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Leukemia Inhibitory Factor Induces DNA Synthesis in Swiss Mouse 3T3 Cells Independently of Cyclin D1 Expression through a Mechanism Involving MEK/ERK1/2 Activation *

Leukemia Inhibitory Factor Induces DNA Synthesis in Swiss Mouse 3T3 Cells Independently of Cyclin... THE JOURNAL OF BIOLOGICAL CHEMISTRY VOL. 281, NO. 10, pp. 6136 –6143, March 10, 2006 © 2006 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in the U.S.A. Leukemia Inhibitory Factor Induces DNA Synthesis in Swiss Mouse 3T3 Cells Independently of Cyclin D Expression through a Mechanism Involving MEK/ERK1/2 Activation Received for publication, May 27, 2005, and in revised form, October 7, 2005 Published, JBC Papers in Press, November 15, 2005, DOI 10.1074/jbc.M505839200 ‡ ‡ ‡ § ‡ ‡ Andres Dekanty , Moira Sauane , Belen Cadenas , Federico Coluccio , Marcela Barrio , Jorgelina Casala , ‡ ‡1 §2 ‡2 ¶ Mercedes Paciencia , Florencia Rogers , Omar A. Coso , Graciela Piwien-Pilipuk , Philip S. Rudland , ‡2,3,4 and Luis Jime´nez de Asu´a ‡ § From the Fundacio ´n Instituto Leloir, Av. Patricias Argentinas 435, 1405 Buenos Aires, Argentina, Laboratorio de Fisiologı´a y Biologı´a Molecular, Facultad de Ciencias Exactas y Naturales, Universidad de Buenos Aires, 1428 Buenos Aires, Argentina, and Molecular Medicine Group, School of Biological Sciences, University of Liverpool, Liverpool L69 3BX, United Kingdom Leukemia inhibitory factor (LIF) and oncostatin M (OSM) induce Depending on the cell type, LIF promotes cellular proliferation or dif- DNA synthesis in Swiss 3T3 cells through common signaling mecha- ferentiation, e.g. embryonic stem cell growth (5, 6), mammalian embryo nism(s), whereas other related cytokines such as interleukin-6 and cil- implantation (2, 4), neuronal differentiation (7, 8), enhancing survival of iary neurotrophic factor do not cause this response. Induction of DNA peripheral neurons (7) and oligodendrocytes (10), promoting bone for- replication by LIF or prostaglandin F (PGF ) occurs, in part, mation (11), and myoblast proliferation (12–14). LIF is also implicated 2 2 through different signaling events. LIF and OSM specifically trigger in a variety of pathophysiological processes (15–19). Cellular responses STAT1 cytoplasmic to nuclear translocation, whereas PGF fails to do to LIF as well as to other cytokines are initiated via heterodimerization so. However, LIF and PGF can trigger increases in ERK1/2 activity, of two members of the cytokine receptor family (8, 20, 21). The resultant which are required for their mitogenic responses because U0126, a signal transduction process involves activation of cytoplasmic Janus MEK1/2 inhibitor, prevents both ERK1/2 activation and induction of kinases (8, 20, 21), which in turn promote tyrosine phosphorylation of DNA synthesis by LIF or PGF treatment. PGF induces cyclin D the signal transducers and activators of transcription (STATs), thereby 2 2 expression and full phosphorylation of retinoblastoma protein. In con- enabling them to translocate to the nucleus and initiate gene transcrip- trast, LIF fails to promote increases in cyclin D mRNA/protein levels; tion of LIF-responsive genes (22). LIF can also trigger alternative signal- consequently, LIF induces DNA synthesis without promoting full ing processes to those causing STAT activation (23). These include phosphorylation of retinoblastoma protein (Rb). However, both LIF activation of the mitogen-activated protein kinase (MAPK) cascade, and PGF increase cyclin E expression. Furthermore, LIF mitogenic 2 including the mitogen-activated protein kinase kinase (MAPKK or action does not involve protein kinase C (PKC) activation, because a MEK), the MAPK isoenzymes (ERK1 and ERK2), and activation of S6 PKC inhibitor does not block this effect. In contrast, PKC activity is protein kinase (8). required for PGF mitogenic action. More importantly, the synergis- 2 We have shown previously that Swiss 3T3 cells are equally responsive ticeffectbetweenLIFandPGF topromoteSphaseentryisindepend- 2 to both sets of growth factors; LIF and PGF are thus equally effective at ent of PKC activation. These results show fundamental differences inducing DNA synthesis (25). The generality of the difference in signal- betweenLIF-andPGF -dependentmechanism(s)thatinducecellular ing events triggered by both cytokines and growth factors in different entryintoSphase.ThesefindingsarecriticalinunderstandinghowLIF cell systems is well established; cytokines trigger activation of Janus and other related cytokine-regulated events participate in normal cell kinases that promote phosphorylation of STATs (8, 20–22), and growth cycle control and may also provide clues to unravel crucial processes factors triggered mitogen-induced Raf/MEK/ERK signaling pathway underlying cancerous cell division. leading to overexpression of cyclin D (26–29, 42–45). However, because these mitogens have been tested in different cellular systems, it is unknown whether this difference is a function of the cell type or is a Leukemia inhibitory factor (LIF) belongs to a closely related group of fundamental difference in the delivery of the transducing signal per se. cytokines, which includes oncostatin M (OSM), ciliary neurotrophic Thus, we have systematically studied LIF-, OSM-, and PGF -depend- factor (CNTF), interleukin 6 (IL-6), and cardiotrophin-1 (1–4). ent mechanisms of control of S phase entry into the Swiss 3T3 cell system. We have shown previously that LIF-triggered signals differ from * This work was supported in part by the Association for International Cancer Research those triggered by classical mitogens, such as PGF , bombesin, or epi- Grant 099-018 (Scotland, UK) and Cancer and Polio Research Fund (Liverpool, UK) (to dermal growth factor (30–32) in Swiss 3T3 cells. LIF and OSM trigger L. J. A. and P. S. R.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertise- initiation of DNA synthesis without the requirement for mevalonic acid ment” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. synthesis because inhibition of the hydroxymethylglutaryl-CoA reduc- Recipient of a GlaxoSmithKline-Argentina fellowship. Investigator of the National Research Council (CONICET) of Argentina. tase by lovostatin does not block LIF- or OSM-induced DNA replication To whom correspondence should be addressed: Fundacio´ n Instituto Leloir, Av. Patri- and cell multiplication (32). Indeed, because LIF triggers cellular entry cias Argentinas 435, C1405BWE Buenos Aires, Argentina. Tel.: 54-11-5238-7500; Fax: into the S phase via a PKC-independent signaling mechanism, it 54-11-5238-7501; E-mail: [email protected]. Visiting Professor from the Royal Society of London, School of Biological Sciences Uni- becomes relevant to investigate which activation cascade (MAPK versity of Liverpool, Liverpool, UK. 5 and/or JAK/STAT) is involved in the onset of DNA replication and cell The abbreviations used are: LIF, leukemia inhibitory factor; OSM, oncostatin M; IL, inter- leukin; CNTF, ciliary neurotrophic factor; MOPS, 4-morpholinepropanesulfonic acid; STAT, signal transducers and activators of transcription; MAPK, mitogen-activated protein kinase; PG, prostaglandin; Rb, retinoblastoma protein; pRb, phosphorylated signal-regulated kinase; MEK, MAPK/ERK kinase; PBS, phosphate-buffered saline; retinoblastoma protein; PKC, protein kinase C; JAK, Janus kinase; ERK, extracellular MBP, myelin basic protein; FBS, fetal bovine serum; TBS, Tris-buffered saline. 6136 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 281 • NUMBER 10 •MARCH 10, 2006 This is an Open Access article under the CC BY license. LIF-triggered Signals and Cell Cycle Control division. Here we show that LIF and OSM stimulate the initiation of Beads (Amersham Biosciences) and washed three times with PBS con- DNA synthesis and cell division through a common signaling mecha- taining 1% (v/v) Nonidet P-40 and 2 mM Na VO , once with 100 mM 3 4 nism that involves MEK/ERK activation as well as STAT1 cytoplasmic- Tris-HCl (pH 7.5), 0.5 M LiCl, and once in kinase reaction buffer (12.5 nuclear translocation. Signals triggered by LIF and OSM are independ- mM MOPS (pH 7.5), 12.5 mM 3-glycerophosphate, 7.5 mM MgCl , 0.5 ent of PKC activation. On the other hand, PGF triggers activation of mM EGTA, 0.5 mM sodium fluoride, 0.5 mM sodium vanadate). The MEK/ERK but fails to activate STAT1 cytoplasmic-nuclear transloca- ERK-2 activity present in the immunoprecipitates was determined by tion. The different signaling pathways involved in the mitogenic resuspension in 30 l of kinase reaction buffer containing 10 Ci of response to cytokines and growth factors cause a major effect on G [- P]ATP per reaction and 20 M of unlabeled ATP, using 20 gof cyclin expression. PGF , as the majority of classical growth factors, myelin basic protein (MBP) as a substrate, as described previously (35). induces the expression of cyclin Ds as an important step in the G to S After 20 min at 30 °C, reactions were terminated by addition of 10 lof phase transition. However, LIF-stimulated S phase entry occurs without 5 Laemmli buffer. Samples were heated 5 min at 95 °C and analyzed by changes in the levels of cyclin Ds but with increases in cyclin E expres- SDS-gel electrophoresis on 12% (w/v) polyacrylamide gels. Autoradiog- sion. These findings are relevant for understanding how LIF, in partic- raphy was performed with the aid of an intensifying screen. Parallel ular, and cytokines, in general, regulate the events involved in control- immunoprecipitates were processed for Western blot analysis using the ling the normal cell cycle. These studies are also important for same antiserum as described previously (35). unraveling critical signaling events capable of underlying unrestricted Indirect Immunofluorescence—It was performed on sparse quiescent cancerous cell division. Swiss 3T3 cells, adhering to coverslips. After stimulation, cells were fixed and permeabilized with 3.7% (v/v) formaldehyde in PBS plus MATERIALS AND METHODS 0.2%(v/v) saponin for 10 min at room temperature. Cells were then blocked for 30 min with 10% (v/v) heat-inactivated goat serum and then Chemicals—The majority of the materials was purchased from Sigma incubated overnight at 4 °C with 20 g/ml of the primary antibody unless otherwise indicated. The mouse recombinant carrier-free LIF, diluted in PBS, 0.1%(v/v) saponin. The primaries antibodies used were OSM, CNTF, and IL-6 were purchased fromR&D(Minneapolis, MN). as follows: STAT1 antibody (G16920), STAT3 antibody (S21320), and PGF was the generous gift from Dr. M. Torkelston, Upjohn (Kalama- STAT5 antibody (S21520) from BD Transduction Laboratories. The zoo, MI), and the GF109203X was kindly provided by GlaxoSmithKline. coverslips were washed three times with PBS and further incubated for U0126 was purchased from Calbiochem. [methyl- H]Thymidine was 1 h with fluorescein isothiocyanate-conjugated goat anti-mouse IgGs purchased from PerkinElmer Life Sciences. (Sigma) diluted 1/50 in PBS. Finally the cells were washed three times Cell Culture—Swiss mouse 3T3 cells were grown in Dulbecco’s mod- with PBS and mounted with 1,4-diazabicyclo[2.2.3]octane solution ified Eagle’s medium containing 100 units of penicillin/ml, 100 gof (Sigma). Images were obtained on a BX-60 Olympus fluorescent micro- streptomycin/ml, and 