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- 10.1074/jbc.271.34.20608
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Abstract
THE JOURNAL OF BIOLOGICAL CHEMISTRY Vol. 271, No. 34, Issue of August 23, pp. 20608–20616, 1996 © 1996 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in U.S.A. MAPK Cyclin D1 Expression Is Regulated Positively by the p42/p44 MAPK and Negatively by the p38/HOG Pathway* (Received for publication, February 21, 1996, and in revised form, May 31, 1996 Jose´e N. Lavoie‡, Gilles L’Allemain, Anne Brunet, Rolf Mu¨ ller§, and Jacques Pouysse´gur From the Centre de Biochimie, CNRS-UMR 134, Faculte´ des Sciences, Parc Valrose, 06108 Nice Cedex 02, France and the §Institut fu¨r Molekularbiologie und Tumorforschung (IMT), Philipps-Universita¨t Marburg, Emil-Mannkopff-Strasse 2, D-35037 Marburg, Federal Republic of Germany We have previously shown that the persistent acti- the modulation of cyclin D1 expression and associated MAPK vation of p42/p44 is required to pass the G restric- cdk activities. tion point in fibroblasts (Page`s, G., Lenormand, P., L’Allemain, G., Chambard, J. C., Meloche, S., and Pouys- se´gur, J. (1993) Proc. Natl. Acad. Sci. U. S. A. 90, 8319– Mammalian cells express multiple mitogen-activated protein 8323) and postulated that MAPKs control the activation (MAP) kinases that mediate the effects of extracellular signals of G cyclin-dependent complexes. We examined the mi- on a wide array of biological processes. In eukaryotic cells, togen-dependent induction of cyclin D1 expression, one three distinct MAPK cascades have been described, which ap- of the earliest cell cycle-related events to occur during pear to be linked to separate signal transduction pathways MAPK the G /G to S-phase transition, as a potential target of 0 1 resulting in the final activation of either p42/p44 , p38/ MAPK regulation. Effects exerted either by the p42/ MAPK HOG , or stress-activated protein kinases (SAPKs) also MAPK MAPK p44 or the p38/HOG cascade on the regulation called Jun kinases (JNKs) (2). Depending on the cellular con- of cyclin D1 promoter activity or cyclin D1 expression text, extracellular signals are thought to elicit a specific cellu- were compared in CCL39 cells, using a co-transfection lar response (proliferation/differentiation/apoptosis) through MAPK procedure. We found that inhibition of the p42/p44 the preferential activation of one of the MAPK cascades, which signaling by expression of dominant-negative forms of have distinct spectra of substrates (3). either mitogen-activated protein kinase kinase 1 MAPK In most cell types, the mitogenic signal is relayed from the (MKK1) or p44 , or by expression of the MAP kinase cytoplasm into the nucleus by the nuclear translocation of the phosphatase, MKP-1, strongly inhibited expression of a MAPK ubiquitously expressed p42/p44 isoforms (also called reporter gene driven by the human cyclin D1 promoter ERK2 and ERK1 for extracellular regulated kinase) (4, 5), as well as the endogenous cyclin D1 protein. Conversely, resulting in activation of a range of transcription factors such activation of this signaling pathway by expression of a constitutively active MKK1 mutant dramatically in- as Elk1 (6–8), c-Ets-1, and c-Ets-2 (9, 10). In fibroblasts, agents creased cyclin D1 promoter activity and cyclin D1 pro- that elicit a short term MAP kinase activation are not mito- tein expression, in a growth factor-independent man- genic, whereas potent mitogens that induce DNA synthesis ner. Moreover, the use of a CCL39-derived cell line that drive long term MAP kinase activation (11). Previous studies MAPKs stably expresses an inducible chimera of the estrogen have shown that a sustained activation of the p42/p44 is receptor fused to a constitutively active Raf-1 mutant required for fibroblasts to pass the G restriction point and MAPK (DRaf-1:ER) revealed that in absence of growth factors, enter S-phase (1, 12). Moreover, activation of the p42/p44 activation of the Raf > MKK1 > p42/p44MAPK cascade is module is sufficient to stimulate early gene transcription and sufficient to fully induce cyclin D1. In marked contrast, to reduce growth factor requirement for DNA synthesis (13, MAPK the p38 cascade showed an opposite effect on the MAPK 14). Therefore, the p42/p44 cascade is likely to regulate regulation of cyclin D1 expression. In cells co-express- some mid-late changes in gene expression that are rate-limit- MAPK ing high levels of the p38 kinase (MKK3) together ing events for S-phase entry, during the G progression of the MAPK 1 with the p38 , a significant inhibition of mitogen- cell cycle. induced cyclin D1 expression was observed. Further- MAPKs In contrast to p42/p44 , which are strongly activated by MAPK more, inhibition of p38 