Activation of Stress-Activated Protein Kinaselc-Jun N-Terminal Kinase by the Non-TPA-Type Tumor Promoter Palytoxin'

Home , Palytoxin

[CANCER RESEARCH56. 637-644. February 1. 1996] Activation of Stress-activated Protein Kinaselc-Jun N-Terminal Kinase by the Non-TPA-type Tumor Promoter Palytoxin'

David W. Kuroki, Gary S. Bignami, and Elizabeth V. Wattenbere

Dis'ision of Ens'ironmental and Occupational Health, University of Minnesota, Minneapolis, Minnesota 55455 ID. W. K., E. V. WI, and Hawaii Biotechnology Group, Inc., Aiea, Hawaii 96701 1G. S. B.]

ABSTRACT blasts (5). Significantly, these later studies also concluded that in the cases where palytoxin and the phorbol esters caused similar cellular Previous studies have shown that structurally diverse tumor promoters effects, they did so by activating different signaling pathways. can modulate protein kinases involved in signal transduction. In this A clue to the nature of the signals that mediate palytoxin action study, we show that palytoxin, a potent non-12-O-tetradecanoylphorbol 13-acetate (TPA)-type skin tumor promoter, induces a signaling pathway came from evidence that palytoxin stimulates sodium influx in every leading to the activation of the stress-activated protein kinases/c-Jun system where it has been tested (6). Palytoxin apparently binds to the N-terminal kinases (iNK) in Swiss 3T3 fibroblasts. Treatment ofcells with Na@,K@-ATPase and either transforms the pump into a sodium chan doses as low as 0.1 flMpalytoxin results in significant activation ofJNK. In nd or forms a sodium channel that is closely associated with this ion contrast to epidermal growth factor, which induces a transient activation pump. Accordingly, studies on the EGF receptor demonstrated that of JNK in Swiss 3T3 cells, palytoxin causes prolonged enzyme activation. palytoxin induces the down-modulation of this receptor through a Since stimulation of ion flux appears to play an important role in the sodium-dependent pathway (7). The specific biochemical steps in mechanism of action of palytoxin in other systems, we investigated the role volved in the novel signal transduction pathways activated by paly of sodium and calcium in the activation of JNK: (a) our results show that toxin, however, remained unclear. incubation of Swiss 3T3 cells in a sodium-free medium dramatically A common theme emerging for the biochemical mechanism of reduced the magnitude of iNK activation by palytoxin; and (b) we found that the sodium ionophore gramicidin activates JNK. Together, these action of structurally diverse tumor promoters is their modulation of results suggest that sodium influx, which is a hallmark ofpalytoxin action, protein kinase signaling cascades. For example, the phorbol esters may play a key role in the activation of JNK by palytoxin. Our results activate protein kinase C (8), okadaic acid is a phosphatase inhibitor indicate that calcium influx is not necessary or sufficient for palytoxin (9), and thapsigargincan induce activation of the p42/p44 MAP induced activation of iNK. In contrast to palytoxin, the TPA-type tumor kinases (10). This raises the question of whether palytoxin can also promoter phorbol 12,13-dibutyrateand the non-TPA-type tumor promot modulate protein kinases involved in signal transduction. A novel era thapsigargin and okadaic acid do not appear to activate JNK in this subgroup of the MAP kinases called the stress-activated protein system. In contrast to phorbol 12,13-dibutyrate, palytoxin does not acti kinases or JNK presented a possible target for palytoxin action (1 1). vate the p42/@ mitogen-activated protein kinases. Our results demon This family of serine/threonine kinases is activated by various types of strate that in Swiss 3T3 fibroblasts, palytoxin can activate a protein kinase cellular stress including UV radiation, proinflammatory cytokines, signaling pathway that is distinct from that activated by the prototypical phorbol ester tumor promoters and other potent skin tumor promoters. and osmotic stress (11). Activation of JNK requires phosphorylation of both tyrosine and threonine residues (12). Once iNK is activated, this kinase can phosphorylate the Ser-63 and Ser-73 residues located INTRODUCTION within the c-Jun amino-terminal, resulting in activation of this impor Palytoxin is a large (Mr 2,681,000), water soluble marine toxin tant component of the transcription factor AP-1 (13). JNK can also isolated from coelenterates belonging to the genus Palythoa (1). regulate the transcription factors Elk-i and ATF2 (14, 15). Since Palytoxin was identified as a skin tumor promoter when a number of palytoxin can dramatically alter the ion content of cells, and therefore natural products were screened to determine if tumor promoters as possibly cause a type of osmotic stress, we hypothesized that this potent as the phorbol esters exist in the environment (2). Early novel tumor promoter might induce a signaling pathway leading to evidence suggested that the biochemical mechanism of action of activation of JNK. palytoxin differs significantly from that of the prototypical phorbol In this study, we report that palytoxin treatment does induce JNK ester tumor promoters; therefore, palytoxin was classified as a non activation in Swiss 3T3 fibroblasts. Our results indicate that sodium TPA3-type tumor promoter. For example, in contrast to the phorbol influx may play an important role in the regulation of JNK by esters, palytoxin does not induce ornithine decarboxylase in mouse palytoxin. This is consistent with previous studies that indicate that a skin, cause HL-60 cell adhesion in vitro, or activate protein kinase C change in ion flux is key to the mechanism of action of palytoxin. In (3). Subsequent studies demonstrated that palytoxin, like other tumor contrast to phorbol esters, palytoxin does not appear to activate the promoters, can induce signaling pathways leading to a variety of p42/p44 MAP kinases. Our results indicate that palytoxin, like other cellular effects, such as arachidonic acid metabolism in rat liver cells tumor promoters, can modulate protein kinase signaling pathways. In (4) anddown-modulationoftheEGFreceptorinSwiss3T3fibro Swiss 3T3 fibroblasts, however, the specific protein kinase signal transduction pathway activated by palytoxin appears to be distinct

