999) and CREB (Lee et al., 1999). NF- B has been shown to control cell growth and differentiation through transcrip- tional regulation of CD1 (Guttridge et al., 1999; Hinz et al., 1999). A nuclear NF- B like DNA-binding activity has been shown to be induced during the G0–G1 transition after serum stimulation in mouse fibroblasts (Baldwin et al., 1991). Despite the fact that the link between NF- B and cell cycle progression is now (at least partly) established, the upstream events leading to NF- B pathway activation upon growth factor stimulation remain to be elucidated. Stimulation of quiescent cells with a variety of growth factors or mitogens induces a transient rise in the intracellular free calcium concentration ([Ca2 ]i) through rapid mobilization from both inositol 1,4,5-triphosphate stores and from the cells’ external environment (McNeil et al., 1985; Takuwa et al., 1995; Ichikawa and Kiyohara, 2001). Such papers have established clear evidence for a role of [Ca2 ]i in the G1/S and G2/M phases of the cell cycle (Takuwa et al., 1995; Cheng et al., 1996; Whitaker and Larman, 2001). However, there have been only a few reports suggesting an important role for [Ca2 ]i at the G0–G1 transition. Regulation of the Abbreviations used in this paper: [Ca2 ]i, intracellular free calcium concentration; CaMK, Ca-calmodulin kinase; CD1, cyclin D1; NF- B, nuclear factor kappa B. 661