10% (v/v) fetal calf serum. Subconfluent cultures scope. The percentage of cells in which STAT1 translocated to the were grown in 100-mm dishes at 37 °C equilibrated with 10% (v/v) CO . nucleus upon LIF or LIF plus Na VO treatment was determined in Initiation of DNA Synthesis Assay—Cells were seeded at 1.5  10 in 3 4 three independent experiments by counting the number of nuclear and 35-mm dishes in 2 ml of Dulbecco’s modified Eagle’s medium supple- non-nuclear stained cells in at least five fields, each containing an aver- mented with low molecular weight nutrients (DEMS), 1% (v/v) newborn age of at least 150 cells, at different time points. calf serum, and 6% (v/v) fetal calf serum. After 3 days of incubation, the SDS-PAGE and Immunoblotting—Proteins were separated on 12% medium was changed to a similar fresh medium, and the cells were (w/v) SDS-polyacrylamide gels and transferred to nitrocellulose filters. further incubated for 3–4 days to allow them to become confluent and Transfers were blocked overnight at 4 °C with 5% (v/v) nonfat milk in quiescent. Cytokines and growth factors were directly added to the con- TBS, 0.1% (v/v) Tween 20 and then incubated for1hat room temper- ditioned medium. Cells were then labeled with [methyl- H]thymidine ature in the primary antibody diluted in 5% (v/v) nonfat milk in TBS, for 28 h before being processed for autoradiography. The percentage of 0.1%(v/v) Tween 20. The primary antibodies used were as follows: phos- cells that initiated DNA synthesis at a given time was determined as MAPK phospecific p42/p44 antibody (sc-7383; diluted 1/1000), p42/ described previously (25, 33). MAPK p44 antibody (sc-154; 1/1000), cyclin D antibody (sc-450; 1/1000), Cell Number—Cells were plated at 1.5 10 in 60-mm dishes in 5 ml cyclin D antibody (sc-593; 1/1000), cyclin D antibody (sc-6283; of medium, for the determination of the initiation of DNA synthesis. 2 3 1/1000), cyclin E antibody (sc-481; 1/1000), CDK4 antibody (sc-260; When cells became quiescent, but were still subconfluent, the corre- 1/1000), and CDK6 antibody (sc-177; 1/1000) from Santa Cruz Biotech- sponding cytokines or prostaglandins were added to the culture medium, and the cell number was determined 60 h later. Cells were nology; cyclin A antibody (C4710) from Sigma. The transfers were rinsed with TBS, 0.1% (v/v) Tween 20 and incubated for1hat room detached from the dish using 5 mM EDTA, 0.05% (v/v) trypsin for 5 min temperature in horseradish peroxidase-conjugated pig anti-rabbit or at 37 °C and counted in a Coulter counter (34). MAPK Assay—MAPK activity assays were performed on subconflu- rabbit anti-mouse serum (Dako) diluted 1/5000 in 5% (v/v) nonfat milk in TBS, 0.1% (v/v) Tween 20. The immunoblots were developed with the ent Swiss 3T3 cells by immunoprecipitation of total endogenous ERK. ECL detection reagent (Amersham Biosciences). Cells were maintained for 16 h in serum-free medium and then treated with agents as indicated in the figure legends, washed with cold PBS, and Northern Blot—Cells were plated at 3.0  10 in 100-mm dishes similar to the assay for DNA synthesis. Under these conditions, cultures lysed at 4 °C in a buffer containing 25 mM HEPES (pH 7.5), 0.3 M NaCl, became confluent and quiescent at a saturating density of 3  10 cells. 1.5 mM MgCl , 0.2 mM EDTA, 0.5 mM dithiothreitol, 1% (v/v) Triton X-100, 0.5% (v/v) sodium deoxycholate, 0.1% (v/v) SDS, 20 mM -glyc- In experiments where the levels of cyclin D mRNA were determined erophosphate, 1 mM sodium vanadate, 1 mM phenylmethylsulfonyl flu- after various times of addition of growth factors, total RNA was pre- oride, 20 g/ml aprotinin, and 20 g/ml leupeptin. ERK was immuno- pared from cells by extracting them with Trizol reagent. Samples (15 precipitated from the cleared lysates by incubation with the specific g) were subjected to 1% (v/v) MOPS/formaldehyde agarose-gel elec- antibody (sc-154 from Santa Cruz Biotechnology) for1hat4 °C.Immu- trophoresis and blotted onto nylon membranes. The membranes were nocomplexes were recovered with the aid of Gamma-bind-Sepharose hybridized with P-labeled cDNA probes for cyclin D and 18 S RNA MARCH 10, 2006• VOLUME 281 • NUMBER 10 JOURNAL OF BIOLOGICAL CHEMISTRY 6137 LIF-triggered Signals and Cell Cycle Control with a Promega kit, washed, and exposed to x-ray film (36). For cyclin D Swiss 3T3 cells with IL-6 or CNTF did not induce cellular entry into the the 1.3-kbp EcoRI fragment of pcBZ04.1 was used. Cyclin D and 18 S S phase (Fig. 1). Taken together, these results suggest that LIF and OSM RNA cDNA probes were generously provided by Drs. C. D. Sherr (St. may share common signaling pathway(s) to induce the entry into S Jude’s Hospital, Memphis, TN) and A. R. Kornblihtt (Physiology and phase. Molecular Biology Laboratory, School of Sciences, University of Buenos To examine whether LIF- or OSM-triggered signals differ from those Aires, Argentina), respectively. elicited by PGF , we analyzed the ability of PGF to enhance LIF- or 2 2 OSM-dependent induction to S phase entry. PGF treatment stimu- RESULTS lated the initiation of DNA synthesis in Swiss 3T3 cells by inducing 7 LIF and OSM Induce DNA Synthesis through Common Signaling and 25% of cells to enter into S phase after 28 h (Fig. 1), similarly to the Mechanism(s)—To investigate whether LIF-triggered signals that effect observed with LIF or OSM. Most interestingly, when PGF and induce cell proliferation can be shared by other related cytokines, such LIF were added together at either subsaturating or saturating concen- as OSM, CNTF, and IL-6, we studied their ability to induce cellular trations, the stimulatory effect of LIF or PGF in inducing DNA syn- entry into S phase in resting Swiss 3T3 cells. Both LIF and OSM, added thesis was potentiated by raising the fraction of cells that entered into S at either subsaturating (10 ng/ml) or saturating (100 ng/ml) concentra- phase to 57 and 67%, respectively (Fig. 1). Similarly, the stimulatory tions, stimulated the initiation of DNA synthesis inducing 8 and 29% of effect of OSM was potentiated by PGF (Fig. 1). These results suggest cells to enter into S phase after 28 h, respectively (Fig. 1), as shown that LIF or OSM in combination with PGF exhibited a synergistic previously (32). When both LIF and OSM were added together, they effect by increasing the percentage of cells entering S phase. Further- caused only an additive effect on the percentage of cells that entered into more, we hypothesized that the synergistic effect observed between LIF S phase at any concentration tested (Fig. 1). In contrast, treatment of or OSM with PGF to induce both DNA synthesis and cell division may be due, at least in part, to the activation of different signaling pathways promoted by LIF and OSM with respect to those activated by PGF . LIF and PGF Activate MAPK Signaling Pathway with a Different Kinetic Pattern—To ascertain whether the mitogenic effect of LIF and PGF on Swiss 3T3 cells involved differential activation of a well char- acterized MAPK cascade (37), we examined the capacity of LIF and PGF to activate ERK. Treatment of Swiss 3T3 cells with LIF or PGF 2 2 resulted in different kinetic patterns of ERK activation. LIF promoted a maximum increase of ERK activity only at 12 min (Fig. 2B), whereas PGF caused a maximum increase in ERK activity within 4 min (Fig. 2A). There was no change in the levels of p44/p42 MAPK throughout this period (Fig. 2, A and B). Because both LIF and PGF promote the activation of ERK1/2, we investigated whether LIF- or PGF -triggered activation of ERK is required for their mitogenic effect. Treatment of cells with U0126, a widely used MEK inhibitor (38), for 1 h before addition of growth factors caused a significant and progressive reduction in either LIF- or PGF - induced ERK phosphorylation without affecting the overall levels of this protein (Fig. 3A). Indeed, U0126 at 10 M inhibited ERK activity by 90% FIGURE 1. Induction of DNA synthesis by LIF and OSM. To measure cellular entry into S phase, quiescent Swiss 3T3 cells were treated with each cytokine and growth factor at in both PGF - and LIF-stimulated cells (Fig. 3B). Moreover, addition of saturating or subsaturating concentrations and then labeled with [methyl- H]thymidine U0126 from 0.5 to 5 M to cells prior to LIF or PGF treatment resulted for 28 h. Radioactive label incorporation was analyzed by autoradiography (see under in a progressive inhibition of DNA replication (Fig. 3C). However, “Materials and Methods”). Additions were as follows: LIF (10 ng/ml), OSM (10 ng/ml), CNTF (10 ng/ml), IL-6 (10 ng/ml), PGF (30 ng/ml) at subsaturating concentrations, and 2 although U0126 at 1 M effectively blocked PGF -stimulated DNA LIF (100 ng/ml), OSM (100 ng/ml), CNTF (100 ng/ml), IL-6 (100 ng/ml), and PGF (300 synthesis by 90%, a higher concentration of 3 M was required to cause ng/ml) at saturating concentrations. Results represent the mean S.E. of four independ- ent experiments. a similar effect with LIF-stimulated cells. In contrast, U0126 did not FIGURE 2. LIF or PGF triggered ERK activation with a different kinetic pattern. Quiescent Swiss 3T3 cells were treated with PGF (300 ng/ml) (A) or LIF (100 ng/ml) (B). Cells were lysed at time inter- vals from 0 to 60 min, and kinase activity was measured as indicated under “Materials and Meth- ods.” Data represent the mean S.E. of three inde- pendent experiments, expressed as fold increase in kinase activity with respect to untreated cells. Upper panel, densitometric analysis expressed as fold increase in kinase activity with respect to untreated cells; middle panel, P-labeled MBP as product of the kinases reaction for one represent- ative experiment; lower panel, equal amounts of ERK protein were immunoprecipitated as shown by Western blot analysis of the immunoprecipi- tated samples using anti-ERK antibodies. 