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Research Article
Smad4-independent, PP2A-dependent apoptoticeffect of exogenous transforming growth factorbeta 1 in lymphoma cells
Anna Sebestyén⁎,1, Melinda Hajdu1, Lilla Kis, Gábor Barna, László KopperSemmelweis University, I. Department of Pathology and Experimental Cancer Research, 1085 Budapest, Üllői út 26, Hungary
Article Chronology:Received 18 October 2006Revised version received10 April 2007Accepted 30 May 2007Available online 29 June 2007
B-lymphoid tumor cells are often less sensitive than their normal counterparts orinsensitive to transforming growth factor beta1 (TGFb) effects. We studied the apoptoticeffect of exogenous TGFb in B-lymphoma cells, focusing on the activity and the role of Smadand protein phosphatase/kinase signals. Recombinant TGFb treatment and Smad4 siRNAtransfection were used in HT58 B-NHL lymphoma cells in vitro. Gene expression andapoptosis were detected by RT–PCR, Western blot analysis and flow cytometry. The role ofMEK1 kinase and PP2A activity – measured with a phosphatase assay – were assessed withthe help of specific inhibitors.Smad4 siRNA treatment completely abolished TGFb-induced early gene upregulation,indicating the absence of the rapid activation of Smad signaling. Moreover, functionalinhibition of Smad4 had no influence on TGFb-induced apoptosis, but it was dependent onPP2A phosphatase activation, ERK1/2 and JNK inactivation in lymphoma cells. The resultsprove that exogenous TGFb uses Smad4-independent, alternative (PP2A/PP2A-likedependent) signaling pathways for apoptosis induction in lymphoma cells. Furtherstudies are needed to clarify the possible role and involvement of Smad4-independenteffects of TGFb in normal and malignant lymphoid cells and in cells of the tumormicroenvironment.
Transforming growth factor beta 1 (TGFb) is a well-knownregulator of different cellular functions, such as proliferation,cell death and differentiation [1,2]. Smads are the leadingtransducers of TGFb signaling, and Smad4 is a centralcomponent of the cascade. Following TGFb1 binding to itsreceptors, R-Smads (Smad2 and 3) become activated, phos-phorylated and form a complex with Smad4. This complex istranslocated into the nucleus, where it interacts with tran-
ebestyén).this work.
er Inc. All rights reserved
scription factors and variousmodulatory co-factors regulatingthe expression of TGFb target genes [3,4]. The simple logic ofthe TGFb–Smad signaling cascade strongly contrasts with themolecular complexity of the cellular processes involved andthe diversity of responses triggered. Since Smad4 appears to bea key element of TGFb signal transduction, it was initiallyaccepted that the loss of Smad4 would completely abolishTGFb responses. However, accumulating data suggests thatSmad4 is in fact dispensable for some TGFb responses, andstriking results from microarray studies identified Smad4-
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dependent and -independent TGFb target genes and functionsin human carcinoma cell lines [4]. Intracellular TGFb signalingis confirmed to be a part of a larger network involving manyother signaling elements (MAPKs, ERK1/2, JNK, PI3K, PP2A/p70S6K, Rho, Pak2, Par6, etc.), which form a “signaling cross-talk/network” [4–13].
The role of TGFb in tumor growth can be bidirectional: itcan act either as a suppressor or a promoter cytokine[1,2,4,13,14]. TGFb-induced apoptosis and growth inhibitionhave been studied in many cell types, including human andmouse lymphocytes and several lymphoma cell lines [14–19].TGFb exerts its inhibitory effect contributing to immunosup-pression in the lymphoid system, however, many lymphoidmalignancies have impaired sensitivity to TGFb. AbnormalSmad3, TGFbRI/II and Smad4 expression – observed occasion-ally – indicates that disruption of the TGFb signaling pathwaycould be involved in the formation and/or progression of somehuman hematological malignancies (e.g. T-ALL, AML, CLL)[14,16,19,20]. The loss of TGFb-dependent apoptotic and anti-proliferative sensitivity is peculiar, because leukemia orlymphoma cells themselves can produce TGFb, in fact,serum TGFb levels in leukemia patients can be elevated. Inspite of resistance to endogenous TGFb, exogenous TGFb canstill induce apoptosis in lymphoma cells in vitro. In ourprevious studies, this induced apoptosis was shown to beindependent of death receptors but dependent on factorsreleased from mitochondria, and was regulated by caspases[21,22,23]. While the effectors of the apoptotic cascade areidentified (at least in certain lymphoid cell lines), the afferentsignaling route is unknown. The aim of this study was toclarify the involvement of different pathways in apoptosisinduction, focusing on the key component of the Smadpathway, i.e. Smad4. The results suggest that in our lymphoidmodel, in HT58 lymphoma cells, TGFb-induced apoptosis isindependent of Smad4 and switched on by phosphatases and/or kinases.
