Proc. Nati. Acad. Sci. USA Vol. 88, pp. 7096-7100, August 1991 Biochemistry

Regulation of c-fos and c-jun protooncogene expression by the Ca2+-ATPase inhibitor thapsigargin (tumor promoter thapsigargin/negative regulator of cell growth)

AXEL SCHONTHAL*t, JEFF SUGARMAN*, JOAN HELLER BROWNt, MICHAEL R. HANLEY§, AND JAMES R. FERAMISCO* *Cancer Center and tDepartment of Pharmacology, University of California at San Diego, La Jolla, CA 92093; and §Department of Biological Chemistry, University of California, Davis, CA 95616

Communicated by J. Edwin Seegmiller, May 22, 1991 (received for review February 27, 1991)

ABSTRACT Thapsigargin, a non-phorbol-ester-type tumor promoter, discharges intracellular Ca21 stores by specific inhibition of the endoplasmic reticulum Ca2+-ATPase. We used this drug to analyze the involvement of Ca2' and Ca21ATPases in the control of growth- and transformation-related genes. Here we show that treatment of mouse NIH 3T3 fibroblasts with thapsigargin induced rapid expression of the c-fos and c-jun protooncogenes. Inhibition or depletion of protein kinase C partially diminished the c-fos but not the c-jun response. Furthermore, thapsigargin could synergize with the tumor promoter phorbol 12-myristate 13-acetate to induce c-fos but not c-jun. However, thapsigargin had no effect on basal or phorbol ester-induced protein kinase C activity. Our results indicate that Ca21 is a potent second messenger that controls expression of growth- and transformation-related genes. Since inhibition of the endoplasmic reticulum Ca2+ATPase results in a strong induction of these genes, our data suggest that this Ca2+ pump may act as a negative regulator of cell growth.

Prototypes of such genes are the c-fos and c-jun protooncogenes. They are induced rapidly and transiently in response to mitogenic stimuli and phorbol ester tumor promoters (16-21). Their protein products are able to form heterodimeric complexes, transcription factor AP-1, that bind to and activate genes via the phorbol 12-myristate 13-acetate ("12-O-tetradecanoylphorbol 13-acetate," TPA) response element (TRE) (for reviews, see refs. 22 and 23). To investigate whether the tumor promoter thapsigargin is able to regulate gene expression, we treated NIH 3T3 cells with this compound and analyzed c-fos and c-jun mRNA levels. Our experiments show that thapsigargin is a potent stimulator of protooncogene expression at nanomolar concentrations. This stimulation does not require newly synthesized proteins or activation of protein kinase C (PKC) but is dependent on intracellular Ca2 . Since inhibition of the endoplasmic reticulum Ca2+-ATPase results in a strong induction of growthand transformation-related genes, these data suggest that this Ca pump might be involved in the down-regulation of growth-stimulating signal-transduction pathways.

Numerous studies have pointed to the involvement of intracellular Ca2" signals in growth control (reviewed in ref. 1). The Ca2+ ionophore A23187 has been widely used to analyze the role of this ion in the regulation of gene expression (2-6). However, the interpretation of these experiments is somewhat hampered by the pleiotropic effects of this drug. For instance, A23187 is known to permeabilize the plasma membrane and its effect often depends upon external Ca2+. A further problem is its propensity to cause the release of eicosanoids, C20 fatty acids. They can be converted to prostaglandins and consequently may stimulate the adenylate cyclase, giving rise to a mixed Ca2+/cAMP signal (7-9). To evade these drawbacks we used a new type of tumor promoter, the sesquiterpene lactone thapsigargin (10, 11). This agent has been shown to specifically inhibit the endoplasmic reticulum Ca2+-ATPase (12). This inhibition is highly selective, as thapsigargin has little or no effect on the Ca2+-ATPases of hepatocyte or erythrocyte plasma membrane or of cardiac muscle sarcoplasmic reticulum (12). It induces acute responses in a large variety of cell types, but in all cases its effects seem to be initiated by a single event: a rapid increase in cytosolic free Ca2+ without hydrolysis of inositolphospholipids (10-14). Since thapsigargin has been shown to be a potent tumor promoter (15), we used it to study a possible involvement of the endoplasmic reticulum Ca2+ATPase in the expression of genes that are thought to contribute to cellular growth regulation and tumorigenic transformation.