Received 9/1 2/95; accepted 11/30/95. from the signaling pathways activated by the phorbol esters and other The costs of publication of this article were defrayed in part by the payment of page potent skin tumor promoters. charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. â€˜Thiswork was supported in part with the assistance of the American Cancer Society MATERIALS AND METHODS Institutional Research Grant IN-13-35-17 and by funds provided through the Grant-in-Aid Program of the Office of the Vice President for Research at the University of Minnesota. 2 To whom requests for reprints should be addressed, at the Division of Environmental Chemicals and Reagents. Palytoxin was isolated from Hawaiian Palythoa and Occupational Health, Box 807 Mayo, 420 Delaware St. SE, Minneapolis, MN 55455. tuberculosa according to published methods (16, 17). EGF, okadaic acid, and 3 The abbreviations used are: TPA, 12-O-tetradecanoylphorbol-13-acetate; EGF, epi myelin basic protein were purchased from Life Technologies (Gaithersburg, dermal growth factor; JNK, c-Jun N-terminal kinase or stress-activated protein kinase; MD). Gramicidin, thapsigargin, and A23187 were purchased from Sigma MAP kinase, mitogen-activated protein kinase; MEK, MAP kinase kinase; SEK, INK kinase; PDBu, phorbol 12, 13-dibutyrate; PMSF, phenylmethylsulfonyl fluoride; GSH, Chemical Co. (St. Louis, MO). [â€˜y-32P]ATPwas purchased from New England glutathione; GST, glutathione S-transferase. Nuclear (Boston, MA). The bacterial expression vector pGEX-2T encoding 637

Downloaded from cancerres.aacrjournals.org on October 1, 2021. © 1996 American Association for Cancer Research. PALYTOXIN ACTIVATES JNK either OST or the GSTcJun (1â€”232amino acids) fusion protein was the ing, the gel was dried and exposed to film. All experiments were performed at generous gift of Dr. Daniel Mueller (Department of Medicine, University of least three times. Results from a representative experiment are shown. Minnesota). The GST fusion protein expression vectors were transformed into the HB1OI strain of Escherichia coli. Protein induction and purification were conducted according to standard procedures (18). RESULTS Cell Culture. Swiss 3T3 cells were grownin a gassed (5%C02), humid Palytoxin Is a Potent Inducer of JNK Activity in Swiss 3T3 ified incubator in DMEM supplemented with 10% FCS. Cells were plated to Fibroblasts. To determine whether palytoxin, like other tumor pro a density of 5 X l0@cells/60-mmplate or 106cells/100-mm plate. After 48 h, confluent cultures were switched to serum-free DMEM for an additional 48 h. moters, can modulate protein kinase signaling cascades, we investi The confluent, quiescent cells were then used for the assays described below. gated the effect of palytoxin on the activation of JNK in Swiss 3T3 Solid Phase iNK Kinase Assay. INK activation was assayed according to fibroblasts. Swiss 3T3 fibroblasts were used because previous studies published methods (13). Briefly, cells were incubated in a gassed (5% C02), demonstrated that palytoxin can activate a novel signal transduction humidified incubator at 37Â°C in DMEM/0.l% FCS in the presence or absence pathway in this cell type (5, 7). The assay for JNK takes advantage of of the appropriate agent. Cells were then washed twice with ice-cold PBS and the fact that JNK binds tightly to the cJun substrate. This assay harvested in lysis buffer [25 misi HEPES (pH 7.7), 300 mr@iNaCl,1.5 m'vi involves mixing whole cell extracts from control or treated cells with MgCl2, 0.2 nmi EDTA, 0.1% Triton X-l00, 0.5 mMDTF, 20 mt@i@-glycero a GSTcJun fusion protein bound to GSH-agarose beads. After exten phosphate, 0.1 mr@iNa3VO4,2 @.tg/mlleupeptin,and 100 jig/mi PMSF). sive washing, the complex is incubated with a kinase buffer in the Whole-cell lysates were rotated for 30 mm at 4Â°C,followedby centrifugation presence of [y-32PJATP and the proteins were resolved by SDS at 10,000 x g for 10 mm. The protein concentration of the supematant was quantitated using the Bradford assay. Lysate containing 50 @gofprotein was PAGE. Phosphorylation of the GSTcJun fusion protein indicates JNK diluted so that the buffer had a final concentration of 20 mr@iHEPES (pH 7.7), activation. When Swiss 3T3 cells were treated for 15 mm with various 75 mMNaC1,2.5 mMMgCl2,0.1 mMEDTA, 0.05% TritonX-100, 0.5 mM doses of palytoxin, we found that palytoxin-induced kinase activation Dli', 20 mM @3-glycerophosphate,0.1mr@iNa3VO4,2 pg/mi leupeptin, and increased from 0.03 nri to 0.3 n@i,decreased slightly at 1 nM, and then 100 @g/mlPMSF. The cell extracts were then mixed with 5 @lofGSH-agarose drop@ dramatically at 3 and 10 ni@palytoxin (Fig. 1, A and B). The beads (Sigma) bound to the GSTcJun fusion protein. The mixture was rotated decrease in kinase activation observed at 3 and 10 nr@ipalytoxin for 3 h at 4Â°C.Thebeads were pelleted and then washed with HEPES binding correlated with signs of cytotoxicity. Swiss 3T3 cells incubated with buffer [20 mMHEPES (pH 7.7), 50 mtsiNaCI, 2.5 mt@iMgC12,0.1 ms@iEDTA, up to 1 aM palytoxin for 15 mm retained greater than 95% viability, 0.05% Triton X-l00, 0.1 mM Na3VO4, 2 g.tg/ml leupeptin, and 100 pg/mi as assessed by trypan blue staining. Cells incubated at higher doses PMSF]. The beads were then resuspended in 30 p3 of kinase buffer [20 mr@i showed signs of cytotoxicity, as indicated both by significantly in HEPES (pH 7.6), 20 mM MgCl2 20 mist f3-glycerophosphate, 20 mM p creased trypan blue staining and by cells lifting off the tissue culture nitrophenylphosphate, 0.1 mi@iNa3VO4, 2 mi@iDTT, 20 @.LMAlP, and 5 pCi [@y-32P]ATP].After a 15-mm incubation at 30Â°C,the beads were washed with plate. Although we found that the dose/response for palytoxin-induced HEPES binding buffer. Proteins were eluted in Laemmli sample buffer and kinase activation can vary from assay to assay, possibly due to the electrophoresed on a 10% SDS-polyacrylamide mini-gel. After Coomassie affinity of palytoxin for plastic (19), we consistently observe that as blue staining, the gel was dried and exposed to film. A Bio-Rad model GS-700 cytotoxicity increases, palytoxin-induced kinase activation decreases. imaging densitometer was used to quantify GSTcJun phosphorylation. All Unless noted otherwise, all of the following assays were conducted experiments were performed at least two times. Results from a representative under noncytotoxic conditions. In any case, these results demonstrate experiment are shown. that palytoxin treatment induces the activation of a kinase that phos In-Gel Kinase Assay for JNK Activation. This assay was performed phorylates GSTcJun. according to published methods (13). cJun binding proteins were isolated from We performed two types of control experiments to provide sup cell extracts using the methods described above for the solid phase INK kinase porting evidence that the GSTcJun phosphorylating activity induced assay. Instead of resuspending the beads in kinase buffer, the proteins were eluted into Laemmli sample buffer and then resolved on a 10% SDS-poly by palytoxin is JNK: (a) we determined that palytoxin specifically acrylamide mini-gel containing 40 @g/m1ofthe GSTcJun fusion protein. The activates an enzyme that binds to and phosphorylates GSTcJun (Fig. gel was washed in 50 mMHEPES (pH 7.6), 5 mM(3-mercaptoethanol,and 20% 1C). When cell lysates from palytoxin-treated cells were incubated 2-propanol and then washed in 50 mM HEPES (pH 7.6). Proteins were with either GST or GSTcJun, we saw the characteristic phosphoryl denatured in 50 mM HEPES (pH 7.6)-6 M urea. The gel was then washed three ation of GSTcJun (Mr @52,000)but did not observe any phosphoryl times in 50 mM HEPES (pH 7.6)-0.5% Tween 20 supplemented first with 3 M ation of GST alone (Mr 26,000); (b) we used an in-gel kinase assay to urea, then 1.5 M urea, and finally 0.75 M urea. The proteins were renatured by show that the GSTcJun phosphorylating activity induced by palytoxin washing the gel overnight at 4Â°Cin50 mMHEPES (pH 7.6)-0.5% Tween 20. runs at the same molecular weight as JNK (Fig. 1D). The kinase reaction was performed by incubating the gel for 1 h at 30Â°Cin For the in-gel kinase assay, cells were treated with either UV light kinase buffer supplemented with 50 @mAlP and 0.25 mCi [y-32P]ATP. The or anisomycin, which are known activators of JNK (20), or with gel was then washed extensively in 5% TCA-10 [email protected] staining and destaining, the gel was dried and exposed to film. All experiments were palytoxin. Proteins were isolated from whole-cell extracts with a performed at least three times. Results from a representative experiment are GSTcJun/GSH-agarose complex and then resolved by SDS-PAGE on shown. gels polymerized in the presence of GSTcJun. A kinase reaction was In-Gel Kinase Assay for p42/p44 MAP Kinase Activation. This assay performed on the proteins renatured within the gel, such that phos was performed according to published methods (10). Briefly, 5â€”10 @gof phorylation of the bands corresponding to the molecular weights of protein from cell lysates were resolved on a 10%SDS polyacrylamide mini-gel either of the two major isoforms of JNK, JNK1 (Mr 46,000) or JNK2 containing 0.2 mg/ml myelin basic protein. The gel was fsrst washed in 50mM (Mr 55,000), indicates enzyme activation (20). The results from the HEPES, 20% isopropanol, and S mr@if3-mercaptoethanolto remove the SDS, in-gel kinase assay indicate that palytoxin induces the activation of a then re-equilibrated in 50 nmi HEPES-5 mrsi @3-meecaptoethanol,andfmally Jun kinase that runs at the same molecular weight as the Jun kinases incubated in 6 Mguanidine-HC1to denature the proteins. The proteins were activated by treatment with UV light and anisomycin, the known renatured by washing the gel in 50 mMHEPES, 0.04% Tween 20, and 5 mt@i activators of JNK (Fig. 1D). The phosphorylated band induced by all @3-mercaptoethanol overnight at 4Â°C. The phosphorylation assay was con ducted by incubating the gel for 30 mm at 30Â°Cina buffer containing 25 mM three agents appears to correspond to the lower molecular weight HEPES (pH 7.5), 10 mM MgC12, 2 mM MnC12, 90 @&MNa3VO4, 5 mM isoform of JNK, JNK1. These data indicate that palytoxin can induce j3-mercaptoethanol, 50 .LMATP, and 0.25 mCi [@y-32P]ATP.Thegel was the activation of JNK in Swiss 3T3 fibroblasts. washed extensively in 5% TCA-lO mittsodium PP1.After staining and destain Uv light and anisomycin have been shown to activate both JNK1 638