Materials and methods
Cell culture
HT58 (a human non-Hodgkin lymphoma B-cell line; EBVnegative, established in our laboratory) [24] cells werecultured in RPMI-1640 (Sigma, St. Louis, MO, USA) with 10%fetal bovine serum (GIBCO-BRL, Grand Island, NY, USA),0.03% glutamine (Sigma) and penicillin–streptomycin (100 U/mL–100 μg/mL, Sigma), at 37 °C, in 5% CO2 atmosphere. Cellsin the exponential growth phase were used for all experi-ments. Cells at a density of 1–2×105/mL were treated with1 ng/mL TGFb1 (R&D Systems, Minneapolis, MN, USA;reconstituted with 4 mM HCl in 0.1% BSA, aliquoted andstored at −20 °C) for 0–72 h in 24-well plates or 25 cm2 flasks.Activity of kinases was blocked by a MEK1 inhibitor (PD98059,1–10 μM, New England Biolabs, Ipswich, MA, USA), phospha-tase activity was inhibited by okadaic acid (100–500 nM,Calbiochem, San Diego, CA, USA) as well as endothall(endothall thioanhydride, 1–10 μM, Sigma). (Both inhibitorsare predominantly PP2A inhibitors in the given dose range.However, neither of them are fully specific for PP2A,
endothall is more specific of the two [25,26].) Since theseinhibitors are highly toxic during a long (48–72 h) treatmentperiod, cells were pretreated for 30 min before a 4-h TGFb1treatment. Cells were then washed twice to remove inhibi-tors and further cultured in fresh medium for 48–72 h. Weconfirmed previously that the apoptotic effect of TGFb1 wassimilar (a) when TGFb1 treatment lasted for 48 h and 72 h(without changing the medium) or (b) when cells werefurther cultured in fresh medium for 48–72 h after a 4-htreatment (Fig. 1a). This is not surprising as the functionalanalysis of TGFb–Smad signaling confirmed that Smadcomplexes accumulate in the nucleus immediately uponligand stimulation, and are directly involved in the regulationof the early (1 h) and late (6 h) target genes before complete,perceptible biological responses (e.g. effect on cell prolifera-tion) occur [4]. Experiments were done in triplicates, andthree independent experiments were performed for eachmeasurement.
Cell cycle analysis and apoptosis measurement
Flow cytometric measurements were performed according toDarzynkievicz et al. [27]. Briefly: for apoptosis detection cellswere fixed in 70% ethanol (−20 °C) followed by alkalicextraction (200 mM Na2HPO4, pH 7.4 and 100 μg/mL RNase(Sigma)) and ethidium bromide staining (10 μg/mL, Calbio-chem). Cell cycle and apoptosis detection was performed ona FACScan flow cytometer (BD Biosciences, San Diego, CA,USA), 10–20 000 events were collected for each sample. Datawas analyzed with WinList software (Verity Software House,Topsman, ME, USA). Cell morphology was evaluated on fixedand H&E-stained cytospin preparations.
Inhibition of Smad4 function
Dominant negative Smad4 construct (DNSmad4; kindly pro-vided by A. Moustakas and CH. Heldin) [9,28] was subclonedinto the p-EGFP-N1 vector (Clontech, Mountain View, CA, USA;Cat 6085-1). Transfection was performed with the Amaxa CellLine Nucleofector Kit T (Amaxa Inc., Gaithersburg, MD, USA).The efficiency of the transfection was detected by flowcytometry after 24–48 h.