activity with the specific growth factors and growth-promoting hormones, JNKs and inhibitor, SB203580, enhanced cyclin D1 transcription MAPK p38 are poorly sensitive to growth signals, and their acti- and protein level. Altogether, these results support the vation is preferentially triggered by pro-inflammatory cyto- notion that MAPK cascades drive specific cell cycle re- kines and environmental stresses (3). The JNK cascade has sponses to extracellular stimuli, at least in part, through been implicated in the modulation of AP-1-regulated gene ex- pression through the phosphorylation of the proto-oncogene * This work was supported by grants from CNRS (Centre National de c-Jun (3, 15, 16). The two stress-activated signaling pathways la Recherche Scientifique), INSERM (Institut National de la Sante´etde can mediate the phosphorylation of the transcription factor la Recherche Me´dicale), from l’Association pour la Recherche contre le Cancer (Grant 1021), and MRC (Medical Research Council of Canada) ATF2 on residues that increase its transcriptional activity in postdoctoral fellowship (to J. N. L.). The costs of publication of this vivo, suggesting that these two cascades may participate in the article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. The abbreviations used are: MAP, mitogen-activated protein; ‡ Present address: Dept. of Biochemistry, Rm. 906B, McIntyre Med- MAPK, mitogen-activated protein kinase; DMEM, Dulbecco’s modified ical Sciences Bldg., McGill University, 3655 Drummond St., Montreal Eagle’s medium; FCS, fetal calf serum; PAGE, polyacrylamide gel elec- H3G 1Y6, Quebec, Canada. Tel.: 514-398-8168; Fax: 514-398-7384. trophoresis; MKK, MAP kinase kinase; MKP-1, MAP kinase phospha- E-mail: [email protected]. tase; IL, interleukin; pRb, Retinoblastoma protein; SAPKs, stress-acti- ¶ To whom correspondence and reprint request should be addressed. vated protein kinases; ERK, extracellular regulated kinase; ATF, E-mail: [email protected]. activating transcription factor. This is an open access article under the CC BY license. 20608 Regulation of Cyclin D1 Expression by MAPKs 20609 was purchased from Pharmingen (Clinisciences, Paris, France), mono- regulation of ATF2-dependent gene expression (17–20). Initial clonal antibody 12CA5, raised to a peptide from influenza HA1 protein, reports suggested that the JNK cascade could be required for was purchased from Babco (Emeryville, CA), and monoclonal antibody mitogenesis in fibroblasts (21). However, a more recent study M2 against the Flag epitope was from Kodak Integra Biosciences. MAPK indicated that activation of JNK and p38 and concurrent Expression Vectors and Reporter Constructs—The cyclin D1 reporter MAPK inhibition of p42/p44 promote the ability of nerve growth construct used for luciferase assays termed D1D-944 contains the hu- factor to induce apoptosis in PC12 cells (22). Nonetheless, JNK man cyclin D1 promoter from residues 2944 to 1139 cloned upstream MAPK of the luciferase gene of the pXP2 reporter construct as described and p38 cascades lie on separate signaling pathways and previously (39). The pCH110 reporter construct contains the SV40 have, in addition to their common substrates, specific cellular promoter upstream of a b-galactosidase gene (Pharmacia Biotech Inc.). targets, i.e. c-Jun and MAPKAPK-2, respectively (23, 24). The expression vectors for MKK1, constitutively active MKK1 mutant These two cascades are thus likely to be involved in the regu- (MKK1-SS/DD), dominant-negative MKK1 (MKK1-S222A) (13, 40), MAPK MAPK MAPK lation of distinct cellular functions; however, it is not clear p38 , dominant-negative p38 (p38 -TY/AF) (kindly pro- MAPK what specific role can be attributed to one or the other stress vided by Dr. R.J. Ulevitch) (41), and p44 , dominant-negative MAPK MAPK p44 (p44 -T192A) (1) were previously cloned into the pECE signaling pathways at the level of cell division mechanisms. and pcDNAneo vectors respectively (InVitrogen), in frame with the Transduction of extracellular signals culminates in the ex- hemagglutinin (HA) epitope. MKP-1 construct (kindly provided by Dr. pression and assembly of different kinase holoenzymes, the N. K. Tonks) was described previously (42). Flag-MKK3 and constitu- cyclin-cdk (cyclin-dependent kinase) complexes, which are tively active Flag-MKK3 mutant (Flag-MKK3-S189G/T193G) (kindly formed and activated at specific stages of the cell division cycle. provided by Dr. R. Davis) have previously been described (23). The Although the kinase-associated activity of these complexes is selection vector used in transient transfection assays termed pNHE3 1 1 contains cDNA encoding the NHE3 cell-specific human Na /H anti- modulated by specific phosphorylation-dephosphorylation porter gene (43). The