A C C) 0 @ -@ -.- .@ â€”74.2kD @ GSTcJun -@. -45.5 kD Dose (nM): 0 0.03 0.1 0.3 1 3 10

B -29.5 kD

â€”18.3kD 15

> D :@ 216 kD V 0 u_ 182 kD 71 .4 kD

o@ 0 0.03 0.1 0.3 1 3 10 JNK1â€”Ã¸- 43.7 kD Dose (nM) C P uv A

Fig. I. Palytoxin activates INK in Swiss 3T3 tibroblasts. A, Swiss 3T3 cells were treated for 15 mm with the indicated doses of palytoxin and then assayed for INK activation using the solid phase kinase assay described in â€œMaterialsandMethods.â€•B, quantification of the GSTcJun phosphorylation shown in A was performed using a Bio-Rad imaging densitometer. C, cells were incubated for 15 mm with 3 nM palytoxin. The cell lysates were incubated with a GSH-agarose suspension, to which was bound either GSTcJun (Mr @52,000)or GST alone (M, 26,000). These complexes were then processed and assayed using the solid phase kinase assay described in â€œMaterialsandMethods.â€•D,INK activation was assayed using the in-gel kinase assay described in â€œMaterialsandMethods.â€•Cells were incubated in the absence (C) or presence of 1 nsi palytoxin for 15 mm (P), UV light as described below (UV), or 25 ng/ml anitomycin for 15 mm (A ). For UV light treatment, the medium was removed from the cultures, and they were then exposed to 40 J/m2 UV for 6 s. The medium was then replaced, and the cells were incubated for 15 mm before lysis. Cell lysates were incubated with a suspension of GSTcJun bound to GSH-agarose. The proteins were eluted from the GSTcJun/GSH-agarose complex, resolved on a gel polymerized in the presence of GSTcJun, and then assayed for INK activation. The width of the phosphorylated bands corresponds to the width of the lanes on the gel as indicated by Coomassie blue staining.

