Oncogene (1998) 17, 1845 ± 1853 1998 Stockton Press All rights reserved 0950 ± 9232/98 $12.00 http://www.stockton-press.co.uk/onc Increased synthesis of phosphocholine is required for UV-induced AP-1 activation Zigang Dong, Chuanshu Huang, Wei-Ya Ma, Barbara Malewicz, Wolfgang J Baumann and Zoltan Kiss The Hormel Institute, University of Minnesota, Austin, Minnesota 55912, USA Exposure of mammalian cells to UV irradiation critical in the process of tumor promotion (Alani et al., stimulates phosphatidylcholine hydrolysis and activates 1991; Dong et al., 1994, 1995; Domann et al., 1994), the transcription factor AP-1. Since phosphocholine such epigenetic modi®cations may play an important (PCho), a phospholipid metabolite, is a potential role in UV-irradiation-mediated tumor promotion regulator of mitogenesis and carcinogenesis, we exam- (Shea and Parrish, 1991; Radler-Pohl et al., 1993; ined the eect of UV exposure on the formation of PCho Angel, 1995; Huang et al., 1996, 1997). The UV and the possible mediatory role of PCho in UVB-and enhanced gene expression mediated by transcriptional UVC-induced activation of AP-1 in mouse JB6 induction is known as `UV response' (Angel, 1995). epidermal cells. We found that both UVB and UVC The two most thoroughly studied transcription irradiation resulted in increased PCho levels. Hemi- factors that have been implicated in mediating this cholinium-3 (HC-3), an inhibitor of choline kinase, `UV response' are AP-1 and NF-kB (Angel, 1995; strongly inhibited UV-induced AP-1 activity. By con- Huang et al., 1996, 1997). Exposure of cells to UV light trast, relatively low levels of PCho (80 mM) or choline rapidly activates an Src-family tyrosine kinase, (20 mM) nearly doubled UV-induced AP-1 activity, while followed by sequential activation of the Ha-Ras, the higher (2 ± 20 mM) concentrations of PCho alone cytoplasmic serine-threonine kinase Raf-1, p42/p44 stimulated AP-1 activity 6 ± 8-fold. Importantly, HC-3 mitogen-activated protein kinases (MAP kinases; also inhibited only the stimulatory eect of choline, but not of known as extracellular signal-regulated kinases, or PCho, on AP-1 activity. Of the mitogen-activated ERK-1 and ERK-2, respectively) and AP-1 (Devary protein (MAP) kinases involved in the regulation of et al., 1992; Radler-Pohl et al., 1993). Since both c-Src AP-1 activity, UVC stimulated the MAP kinase family and Ha-Ras are involved in the UV-induced signal ERK-1/ERK-2, JNK as well as p38 kinase activity. transduction pathway, the primary signal generated in These UVC eects were all inhibited by HC-3. With the plasma membrane by UV must be initiated UVB, by contrast, only the activation of ERK-1/ERK-2 upstream from Ras/Raf. In this context, it is was inhibited by HC-3. The data suggest that increased important to note that UV irradiation has also been formation of PCho is required for UV-induced activation shown to enhance phospholipid hydrolysis by both of AP-1 by an ERK-1/ERK-2-dependent mechanism. phospholipase C and D (Carsberg et al., 1995). Based on this, it was suggested that the signaling cascade Keywords: AP-1; UV irradiation; phosphocholine leading to the activation of AP-1 by UV may be phosphatidylcholine; MAP kinase generated at the plasma membrane (Devary et al., 1993; Angel, 1995; Karin and Hunter, 1995; Rosette and Karin, 1996). We have recently demonstrated that atypical protein Introduction kinase C (aPKC) isoforms are required for UV-induced AP-1 activation by using cells transfected with an Exposure to ultraviolet (UV) irradiation accounts for a antisense sequence of mouse PKCz or by a dominant high proportion of human skin cancers (IARC, 1992). negative mutant construct of Xenopus PKC l/i and Radiation in the UVC (180 ± 290 nm), UVB (290 ± cells in which the conventional (a, bI, bII and g) and 320 nm) and UVA (320 ± 400 nm) regions induces novel (d, e, Z and y) PKC isoforms were down- tumor formation in mouse skin (Staberg et al., 1983; regulated by chronic treatment with 12-0-tetradecanoyl Strickland, 1986; IARC, 1992). UV irradiation has been phorbol 13-acetate (TPA) (Huang et al., 1996, 1997). shown to act both as a tumor initiator and a tumor Although the upstream eector of aPKC in the UV promoter (Staberg et al., 1983; Strickland, 1986). It has signal transduction cascade is not clear, some lipids or been proposed that damage of important regulatory their metabolites, such as ceramide, phosphatidic acid genes, such as p53 or ras, plays a role in UV-induced and phosphatidylinositol-3,4,5-P3, are believed to be tumor initiation. UV-induced modulation of signaling responsible for the activation of these enzymes molecules may enhance or decrease gene expression (Nakanishi and Exton, 1992; Nakanishi et al., 1993; leading to increased cell proliferation or apoptosis Dominguez