6138 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 281 • NUMBER 10 •MARCH 10, 2006 LIF-triggered Signals and Cell Cycle Control FIGURE 3. ERK activation is required for LIF- or PGF -dependent DNA synthesis. A, cells were treated with vehicle (lane C), LIF (100 ng/ml), or PGF (300 ng/ml) for 12 and 4 min, respectively, in the absence or presence of U0126. Cells were lysed, and equal amounts of protein were analyzed by Western blot using a specific phospho-ERK1/2 antibody (upper panel) or a specific ERK2 antibody (lower panel). The results are repre- sentative of three independent experiments. B, cells were left untreated or treated with LIF (100 ng/ml) or PGF (300 ng/ml) for 12 and 4 min, respectively, in the absence or presence of U0126 (10M). Endogenous ERK2 protein was immunoprecipitated from the cell extracts and kinase activity assayed using MBP as substrate. Data represent the mean  S.E. of three independent experiments expressed as fold increase in kinase FIGURE 4. LIF, but not PGF , promotes nuclear translocation of STAT1. A, subcon- activity with respect to untreated cells. C, to measure the effect of ERK inhibition on LIF- fluent Swiss 3T3 cells were treated with vehicle (A), LIF (100 ng/ml) (B), OSM (ng/ml) (D), or PGF -stimulated DNA synthesis, quiescent Swiss 3T3 fibroblasts were treated for 28 h or PGF (300 ng/ml) (C) for 15 min. Cells were fixed, and STAT1 localization was visual- with LIF (100 ng/ml), PGF (300 ng/ml), and FBS (10%v/v) in the absence or presence of ized by indirect immunofluorescence with an anti-STAT1 monoclonal antibody as the indicated concentrations of U0126 and then labeled with [methyl- H]thymidine. described under “Materials and Methods.” Insets in panels show a higher magnification U0126 was added 1 h prior to the growth factors. The percentage of cells that entered of a cell for better visualization. Percentage of cells with STAT1 nuclear staining is as into S phase was determined by autoradiography, as described under “Materials and follows: control, 10  4%; LIF, 89  5%; OSM, 85  7%; and PGF 15  6%. B, kinetic 2, Methods.” The results are presented as the percentage of labeled nuclei with respect to pattern of STAT1 translocation was determined as follows. Swiss 3T3 cells were stimu- the growth factor alone and are representative of four independent experiments. lated with LIF in the absence or presence of Na VO (30 M). This inhibitor was added 1 h 3 4 prior to cell treatment. Cells were fixed, and the STAT1 localization was visualized by affect DNA synthesis stimulated by fetal bovine serum (FBS). These indirect immunofluorescence as described under “Materials and Methods.” The percent- age of STAT1-immunostained nuclei was determined at different times after cytokine results showed that ERK activation is required for both LIF- and PGF - addition. At least 750 cells were counted per time point. The results represent the triggered cellular entry into S phase, although with a different kinetic means  S.E. of three independent experiments. pattern. Role of JAK/STAT Signaling Pathway in LIF/PGF -induced DNA cence were obtained when the subcellular localization of STAT1 was Synthesis—To investigate whether LIF, OSM, and PGF exert a differ- determined by analyzing cytoplasmic and nuclear fractions of LIF-stim- ential stimulation of the JAK/STAT pathway, we examined their ability ulated cells by Western blotting (data not shown). Furthermore, LIF, to cause cytoplasmic to nuclear translocation of the different STATs by OSM, and PGF could not promote translocation of STAT3 or STAT5 indirect immunofluorescence upon treatment of subconfluent resting Swiss 3T3 cells. In control, nonstimulated cells, STAT1 was mainly to the nucleus in the 3T3 cells (not shown). As positive controls, anti- STAT3 and STAT5 antibodies were able to detect STAT3 and STAT5 diffusely distributed within the cytoplasm (Fig. 4A). LIF and OSM treat- cytoplasmic to nuclear translocation in 3T3 L1 pre-adipocytes stimu- ment promoted nuclear translocation of STAT1 as demonstrated by the nuclear immunostaining of STAT1 (Fig. 4, panels A, B, and D, respec- lated by different cytokines, growth factors, and hormones (data not tively). In contrast, PGF did not trigger STAT1 nuclear localization shown) (39). No translocation was observed with either IL-6 or CNTF, (Fig. 4A, panel C). Results similar to those found by immunofluores- which are not mitogenic for Swiss 3T3 cells (Fig. 4B). MARCH 10, 2006• VOLUME 281 • NUMBER 10 JOURNAL OF BIOLOGICAL CHEMISTRY 6139 LIF-triggered Signals and Cell Cycle Control FIGURE 5. Inhibition of tyrosine phosphatases enhances LIF induction of DNA syn- thesis. To analyze the effect of tyrosine phosphatase inhibition on LIF stimulation of DNA synthesis, quiescent Swiss 3T3 cells were treated for 28 h with LIF (100 ng/ml) or OSM (100 ng/ml) in the absence or presence of 30 M Na VO and then labeled with 3 4 [methyl- H]thymidine. C, control. The Na VO was added 1 h before the cytokine treat- 3 4 ment. The percentage of cells that entered into S phase was determined by autoradiog- raphy, as described under “Materials and Methods.” Results represent the means  S.E. of three independent experiments. To ascertain whether regulation of tyrosine phosphatases is impli- cated in LIF-dependent nuclear localization of STAT1, Swiss 3T3 cells were incubated in the presence or absence of Na VO , a general inhib- 3 4 itor of tyrosine phosphatases (40, 41), prior to and during cytokine treat- ment. LIF-promoted cytoplasmic-nuclear translocation of STAT1 occurred as rapidly as 2 min after addition of LIF, and its nuclear local- ization attained a plateau at 10–15 min and declined to the basal level by 60 min (Fig. 4B). In the presence of Na VO , LIF also promoted the 3 4 rapid translocation of STAT1 to the nucleus. However, in the presence of Na VO , STAT1 remains in the nucleus after 60 min of LIF treatment 3 4 (Fig. 4B). This observation is consistent with findings that showed that a phosphatase inhibitor could prolong the activation of STAT1 and thus its nuclear retention (42). Indeed, Na VO also markedly enhanced LIF- 3 4 induced cellular entry into S phase, raising the percentage of cells under- going DNA synthesis over 2.5-fold (Fig. 5). Na VO also enhanced 3 4 FIGURE 6. The mitogenic effect of LIF is independent of cyclin D expression. A, OSM- and PGF -dependent cellular entry into the S phase (Fig. 5). 2 Swiss3T3 cells were treated with vehicle or saturating concentrations of LIF (100 ng/ml) or PGF (300 ng/ml) for different periods of time. Cell extracts were prepared, and equal These experiments indicated that LIF-dependent activation of STAT1 2 amounts of protein were analyzed by immunoblotting using specific antibodies for and initiation of DNA replication might both involve tyrosine kinase cyclin D , cyclin D , cyclin D , CDK4, and CDK6 (see “Materials and Methods”). Data are 1 2 3 activation. In contrast, the PGF -dependent mitogenic effect appears representative of three independent experiments. B, cyclin D mRNA levels were deter- mined at various times after cytokine or PGF treatment. Swiss 3T3 cells were treated not to require STATs activation. with vehicle or saturating concentrations of LIF (100 ng/ml), OSM (100 ng/ml), CNTF (100 Effect of LIF and PGF on G Cyclin Expression and pRb Phosphoryl- ng/ml), IL-6 (100 ng/ml), or PGF (300 ng/ml). After 8 –10 h, total RNA was extracted from 2 1 2 cells; Northern blot was performed as described under “Materials and Methods.” North- ation—To determine whether differences between LIF/OSM and ern blot densitometric analysis was standardized to 18 S RNA. Similar results were PGF2 signaling pathways have major consequences on expression of obtained in three independent experiments. C, Swiss 3T3 cells were treated with vehicle, LIF, or FBS for various times. Cell extracts were prepared, analyzed by SDS-PAGE, and key G cyclins and their effector kinases involved in executing the G /S 1 1 immunoblotted using specific antibodies against cyclin D , cyclin E, and cyclin A. Results transition, the expression of cyclins was assessed at different times upon are representative of three independent experiments. D, extracts from cells treated with treatment of quiescent 3T3 cells with LIF or PGF . Fig. 6 shows that vehicle (lane C), PGF , LIF, or FBS for the indicated periods of time were separated by SDS-PAGE and subjected to immunoblot analysis for pRb. Arrows indicate pRb with dif- PGF raised cyclin D protein levels within 9 h, reaching a plateau after 2 1 ferent levels of phosphorylation. 12–15 h, and these levels remained relatively high for up to 21 h (Fig. 6A). PGF also raised cyclin D protein levels at later times (within PGF increases cyclin E and A protein levels similar to those for LIF 2 2 15–21 h of treatment) but failed to increase cyclin D protein levels (Fig. 3 (data not shown). 6A). In contrast, LIF as well as OSM failed to increase cyclin D protein Cyclin D-CDK complexes were shown to phosphorylate the retino- and mRNA levels (Fig. 6, A and B), as well as failing to raise cyclin D or blastoma tumor suppressor protein (pRb), leading to inactivation of pRb cyclin D protein levels (Fig. 6A). IL-6 and CNTF were also unable to (44). It is well documented that inactivation of pRb results in release or induce cyclin D mRNA levels (Fig. 6B). The levels of the corresponding derepression of the E2F transcription factors and drives cell entry into partner CDK4/6 kinases (43) did not show any increase upon LIF or the S phase (44). To examine whether differences in cyclin D expression PGF treatments (Fig. 6A). However, addition of LIF induced an mediated by LIF and PGF result in differences in pRb phosphoryla- 2 2 increase in cyclin E and cyclin A protein levels after 14 and 28 h to levels tion, quiescent Swiss 3T3 cells were induced to enter S phase, and phos- comparable with that induced by 10% FBS, at least for cyclin E (Fig. 6C). phorylation of pRb was assessed by immunoblotting. As shown in Fig. 6140 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 281 • NUMBER 10 •MARCH 10, 2006 LIF-triggered Signals and Cell Cycle Control FIGURE 7. The synergistic effect between LIF and PGF to induce S phase entry is independ- ent of PKC activation. A, quiescent Swiss 3T3 cells were treated for 28 h with LIF or PGF in the absence or presence of GF109203X added 1 h prior to the addition of growth factors and then labeled with [methyl- H]thymidine. The percent- age of cells that entered into S phase was deter- mined by autoradiography, as described under “Materials and Methods.” B, quiescent Swiss 3T3 cells were treated for 28 h with vehicle, LIF, PGF , or LIF and PGF in the absence or presence of GF109203X (10 M) added 1 h prior to addition of growth factors. The percentage of cells that entered into S phase was determined by autora- diography. Results represent the mean  S.E. of four independent experiments. 6D, LIF did not promote full phosphorylation of pRb (lane 4 versus lane However, because these mitogens have been tested in different cellular 1). In contrast, PGF induced hyperphosphorylation of pRb (Fig. 6D, systems, it is unknown whether this difference is a function of the cell lanes 2 and 3 versus lane 1), consistent with the induction of expression type or is a fundamental difference in the delivery of the transducing of cyclins Ds. Phosphorylation of pRB induced by PGF is comparable signal per se. In this study we show that LIF-triggered signaling mecha- with the level of pRb phosphorylation induced by FBS (Fig. 6D, lane 5) nism(s) for inducing cellular entry into S phase are shared only by OSM upon cell entry into S phase. In summary, LIF induces neither expres- and not by PGF in the same quiescent Swiss mouse 3T3 cell system. sion of cyclin Ds nor hyperphosphorylation of pRb, whereas PGF Their differences are not due to subpopulations of 3T3 cells that are promotes both the increase in the expression of cyclin Ds and the hyper- primarily responsive to LIF/OSM or to PGF , because repeated sub- phosphorylation of pRb to exert its mitogenic effect. Taken together, cloning of 3T3 cells and addition of LIF/OSM or PGF yield the same these results provide further evidence that LIF and PGF may act percentages of cells stimulated to synthesize DNA in the given time through different signaling and molecular events to control the initia- period. Treatment of Swiss 3T3 cells with LIF or OSM together with tion of cellular entry into S phase. PGF mutually potentiated their ability to induce cellular DNA synthe- The Synergistic Effect of LIF and PGF to Induce S Phase Entry Is sis, whereas co-treatment of cells with LIF and OSM rendered no fur- Independent of PKC Activation—We have shown previously that LIF ther increase. Experimentally cellular DNA synthesis is measured