and JNK2 in other systems (20). Therefore, although these results we began to observe some cell lift off. Under conditions where suggest that palytoxin selectively activates JNK1, it is possible that cytotoxicity was not limiting, we have observed elevated JNK activity activation of JNK2 by palytoxin may not be detected in this system. through 360 mm of palytoxin treatment (data not shown). By contrast, For example, Swiss 3T3 cells may express only low levels of this activation of JNK by EGF peaked within 15 mm and decreased to isoform. background levels by 60 mm (Fig. 2, B and C). Furthermore, paly To further characterize the effect of palytoxin on JNK, we exam toxin appears to activate JNK much more effectively than EGF. In this med the kinetics of enzyme activation. Since EGF is an endogenous assay, palytoxin maximally activated iNK approximately 70-fold agent that activates JNK in Swiss 3T3 fibroblasts, we compared above control levels compared to an approximate 5-fold maximal palytoxin to EGF with respect to the time course of enzyme activation. activation by EGF. We have also found that the kinetics of palytoxin We found that palytoxin induced prolonged enzyme activation, induced activation of JNK is dose dependent, such that the higher the whereas EGF induced relatively rapid, transient JNK activation (Fig. dose of palytoxin, the more rapidly JNK activation is detected, and 2). Significant activation of JNK was detected within 15 mm of conversely, the lower the dose of palytoxin, the greater the delay treatment with 0.1 nMpalytoxin (Fig. 2, A and C). In palytoxin-treated before JNK activation is detected (data not shown). These dose cells, JNK activation peaked within approximately 45 mm and began dependent kinetics are consistent with what has been observed with to decrease by 60 mm but was still significantly induced at 240 mm. respect to the dose-dependent kinetics of the down-modulation of the Cell viability was greater than 95% through 120 mm of treatment with EGF receptor by palytoxin (5). Our results indicate that the tumor 0.1 BM palytoxin, as assessed by trypan blue staining. By 240 mm, promoter palytoxim has a much more dramatic effect on JNK activa however, there was a significant increase in trypan blue staining, and tion than the endogenous growth factor EGF in terms of both sustain 639

Downloaded from cancerres.aacrjournals.org on October 1, 2021. © 1996 American Association for Cancer Research. PALYTOXIN ACI'IVATES INK A palytoxin activated JNK approximately 20-fold above background, Palytoxi n whereas in the absence of sodium, 0. 1 nr@iactivated JNK less than 3-fold above background. An even more dramatic difference in JNK activation was observed when cells were treated with 0.3 nrvipaly GSTcJunâ€”. @,. toxin.Inthepresenceofsodium,0.3nr@ipalytoxinactivatedJNKto levels approximately 70-fold over background, whereas in the absence of sodium, 0.3 flM palytoxin only activated JNK to approximately Time (mm): 0 5 10 15 20 30 45 60 120240 5-fold above background. In sodium-free medium, 1 and 3 nisi paly toxin induced an increase in JNK activation relative to the lower doses of palytoxin, although the magnitude of activation at 1 and 3 nr@i palytoxin was still significantly lower than that observed for the same doses in the presence of sodium. These results indicate that the B EGF activation of JNK by palytoxin appears to be largely sodium depend ent, although under some conditions palytoxin can also activate JNK to some extent through a sodium-independent mechanism. We have GSTcJun also found that the cytotoxic effects of palytoxin were decreased in the absence of sodium. In the assay represented in Fig. 3, we observed some signs of cytotoxicity at 3 flMpalytoxin in the presence of sodium Time(mm): 0 5 10 15 20 30 45 60 and total cell lift off when cells were treated with 10 flMpalytoxin in this medium (data not shown). In the absence of sodium, 3 n@i palytoxin did not appear to be cytotoxic, but at 10 flM palytoxin, we C observed signs of cytotoxicity that corresponded to those noted for 3 aMin the presenceofsodium(datanot shown).Therefore,the decrease in activation of JNK by palytoxin is not simply due to an increase in cytotoxicity. Rather, these data indicate that palytoxin activates JNK predominantly through a sodium-dependent pathway. C The observation that activation of JNK by palytoxin is dramatically 0 reduced in the absence of extracellular sodium suggests that enzyme > activation might be triggered by sodium influx. To test this possibility, 0 we determined if the sodium ionophore gramicidin could activate JNK. We chose gramicidin because it is a pore-forming iomophore -C 0 and, therefore, would probably more closely mimic palytoxin-induced U- sodium influx than monensin, which is a sodium-hydrogen exchanger. Like palytoxin, gramicidin also activated JNK in Swiss 3T3 fibro blasts (Fig. 3D). Similarly, activation of JNK by gramicidin was significantly diminished in the absence of extracellular sodium. These results indicate that sodium influx may play an important role in the 0 5 10 15 20 30 45 60 120240 activation of JNK by palytoxin. Although the primary effect of palytoxin is to induce sodium influx, Time(mm) under certain conditions palytoxin can also cause calcium influx. For example, palytoxin can activate sodium-dependent voltage-gated cal Fig. 2. Time course of palytoxin-induced INK activation. Cells were incubated for the indicated times with either 0. 1 nsi palytoxin (A) or 8 nM EGF (B) and then assayed for cium channels in excitable cells and may activate sodium/calcium iNK activation using the solid phase kinase assay described in â€œMaterialsandMethods.â€• exchangers in other systems (22, 23). It has also been reported that in C, quantification of the GSTcJun phosphorylation shown in A and B was performed using some systems, the palytoxin-induced sodium channel may be slightly a Bio-Rad imaging densitometer. S. palytoxin treatment; @,EGFtreatment. permeable to calcium (23). Therefore, we wanted to determine if activation of JNK by palytoxin requires calcium. We determined if ing kinase activity and the magnitude of maximal kinase activation. activation of JNK by palytoxin requires extracellular calcium. Swiss This suggests that the mechanism by which palytoxin activates JNK 3T3 cells were incubated with 0.1â€”100flM palytoxin in regular me differs significantly from that of EGF. dium (Fig. 4, A and C) or medium containing the calcium chelator Sodium Influx May Play a Role in the Activation of JNK by EGTA (Fig. 4, B and C). Our results indicate that activation of JNK Palytoxin. The primary effect of palytoxin in every system where it by palytoxin does not require calcium influx. When cells were incu has been tested, including Swiss 3T3 fibroblasts, is to cause sodium bated in standard DMEM, 0.1 BMpalytoxin induced significant JNK influx (6, 21). Since we had shown previously that palytoxin activates activity, and 1 nr@tpalytoxin induced maximal JNK activation. In the a sodium-dependent signal transduction pathway in Swiss 3T3 fibro assay shown, we observed some cell lift off at 3 n@ipalytoxin and blasts leading to down-modulation of the EGF receptor (7, 21), we almost complete cell lift off at higher doses (data not shown). In the investigated the role of sodium in the activation of JNK by palytoxin. presence of EGTA, we observed a shift in dose/response such that We determined if activation of JNK by palytoxin requires extracellu significant activation of JNK was not observed at doses lower than 1 lar sodium. To make a sodium-free medium with the same osmolarity mM palytoxin, and 3 flM palytoxin induced the same maximal level of as the regular medium, we reconstituted DMEM, replacing all of the JNK activation induced by 1 flM palytoxin in the absence of EGTA. sodium salts with the analogous potassium salts. When we incubated Likewise, we observed a shift in the dose/response for cytotoxicity, cells with various concentrations of palytoxin in the sodium-free with signs of cytotoxicity only appearing after treatment with 100 flM medium, we observed a striking decrease in the magnitude of JNK palytoxin (data not shown). Previous studies have shown that extra activation (Fig. 3, A, B, and C). In the presence of sodium, 0.1 nM cellular calcium increases the affinity of palytoxin for a cellular 640