MATERIALS AND METHODS Cell Culture. NIH 3T3 cells were grown in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal calf serum (FCS). For experiments where starvation of cells was required, the monolayers were washed twice with phosphate-buffered saline and incubated further in DMEM with 0.5% FCS. Stably Transfected Cell Lines. For transfection we used the pBLCAT4 vector (2) with synthetic oligonucleotides inserted at the HindIII/Xba I sites of the polylinker. The inserted sequences were representative of multimers of the serum response element (SRE) and the cAMP response element (CRE) of the c-fos promoter, and the TRE of the human collagenase promoter (21, 24-27). As a control a "nonsense" oligonucleotide (NON) of the same length was used. Five micrograms of these chloramphenicol acetyltransferase (CAT) constructs and 0.5 jig of pSV2neo (28) were cotransfected into NIH 3T3 cells, and resistant clones were selected in medium containing the neomycin analogue G418 at 600 gg/ml. Approximately 1000 clones per transfection were pooled and analyzed for their specific response to serum stimulation, TPA, or cAMP induction. Isolation of Poly(A)+ RNA. Cells were grown in 15-cm tissue culture dishes (Falcon) to 75-80% confluency. Two plates for each time point were harvested, and poly(A)+ RNA Abbreviations: PKC, protein kinase C; TPA, "12-0-tetradecanoylphorbol 13-acetate" (phorbol 12-myristate 13-acetate); TRE, TPA response element; SRE, serum response element; CRE, cAMP response element; CAT, chloramphenicol acetyltransferase; FCS, fetal calf serum; DMSO, dimethyl sulfoxide. tTo whom reprint requests should be addressed.

The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact. 7096

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Proc. Natl. Acad. Sci. USA 88 (1991)

was selected from total cell lysates by affinity chromatography on oligo(dT)-cellulose essentially as described elsewhere

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RESULTS To investigate whether thapsigargin affects gene expression, we treated NIH 3T3 cells with various concentrations of this drug and then analyzed the mRNA by Northern blot analyses. Thapsigargin effectively induced accumulation of c-fos and c-jun mRNA at doses as low as 10 nM (Fig. 1A). Induction of c-fos was nearly maximal with 100 nM, although there was some additional stimulation with higher concentrations of thapsigargin. In contrast, c-jun induction had a maximum at 100 nM and declined slightly with higher doses. These results are consistent with those of others (11), who found that the increase in intracellular Ca2l levels was maximal with 170 nM thapsigargin. To confirm that c-fos induction was due to changes in the intracellular Ca2+ level, we pretreated cells with the tetrakis(acetoxymethyl) ester of the intracellular Ca2+ chelator BAPTA, to block increases in cytosolic free Ca2'. Under these conditions the induction of c-fos expression was completely inhibited (Fig. 1B). In contrast, TPA was still able to induce c-fos, although to a reduced level. This result suggests that inhibition of the Ca2+-ATPase by thapsigargin might indeed be the main mechanism by which this tumor promoter initiates expression of the c-fos protooncogene. Induction of c-fos in response to serum is greater when the cells have been deprived of serum for -24 hr (16, 17, 31). Furthermore, simultaneous addition of an inhibitor of protein synthesis strongly enhances c-fos expression in response to serum or TPA ("superinduction"; refs. 31 and 34). We found that c-fos expression after treatment of cells with thapsigargin was 1.5-fold stronger in logarithmically growing cells (10%6 FCS) than in serum-starved cells (0.5% FCS) (Fig. 1C).

(29).