et al., 1993; Berra et al., 1995; MuÈ ller et (Ronai and Weinstein, 1988; Stein et al., 1989; Devary al., 1995). et al., 1991). Since the expression of these genes is Phosphocholine (PCho), an intermediate and product of phospholipid metabolism, has recently been shown to stimulate DNA synthesis in NIH3T3 ®broblasts (Cuadrado et al., 1993; Jime nez et al., 1995; Tomono Correspondence: Z Dong Received 1 December 1997; revised 27 April 1998; accepted 28 April et al., 1995; Kiss, 1996; Chung et al., 1997; Kiss and 1998 Mukherjee, 1997). It has also been reported that PCho Phosphocholine in UV-induced signal transduction ZDonget al 1846 levels are regulated by growth factors (Warden and UVB (3 KJ/m2) was about 30 ± 40% more eective Friedkin, 1985), oncogenes (Macara, 1989; Ratnam and than was UVC (60 J/m2) in stimulating the formation Kent, 1995; Kiss and Crilly, 1995) and chemical of [14C]PCho (Figure 2). Interestingly, HC-3 had a carcinogens (Ishidate et al., 1980; Paulson et al., 1989; somewhat greater inhibitory eect on UVB-induced Kiss and Tomono, 1995). It has further been than on UVC-induced formation of [14C]PCho (Figure demonstrated that most human tumors are character- 2). It should be mentioned here again that the cells ized by elevated levels of PCho (Navon et al., 1977; were irradiated with UVC for 45 s, but with UVB for Daly et al., 1987; Evans and Kaplan, 1997) and that 16 min which may in part account for observed high expression of choline kinase activity is required for dierences. When the incubations were terminated, cancer cell growth (HernaÁ ndez-Alcoceba et al., 1997). only a small portion (*1%) of the newly formed Taken together this may suggest that PCho is involved cellular [14C]PCho was detected in the medium of both in the regulation of cell growth and perhaps in the UV-irradiated and the non-irradiated cultures. The carcinogenesis in vivo. Based on the indicated impor- amount of [14C]PCho released was proportional to the tance of PCho as a growth regulator in cells, and on the cellular [14C]PCho levels formed, indicating that observed eects of UV on phospholipid hydrolysis, we [14C]PCho release from the cells was not stimulated asked the question whether PCho might be a mediator either by UVC or UVB (data not shown). in UV-induced signal transduction leading to the UVC, and particularly UVB, also enhanced the activation of AP-1. incorporation of [14C]choline into the cellular PtdCho In the present study, we used mouse JB6 epidermal pool (Figure 3). As expected, HC-3 strongly inhibited cells to assess the potential role of PCho and choline PtdCho synthesis in both UV-irradiated and non- kinase in UV-induced AP-1 activation. Hemicholinium- irradiated cultures (Figure 3). Since UV induced similar 3 (HC-3) (Cuadrado et al., 1993) was utilized to inhibit increases in [14C]PCho after 30 min, it is presently not choline kinase activity. Changes in water-soluble clear whether increased labeling of PtdCho was due to phospholipid metabolites were followed by radiolabel- increased de novo synthesis or merely re¯ected the ing and by phosphorus-31 NMR. Our data strongly increase in the cellular [14C]PCho pool available for suggest that activation of AP-1 by UV requires PtdCho synthesis. increased PCho formation. While the [14C]choline experiments strongly sug- gested that choline kinase activity was stimulated by UV irradiation, these experiments did not provide unambiguous information on the total PCho content of Results the irradiated and non-irradiated cells. We therefore used 31P NMR to directly measure the short-term PCho formation in UV-irradiated JB6 cells eects of UVB and UVC on cell PCho levels. Figure 4 To evaluate the possible contribution of PCho to UV- induced signal transduction, we ®rst compared the relative cellular levels of PCho as well as the amounts of PCho released from the cells into the medium in UV-irradiated and non-irradiated JB6 cells incubated in the presence of [14C]choline. Preliminary experiments had shown that radiolabeling can detect rapid eects of UV-irradiation on the formation of [14C]PCho provided the cells are preincubated for 60 min with [14C]choline. Preincubation with 0.4 mM HC-3 for 30 min did not have an eect on cellular [14C]choline levels (data not shown). Since HC-3 is known to inhibit high-anity choline uptake as well as choline kinase (referenced in Cuadrado et al., 1993), we conclude that at the [14C]choline concentration (25 mM) used, choline entered the cells by a predominantly HC-3-insensitive mechanism. Due to the inhibitory eect of HC-3 on choline kinase activity, the control cells contained about 2.7- times higher [14C]PCho levels than did cells treated with HC-3 for 30 min prior to brief (45 s) UVC exposure (60 J/m2; UVC treatment was terminated at 0 time; Figure 1).