@ GSTcJunâ€”Ã¸ .: C Dose (nM): 0 0.1 0.3 1 3

60 B -Sodium C 0

GSTcJun 40

Dose (nM): 0 0.1 0.3 1 3 10 20 D

GSTcJunâ€”@ 0@ 100

Dose (nM)

Gramicidin: + - +

Sodium: + + - -

Fig. 3. Palytoxin activates INK through a sodium-dependent mechanism. Cells were incubated for 15 mm with the indicated doses of palytoxin in either a medium that contains sodium (A: + Sodium) or a sodium-free medium (B: â€”Sodium)and then assayed for INK activation using the solid phase kinase assay described in â€œMaterialsandMethods.â€•The sodium-free medium was made by reconstituting DMEM, replacing all of the sodium salts with the analogous potassium salts. C. quantification of the GSTcJun phosphorylation shown in A and B was performed using a Bio-Rad imaging densitometer. 0, activation of INK in sodium-containing medium; â€¢,enzymeactivation in sodium-free medium. D, cells were incubated for IS mm as indicated in the presence ( + ) or absence ( â€”)of I psi gramicidin in either a sodium-containing (+) or sodium-free (â€”)medium and then assayed for INK activation as described for A and B.

receptor (6). Therefore, the shift in the dose/response observed in the course with 100 ni@iokadaic acid or for 15 mm with 1 flM palytoxin, presence of EGTA for both palytoxin-induced activation of JNK and only palytoxin induced significant activation of JNK (Fig. 5). cytotoxicity may be due to a decrease in the binding affinity of Similarly, we did not observe JNK activation when Swiss 3T3 cells palytoxin because of the reduction in free extracellular calcium. To were incubated with okadaic acid for periods less than 30 mm (data investigate whether calcium primarily plays a role in palytoxin action not shown). The observation that thapsigargin alone does not activate at the level of palytoxin binding, as opposed to directly mediating the JNK in Swiss 3T3 cells, at a dose high enough to activate the p42/p44 activation of JNK, we determined if JNK could be activated by an MAP kinases in human foreskin fibroblasts (10), is consistent with the increase in calcium influx. When cells were treated with the calcium suggestion that the primary mechanism of action of thapsigargin is to ionophore A23l87, we did not observe significant activation of JNK cause a rise in intracellular calcium (10). As shown in Fig. 4B, a rise (Fig. 4D). These results indicate that calcium influx is not sufficient in intracellular calcium alone does not appear to activate JNK in this to activate JNK to the levels observed with palytoxin. Although it is system. Activation of JNK by phorbol esters appears to be cell type possible that calcium influx can play some role in modulating the specific (24). In T lymphocytes, phorbol esters cause modest activa activation of JNK by palytoxin, these results indicate that calcium tion of JNK, which is potentiated by cotreatment with A23l87 (24). influx is not necessary or sufficient for the activation of JNK by Consistent with what has been observed in other fibroblast cell lines, palytoxin. we find that even the combination of A23l87 with PDBu does not Activation of JNK Distinguishes Palytoxin from Other Potent activate JNK in Swiss 3T3 cells (data not shown). Finally, okadaic Skin Tumor Promoters. The phorbol esters represent TPA-type acid did not appear to activate JNK at concentrations sufficient to tumor promoters, and thapsigargin and okadaic acid represent non significantly increase the phosphorylation of proteins in hepato TPA-type tumor promoters that have been shown to modulate protein cytes and adipocytes (25) via inhibition of the protein phosphata kinase signaling pathways in other systems (8â€”10).Therefore, we ses-1 and -2A. Although it is possible that PDBu, thapsigargin, or wanted to determine if these skin tumor promoters, which act through okadaic acid alone can activate iNK in Swiss 3T3 cells under various cellular mechanisms of action, could activate iNK in Swiss conditions we have not tested, our results indicate the kinetics of 3T3 cells. When Swiss 3T3 cells were incubated over a 60-mm time activation would have to differ significantly from those of paly course with I.5 @.LMthapsigarginor 100 nM PDBu, or a 90-mm time toxin or require doses over 1000-fold greater than required for 641