Northern Blotting. Five micrograms of poly(A)+ RNA per lane was electrophoresed in 1% agarose gels containing 1.1 M formaldehyde, and the size-separated RNA was blotted onto nitrocellulose filters (30). After baking, the filters were prehybridized for 3 hr at 650C, then hybridization was carried out for 20 hr at 650C as described (30). For hybridization we used the following probes, which were labeled with [a-32P]dCTP (multiprime labeling kit, Amersham): fos, a 1-kilobase (kb) Pst I fragment of pv-fos (31); jun, a 0.9-kb EcoRI-HindIII fragment ofpc-jun 3' untranslated region (32); cho A, a 0.7-kb Pst I fragment of CHO-A [clone A of some highly abundant mRNAs originally isolated from Chinese hamster ovary (CHO) cells; ref. 33]. After hybridization to thefos or thejun probe, the filters were stripped [30 min at 95°C in 0.1 x standard saline citrate (SSC)] and rehybridized with the cho A probe to control for the amounts of RNA loaded in each lane. S1 Nuclease Protection Assay. Five micrograms of poly(A)+ RNA was hybridized in solution to DNA fragments that were end-labeled with [y-32P]ATP as described (2). The hybridization mixture contained 80%o (vol/vol) formamide, 0.4 M NaCl, 40 mM Pipes, 1 mM EDTA, and 2 x 105 cpm of labeled probe in a final volume of 10 ,l. Hybridization was for 16 hr at 47.5°C. Digestion with S1 nuclease was done at 30°C for 45 min in a final volume of 110 IlI containing 0.3 M NaCl, 30 mM sodium acetate, 3 mM ZnSO4, and 100 units of S1 nuclease (BRL). The reaction products were analyzed in a 5% polyacrylamide/7 M urea gel. A

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FIG. 1. Accumulation ofc-fos and c-jun mRNA after thapsigargin (Tg) treatment ofNIH 3T3 cells. Shown are Northern blots of poly(A)+ RNA. After hybridization with c-fos or c-jun probes the filters were stripped and rehybridized with the cho A probe (see Materials and Methods). (A) Concentration dependence. Cells were treated with the indicated concentrations of Tg for 1 hr. (B) Divalent cation dependence. Cells were preincubated with 20 ,uM bis(o-aminophenoxy)ethane-NNN',N'-tetraacetic acid (BAPTA) tetrakis(acetoxymethyl) ester or the same volume of dimethyl sulfoxide (DMSO) for 30 min. Then Tg (300 nM), TPA (300 nM), or the same volume of DMSO (control, Co) was added for 50 min. Numbers at the bottom show the fold induction with respect to control cells. (C) Serum and protein synthesis dependence. Cells were grown in medium supplemented with either 0.5% or 10%6 FCS for 24 hr. Tg (100 nM) with or without anisomycin (An, 100 ,g/ml) was then added for 1 hr. (D) Time dependence. Cells were incubated with 300 nM Tg for the indicated times.

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Proc. Natl. Acad. Sci. USA 88 (1991)

Under both conditions, simultaneous addition of anisomycin, an inhibitor of protein synthesis, resulted in a superinduction of c-fos expression. This demonstrates that the induction of c-fos gene expression by thapsigargin is independent of new protein synthesis. The time course of c-fos and c-jun mRNA accumulation was also studied. The induction of c-fos mRNA expression was rapid, with a maximum around 1 hr (Fig. 1D). The response was also transient: 2 hr after stimulation most of the c-fos mRNA already had disappeared. In contrast, the c-jun mRNA level reached a maximum after 1-2 hr and remained elevated 4 hr after induction. To learn more about the molecular pathways involved in thapsigargin effects, we analyzed whether PKC, the receptor for phorbol ester tumor promoters (35), might contribute to thapsigargin-induced c-fos or c-jun expression. First, we used the isoquinoline sulfonamide H7 to block PKC activation. Treatment of cells with H7 partially reduced thapsigargin-induced c-fos expression (to 62%; Fig. 2A). H7 did not influence the induction of c-fos by cAMP. Interestingly, simultaneous addition of thapsigargin and cAMP resulted in a synergistic effect on c-fos expression. This might reflect the synergism between Ca2+ and cAMP signals that has been found for a number of other systems (10, 38-40). We next used chronic phorbol ester treatment to deplete cells of PKC. Treatment of cells with high doses of TPA for 24 hr reduced the magnitude of c-fos induction by thapsigargin only slightly (to 65%; Fig. 23). In contrast, the cells were unresponsive to a second treatment with TPA. Furthermore, simultaneous addition of thapsigargin and TPA resulted in a strong synergistic effect on c-fos A

caused a 10-fold activation of PKC. This demonstrates that thapsigargin does not activate PKC and supports the view that PKC is not activated solely by Ca21 in vivo (35). To study these signal-transduction pathways further, we analyzed defined cis-acting elements ofthe c-fos promoter for their responsiveness to thapsigargin. The SRE has been shown to mediate induction of the c-fos gene by serum, growth factors (such as platelet-derived growth factor or epidermal growth factor), insulin, TPA, and UV light (2, 3, 24, 42-44). In addition, the c-fos promoter contains several C