by the and PGF differ markedly in the requirement for PKC in stimulating fraction of cells with [ H]DNA in their nuclei after 28 h. A longer expo- DNA synthesis (25). LIF triggers cellular entry into S phase via a PKC- sure of cells to [ H]thymidine with one mitogen increases this fraction, independent signaling mechanism, whereas PGF requires the activa- eventually reaching almost 100% of the cell population (22, 25, 27, 28, 33, tion of the PKC signaling pathway (25). Therefore, we examined 34). The synergy observed between LIF and PGF merely increases the whether PGF -dependent activation of PKC plays a role in the syner- rate of cellular entry into S phase but not the absolute fraction of cells gistic effect observed between LIF and PGF in the induction of DNA that are responding. These observations suggest that LIF and OSM trig- synthesis in Swiss 3T3 cells. We tested the effect of increasing concen- ger common signaling pathways that may differ from those activated by trations of GF109203X, a specific inhibitor of PKC, on the ability of LIF PGF for the induction of DNA synthesis. The importance of the study and PGF alone or in combination to trigger DNA replication. As therefore lies in the uncovering of the biochemical differences in the shown in Fig. 7A, GF109203X progressively inhibited PGF -dependent signal transduction pathways of these two groups of mitogens in the DNA replication but completely failed to block LIF-dependent DNA same cell system. replication. These results are consistent with our previous findings ERK1/2 are components of the well known MAPK signaling cascade obtained with 12-O-tetradecanoylphorbol-13-acetate indicating that activated by mitogens and are thus involved in controlling cell prolifer- LIF and PGF differed markedly in the requirement for PKC in stimu- ation (37). Here we show that both LIF or PGF by themselves can 2 2 lating DNA synthesis (25). Most interestingly, GF109203X did not pre- promote ERK activation. However, LIF and PGF differ in their timing vent the synergistic effect between PGF and LIF in increasing the of MAPK activation. Although PGF induced a maximum at 4 min, LIF 2 2 percentage of cells that entered into S phase (Fig. 7B). These results did so only after 12 min after addition, a result that suggests that LIF and suggest that the synergistic effect of LIF and PGF to promote S phase PGF cause ERK activation via two separate upstream signaling events. 2 2 entry is independent of PKC activation. U0126, a highly specific MEK inhibitor, blocked both LIF- and PGF - triggered MAPK activation and their mitogenic responses, strongly sug- DISCUSSION gesting that MAPK activation is required for the initiation of both LIF It has been shown previously that Swiss 3T3 cells are equally respon- and PGF -dependent DNA synthesis. However, how LIF increases sive to both sets of growth factors; LIF and PGF are thus equally ERK activity and the consequent stimulation of DNA synthesis in these effective at inducing DNA synthesis (25). The generality of the differ- cells is still unknown. MAPK activation is more complex than a simple ence in signaling events triggered by both cytokines and growth factors linear pathway. For example, LIF-triggered ERK1/2 activity in 3T3-L1 in different cell systems is well established; cytokines trigger activation adipocytes can occur via both Raf-1-dependent and -independent pro- of Janus kinases that promote phosphorylation of STATs (8, 23–25), and growth factors trigger the mitogen-induced Raf/MEK/ERK signal- ing pathway leading to overexpression of cyclin D (26–29, 42–45). P. Rudland and L. Jime´nez de Asu´ a, unpublished results. MARCH 10, 2006• VOLUME 281 • NUMBER 10 JOURNAL OF BIOLOGICAL CHEMISTRY 6141 LIF-triggered Signals and Cell Cycle Control cesses (22). In addition, it has been shown that an increase in phosphati- other growth factors, which must increase the levels of cyclin Ds to dylinositol 3-kinase activity may be involved in ERK activation (45) and induce S phase entry (36, 55, 56). Whether the low basal levels of cyclin D/CDK4 are sufficient to trigger the next step, phosphorylation of Rb that phosphatidylinositol 3-kinase may play a role in prolonging ERK activity (46). Moreover, cytokines such as interferon  or OSM can (57), or whether this first stage of Rb phosphorylation is bypassed is activate Raf-1 in a Ras-independent manner via increased activity of unknown at present. Moreover, different findings reveal that cyclin E/CDK2 can induce cellular entry into S phase in the absence of cyclin JAK1 or Tyk2 (47). These findings and our present results support the D/CDK4 activation (56, 58) and that c-Myc and Cdc25A can participate notion that multiple, temporally distinct pathways can converge on MAPK and that these pathways can be utilized differentially by various in the activation of the cyclin E-CDK2 complex (59–61). A recent report demonstrates that proliferation of mouse embryonic fibroblasts stimuli and cell types. proceeds relatively normally in the absence of the D-cyclins (62). Kozar Cyclin D expression is generally regulated by a mitogen-induced et al. (62) shows that mouse embryonic fibroblasts from cyclin D Raf/MEK/ERK signaling pathway (26–29). Indeed, the duration of an 1 / / D D mice critically depend on CDK2, suggesting that cyclin ERK signal appears to determine whether cells will induce cyclin D 2 3 D-CDK4/6 and cyclin E-CDK2 complexes may perform overlapping expression. Mitogens that only produce a transient ERK activation fail functions in “cyclin D-independent” systems. It is established that the to induce cyclin D , whereas growth factors that induce a sustained ERK initial phosphorylation of the pRb by cyclin D-CDK complexes is activation cause continuous maintenance of cyclin D expression (48, required to allow full phosphorylation of the pRb by the cyclin E- and 49). Thus, the critical determinant in the induction of cyclin D is the cyclin A-associated kinases (9, 63). However, Kozar et al. (62) shows that duration, rather than the intensity, of the ERK signal. Our studies show phosphorylation of pRb on cyclin D-specific sites is not required for that LIF-stimulated DNA synthesis requires an intact MEK/ERK signal- further phosphorylation and that cyclin E- and cyclin A-driven phos- ing cascade. However, LIF-stimulated ERK activation is likely not to be phorylation is sufficient to allow the expression of E2Fs target genes linked to the increase in cyclin D expression. In contrast, PGF -stim- 1 2 during cell cycle re-entry. Our study shows that LIF promotes expres- ulated ERK activation may be directly involved with increasing the sion of cyclin E and A but not cyclin Ds, as shown in Fig. 6. Furthermore, expression of cyclin D and ultimately with its mitogenic response. How full phosphorylation of pRb does not take place as cells re-enter the cell LIF promotes S phase entry in an ERK-dependent manner and how ERK cycle. Therefore, our future goal will be to elucidate the role of cyclin activation does not result in an increase in cyclin D expression have yet E/CDK2 in regulating LIF induction of DNA replication in a cyclin to be elucidated. The results presented here suggest that the different D-independent manner. kinetics of MAPK activation may result in a different pattern of G In summary, our present work demonstrates that LIF and PGF cyclin expression, although alternative explanations based on ERK1/2- trigger different signaling and molecular events prior to cellular entry independent activation of the cyclin D promoter by PGF and not by 1 2 into S phase in the same cell system. The importance of this work estab- LIF may be possible. lishes that stem cell factors like LIF can bypass the normal growth fac- JAK/STAT signal cascades are known to be involved in responses to tor-induced Raf/MEK/ERK signaling pathway to cyclin D and activation cytokines. Our immunofluorescence studies reveal that LIF and OSM of CDK4/6, the key event that normally allows progress through the trigger a similar pattern of STAT1 cytoplasmic to nuclear translocations Restriction Point R and commitment to enter the cell cycle (54). LIF and, after 2 min, attaining a maximum at 10–15 min and declining at 60 min by implication, stem cell growth factors in general then trigger cellular to the basal level, whereas CNTF, IL-6, and PGF were without effect. entry into S phase by partial phosphorylation of Rb through increases in Our experiments to understand the role of LIF induction of DNA syn- cyclin E and activation of CDK2. Thus the enhancing effect of PGF on thesis indicated that the effect of LIF is mediated via tyrosine kinase the induction of cellular entry into S phase mediated by LIF is probably because Na VO potentiates LIF’s stimulation of DNA synthesis. Fur- 3 4 because of the ability of the former mitogen to phosphorylate Rb com- thermore, this result is correlated with the prolonged localization of pletely and thereby further reduce its inhibitory activity for the E2F STAT1 in the nucleus. However, it will be important to elucidate transcription factor required for G to S phase transition (54). Presum- whether STAT1 cytoplasmic-nuclear translocation in conjunction or ably, this synergy is mediated by interactions between those signals not with MAPK activation is required for LIF stimulation of DNA generated from the PGF receptor that are different from those of LIF, synthesis. in particular the very early rise in ERK activation and the activation of LIF is overproduced and secreted by several cancer cells (50, 51) and STAT1. By understanding the molecular mechanisms by which LIF in thus may act as an autocrine stimulator. Moreover, it is known that particular and cytokines in general control normal cell cycle constitutes during oncogenesis different STAT proteins are continuously activated the basis to unravel the critical cytokine-related signaling events under- in a variety of cancer cells types (52, 53), and if inhibited the resultant lying unrestricted cancerous cell division, as well as providing possible cancer cells grow much more slowly (52, 53, 60, 61). The fact that cyto- therapeutic targets to blockade this second signal transduction pathway kines stimulate DNA replication through different signaling events may in the event that it is necessary to inhibit both growth factor and cyto- result in carcinomas rapidly eluding the control of growth factor signals, kine signaling pathways to prevent cancer cell growth. and therefore any therapy targeted to the early signaling events trig- gered by growth factors may become rapidly ineffective. Thus the elu- Acknowledgments—We thank Liz Shannon of BD Biosciences for the generous cidation of the molecular mechanism of cytokine action is an important gift of tissue culture material. We thank Drs. Eduardo Passeron and Israel step in dissecting deranged regulatory events leading to malignant Algranati for the revision of the manuscript and for encouragement in our transformation and as a second parallel target for mounting a therapeu- research. 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S., and Jimenez de Cell 118, 477–491 Asu´a, L. (2004) Biochem. Biophys. Res. Commun. 313, 926–930 63. Lundberg, A. S., and Weinberg, R. A. (1998) Mol. Cell. Biol. 1, 753–761 MARCH 10, 2006• VOLUME 281 • NUMBER 10 JOURNAL OF BIOLOGICAL CHEMISTRY 6143 http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Journal of Biological Chemistry American Society for Biochemistry and Molecular Biology