Palytoxin Does Not Activate the p42/p44 MAP Kinases. Recent A No EGTA studies indicate that JNK is activated through a signaling pathway that is distinct from the pathway leading to activation of the p42/p44 MAP kinases (26). Likewise, we found that although palytoxin is a potent

@ GSTcJunâ€”a@ :@,;@.-, activator of JNK in Swiss 3T3 cells, palytoxin does not appear to activate the p42/p44 MAP kinases. We treated cells with either 0.1 nr@i Dose (nM): 0 0.1 0.3 1 3 palytoxin over a 2-h time course or for single time points with PDBu, EGF, or A23 187. Cell lysates were then assayed for p42/p44 MAP kinase activation using an in-gel kinase assay in which the kinase substrate myelin basic protein was polymerized in the gel (Fig. 6). In contrast to PDBu, EGF, and A23187, which clearly activate the B +EGTA p42/p44 MAP kinases in these cells, palytoxin did not activate the p42/p44 MAP kinases. Similarly, we have found that higher concen GSTcJun trations of palytoxin also do not activate the p42/p44 MAP kinases (data not shown): (a) these results indicate that palytoxin selectively activates a signaling pathway that leads to JNK activation, but not to Dose (nM): 0 0.1 0.3 3 10 30 100 p42/p44 MAP kinase activation; (b) the observation that the calcium ionophore A23187 can activate the p42/p44 MAP kinases, but that palytoxin does not, supports the suggestion that in this system paly C toxin action is not mediated by a rise in intracellular calcium; and (c) these results demonstrate that in Swiss 3T3 fibroblasts, palytoxin can

Thapsigargin PDBu

C I I I Paly 0

@ > GSTcJun â€”â€˜. .

@0 0 U- Time(min): 0 10 20 30 60 10 20 30 60 15

OkadaicAcid 1000 Dose (nM) GSTcJun D \ Time (mm): 0 30 60 90 @,0 â€˜@ C, q 4, Fig. 5. Treatment of Swiss 3T3 cells with other potent skin tumor promoters does not activate INK. Cells were incubated for the indicated times with 1.5 @.LMthapsigargin,100 tIM PDBu, 1 nM palytoxin (Paly), or 100 tm okadaic acid. Cell lysates were then assayed for INK activation using the solid phase kinase assay described in â€œMaterialsand GSTcJunâ€”.@ Methods.â€•

Fig. 4. Activation of INK by palytoxin does not require extracellular calcium. Cells @&______Palytoxin â€˜)â€˜< were incubated for 15 mm with the indicated doses of palytoxin in A, the absence of EGTA or B, the presence of 4 ms@EGTA, and then assayed for INK activation using the ()d:'1 I solid phase kinase assay described in â€œMaterialsand Methods.â€•Under the conditions of our experiments, 4 msi EGTA reduces the concentration of extracellular free calcium in DMEM from 1.8 ms@to approximately 100 nsf. as calculated using MAXCHELATOR (version 6.63)software. C, quantification ofthe GSTcJun phosphorylation shown mA and B was performed using a Bio-Rad imaging densitometer. 0. activation of INK in medium that does not contain EGTA; S. enzyme activation in the presence ofEGTA. D, cells were P44 MAPK@ incubated for 15 mm in the absence (Control) or presence of 10 @siA23187, 1 nsi p42 MAPK-' palytoxin, or 8 nMEGF and then assayed using the solid phase kinase assay described in ______â€œMaterialsand Methods.â€• Time (mm): 4 15 30 60 120

Fig. 6. Palytoxin treatment does not induce activation of the p42/p44 MAP kinases. activation of JNK by palytoxin. Altogether, these results indicate Cells were incubated in the presence or absence of either 0. 1 n@i palytoxin for the that in Swiss 3T3 fibroblasts, palytoxin can activate a protein indicated times, 100 nsi PDBu for 4 mist, 8 nat EGF for 4 mm, or 10 psi A23187 for 10 mm. Cell lysates (9 @.tgofprotein) were resolved on a gel polymerized in the presence of kinase signaling cascade that is distinct from those activated by myelin basic protein, and p42/p44 MAP kinase activity was assayed according to the other potent skin tumor promoters. in-gel kinase assay described in â€œMaterialsand Methods.â€• 642