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expression (Fig. 2B). Induction of the c-jun gene by thapsigargin was not at all affected by TPA pretreatment. And simultaneous addition of thapsigargin and TPA induced c-jun only minimally more than either agent alone (Fig. 2B). Taken together, these results suggest that c-fos and c-jun exhibit a differential response to thapsigargin. While PKC seems to play a minor role in thapsigargin-induced c-fos expression, it does not appear to contribute to elevated c-jun expression in response to thapsigargin. Previous studies classified thapsigargin as a non-phorbolester-type tumor promoter because of its inability to bind to PKC (15). However, this did not exclude the possibility that thapsigargin might activate or "prime" the activation of PKC by high Ca2l levels (41). To analyze this further, we examined the kinase activity of PKC after treatment of cells with thapsigargin and/or TPA. This was done by measuring the phosphorylation of a peptide (derived from the epidermal growth factor receptor) that served as a specific substrate for PKC. Thapsigargin had no effect on PKC activity whether alone or in combination with TPA (Fig. 2C). In contrast, TPA

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FIG. 2. Involvement of PKC in thapsigargin (Tg)-mediated signal transduction. (A) Pretreatment of cells with the PKC inhibitor H7. Cells were incubated with or without 30 ,uM H7 for 30 min. ThenTg(300nM), cAMP (cA, 1 mM), orTPA (300 nM) was added for 50 min. Co, control (no pretreatment or treatment). (B) Down-regulation ofPKC with chronic phorbol ester treatment. Cells were incubated with TPA (1.5 ,uM) or the same volume of solvent alone (DMSO) for 24 hr. Then Tg (300 nM), TPA (1.5 ,uM), or DMSO (-) was added for 50 min. Simultaneous treatment with Tg plus TPA (right lane) was done only with cells pretreated with DMSO. A and B show Northern blots of poly(A)l RNA. Numbers in A and B show the relative intensities of the bands as determined by scanning appropriately exposed autoradiographs. The filters were rehybridized with the cho A probe. (C) Phosphorylation of a PKC substrate. Cells were treated with Tg (300 nM), TPA (300 nM), or both for 10 min. Then assays of VRKRTLRRL peptide phosphorylation in permeabilized cells were conducted by the method of Heasley and Johnson (36) with modifications as described (37). Data represent the mean + SE of four to five samples.

Proc. Natl. Acad. Sci. USA 88 (1991)

Biochemistry: Sch6nthal et A CREs that confer responsiveness of the gene to cAMP (2, 3, 25, 45, 46). We analyzed whether the SRE or CRE could be activated by thapsigargin. For this purpose we used stably transfected cells that contained multimers of these elements fused to a herpes simplex thymidine kinase promoter-CAT gene construct. The transcriptional activity of these constructs in the presence or absence of thapsigargin was determined by S1 nuclease protection analysis (Fig. 3). Thapsigargin transiently induced expression of the 2x SRE CAT construct. The 4x CRE CAT construct, however, was not more inducible than the same backbone vector with a nonsense oligonucleotide in place of the response elements (4x NON), suggesting that the CRE does not mediate thapsigargin-induced gene expression in these cells (Fig. 3). A construct that contained five copies of the human collagenase TRE (5x TRE) was inducible by thapsigargin. This indicates that the elevated c-fos and c-jun mRNA levels might indeed result in active Fos/Jun (AP-1) complexes. These results were confirmed in cell lines that were stably transfected with SRE, CRE, or TRE sequences fused to a /3-galactosidase reporter gene (ref. 49; data not shown). 5x TRE 4x NON 4x CRE 014014014

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FIG. 3. Cis-acting promoter elements responsive to thapsigargin (Tg). Mass cultures of NIH 3T3 cell lines that were stably transfected with various CAT reporter plasmids were treated with 100 nM Tg for 0, 1, or 4 hr. Then poly(A)' RNA was harvested and subjected to S1 protection analysis. For the detection of correctly initiated CAT transcripts we used a 450-bp Pvu II fragment of pBLCAT2 (47) that spans 215 bp of 5' transcribed plus 235 bp of nontranscribed upstream sequence. For the detection of endogenous c-fos mRNA we used a 633-bp genomic sequence from position 711 (Xho I) to 1344 (BstXl) that spans the intron 1 and 165 bp of the exon 11 (48). The marker (lane M) was 4X174 DNA digested with Hinfl (purchased from Stratagene); lengths (bp) are at left. In independent experiments we confirmed that each transfected cell line responded to serum, TPA, or cAMP in a specific manner (data not shown). fragm., Fragment.