Leukemia Inhibitory Factor Induces DNA Synthesis in Swiss Mouse 3T3 Cells Independently of Cyclin D1 Expression through a Mechanism Involving MEK/ERK1/2 Activation *

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Abstract

THE JOURNAL OF BIOLOGICAL CHEMISTRY VOL. 281, NO. 10, pp. 6136 –6143, March 10, 2006 © 2006 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in the U.S.A. Leukemia Inhibitory Factor Induces DNA Synthesis in Swiss Mouse 3T3 Cells Independently of Cyclin D Expression through a Mechanism Involving MEK/ERK1/2 Activation Received for publication, May 27, 2005, and in revised form, October 7, 2005 Published, JBC Papers in Press, November 15, 2005, DOI 10.1074/jbc.M505839200 ‡ ‡ ‡ § ‡ ‡ Andres Dekanty , Moira Sauane , Belen Cadenas , Federico Coluccio , Marcela Barrio , Jorgelina Casala , ‡ ‡1 §2 ‡2 ¶ Mercedes Paciencia , Florencia Rogers , Omar A. Coso , Graciela Piwien-Pilipuk , Philip S. Rudland , ‡2,3,4 and Luis Jime´nez de Asu´a ‡ § From the Fundacio ´n Instituto Leloir, Av. Patricias Argentinas 435, 1405 Buenos Aires, Argentina, Laboratorio de Fisiologı´a y Biologı´a Molecular, Facultad de Ciencias Exactas y Naturales, Universidad de Buenos Aires, 1428 Buenos Aires, Argentina, and Molecular Medicine Group, School of Biological Sciences, University of Liverpool, Liverpool L69 3BX, United Kingdom Leukemia inhibitory factor (LIF) and oncostatin M (OSM) induce Depending on the cell type, LIF promotes cellular proliferation or dif- DNA synthesis in Swiss 3T3 cells through common signaling mecha- ferentiation, e.g. embryonic stem cell growth (5, 6), mammalian embryo nism(s), whereas other related cytokines such as interleukin-6 and cil- implantation (2, 4), neuronal differentiation (7, 8), enhancing survival of iary neurotrophic factor do not cause this response. Induction of DNA peripheral neurons (7) and oligodendrocytes (10), promoting bone for- replication by LIF or prostaglandin F (PGF ) occurs, in part, mation (11), and myoblast proliferation (12–14). LIF is also implicated 2 2 through different signaling events. LIF and OSM specifically trigger in a variety of pathophysiological processes (15–19). Cellular responses STAT1 cytoplasmic to nuclear translocation, whereas PGF fails to do to LIF as well as to other cytokines are initiated via heterodimerization so. However, LIF and PGF can trigger increases in ERK1/2 activity, of two members of the cytokine receptor family (8, 20, 21). The resultant which are required for their mitogenic responses because U0126, a signal transduction process involves activation of cytoplasmic Janus MEK1/2 inhibitor, prevents both ERK1/2 activation and induction of kinases (8, 20, 21), which in turn promote tyrosine phosphorylation of DNA synthesis by LIF or PGF treatment. PGF induces cyclin D the signal transducers and activators of transcription (STATs), thereby 2 2 expression and full phosphorylation of retinoblastoma protein. In con- enabling them to translocate to the nucleus and initiate gene transcrip- trast, LIF fails to promote increases in cyclin D mRNA/protein levels; tion of LIF-responsive genes (22). LIF can also trigger alternative signal- consequently, LIF induces DNA synthesis without promoting full ing processes to those causing STAT activation (23). These include phosphorylation of retinoblastoma protein (Rb). However, both LIF activation of the mitogen-activated protein kinase (MAPK) cascade, and PGF increase cyclin E expression. Furthermore, LIF mitogenic 2 including the mitogen-activated protein kinase kinase (MAPKK or action does not involve protein kinase C (PKC) activation, because a MEK), the MAPK isoenzymes (ERK1 and ERK2), and activation of S6 PKC inhibitor does not block this effect. In contrast, PKC activity is protein kinase (8). required for PGF mitogenic action. More importantly, the synergis- 2 We have shown previously that Swiss 3T3 cells are equally responsive ticeffectbetweenLIFandPGF topromoteSphaseentryisindepend- 2 to both sets of growth factors; LIF and PGF are thus equally effective at ent of PKC activation. These results show fundamental differences inducing DNA synthesis (25). The generality of the difference in signal- betweenLIF-andPGF -dependentmechanism(s)thatinducecellular ing events triggered by both cytokines and growth factors in different entryintoSphase.ThesefindingsarecriticalinunderstandinghowLIF cell systems is well established; cytokines trigger activation of Janus and other related cytokine-regulated events participate in normal cell kinases that promote phosphorylation of STATs (8, 20–22), and growth cycle control and may also provide clues to unravel crucial processes factors triggered mitogen-induced Raf/MEK/ERK signaling pathway underlying cancerous cell division. leading to overexpression of cyclin D (26–29, 42–45). However, because these mitogens have been tested in different cellular systems, it is unknown whether this difference is a function of the cell type or is a Leukemia inhibitory factor (LIF) belongs to a closely related group of fundamental difference in the delivery of the transducing signal per se. cytokines, which includes oncostatin M (OSM), ciliary neurotrophic Thus, we have systematically studied LIF-, OSM-, and PGF -depend- factor (CNTF), interleukin 6 (IL-6), and cardiotrophin-1 (1–4). ent mechanisms of control of S phase entry into the Swiss 3T3 cell system. We have shown previously that LIF-triggered signals differ from * This work was supported in part by the Association for International Cancer Research those triggered by classical mitogens, such as PGF , bombesin, or epi- Grant 099-018 (Scotland, UK) and Cancer and Polio Research Fund (Liverpool, UK) (to dermal growth factor (30–32) in Swiss 3T3 cells. LIF and OSM trigger L. J. A. and P. S. R.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertise- initiation of DNA synthesis without the requirement for mevalonic acid ment” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. synthesis because inhibition of the hydroxymethylglutaryl-CoA reduc- Recipient of a GlaxoSmithKline-Argentina fellowship. Investigator of the National Research Council (CONICET) of Argentina. tase by lovostatin does not block LIF- or OSM-induced DNA replication To whom correspondence should be addressed: Fundacio´ n Instituto Leloir, Av. Patri- and cell multiplication (32). Indeed, because LIF triggers cellular entry cias Argentinas 435, C1405BWE Buenos Aires, Argentina. Tel.: 54-11-5238-7500; Fax: into the S phase via a PKC-independent signaling mechanism, it 54-11-5238-7501; E-mail: [email protected]. Visiting Professor from the Royal Society of London, School of Biological Sciences Uni- becomes relevant to investigate which activation cascade (MAPK versity of Liverpool, Liverpool, UK. 5 and/or JAK/STAT) is involved in the onset of DNA replication and cell The abbreviations used are: LIF, leukemia inhibitory factor; OSM, oncostatin M; IL, inter- leukin; CNTF, ciliary neurotrophic factor; MOPS, 4-morpholinepropanesulfonic acid; STAT, signal transducers and activators of transcription; MAPK, mitogen-activated protein kinase; PG, prostaglandin; Rb, retinoblastoma protein; pRb, phosphorylated signal-regulated kinase; MEK, MAPK/ERK kinase; PBS, phosphate-buffered saline; retinoblastoma protein; PKC, protein kinase C; JAK, Janus kinase; ERK, extracellular MBP, myelin basic protein; FBS, fetal bovine serum; TBS, Tris-buffered saline. 6136 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 281 • NUMBER 10 •MARCH 10, 2006 This is an Open Access article under the CC BY license. LIF-triggered Signals and Cell Cycle Control division. Here we show that LIF and OSM stimulate the initiation of Beads (Amersham Biosciences) and washed three times with PBS con- DNA synthesis and cell division through a common signaling mecha- taining 1% (v/v) Nonidet P-40 and 2 mM Na VO , once with 100 mM 3 4 nism that involves MEK/ERK activation as well as STAT1 cytoplasmic- Tris-HCl (pH 7.5), 0.5 M LiCl, and once in kinase reaction buffer (12.5 nuclear translocation. Signals triggered by LIF and OSM are independ- mM MOPS (pH 7.5), 12.5 mM 3-glycerophosphate, 7.5 mM MgCl , 0.5 ent of PKC activation. On the other hand, PGF triggers activation of mM EGTA, 0.5 mM sodium fluoride, 0.5 mM sodium vanadate). The MEK/ERK but fails to activate STAT1 cytoplasmic-nuclear transloca- ERK-2 activity present in the immunoprecipitates was determined by tion. The different signaling pathways involved in the mitogenic resuspension in 30 l of kinase reaction buffer containing 10 Ci of response to cytokines and growth factors cause a major effect on G [- P]ATP per reaction and 20 M of unlabeled ATP, using 20 gof cyclin expression. PGF , as the majority of classical growth factors, myelin basic protein (MBP) as a substrate, as described previously (35). induces the expression of cyclin Ds as an important step in the G to S After 20 min at 30 °C, reactions were terminated by addition of 10 lof phase transition. However, LIF-stimulated S phase entry occurs without 5 Laemmli buffer. Samples were heated 5 min at 95 °C and analyzed by changes in the levels of cyclin Ds but with increases in cyclin E expres- SDS-gel electrophoresis on 12% (w/v) polyacrylamide gels. Autoradiog- sion. These findings are relevant for understanding how LIF, in partic- raphy was performed with the aid of an intensifying screen. Parallel ular, and cytokines, in general, regulate the events involved in control- immunoprecipitates were processed for Western blot analysis using the ling the normal cell cycle. These studies are also important for same antiserum as described previously (35). unraveling critical signaling events capable of underlying unrestricted Indirect Immunofluorescence—It was performed on sparse quiescent cancerous cell division. Swiss 3T3 cells, adhering to coverslips. After stimulation, cells were fixed and permeabilized with 3.7% (v/v) formaldehyde in PBS plus MATERIALS AND METHODS 0.2%(v/v) saponin for 10 min at room temperature. Cells were then blocked for 30 min with 10% (v/v) heat-inactivated goat serum and then Chemicals—The majority of the materials was purchased from Sigma incubated overnight at 4 °C with 20 g/ml of the primary antibody unless otherwise indicated. The mouse recombinant carrier-free LIF, diluted in PBS, 0.1%(v/v) saponin. The primaries antibodies used were OSM, CNTF, and IL-6 were purchased fromR&D(Minneapolis, MN). as follows: STAT1 antibody (G16920), STAT3 antibody (S21320), and PGF was the generous gift from Dr. M. Torkelston, Upjohn (Kalama- STAT5 antibody (S21520) from BD Transduction Laboratories. The zoo, MI), and the GF109203X was kindly provided by GlaxoSmithKline. coverslips were washed three times with PBS and further incubated for U0126 was purchased from Calbiochem. [methyl- H]Thymidine was 1 h with fluorescein isothiocyanate-conjugated goat anti-mouse IgGs purchased from PerkinElmer Life Sciences. (Sigma) diluted 1/50 in PBS. Finally the cells were washed three times Cell Culture—Swiss mouse 3T3 cells were grown in Dulbecco’s mod- with PBS and mounted with 1,4-diazabicyclo[2.2.3]octane solution ified Eagle’s medium containing 100 units of penicillin/ml, 100 gof (Sigma). Images