Downloaded from cancerres.aacrjournals.org on October 1, 2021. © 1996 American Association for Cancer Research. PALYTOXIN ACTIVATES JNK activate a protein kinase signaling pathway that is distinct from that JNK activation through a related mechanism. Experiments are cur activated by the phorbol ester tumor promoters. rently under way to detennine more specifically how sodium influx triggers JNK activation. DISCUSSION JNK is activated by mechanistically diverse carcinogenic agents, including UV radiation, the genotoxic carcinogen methyl methane The results presented here demonstrate that the novel tumor pro sulfonate, and the tumor promoter palytoxin (13, 30). The proinflam moter palytoxin can induce a signal transduction pathway leading to matory cytokine tumor necrosis factor a also activates JNK (3 1). This activation of the protein kinase JNK. This study also indicates that in is particularly intriguing, since a set of studies indicates that tumor Swiss 3T3 fibroblasts, palytoxin can activate a protein kinase signal necrosis factor a itself may be an endogenous tumor promoter (32, ing pathway that is distinct from that activated by the phorbol esters. 33). Collectively, these findings suggest that JNK may play a signif These data support previous studies that indicated that the cellular icant role in carcinogenesis. Palytoxin may prove to be a useful tool mechanism of action of palytoxin is biochemically distinct from that for investigating both the mechanisms that regulate JNK activity and of the prototypical phorbol ester tumor promoters (3â€”5).Forexample, the role this enzyme might play in carcinogenesis. in contrast to the phorbol esters, palytoxin is not mitogenic in Swiss 3T3 cells (5, 27). Accordingly, we find that in Swiss 3T3 cells, ACKNOWLEDGMENTS palytoxin activates iNK, but not the p42/p44 MAP kinases, which are activated by a variety of mitogenic agents. The converse is true for the We would like to thank Dr. Marsha Rich Rosner of the Ben May phorbol esters. These results support our hypothesis that palytoxin Institute, University of Chicago, Dr. Daniel Mueller of the Department of activates a stress-induced signaling pathway as opposed to a mito Medicine, University of Minnesota. and Dr. Wei Li of the Department of genic signaling pathway. Palytoxin, like other tumor promoters, Medicine, University of Minnesota for helpful discussions. causes various types of effects in different cell types, including the sodium-dependent down-modulation of EGF receptors in Swiss 3T3 REFERENCES cells. It remains to be determined whether activation of JNK mediates the other cellular effects of palytoxin. Finally, our results place 1. Moore, R. E., and Bartolini, G. Structure of palytoxin. J. Am. Chem. Soc., 103: 2491â€”2494,1981. palytoxin in a class of potent skin tumor promoters, including okadaic 2. Fujiki, H., Suganuma,M., Tahira, T., Yoshioka,A., Nakayasu,M., Endo, Y., Shudo, acid, thapsigargin, and the phorbol esters, which all modulate protein K., Takayama, S., Moore, R. E., and Sugimura, T. Nakahara memorial lecture. New kinases. Protein phosphorylation is certainly a fundamental mecha classes of tumor promoters: teleocidin, aplysiatoxin and palytoxin. In: H. Fujiki, E. Hecker, R. E. Moore, T. Sugimura, and I. B., Weinstein (eds.), Cellular Interactions nism by which cells regulate enzyme activity and, therefore, tightly by Environmental Tumor Promoters, pp. 37â€”45.Japan Sci. Soc. Press: Utrecht: control various events involved in cell growth, death, and differenti Tokyo/VNU Science Press, 1984. 3. Fujiki, H., Suganuma, M., Nakayasu, M., Hakii, H., Horiuchi, 1., Takayama, S., and ation. This common theme among mechanistically diverse tumor Sugimura, 1. Palytoxin is a non-12-O-tetradecanoylphorbol-l3-acetate type tumor promoters supports the hypothesis that tumor promoters in general promoter in two-stage mouse skin carcinogenesis. Carcinogenesis (Land.), 7: may contribute to carcinogenesis by inappropriately regulating critical 707â€”710,1986. 4. Levine, L., and Fujiki, H. Stimulation of arachidonic acid metabolism by different cellular switches. types of tumor promoters. Carcinogenesis (Land.), 6: 1631â€”1634,1985. The finding that palytoxin activates JNK raises questions about the 5. Wattenberg, E. V., Fujiki, H., and Rosner, M. R. Heterologous regulation of the biochemical steps in the signaling pathway leading from palytoxin epidermal growth factor receptor by palytoxm, a non-l2-O-tetradecanoylphorbol-13- acetate-type tumor promoter. Cancer Res., 47: 4618â€”4622,1987. treatment to enzyme activation. It has been shown recently that JNK 6. Habermann, E. Palytoxin acts through Na@/K@-ATPase. Toxicon, 27: 1171â€”1187, activation is regulated by a protein kinase cascade that appears to be 1989. 7. Wattenberg, E. V., Byron, K. L., Villereal, M. L., Fujiki, H., and Rosner, M. R. largely parallel but analogous to the cascade of protein kinases that Sodium as a mediator of non-phorbol tumor promoter action. J. Biol. Chem., 264: regulate the p42/p44 MAP kinases. Just as there is a MEK that lies 14668â€”14673,1989. upstream of the p42/p44 MAP kinases, there also appears to be a SEK 8. Nishizuka, Y. The role of protein kinase C in cell surface signal transduction and tumour promotion. Nature (Land.), 308 693-698, 1984. that lies upstream of JNK (28). Likewise, whereas Raf phosphorylates 9. Suganuma, M., Fujiki, H., Okabe, S., Nishiwaki, S., Brautigan, D., Ingebritsen, T. S., and activates MEK, which in turn phosphorylates and activates the and Rosner, M. R. Structurally different members ofthe okadaic acid class selectively p42/p44 MAP kinases (the RafIMEKIMAP kinase pathway), a kinase inhibit protein serine/threonine but not tyrosine phosphatase activity. Toxicon, 30: 873â€”878, 1992. identified as MEK kinase-l (MEKK) phosphorylates and activates 10. Chao, T. 0., Byron, K. L., Lee, K., Villereal, M., and Rosner, M. R. Activation of SEK, which in turn phosphorylates and activates JNK (the MEKK/ MAP kinases by calcium-dependent and calcium-independent pathways. J. Biol. Chem., 267: 19876-19883, 1992. SEK/JNK pathway; Ref. 26). We are currently investigating if paly 11. Davis, R. J. MAPKs: new INK expands the group. Trends Biochem. Sci., 19: toxin activates JNK via the MEKK/SEK pathway or whether paly 470â€”473, 1994. toxin regulates JNK activity through an alternate signaling cascade. 12. DÃ©rijard,B.,Hibi, M., Wu, I., Barrert,T., Su, B., Deng, T., Karin, M., and Davis, R. J. INKI: a protein kinase stimulated by UV light and Ha-Rat that binds and phospho In terms of the primary signals that trigger JNK activation, our rylates the c-Jun activation domain. Cell, 76: 1025-1037, 1994. results indicate that sodium influx may play a role in the activation of 13. Hibi, M., Lin, A., Smeal, 1., Minden, A., and Karin M. Identification of an onco JNK by palytoxin. The mechanism by which sodium influx activates protein- and UV-responsive protein kinase that binds and potentiates the c-Jun activation domain. Genes & Dev., 7: 2135â€”2148, 1993. this kinase is not yet clear. Sodium influx can have a number of 14. Whitmarsh, A. J., Shore, P., Sharrocks, A. D., and Davis, R. I. Integration of MAP secondary effects, including depolarization of the membrane, a kinase signal transduction pathways at the serum response element. Science (Wash ington DC), 269: 403â€”407, 1995. change in intracellular pH, and ultimately cell swelling and lysis. It is 15. Gupta, S. Campbell, D., DÃ©rijard,B.and Davis, R. J. Transcription factor ATF2 unlikely that palytoxin-induced depolarization of the membrane is the regulation by the INK signal transduction pathway. Science (Washington DC), 267: primary trigger of JNK activation since incubation of the cells in a 389â€”393,1995. 16. Corpuz, G. P., Grothaus, P. G., Waller, D. F., and Bignami, G. S. An HPLC method high potassium medium, which depolarizes the membrane (7), did not for rapid analysis of palytoxin congeners and prodrugs. In: Y. Hokama, P. 1. Scheuer, cause extensive JNK activation. Palytoxin can cause intracellular and T. Yasumoto (eds.), Proceedings of the International Symposium on Ciguatera acidification, and therefore, it is possible that this change in pH and Marine Natural Products, pp. 143â€”151.Honolulu: Asian Pacific Research Foun dation, 1995. triggers iNK activation. Cell swelling could also trigger iNK activa 17. Moore, R. E., and Scheuer, P. I. Palytoxin: a new marine toxin from a coclenterate. tion. Curiously, incubating rat fibroblasts in a hyperosmotic medium, Science (Washington DC), 172: 495â€”498, 1971. 18. Smith, S. B., and Johnson, K. S. Single-step purification of polypeptides expressed in which should cause cells to shrink, activates JNK (29). Conceivably, Escherichia coli as fusions with glutathione S-transferase. Gene (Amst.), 67: 31â€”40, changes in extracellular and intracellular osmolanty could trigger 1988. 643