estradiol receptor/constitutively active Raf-1 chi- events on the catalytic subunits, the temporal activation of the meric construct (DRaf-1:ER) was provided by Dr. M. McMahon (44). holoenzymes is primarily dependent on the synthesis and ac- Cell Culture—The established Chinese hamster lung fibroblast cell cumulation of specific regulatory subunits, the cyclins (25). line CCL39 (American Type Culture Collection) and its derivative Cyclins D1, D2, and D3, in conjunction with their catalytic CCL39-DRaf-1:ER cell line that stably expresses the DRaf-1:ER chime- partners cdk4 and cdk6, appear to regulate the initial phases of ra were maintained in Dulbecco’s modified Eagle’s medium (DMEM, Life Technologies, Inc.), supplemented with 7.5% fetal calf serum (Life G progression (26–29). In normal untransformed cells, the Technologies, Inc.), penicillin (50 units/ml), and streptomycin sulfate growth factor-dependent accumulation of cyclin D1 has been (50 mg/ml). Cells were grown in a humidified 5% CO atmosphere at shown to be required to allow cells to pass the G restriction 37 °C. Cells were rendered quiescent (G -arrested) by a 24 h-incubation point (27, 30, 31). However, cyclin D1 requirement becomes in serum-free DMEM. dispensable in a cell background deficient for the tumor sup- Transient Transfection and Luciferase Assay—CCL39 cells were pressor (pRb) function (32). This observation further empha- transfected by the calcium phosphate method. For the cyclin D1-lucif- erase expression assays, CCL39 cells were seeded at a density of sizes the functional link between pRb and cyclin D1-associated 150,000 cells per well in 24-well plates and co-transfected with 0.25 mg kinase activity, which has been shown to be responsible in part of the reporter D1D-944 together with 0.75 mg of relevant expression for hyperphosphorylating pRb (33–37). Therefore, early ap- vector, or the corresponding empty vector. For experiments performed pearance of cyclin D1 upon growth factor stimulation of resting in exponentially growing cells, luciferase activity was measured 48 h fibroblasts plays a central role in regulating the G -G transi- after transfection. Normalization was achieved by co-transfecting 0.1 0 1 tion of the cell cycle. mg of pCH110, a b-galactosidase reporter construct as an internal control for the transfection efficiency. For mitogen-stimulated cyclin Because of the strict requirement for a persistent p42/ MAPK D1-luciferase reporter expression, 1 day following the transfection cells p44 activation to successfully pass the G restriction were serum-starved for 30 h and then stimulated with 10% FCS for point, we hypothesized that these MAPKs control cyclin D1- 18 h. Luciferase and b-galactosidase activities were measured accord- associated kinase activity. We have thus drawn our attention ing to the Promega protocol. Data are representative for at least three to cyclin D1 as a potential “nuclear sensor” of extracellular independent experiments performed in duplicate and are expressed as signals. Here we report that cyclin D1 expression is positively “fold increase in luciferase activity,” which was calculated relative to MAPK the basal level of cyclin D1 reporter activity set to 1 unit and corrected controlled by the Raf . MKK1 . p42/p44 cascade in the for empty vector effects for each expression vector. Cyclin D1 protein Chinese hamster fibroblast cell line CCL39, whereas the expression analyses were performed in CCL39 cells seeded at a density MAPK stress-activated p38/HOG cascade antagonizes this ex- of 600,000 cells per well in six-well plates, co-transfected by the calcium pression. This report that establishes a link between persistent phosphate technique with 6 mg of the selection vector pNHE3, together MAPKs activation of p42/p44 and cyclin D1 expression provides a with 14 mg of relevant expression vector. Two days following the trans- key element to the understanding of the temporal action of fection, cells were submitted to an acid-load selection (45) in the pres- ence of the HOE694 compound to inhibit the endogenous NHE1 anti- growth factors. porter activity, so that only transfected cells expressing the NHE3 EXPERIMENTAL PROCEDURES isoform survived (46). Cells were then allowed to recover for at least 4 h before serum starvation. Cells were lysed 3 days following the trans- Materials—Highly purified a-thrombin (3209 NIH units/mg) was fection, and cyclin D1 expression was monitored by immunoblotting kindly provided by Dr. J. W. Fenton II (New York State Department of using the antisera described above. Expression of the transfected Health, Albany, NY). Epidermal growth factor and insulin were from MAPK constructs was checked for each experiment using the appropri- Sigma. IL-1b was purchased from Boehringer Mannheim. The