Synthetic Smad4 siRNA (s: r(CAU-CCU-AGU-AAA-UGU-GUUA)dTdT; as: r(UAA-CAC-AUU-UAC-UAG-GAUG)dAdG)(Qiagen GmbH, Hilden, Germany) was used to silence Smad4,a fluorescein-labeled negative control siRNA was used as acontrol. 3×106 cells (7.5×105 cells/mL)were transfectedwith 5–10 μl (20 μM) siRNA and 24 μl HiPerFect reagent (Qiagen) in4mLmedium. Transfection efficiencywas determined by flowcytometry after 6–74 h in Smad4 siRNA and fluorescent controlsiRNA (1:1) co-treated samples. Smad4 expression wasdetected by Smad4 RT–PCR and Western blot analysis, andSmad4 activity was determined by screening TIEG mRNAupregulation in TGFb-treated (1–2 h) cultures by RT–PCR.
RT–PCR
Total RNA was isolated from 5 to 10×106 cells with QiagenRNeasy kit. RNA was reverse transcribed using MMLVReverse Transcriptase and random primers (Invitrogen).
Fig. 1 – Effect of Smad4 siRNA on TGFb-dependent apoptosis in HT58 lymphoma cells. (a) Flow cytometric analysis andmorphology (cytospin, H&E staining) of control (Co) and TGFb treated HT58 cells (72 h TGFb; 4 h TGFb treatment followed bymedium change and 72 h of incubation). (b) Time-dependent accumulation of fluorescein-labeled negative control siRNA afterSmad4 and negative control siRNA (1:1) co-transfection detected by flow cytometry. Percentages of siRNA-positive cells areindicated on the diagrams. (c) The effect of control and Smad4 siRNA transfection/co-transfection on mRNA expression ofSmad4 (28 cycles, RT–PCR). (d) Reduction of Smad4 protein expression and function after Smad4 siRNA transfection. The loss ofSmad4 protein expression (Western blot) and the inhibition of Smad4 function (TIEG RT–PCR)were detected in cells treatedwithSmad4 siRNA (6 h) and then with TGFb (2 h treatment, 6 h after siRNA transfection).
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100 ng cDNA was used for PCR. Equal quantity of cDNA wasconfirmed by β-actin (b-actin) amplification in control andTGFb-treated samples in semiquantitave RT–PCR. PCR con-ditions were as follows: 94 °C 1 min, 55 °C or 57 °C or 60 °C30 s, 72 °C 45 s; 26–30 cycles using RedTaq polymerase(Sigma). PCR products were resolved by agarose gel (1.5%)electrophoresis, stained with ethidium bromide and ana-
lyzed with an Eagle Eye video densitometer (Stratagene, LaJolla, CA, USA). Primers: Smad4 (205 bp, 60 °C, 26–28 cycles) 5′GTG GAA TAG CTC CAG CTA TC3′, 5′CGG CAT GGT ATG AAGTAC TCC3′; TIEG (229 bp, 60 °C, 28 cycles) 5′ACA GGA GAAAAG CCT TTC AGC3′, 5′TTT TAC ATC ACC ACT GGC TCC3′;b-actin (538 bp) 5′GTG-GGG-CGC-CCC-AGG-CAC-CA3′, 5′CTC-CTT-AAT-GTC-ACG-CAC-GAT-TTC3′.
Fig. 2 – Functional Smad4 inhibition does not influenceTGFb-induced apoptosis in HT58 lymphoma cells.(a) Apoptotic effect detected by flow cytometry after 72 h TGFbtreatment, following control and Smad4 siRNA transfection
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Western blotting
2×106 cells were lysed on ice in 100 μl SDS sample buffercontaining 50 mM Tris–HCl, pH 7.5, 150 mM NaCl, 1% NP40,1 mM PMSF, 10 mM NaF, 0.5 mM sodium vanadate, 10 μg/mLleupeptide and 10% glycerol. Lysates were kept on ice for10 min and centrifuged at 15000×g for 20 min to collect thesupernatant. Protein concentration was estimated with theBradford assay. Equal amounts of protein were diluted with2× SDS protein sample buffer (60 mM Tris–HCl, 2% SDS, 20%glycerol, 2% β-mercaptoethanol, bromophenol blue), sepa-rated on 12.5% SDS–PAGE gels and blotted onto PVDFmembranes (Bio-Rad, Hercules, CA, USA). Membranes wereincubated with polyclonal anti-Smad4/DPC4, monoclonalanti-ERK1 or anti-JNK2 (D-2) antibodies (Santa Cruz Biotech.,Santa Cruz, CA, USA; 1:200), followed by the appropriatesecondary antibodies (HRP-conjugated anti-mouse or HRP-conjugated anti-rabbit, Santa Cruz) and ECL (Pierce, Rock-ford, IL, USA). Phosphoproteins were visualized with aphospho-MAPK antibody sampler [New England BioLabspolyclonal primary antibodies: anti-phospho MAPK–ERK1/2(Thr202/Tyr204), anti-phospho-p38 MAPK (Thr180/Tyr182), anti-phospho SAPK/JNK (Thr183/Tyr185)] and the appropriatesecondary antibody, developed with ECL.