19. Taylor, T. J., Parker, G. W., Fajer, A. B., and Mereish, K. A. Non-specific binding of 27. Rozengurt, E. Early signals in the mitogenic response. Science (Washington DC), palytoxin to plastic surfaces. Toxicol. Lett., 57: 291â€”296,1991. 234: 161â€”166,1986. 20. Kallunki, T., Su, B., Tsigelny, I., Sluss, H. K., DÃ©rijard,B.,Moore, G., Davis, R., and 28. Sanchez, I., Hughes, R. 1., Mayer, B. J., Yee, K., Woodgett, J. R., Avruch, J., Karin, M. JNK2 contains a specificity-determining region responsible for efficient Kyriakis, I. M., and Zon, L. I. Role of SAPK/ERK kinase-l in the stress-activated c-Jun binding and phosphorylation. Genes & Dev., 8: 2996â€”3007,1994. pathway regulating transcription factor c-Jun. Nature (Lond.), 372: 794â€”798, 1994. 21. Wauenberg, E. V., McNeil, P. L., Fujiki, H., and Rosner, M. R. Palytoxin down 29. Moriguchi, T., Kawasaki, H., Matsuda, S., Gotoh, Y., and Nishida, E. Evidence for modulates the epidermal growth factor receptor through a sodium-dependent path multiple activators for stress-activated protein kinases/c-Jun amino-terminal kinases. way. J. Biol. Chem., 264: 213â€”219,1989. I. Biol. Chem., 270: 12969â€”12972,1995. 22. Sauviat, M. Palytoxin: characterization of mode of action in excitable cells. Methods 30. Liu, Y., Gorospe, M., Yang, C., and Holbrook, N. J. Role of mitogen-activated protein Neurosci., 8: 332â€”346,1992. kinase phosphatase during the cellular response to genotoxic stress. I. Biol. Chem., 23. Yoshizumi, M., Nakanishi, A., Houchi, H., Morita, K., Katoh, I., and Oka, M. Characterization of palytoxin-induced catecholamine secretion from cultured bovine 270: 8377â€”8380,1995. 31. Sluss, H. K., Barrett, T., DÃ©rijard,B., and Davis, R. I. Signal transduction by tumor adrenal chromaftin cells. Biochem. Pharmacol., 42: 17â€”23,1991. 24. Su, B., Jacinto, E., Hibi, M., Kallunki, T., Karin, M., and Ben-Neriah, Y. INK is necrosis factor mediated by iNK protein kinases. Mol. Cell. Biol., 14: 8376â€”8384, involved in signal integration during costimulation of I lymphocytes. Cell, 77: 1994. 727â€”736.1994. 32. Komori, A., Yatsunami, I., Suganuma, M., Okabe, S., Abe, S., Sakai, A., Sasaki, K., 25. Haystead, T. A., Sim, A. 1. R., Carling, D., Honnor, R. C., Tsukitani, Y., Cohen, P., and Fujiki,H. Tumornecrosisfactoracts as a tumorpromoterin BALB/3T3cell and Hardie, D. 0. Effects of the tumour promoter okadaic acid on intracellular protein transformation. Cancer Res., 53: 1982â€”1985,1993. phosphorylation and metabolism. Nature (Lond.), 337: 78â€”81, 1989. 33. Komori, A., Suganuma, M., Okabe, S., Zou, X., Tius, M. A., and Fujiki, H. 26. Yan, M., Dai, 1., Deak, I. C., Kyriakis, J. M., Zon, L. I., Woodgett, I. R., and Canventol inhibits tumor promotion in CD-l mouse skin through inhibition of Templeton, D. J. Activation of stress-activated protein kinase by MEKK1 phospho tumor necrosis factor a release and of protein isoprenylation. Cancer Res., 53: rylation of its activator SEK1. Nature (Land.), 372: 798â€”800,1994. 3462â€”3464, 1993.

644

Downloaded from cancerres.aacrjournals.org on October 1, 2021. © 1996 American Association for Cancer Research. Activation of Stress-activated Protein Kinase/c-Jun N-Terminal Kinase by the Non-TPA-type Tumor Promoter Palytoxin

David W. Kuroki, Gary S. Bignami and Elizabeth V. Wattenberg

Cancer Res 1996;56:637-644.

Updated version Access the most recent version of this article at: http://cancerres.aacrjournals.org/content/56/3/637

E-mail alerts Sign up to receive free email-alerts related to this article or journal.

Reprints and To order reprints of this article or to subscribe to the journal, contact the AACR Publications Subscriptions Department at [email protected].

Permissions To request permission to re-use all or part of this article, use this link http://cancerres.aacrjournals.org/content/56/3/637. Click on "Request Permissions" which will take you to the Copyright Clearance Center's (CCC) Rightslink site.