were obtained on a BX-60 Olympus fluorescent micro- streptomycin/ml, and 10% (v/v) fetal calf serum. Subconfluent cultures scope. The percentage of cells in which STAT1 translocated to the were grown in 100-mm dishes at 37 °C equilibrated with 10% (v/v) CO . nucleus upon LIF or LIF plus Na VO treatment was determined in Initiation of DNA Synthesis Assay—Cells were seeded at 1.5  10 in 3 4 three independent experiments by counting the number of nuclear and 35-mm dishes in 2 ml of Dulbecco’s modified Eagle’s medium supple- non-nuclear stained cells in at least five fields, each containing an aver- mented with low molecular weight nutrients (DEMS), 1% (v/v) newborn age of at least 150 cells, at different time points. calf serum, and 6% (v/v) fetal calf serum. After 3 days of incubation, the SDS-PAGE and Immunoblotting—Proteins were separated on 12% medium was changed to a similar fresh medium, and the cells were (w/v) SDS-polyacrylamide gels and transferred to nitrocellulose filters. further incubated for 3–4 days to allow them to become confluent and Transfers were blocked overnight at 4 °C with 5% (v/v) nonfat milk in quiescent. Cytokines and growth factors were directly added to the con- TBS, 0.1% (v/v) Tween 20 and then incubated for1hat room temper- ditioned medium. Cells were then labeled with [methyl- H]thymidine ature in the primary antibody diluted in 5% (v/v) nonfat milk in TBS, for 28 h before being processed for autoradiography. The percentage of 0.1%(v/v) Tween 20. The primary antibodies used were as follows: phos- cells that initiated DNA synthesis at a given time was determined as MAPK phospecific p42/p44 antibody (sc-7383; diluted 1/1000), p42/ described previously (25, 33). MAPK p44 antibody (sc-154; 1/1000), cyclin D antibody (sc-450; 1/1000), Cell Number—Cells were plated at 1.5 10 in 60-mm dishes in 5 ml cyclin D antibody (sc-593; 1/1000), cyclin D antibody (sc-6283; of medium, for the determination of the initiation of DNA synthesis. 2 3 1/1000), cyclin E antibody (sc-481; 1/1000), CDK4 antibody (sc-260; When cells became quiescent, but were still subconfluent, the corre- 1/1000), and CDK6 antibody (sc-177; 1/1000) from Santa Cruz Biotech- sponding cytokines or prostaglandins were added to the culture medium, and the cell number was determined 60 h later. Cells were nology; cyclin A antibody (C4710) from Sigma. The transfers were rinsed with TBS, 0.1% (v/v) Tween 20 and incubated for1hat room detached from the dish using 5 mM EDTA, 0.05% (v/v) trypsin for 5 min temperature in horseradish peroxidase-conjugated pig anti-rabbit or at 37 °C and counted in a Coulter counter (34). MAPK Assay—MAPK activity assays were performed on subconflu- rabbit anti-mouse serum (Dako) diluted 1/5000 in 5% (v/v) nonfat milk in TBS, 0.1% (v/v) Tween 20. The immunoblots were developed with the ent Swiss 3T3 cells by immunoprecipitation of total endogenous ERK. ECL detection reagent (Amersham Biosciences). Cells were maintained for 16 h in serum-free medium and then treated with agents as indicated in the figure legends, washed with cold PBS, and Northern Blot—Cells were plated at 3.0  10 in 100-mm dishes similar to the assay for DNA synthesis. Under these conditions, cultures lysed at 4 °C in a buffer containing 25 mM HEPES (pH 7.5), 0.3 M NaCl, became confluent and quiescent at a saturating density of 3  10 cells. 1.5 mM MgCl , 0.2 mM EDTA, 0.5 mM dithiothreitol, 1% (v/v) Triton X-100, 0.5% (v/v) sodium deoxycholate, 0.1% (v/v) SDS, 20 mM -glyc- In experiments where the levels of cyclin D mRNA were determined erophosphate, 1 mM sodium vanadate, 1 mM phenylmethylsulfonyl flu- after various times of addition of growth factors, total RNA was pre- oride, 20 g/ml aprotinin, and 20 g/ml leupeptin. ERK was immuno- pared from cells by extracting them with Trizol reagent. Samples (15 precipitated from the cleared lysates by incubation with the specific g) were subjected to 1% (v/v) MOPS/formaldehyde agarose-gel elec- antibody (sc-154 from Santa Cruz Biotechnology) for1hat4 °C.Immu- trophoresis and blotted onto nylon membranes. The membranes were nocomplexes were recovered with the aid of Gamma-bind-Sepharose hybridized with P-labeled cDNA probes for cyclin D and 18 S RNA MARCH 10, 2006• VOLUME 281 • NUMBER 10 JOURNAL OF BIOLOGICAL CHEMISTRY 6137 LIF-triggered Signals and Cell Cycle Control with a Promega kit, washed, and exposed to x-ray film (36). For cyclin D Swiss 3T3 cells with IL-6 or CNTF did not induce cellular entry into the the 1.3-kbp EcoRI fragment of pcBZ04.1 was used. Cyclin D and 18 S S phase (Fig. 1). Taken together, these results suggest that LIF and OSM RNA cDNA probes were generously provided by Drs. C. D. Sherr (St. may share common signaling pathway(s) to induce the entry into S Jude’s Hospital, Memphis, TN) and A. R. Kornblihtt (Physiology and phase. Molecular Biology Laboratory, School of Sciences, University of Buenos To examine whether LIF- or OSM-triggered signals differ from those Aires, Argentina), respectively. elicited by PGF , we analyzed the ability of PGF to enhance LIF- or 2 2 OSM-dependent induction to S phase entry. PGF treatment stimu- RESULTS lated the initiation of DNA synthesis in Swiss 3T3 cells by inducing 7 LIF and OSM Induce DNA Synthesis through Common Signaling and 25% of cells to enter into S phase after 28 h (Fig. 1), similarly to the Mechanism(s)—To investigate whether LIF-triggered signals that effect observed with LIF or OSM. Most interestingly, when PGF and induce cell proliferation can be shared by other related cytokines, such LIF were added together at either subsaturating or saturating concen- as OSM, CNTF, and IL-6, we studied their ability to induce cellular trations, the stimulatory effect of LIF or PGF in inducing DNA syn- entry into S phase in resting Swiss 3T3 cells. Both LIF and OSM, added thesis was potentiated by raising the fraction of cells that entered into S at either subsaturating (10 ng/ml) or saturating (100 ng/ml) concentra- phase to 57 and 67%, respectively (Fig. 1). Similarly, the stimulatory tions, stimulated the initiation of DNA synthesis inducing 8 and 29% of effect of OSM was potentiated by PGF (Fig. 1). These results suggest cells to enter into S phase after 28 h, respectively (Fig. 1), as shown that LIF or OSM in combination with PGF exhibited a synergistic previously (32). When both LIF and OSM were added together, they effect by increasing the percentage of cells entering S phase. Further- caused only an additive effect on the percentage of cells that entered into more, we hypothesized that the synergistic effect observed between LIF S phase at any concentration tested (Fig. 1). In contrast, treatment of or OSM with PGF to induce both DNA synthesis and cell division may be due, at least in part, to the activation of different signaling pathways promoted by LIF and OSM with respect to those activated by PGF . LIF and PGF Activate MAPK Signaling Pathway with a Different Kinetic Pattern—To ascertain whether the mitogenic effect of LIF and PGF on Swiss 3T3 cells involved differential activation of a well char- acterized MAPK cascade (37), we examined the capacity of LIF and PGF to activate ERK. Treatment of Swiss 3T3 cells with LIF or PGF 2 2 resulted in different kinetic patterns of ERK activation. LIF promoted a maximum increase of ERK activity only at 12 min (Fig. 2B), whereas PGF caused a maximum increase in ERK activity within 4 min (Fig. 2A). There was no change in the levels of p44/p42 MAPK throughout this period (Fig. 2, A and B). Because both LIF and PGF promote the activation of ERK1/2, we investigated whether LIF- or PGF -triggered activation of ERK is required for their mitogenic effect. Treatment of cells with U0126, a widely used MEK inhibitor (38), for 1 h before addition of growth factors caused a significant and progressive reduction in either LIF- or PGF - induced ERK phosphorylation without affecting the overall levels of this protein (Fig. 3A). Indeed, U0126 at 10 M inhibited ERK activity by 90% FIGURE 1. Induction of DNA synthesis by LIF and OSM. To measure cellular entry into S phase, quiescent Swiss 3T3 cells were treated with each cytokine and growth factor at in both PGF - and LIF-stimulated cells (Fig. 3B). Moreover, addition of saturating or subsaturating concentrations and then labeled with [methyl- H]thymidine U0126 from 0.5 to 5 M to cells prior to LIF or PGF treatment resulted for 28 h. Radioactive label incorporation was analyzed by autoradiography (see under in a progressive inhibition of DNA replication (Fig. 3C). However, “Materials and Methods”). Additions were as follows: LIF (10 ng/ml), OSM (10 ng/ml), CNTF (10 ng/ml), IL-6 (10 ng/ml), PGF (30 ng/ml) at subsaturating concentrations, and 2 although U0126 at 1 M effectively blocked PGF -stimulated DNA LIF (100 ng/ml), OSM (100 ng/ml), CNTF (100 ng/ml), IL-6 (100 ng/ml), and PGF (300 synthesis by 90%, a higher concentration of 3 M was required to cause ng/ml) at saturating concentrations. Results represent the mean S.E. of four independ- ent experiments. a similar effect with LIF-stimulated cells. In contrast, U0126 did not FIGURE 2. LIF or PGF triggered ERK activation with a different kinetic pattern. Quiescent Swiss 3T3 cells were treated with PGF (300 ng/ml) (A) or LIF (100 ng/ml) (B). Cells were lysed at time inter- vals from 0 to 60 min, and kinase activity was measured as indicated under “Materials and Meth- ods.” Data represent the mean S.E. of three inde- pendent experiments, expressed as fold increase in kinase activity with respect to untreated cells. Upper panel, densitometric analysis expressed as fold increase in kinase activity with respect to untreated cells; middle panel, P-labeled MBP as product of the kinases reaction for one represent- ative experiment; lower panel, equal amounts of ERK protein were immunoprecipitated as shown by Western blot analysis of the immunoprecipi- tated samples using anti-ERK antibodies. 