Membranes were stained with PonceauS (Sigma), and alsostripped and re-probed with anti-β-actin antibody (Sigma) toconfirm equal protein loading.
Phosphatase activity measurement
Nonradioactive Serine/Threonine Phosphatase Assay System(V2460, Promega, Madison, WI, USA) was used according tothe manufacturer's instructions. Cell lysates were preparedfrom 107 cells in 0.5 mL lysis buffer (10 mM Tris, pH 7.5; 0.1%Triton X-100; 140 mM NaCl; 1 mM PMSF; protease inhibitorcocktail) and passed through Sephadex G-25 columns toremove free phosphate. The activity of the extract (corre-sponding to 2 μg protein) was measured in an enzyme-specific reaction buffer (250 mM imidazole pH 7.2; 1 mMEGTA, 0.1% β-mercaptoethanol; 0.5 mg/mL BSA) with 1 mMphosphopeptide and Molybdate Dye/Additive incubation.Colorimetric OD results were read at 620 nm. Calculationswere done from parallel measurements of standard free phos-phate reactions.
Statistics
Descriptive statistics (n, mean and SD) was applied for dataanalysis. Student's t test was applied for evaluating signifi-cance. Statistical analysis of data was performed using SPSSsoftware (SPSS Inc., Chicago, IL, USA).
for 6 h or 24 h. (b) Inhibition of Smad4 protein expressionwasconfirmed by Western blotting after 6–24 h siRNAtransfection, followed by 2 h of TGFb treatment. (c) Functionalinhibition of Smad4 was confirmed by the lack ofTGFb-induced TIEG mRNA expression (RT–PCR) in Smad4siRNA-transfected cells. (Cells were collected for RT–PCR andWestern blotting from the adequate apoptotic experimentsafter 1–2 h TGFb treatment following 6–24 h Smad4 siRNAtransfection.)
Results
Knockdown of Smad4 in HT58 lymphoma cells
To identify TGFb signaling pathways which lead to apoptosisinduction in lymphoma cells, first we focused on Smad4, thecentral element in the canonical pathway. Functional
inhibition of Smad4 in HT58 lymphoma cells was achievedby transfection of Smad4 siRNA (efficiency: ∼95–98%). Cellviability was not significantly reduced after siRNA transfec-tion by HiPerFect or the Amaxa Nucleofector system. siRNAwas continuously present during the first period of ourexperiments, when TGFb signaling was turned on (Fig. 1b).Decreased Smad4 mRNA expression and reduction of theprotein was observed even at 6 h after transfection. Smad4mRNA expression remained inhibited longer than the timerequired for exogenous TGFb to turn on direct signalingpathways (transcriptional effects of TGFb usually appearvery early); furthermore, loss of Smad4 mRNA expressionwas detected during the full period of TGFb treatment(Figs. 1c, d). TGFb up-regulated TIEG expression – an earlymarker of Smad4-dependent signaling – in control siRNA-treated samples, and this effect was abolished in Smad4siRNA-transfected samples, indicating the functional inhibi-tion of Smad4 as early as 6 h after Smad4 siRNA transfection
Table 1 – Effect of MEK1 kinase inhibitor(MEK1I–PD98059) on TGFb (1 ng/mL)-induced apoptosis inHT58 lymphoma cells
The role of MEK1 kinase activity in TGFb-induced apoptosis in HT58lymphoma cells.Effect of the PD98059 MEK1 kinase inhibitor (MEK1I) on TGFb-induced apoptosis in HT58 lymphoma cells. Apoptosis wasdetected by flow cytometry; n.e.: not evaluated by flow cytometry(∼100% cell death and cell debris); 72 h co-treated culturescontained apoptotic and necrotic cells.⁎ MEK1I and TGFb were used in combination in the first 4 h oftreatment.