7099

DISCUSSION The results demonstrate that the tumor promoter thapsigargin is able to induce expression of the c-fos and c-jun protooncogenes. Since these genes have been implicated in the regulation of cell proliferation and tumorigenesis, it is likely that they contribute to tumor promotion induced by thapsigargin. The major mechanism by which thapsigargin induces c-fos and c-jun expression appears to be independent of PKC. This is in keeping with the finding that the activation of Ca2" channels in rat pituitary cells induces c-fos expression without activation of PKC (50). Because of the increased intracellular Ca2" induced by thapsigargin, pathways affected might involve Ca2"-dependent protein kinases such as the Ca2 /calmodulin-dependent protein kinases. Indeed, previous pharmacological studies have implicated calmodulin in Ca2+ regulation of c-fos expression (51). PKCindependent pathways of c-fos gene induction have also been described by other laboratories (52-56). Our data show that the c-fos SRE can mediate transcriptional activation in response to thapsigargin. In rat PC12 pheochromocytoma cells, it has been shown that increased intracellular Ca2+ in response to membrane depolarization or ionophore leads to the transcriptional activation of c-fos via the CRE (39, 46). The difference between these results and the present ones may simply reflect differences in the cell types studied, fibroblasts (3T3) versus neuronal (PC12) cells. This is further supported by our finding that thapsigargin induces c-jun in fibroblasts. This gene has not been found to be activated by increased Ca2+ in neuronal cells (57). The lack of induction in the PC12 cells is thought to be due to the absence of a CRE in the c-jun promoter (39). These data suggest that increased Ca2+ in fibroblasts may trigger signaling pathways distinct from those in neuronal cells. This might explain the different promoter elements mediating the transcriptional response of c-fos in the two cell types. Furthermore, the kinetic differences in the induction of c-fos and c-jun in fibroblasts treated with thapsigargin may reflect the response of different regulatory elements in the promoters of these protooncogenes. Since the c-jun gene contains a TRE that is able to bind AP-1 (58), it is conceivable that thapsigargin activates c-jun via this element. In the same vein, it has been shown that thapsigargin is able to enhance the activity of a TRE-like sequence in combination with TPA (59). In conclusion, our results indicate that Ca2+ is a potent second messenger that might be causally related to tumor promotion by thapsigargin. If we assume that the action of thapsigargin is mediated by inhibition of the endoplasmic reticulum Ca2+-ATPase, then the present experiments suggest that this Ca2+ pump may act as a negative regulator of cell growth. We thank David Goldstein for excellent technical assistance and Kristin Martin and Audrey Majors for help in preparation of the manuscript. A.S. is supported by a fellowship from the Human Frontier Science Program Organization. J.R.F. is supported by grants from the National Cancer Institute and the Council for Tobacco Research. 1. Whitaker, M. & Patel, R. (1990) Development 108, 525-542. 2. Buscher, M., Rahmsdorf, H. J., Litfin, M., Karin, M. & Herrlich, P. (1988) Oncogene 3, 301-311. 3. Fisch, T. M., Prywes, R. & Roeder, R. G. (1987) Mol. Cell. Biol. 7, 3490-3502. 4. Bravo, R., Burckhardt, J., Curran, T. & Muller, R. (1985) EMBO J. 4, 1193-1197. 5. Shibanuma, M., Kuroki, T. & Nose, K. (1987) Eur. J. Biochem. 164, 15-19. 6. Cutry, A. F., Kinniburgh, A. J., Krabak, M. J., Hui, S. W. & Wenner, C. E. (1989) J. Biol. Chem. 264, 19700-19705. 7. Smith, P. L. & McCabe, R. D. (1984) Am. J. Physiol. 247,

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