6138 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 281 • NUMBER 10 •MARCH 10, 2006 LIF-triggered Signals and Cell Cycle Control FIGURE 3. ERK activation is required for LIF- or PGF -dependent DNA synthesis. A, cells were treated with vehicle (lane C), LIF (100 ng/ml), or PGF (300 ng/ml) for 12 and 4 min, respectively, in the absence or presence of U0126. Cells were lysed, and equal amounts of protein were analyzed by Western blot using a specific phospho-ERK1/2 antibody (upper panel) or a specific ERK2 antibody (lower panel). The results are repre- sentative of three independent experiments. B, cells were left untreated or treated with LIF (100 ng/ml) or PGF (300 ng/ml) for 12 and 4 min, respectively, in the absence or presence of U0126 (10M). Endogenous ERK2 protein was immunoprecipitated from the cell extracts and kinase activity assayed using MBP as substrate. Data represent the mean  S.E. of three independent experiments expressed as fold increase in kinase FIGURE 4. LIF, but not PGF , promotes nuclear translocation of STAT1. A, subcon- activity with respect to untreated cells. C, to measure the effect of ERK inhibition on LIF- fluent Swiss 3T3 cells were treated with vehicle (A), LIF (100 ng/ml) (B), OSM (ng/ml) (D), or PGF -stimulated DNA synthesis, quiescent Swiss 3T3 fibroblasts were treated for 28 h or PGF (300 ng/ml) (C) for 15 min. Cells were fixed, and STAT1 localization was visual- with LIF (100 ng/ml), PGF (300 ng/ml), and FBS (10%v/v) in the absence or presence of ized by indirect immunofluorescence with an anti-STAT1 monoclonal antibody as the indicated concentrations of U0126 and then labeled with [methyl- H]thymidine. described under “Materials and Methods.” Insets in panels show a higher magnification U0126 was added 1 h prior to the growth factors. The percentage of cells that entered of a cell for better visualization. Percentage of cells with STAT1 nuclear staining is as into S phase was determined by autoradiography, as described under “Materials and follows: control, 10  4%; LIF, 89  5%; OSM, 85  7%; and PGF 15  6%. B, kinetic 2, Methods.” The results are presented as the percentage of labeled nuclei with respect to pattern of STAT1 translocation was determined as follows. Swiss 3T3 cells were stimu- the growth factor alone and are representative of four independent experiments. lated with LIF in the absence or presence of Na VO (30 M). This inhibitor was added 1 h 3 4 prior to cell treatment. Cells were fixed, and the STAT1 localization was visualized by affect DNA synthesis stimulated by fetal bovine serum (FBS). These indirect immunofluorescence as described under “Materials and Methods.” The percent- age of STAT1-immunostained nuclei was determined at different times after cytokine results showed that ERK activation is required for both LIF- and PGF - addition. At least 750 cells were counted per time point. The results represent the triggered cellular entry into S phase, although with a different kinetic means  S.E. of three independent experiments. pattern. Role of JAK/STAT Signaling Pathway in LIF/PGF -induced DNA cence were obtained when the subcellular localization of STAT1 was Synthesis—To investigate whether LIF, OSM, and PGF exert a differ- determined by analyzing cytoplasmic and nuclear fractions of LIF-stim- ential stimulation of the JAK/STAT pathway, we examined their ability ulated cells by Western blotting (data not shown). Furthermore, LIF, to cause cytoplasmic to nuclear translocation of the different STATs by OSM, and PGF could not promote translocation of STAT3 or STAT5 indirect immunofluorescence upon treatment of subconfluent resting Swiss 3T3 cells. In control, nonstimulated cells, STAT1 was mainly to the nucleus in the 3T3 cells (not shown). As positive controls, anti- STAT3 and STAT5 antibodies were able to detect STAT3 and STAT5 diffusely distributed within the cytoplasm (Fig. 4A). LIF and OSM treat- cytoplasmic to nuclear translocation in 3T3 L1 pre-adipocytes stimu- ment promoted nuclear translocation of STAT1 as demonstrated by the nuclear immunostaining of STAT1 (Fig. 4, panels A, B, and D, respec- lated by different cytokines, growth factors, and hormones (data not tively). In contrast, PGF did not trigger STAT1 nuclear localization shown) (39). No translocation was observed with either IL-6 or CNTF, (Fig. 4A, panel C). Results similar to those found by immunofluores- which are not mitogenic for Swiss 3T3 cells (Fig. 4B). MARCH 10, 2006• VOLUME 281 • NUMBER 10 JOURNAL OF BIOLOGICAL CHEMISTRY 6139 LIF-triggered Signals and Cell Cycle Control FIGURE 5. Inhibition of tyrosine phosphatases enhances LIF induction of DNA syn- thesis. To analyze the effect of tyrosine phosphatase inhibition on LIF stimulation of DNA synthesis, quiescent Swiss 3T3 cells were treated for 28 h with LIF (100 ng/ml) or OSM (100 ng/ml) in the absence or presence of 30 M Na VO and then labeled with 3 4 [methyl- H]thymidine. C, control. The Na VO was added 1 h before the cytokine treat- 3 4 ment. The percentage of cells that entered into S phase was determined by autoradiog- raphy, as described under “Materials and Methods.” Results represent the means  S.E. of three independent experiments. To ascertain whether regulation of tyrosine phosphatases is impli- cated in LIF-dependent nuclear localization of STAT1, Swiss 3T3 cells were incubated in the presence or absence of Na VO , a general inhib- 3 4 itor of tyrosine phosphatases (40, 41), prior to and during cytokine treat- ment. LIF-promoted cytoplasmic-nuclear translocation of STAT1 occurred as rapidly as 2 min after addition of LIF, and its nuclear local- ization attained a plateau at 10–15 min and declined to the basal level by 60 min (Fig. 4B). In the presence of Na VO , LIF also promoted the 3 4 rapid translocation of STAT1 to the nucleus. However, in the presence of Na VO , STAT1 remains in the nucleus after 60 min of LIF treatment 3 4 (Fig. 4B). This observation is consistent with findings that showed that a phosphatase inhibitor could prolong the activation of STAT1 and thus its nuclear retention (42). Indeed, Na VO also markedly enhanced LIF- 3 4 induced cellular entry into S phase, raising the percentage of cells under- going DNA synthesis over 2.5-fold (Fig. 5). Na VO also enhanced 3 4 FIGURE 6. The mitogenic effect of LIF is independent of cyclin D expression. A, OSM- and PGF -dependent cellular entry into the S phase (Fig. 5). 2 Swiss3T3 cells were treated with vehicle or saturating concentrations of LIF (100 ng/ml) or PGF (300 ng/ml) for different periods of time. Cell extracts were prepared, and equal These experiments indicated that LIF-dependent activation of STAT1 2 amounts of protein were analyzed by immunoblotting using specific antibodies for and initiation of DNA replication might both involve tyrosine kinase cyclin D , cyclin D , cyclin D , CDK4, and CDK6 (see “Materials and Methods”). Data are 1 2 3 activation. In contrast, the PGF -dependent mitogenic effect appears representative of three independent experiments. B, cyclin D mRNA levels were deter- mined at various times after cytokine or PGF treatment. Swiss 3T3 cells were treated not to require STATs activation. with vehicle or saturating concentrations of LIF (100 ng/ml), OSM (100 ng/ml), CNTF (100 Effect of LIF and PGF on G Cyclin Expression and pRb Phosphoryl- ng/ml), IL-6 (100 ng/ml), or PGF (300 ng/ml). After 8 –10 h, total RNA was extracted from 2 1 2 cells; Northern blot was performed as described under “Materials and Methods.” North- ation—To determine whether differences between LIF/OSM and ern blot densitometric analysis was standardized to 18 S RNA. Similar results were PGF2 signaling pathways have major consequences on expression of obtained in three independent experiments. C, Swiss 3T3 cells were treated with vehicle, LIF, or FBS for various times. Cell extracts were prepared, analyzed by SDS-PAGE, and key G cyclins and their effector kinases involved in executing the G /S 1 1 immunoblotted using specific antibodies against cyclin D , cyclin E, and cyclin A. Results transition, the expression of cyclins was assessed at different times upon are representative of three independent experiments. D, extracts from cells treated with treatment of quiescent 3T3 cells with LIF or PGF . Fig. 6 shows that vehicle (lane C), PGF , LIF, or FBS for the indicated periods of time were separated by SDS-PAGE and subjected to immunoblot analysis for pRb. Arrows indicate pRb with dif- PGF raised cyclin D protein levels within 9 h, reaching a plateau after 2 1 ferent levels of phosphorylation. 12–15 h, and these levels remained relatively high for up to 21 h (Fig. 6A). PGF also raised cyclin D protein levels at later times (within PGF increases cyclin E and A protein levels similar to those for LIF 2 2 15–21 h of treatment) but failed to increase cyclin D protein levels (Fig. 3 (data not shown). 6A). In contrast, LIF as well as OSM failed to increase cyclin D protein Cyclin D-CDK complexes were shown to phosphorylate the retino- and mRNA levels (Fig. 6, A and B), as well as failing to raise cyclin D or blastoma tumor suppressor protein (pRb), leading to inactivation of pRb cyclin D protein levels (Fig. 6A). IL-6 and CNTF were also unable to (44). It is well documented that inactivation of pRb results in release or induce cyclin D mRNA levels (Fig. 6B). The levels of the corresponding derepression of the E2F transcription factors and drives cell entry into partner CDK4/6 kinases (43) did not show any increase upon LIF or the S phase (44). To examine whether differences in cyclin D expression PGF treatments (Fig. 6A). However, addition of LIF induced an mediated by LIF and PGF result in differences in pRb phosphoryla- 2 2 increase in cyclin E and cyclin A protein levels after 14 and 28 h to levels tion, quiescent Swiss 3T3 cells were induced to enter S phase, and phos- comparable with that induced by 10% FBS, at least for cyclin E (Fig. 6C). phorylation of pRb was assessed by immunoblotting. As shown in Fig. 6140 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 281 • NUMBER 10 •MARCH 10, 2006 LIF-triggered Signals and Cell Cycle Control FIGURE 7. The synergistic effect between LIF and PGF to induce S phase entry is independ- ent of PKC activation. A, quiescent Swiss 3T3 cells were treated for 28 h with LIF or PGF in the absence or presence of GF109203X added 1 h prior to the addition of growth factors and then labeled with [methyl- H]thymidine. The percent- age of cells that entered into S phase was deter- mined by autoradiography, as described under “Materials and Methods.” B, quiescent Swiss 3T3 cells were treated for 28 h with vehicle, LIF, PGF , or LIF and PGF in the absence or presence of GF109203X (10 M) added 1 h prior to addition of growth factors. The percentage of cells that entered into S phase was determined by autora- diography. Results represent the mean  S.E. of four independent experiments. 6D, LIF did not promote full phosphorylation of pRb (lane 4 versus lane However, because these mitogens have been tested in different cellular 1). In contrast, PGF induced hyperphosphorylation of pRb (Fig. 6D, systems, it is unknown whether this difference is a function of the cell lanes 2 and 3 versus lane 1), consistent with the induction of expression type or is a fundamental difference in the delivery of the transducing of cyclins Ds. Phosphorylation of pRB induced by PGF is comparable signal per se. In this study we show that LIF-triggered signaling mecha- with the level of pRb phosphorylation induced by FBS (Fig. 6D, lane 5) nism(s) for inducing cellular entry into S phase are shared only by OSM upon cell entry into S phase. In summary, LIF induces neither expres- and not by PGF in the same quiescent Swiss mouse 3T3 cell system. sion of cyclin Ds nor hyperphosphorylation of pRb, whereas PGF Their differences are not due to subpopulations of 3T3 cells that are promotes both the increase in the expression of cyclin Ds and the hyper- primarily responsive to LIF/OSM or to PGF , because repeated sub- phosphorylation of pRb to exert its mitogenic effect. Taken together, cloning of 3T3 cells and addition of LIF/OSM or PGF yield the same these results provide further evidence that LIF and PGF may act percentages of cells stimulated to synthesize DNA in the given time through different signaling and molecular events to control the initia- period. Treatment of Swiss 3T3 cells with LIF or OSM together with tion of cellular entry into S phase. PGF mutually potentiated their ability to induce cellular DNA synthe- The Synergistic Effect of LIF and PGF to Induce S Phase Entry Is sis, whereas co-treatment of cells with LIF and OSM rendered no fur- Independent of PKC Activation—We have shown previously that