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as well (Fig. 1d). The status of Smad4-dependent TGFbsignaling was also confirmed 24 h after Smad4 siRNAtransfection, and we found that TGFb failed to up-regulateTIEG expression in the absence of Smad4, thus, Smad4-dependent signaling remained silenced for a longer period(Fig. 2c).
Functional inhibition of Smad4 has no effect on apoptosisinduced by TGFb
There was no significant difference between the percentageof apoptotic cells in Smad4 siRNA- or control siRNA-treatedlymphoma cells at 72 h after TGFb treatment (Fig. 2a).Functional inhibition of Smad4 was confirmed in theadequate samples of siRNA-treated cultures. The loss ofSmad4 protein expression and the consequent functionalinhibition of Smad4 prevented TGFb-induced TIEG expres-sion, but it did not abolish apoptosis (Fig. 2). DNSmad4vector transfection was not as effective as siRNA transfec-tion: transfection efficiency was ∼65%. However, vectortransfection yielded similar results as siRNA transfection:Smad4 did not influence the apoptotic effect; the extent ofTGFb-induced apoptosis in transient DNSmad4 vector-transfected HT58 cells was the same as in control vector-transfected cells (data not shown). We can conclude thatSmad4 is not required for TGFb-induced cell death in HT58lymphoma cells.
Fig. 3 – MAPK kinase and PP2A activity in TGFb-dependentapoptosis in HT58 lymphoma cells. (a) Activity of p38MAPK,ERK1/2 and JNK was detected by Western blotting usingphospho-p38MAPK, phospho-ERK1/2 and phospho-JNKspecific antibodies. (b) Time-dependent PP2A activity afterTGFb treatment in 2 μg protein extracts measured by a PP2Aactivity assay.
TGFb-induced early signaling involves ERK1/2 and JNKkinases and PP2A phosphatase activity
To study the role of the MAPK kinase pathways, TGFb-treatedcellswere assayed for p38MAPK, ERK1/2 and JNK kinase activityby determining the amount of their phosphorylated forms. Thelevel of p-ERK1/2 and p-JNK proteins rapidly decreased (Fig. 3a),but the phosphorylated form of p38MAPK and the total amountof JNK and ERK kinases did not change after TGFb treatment. Toconfirm the functional role of ERK/JNK inactivation, cells wereco-treated with a MEK1 kinase inhibitor and TGFb for 4 h. Thiscombinational treatment enhanced the apoptotic effect ofTGFb: apoptosis induced after 48 h was as high as that inducedby72h treatmentwithTGFbonly.At72hcell culturescontained
Table 2 – Effect of phosphatase inhibitors on TGFb(1 ng/mL)-dependent apoptosis in HT58 lymphoma cells
% ofapoptotic
cells
% ofapoptotic
cells
48 h(%)
72 h(%)
48 h(%)
72 h(%)
Control 11±3 12±3 Control 9±0 15±3100 nM OKAa 11±2 3±3 1 μM ETAb 8±0 17±3500 nM OKA 12±2 15±4 10 μM ETA 17±1 20±41 ng/mL TGFb 40±2 58±4 1 ng/mL TGFb 38±2 68±7100 nMOKA+TGFb
18±6 23±3 1 μM ETA+TGFb 13±1 20±4
500 nMOKA+TGFb
16±3 21±2 10 μM ETA+TGFb 23±2 25±5
The role of PP2A phosphatase activation in TGFb-induced apoptosisin HT58 lymphoma cells.Effect of phosphatase inhibitors on TGFb-dependent apoptosis inHT58 lymphoma cells. Apoptotic effect was detected by flowcytometry.a Okadaic acid and TGFb; andb Endothall combination were used in the first 4 h of treatment.
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mainly apoptotic and necrotic cells, therefore they were notevaluated by flow cytometry; ∼100% cell death and cell debriswas seen on cytospin preparations (Table 1). This suggests thatthe inhibition of kinases promoted and accelerated theapoptosis program.
The activation of cellular PP2A and its role was alsoevaluated. TGFb induced a biphasic increase in PP2A activity:at an early phase (20 min and 1 h after treatment with TGFb)and at a later period (48 h after TGFb treatment) (Fig. 3b). Theformer could be a signaling/initiator phase, whereas the lattermight represent the effector phase of apoptosis. The role ofPP2A activation was assayed by treating cells with phospha-tase inhibitors. Okadaic acid and endothall (mainly PP2Ainhibitors in the applied dose range) given in the first 4 h ofTGFb treatment almost completely abolished the apoptoticeffect of TGFb in lymphoma cells (Table 2).
Discussion
We previously reported that exogenous TGFb elicited apopto-sis in a TGFb producing B-cell lymphoma in vitro. Our resultsraised two questions: (a) what pathways are activated by theligand, and (b) what is the mechanism of apoptosis? Experi-ments designed to answer the latter question indicated thatthe induced apoptosis was not dependent on cell-deathreceptors, but required factors from the mitochondria andthe activation of caspases [21–23,29]. In this work, we tried toidentify the events between ligand binding and the “decision”point of apoptosis.
The functional analysis of TGFb signaling confirmed thatthe activation of Smads is an early event in the cascade, andthat Smad signaling activity is directly involved in theregulation of several early (1 h) and late (6 h) target genesafter TGFbR stimulation [4]. An increasing number of studiesdescribe Smad4-independent responses to TGFb in Smad4-deficient carcinoma cells. These studies involve mutantSmad4 cells, dominant negative Smad4-transfected celllines, Smad4-deficient and Smad4 knock-out cells as well[30–33]. These systems are maintained in the absence ofSmad4 for extended periods, allowing cells to accumulatemutations or adjust the expression level of other genes thatmight compensate for the loss of Smad4. We used transientinhibition with synthetic siRNA in order to silence Smad4-dependent TGFb signals during the time period when theafferent pathway of TGFb-induced apoptosis is switched on.This period is less than 24 h in lymphoid cells [21–23]. The“point of no return” in the apoptoticmachinery (i.e. mitochon-drial depolarization and the activation of caspase 3) is reached20–24 h after TGFb treatment in HT58 lymphoma cells;however, the appearance of detectable DNA fragmentationand morphological changes needs longer time [21,22]. TIEGwas reported to be a Smad4-dependent early target gene ofTGFb, therefore, TIEG expression can be used as a marker ofSmad4-dependent signaling in vitro [4]. Our results indicatedthat early upregulation of TIEG mRNA expression by TGFb iscompletely abolished in Smad4 siRNA-transfected cells. How-ever, Smad4 silencing did not influence apoptosis in HT58lymphoma cells. Consequently, apoptosis induced by exogen-ous TGFb is Smad4-independent in our system.
Different microarray studies described both Smad4-depen-dent and -independent regulation of certain apoptotic genes(e.g. death-associated protein 6, TRAIL, TNSF10, GADD45B)[4,28,30]. Smad7 is also considered as a Smad4-independentlyregulated TGFb responsive gene by some studies. TGFb-induced Smad7 mRNA upregulation was found to be requiredfor apoptosis induction, and TGFb-induced apoptosis wasabolished in Smad7 knock-out epithelial cells [34]. We showedpreviously that Smad7 expression was induced during TGFb-mediated apoptosis in HT58 lymphoma cells [29]. These factsfurther support the presence of Smad4-independent signalingmechanisms in TGFb-mediated apoptosis in lymphoma cells.
How can TGFb signaling occur without the central compo-nent, Smad4, or without Smads at all? (a) The existence of anunknown co-Smad cannot be excluded; however, no addi-tional Smad-family member has been found by sequencehomology; (b) Smad4-independent responses could bemediated via activated R-Smads translocated into the nucleus[35–37]; (c) Finally, alternative signaling pathways may existwithout the activation of Smad4 or any other Smads [4,8–12].Our results and other recent reports support the latter twopossibilities.