LIF ther increase. Experimentally cellular DNA synthesis is measured by the and PGF differ markedly in the requirement for PKC in stimulating fraction of cells with [ H]DNA in their nuclei after 28 h. A longer expo- DNA synthesis (25). LIF triggers cellular entry into S phase via a PKC- sure of cells to [ H]thymidine with one mitogen increases this fraction, independent signaling mechanism, whereas PGF requires the activa- eventually reaching almost 100% of the cell population (22, 25, 27, 28, 33, tion of the PKC signaling pathway (25). Therefore, we examined 34). The synergy observed between LIF and PGF merely increases the whether PGF -dependent activation of PKC plays a role in the syner- rate of cellular entry into S phase but not the absolute fraction of cells gistic effect observed between LIF and PGF in the induction of DNA that are responding. These observations suggest that LIF and OSM trig- synthesis in Swiss 3T3 cells. We tested the effect of increasing concen- ger common signaling pathways that may differ from those activated by trations of GF109203X, a specific inhibitor of PKC, on the ability of LIF PGF for the induction of DNA synthesis. The importance of the study and PGF alone or in combination to trigger DNA replication. As therefore lies in the uncovering of the biochemical differences in the shown in Fig. 7A, GF109203X progressively inhibited PGF -dependent signal transduction pathways of these two groups of mitogens in the DNA replication but completely failed to block LIF-dependent DNA same cell system. replication. These results are consistent with our previous findings ERK1/2 are components of the well known MAPK signaling cascade obtained with 12-O-tetradecanoylphorbol-13-acetate indicating that activated by mitogens and are thus involved in controlling cell prolifer- LIF and PGF differed markedly in the requirement for PKC in stimu- ation (37). Here we show that both LIF or PGF by themselves can 2 2 lating DNA synthesis (25). Most interestingly, GF109203X did not pre- promote ERK activation. However, LIF and PGF differ in their timing vent the synergistic effect between PGF and LIF in increasing the of MAPK activation. Although PGF induced a maximum at 4 min, LIF 2 2 percentage of cells that entered into S phase (Fig. 7B). These results did so only after 12 min after addition, a result that suggests that LIF and suggest that the synergistic effect of LIF and PGF to promote S phase PGF cause ERK activation via two separate upstream signaling events. 2 2 entry is independent of PKC activation. U0126, a highly specific MEK inhibitor, blocked both LIF- and PGF - triggered MAPK activation and their mitogenic responses, strongly sug- DISCUSSION gesting that MAPK activation is required for the initiation of both LIF It has been shown previously that Swiss 3T3 cells are equally respon- and PGF -dependent DNA synthesis. However, how LIF increases sive to both sets of growth factors; LIF and PGF are thus equally ERK activity and the consequent stimulation of DNA synthesis in these effective at inducing DNA synthesis (25). The generality of the differ- cells is still unknown. MAPK activation is more complex than a simple ence in signaling events triggered by both cytokines and growth factors linear pathway. For example, LIF-triggered ERK1/2 activity in 3T3-L1 in different cell systems is well established; cytokines trigger activation adipocytes can occur via both Raf-1-dependent and -independent pro- of Janus kinases that promote phosphorylation of STATs (8, 23–25), and growth factors trigger the mitogen-induced Raf/MEK/ERK signal- ing pathway leading to overexpression of cyclin D (26–29, 42–45). P. Rudland and L. Jime´nez de Asu´ a, unpublished results. MARCH 10, 2006• VOLUME 281 • NUMBER 10 JOURNAL OF BIOLOGICAL CHEMISTRY 6141 LIF-triggered Signals and Cell Cycle Control cesses (22). In addition, it has been shown that an increase in phosphati- other growth factors, which must increase the levels of cyclin Ds to dylinositol 3-kinase activity may be involved in ERK activation (45) and induce S phase entry (36, 55, 56). Whether the low basal levels of cyclin D/CDK4 are sufficient to trigger the next step, phosphorylation of Rb that phosphatidylinositol 3-kinase may play a role in prolonging ERK activity (46). Moreover, cytokines such as interferon  or OSM can (57), or whether this first stage of Rb phosphorylation is bypassed is activate Raf-1 in a Ras-independent manner via increased activity of unknown at present. Moreover, different findings reveal that cyclin E/CDK2 can induce cellular entry into S phase in the absence of cyclin JAK1 or Tyk2 (47). These findings and our present results support the D/CDK4 activation (56, 58) and that c-Myc and Cdc25A can participate notion that multiple, temporally distinct pathways can converge on MAPK and that these pathways can be utilized differentially by various in the activation of the cyclin E-CDK2 complex (59–61). A recent report demonstrates that proliferation of mouse embryonic fibroblasts stimuli and cell types. proceeds relatively normally in the absence of the D-cyclins (62). Kozar Cyclin D expression is generally regulated by a mitogen-induced et al. (62) shows that mouse embryonic fibroblasts from cyclin D Raf/MEK/ERK signaling pathway (26–29). Indeed, the duration of an 1 / / D D mice critically depend on CDK2, suggesting that cyclin ERK signal appears to determine whether cells will induce cyclin D 2 3 D-CDK4/6 and cyclin E-CDK2 complexes may perform overlapping expression. Mitogens that only produce a transient ERK activation fail functions in “cyclin D-independent” systems. It is established that the to induce cyclin D , whereas growth factors that induce a sustained ERK initial phosphorylation of the pRb by cyclin D-CDK complexes is activation cause continuous maintenance of cyclin D expression (48, required to allow full phosphorylation of the pRb by the cyclin E- and 49). Thus, the critical determinant in the induction of cyclin D is the cyclin A-associated kinases (9, 63). However, Kozar et al. (62) shows that duration, rather than the intensity, of the ERK signal. Our studies show phosphorylation of pRb on cyclin D-specific sites is not required for that LIF-stimulated DNA synthesis requires an intact MEK/ERK signal- further phosphorylation and that cyclin E- and cyclin A-driven phos- ing cascade. However, LIF-stimulated ERK activation is likely not to be phorylation is sufficient to allow the expression of E2Fs target genes linked to the increase in cyclin D expression. In contrast, PGF -stim- 1 2 during cell cycle re-entry. Our study shows that LIF promotes expres- ulated ERK activation may be directly involved with increasing the sion of cyclin E and A but not cyclin Ds, as shown in Fig. 6. Furthermore, expression of cyclin D and ultimately with its mitogenic response. How full phosphorylation of pRb does not take place as cells re-enter the cell LIF promotes S phase entry in an ERK-dependent manner and how ERK cycle. Therefore, our future goal will be to elucidate the role of cyclin activation does not result in an increase in cyclin D expression have yet E/CDK2 in regulating LIF induction of DNA replication in a cyclin to be elucidated. The results presented here suggest that the different D-independent manner. kinetics of MAPK activation may result in a different pattern of G In summary, our present work demonstrates that LIF and PGF cyclin expression, although alternative explanations based on ERK1/2- trigger different signaling and molecular events prior to cellular entry independent activation of the cyclin D promoter by PGF and not by 1 2 into S phase in the same cell system. The importance of this work estab- LIF may be possible. lishes that stem cell factors like LIF can bypass the normal growth fac- JAK/STAT signal cascades are known to be involved in responses to tor-induced Raf/MEK/ERK signaling pathway to cyclin D and activation cytokines. Our immunofluorescence studies reveal that LIF and OSM of CDK4/6, the key event that normally allows progress through the trigger a similar pattern of STAT1 cytoplasmic to nuclear translocations Restriction Point R and commitment to enter the cell cycle (54). LIF and, after 2 min, attaining a maximum at 10–15 min and declining at 60 min by implication, stem cell growth factors in general then trigger cellular to the basal level, whereas CNTF, IL-6, and PGF were without effect. entry into S phase by partial phosphorylation of Rb through increases in Our experiments to understand the role of LIF induction of DNA syn- cyclin E and activation of CDK2. Thus the enhancing effect of PGF on thesis indicated that the effect of LIF is mediated via tyrosine kinase the induction of cellular entry into S phase mediated by LIF is probably because Na VO potentiates LIF’s stimulation of DNA synthesis. Fur- 3 4 because of the ability of the former mitogen to phosphorylate Rb com- thermore, this result is correlated with the prolonged localization of pletely and thereby further reduce its inhibitory activity for the E2F STAT1 in the nucleus. However, it will be important to elucidate transcription factor required for G to S phase transition (54). Presum- whether STAT1 cytoplasmic-nuclear translocation in conjunction or ably, this synergy is mediated by interactions between those signals not with MAPK activation is required for LIF stimulation of DNA generated from the PGF receptor that are different from those of LIF, synthesis. in particular the very early rise in ERK activation and the activation of LIF is overproduced and secreted by several cancer cells (50, 51) and STAT1. By understanding the molecular mechanisms by which LIF in thus may act as an autocrine stimulator. Moreover, it is known that particular and cytokines in general control normal cell cycle constitutes during oncogenesis different STAT proteins are continuously activated the basis to unravel the critical cytokine-related signaling events under- in a variety of cancer cells types (52, 53), and if inhibited the resultant lying unrestricted cancerous cell division, as well as providing possible cancer cells grow much more slowly (52, 53, 60, 61). The fact that cyto- therapeutic targets to blockade this second signal transduction pathway kines stimulate DNA replication through different signaling events may in the event that it is necessary to inhibit both growth factor and cyto- result in carcinomas rapidly eluding the control of growth factor signals, kine signaling pathways to prevent cancer cell growth. and therefore any therapy targeted to the early signaling events trig- gered by growth factors may become rapidly ineffective. Thus the elu- Acknowledgments—We thank Liz Shannon of BD Biosciences for the generous cidation of the molecular mechanism of cytokine action is an important gift of tissue culture material. We thank Drs. Eduardo Passeron and Israel step in dissecting deranged regulatory events leading to malignant Algranati for the revision of the manuscript and for encouragement in our transformation and as a second parallel target for mounting a therapeu- research. 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Journal of Biological Chemistry – American Society for Biochemistry and Molecular Biology

Published: Mar 10, 2006

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