Numerous observations suggest that PP2A plays a majorrole in the downregulation of the ERK–MAPK pathway. PP2Ahas been found to form stable complexeswith several kinases,and numerous kinases have been identified as substrates ofPP2A, including ERK1/2 and JNK [38,39]. In vitro PP2A candephosphorylate and inactivateMEK1 and ERK family kinases,and both of them are activated after treatment with okadaicacid [38–40]. We showed that ERK1/2 and JNK are rapidlyinactivated in TGFb-treated HT58 cells. The inhibition of anupstream kinase, MEK1, increased the percentage of apoptoticcells, and the time required for the onset of apoptosis wasshortened. PP2A activity changed in a time-dependent man-ner during apoptosis; furthermore, two potent PP2A inhibitors– okadaic acid and endothall – significantly decreased TGFb-induced apoptosis in HT58 cells. Thus, our results indicatethat the activation of PP2A and the inactivation of ERK1/2 andJNK play an important role in Smad4-independent TGFb-mediated apoptosis in HT58 lymphoma cells. Whether thereduction in ERK and JNK activity is directly linked to PP2Aactivity in TGFb-induced apoptosis is not yet determined.TGFb-induced inhibition of EGF-dependent proliferation wasdescribed in a Smad4-deficient pancreatic carcinoma, whereTGFb-induced serine/threonine phosphatase activity inacti-vated ERK2 [41]. Although these phosphatases are not yetidentified, we and others suggest that PP2A may have thisfunction, as it was found to interact with the TGFb receptorcomplex and to dephosphorylate activated MEK and ERK[41,42].
MAPK activity has an important role in B-cell receptorregulated survival in B-lymphoid cells, especially in B-lymphoma cells [43–46], which is completely different fromTGFbR-activated, JNK-mediated cellular responses in non-Blymphoid cell lines [8]. The disruption of this regulation canshift cellular decisions towards cell death/apoptosis. HowTGFb signaling inactivates MAPK kinases – directly or indir-ectly – remains to be determined. Early activation of PP2A orother phosphatases directly inactivate ERK and/or JNK. ERK/JNK inactivation and PP2A or PP2A-like phosphatase activity
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can also participate in alternative signaling routes indepen-dently of each other [41,42]. It was shown that TGFb-mediatedearly PP2A activation might regulate p70S6K [47], and wedetected a reduction in p70S6K activity upon TGFb treatmentas well (data not shown).
It is well known that the phosphorylation state and –subsequently – the activity of Smad2/3 is regulated by not onlythe TGFbR, but also by dynamic interplay between kinases andphosphatases. The serine–threonine-rich linker region of R-Smads can be phosphorylated by MAPKs and CDKs (both in aninhibitory and activating manner), which may contribute toSmad4-dependent and -independent signaling mechanisms[1,6–8]. Recently, SCPs (small C-terminal domain phospha-tases) have been identified, which are able to dephosphorylatethe Smad2/3 linker region after TGFb treatment and enhanceSmad signaling activity [48,49]. SCP1/2/3 are likely to beinvolved in modulating crosstalk between signaling pathwaysthat converge on R-Smads.
Cell survival depends on the balance between anti-apoptotic survival and proapoptotic signals. This balance canbe shifted towards apoptosis by exogenous TGFb, lowering thesurvival threshold through the regulation of PP2A, ERK1/2 andJNK activity, providing Smad4-independent alternative signal-ing routes in lymphoma cells. Our results confirm that TGFbsignaling comprises a large network and is a part of a signalingcrosstalk, which is responsible for the complexity of TGFb-mediated biological effects. The mapping of Smad4-depen-dent and -independent components of this complex, cell type-dependent network in normal and malignant lymphoid cells(or in the microenvironment of other tumors) should behelpful in developing future therapies by targeting tumor-promoting functions of TGFb.
Acknowledgments
We thank C.H. Heldin and A. Moustakas for the DN-Smad4construct and I. Kovalszky for GFP-vectors. We thank P.I.Bauer for technical advice and Gézáné Csorba for technicalassistance. We also thank R. Mihalik for valuable discussionduring the preparation of the manuscript. This work wassupported by grants from the National Science Foundation ofHungary, Ministry of Welfare and Ministry of Education andCulture, Hungary (OTKA (F048380, TS049887), NKFP1A/00-04and Békésy Foundation (118/2001)).
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