specific MAPK ated antibody. p38 inhibitor SB203580 was provided by SmithKline Beecham Immunoblotting—Cells were lysed in SDS sample buffer (62.5 mM Pharmaceuticals (King of Prussia, PA). Rabbit polyclonal cyclin D1 Tris-HCl, pH 6.8, 2.3% SDS, 10% glycerol, 5% b-mercaptoethanol, antibody was raised against a peptide from recombinant human cyclin 0.005% bromphenol blue, 1 mM phenylmethylsulfonyl fluoride), and D1 and was a generous gift from Dr. V. Baldin (30). Mouse monoclonal proteins from whole cell lysates were separated by SDS-PAGE in 10% antibodies against cyclin D2 and cyclin D3 were kindly provided by Dr. gels or in 7.5% gels for pRb detection. For p42MAPK “shift up” exper- J. Bartek and have been previously described (38). Rabbit polyclonal MAPK iments, the percentage of bisacrylamide was reduced to 0.07% (final), antibody E1B against p42/p44 was raised against a C-terminal and samples were run on 12.5% SDS-polyacrylamide gels. Proteins peptide of mouse ERK2. Rabbit polyclonal antibody against MKK1 were detected immunologically following electrotransfer onto nitrocel- (MKK13) was described previously (5). Rabbit polyclonal antibody MAPK lulose membranes as described previously (47). Horseradish peroxi- SAM2 against p38 was raised against a 14-residue C-terminal dase-linked goat anti-rabbit or anti-mouse immunoglobulin G (Sigma), peptide of the human p38MAPK. Monoclonal antibody against pRb 2 4 F. McKenzie, unpublished data. Lenormand, P., McMahon, M., and Pouysse´gur, J. (1996) J. Biol. A. Brunet, unpublished data. Chem. 271, 15762–15768. 20610 Regulation of Cyclin D1 Expression by MAPKs lower panel) and when the first cells enter S-phase. The re- sults suggest that cyclin D1 is the major mitogen-regulated D-type cyclin identifiable in this cell system and, therefore, that its associated kinase activity is likely to participate in pRb inactivation by phosphorylation. Previous studies demonstrated that in fibroblasts only po- tent mitogens that drive the G to S-phase transition can MAPK maintain a long term activation of the p42/p44 cascade (11). Initial experiments to link this signal transduction path- way to mitogen regulation of cyclin D1 expression revealed a tight correlation between the extent of cyclin D1 accumulation MAPK and the ability of various agents to induce sustained p42 activation. Cells were serum-starved for 24 h and stimulated for9hinthe presence of various agonists, and whole cell extracts were analyzed for cyclin D1 protein levels, pRb phos- MAPK phorylation, and p42 activity. The polyclonal anti- MAPK p42 antibody used detected three electrophoretically dis- FIG.1. A, time course of D-type cyclin expression and pRb phospho- tinct forms of the kinase: a fast-migrating band representing rylation in CCL39 cells. CCL39 were serum-starved for 24 h and then the inactive unphosphorylated form of the endogenous stimulated with 20% FCS for the indicated period (hours). Equal MAPK amounts of whole cell lysates were separated by SDS-PAGE, and pro- p42 , an intermediate band that corresponds to the phos- teins were electrotransfered onto nitrocellulose. Western blot detection phorylated and active enzyme, and an upper, slow-migrating of cyclin D1, cyclin D3, and pRb proteins was performed using the form that corresponds to cross-reactivity of the antibody with appropriate specific antibodies. B, cyclin D1 accumulation, pRb phos- MAPK MAPK the p44 isoform (Fig. 1B, lower panel). Cyclin D1-associ- phorylation, and p42 activity in G -arrested CCL39 stimulated with various agonists. Cells were serum-starved for 24 h and then ated kinase activity that is mainly dependent on the level of stimulated for9hinthe presence of various agonists as indicated. cyclin D1 was also evaluated in each sample by analyzing pRB Equal amounts of whole cell lysates were separated by SDS-PAGE, and phosphorylation. The ability of the various mitogens to induce Western blot detection was performed using either antibody against MAPK cyclin D1 expression and pRb hyperphosphorylation was cyclin D1, pRb, or p42 , following electrotransfer of total proteins MAPK onto nitrocellulose. closely related to their ability to maintain p42 activities throughout G progression of the cell cycle (Fig. 1B). Only revealed by the ECL detection system (Amersham), was used to detect potent mitogens, such as whole serum and thrombin, which the antigen-antibody complexes. Protein concentrations were measured potently initiate DNA synthesis in arrested CCL39 cells, were using a modified Lowry procedure (48) with bovine serum albumin as able to promote high levels of cyclin D1 accumulation and allow standard. Quantitative analyses of protein levels were performed by pRb phosphorylation (appearance of a minor slow-migrating scanning of the autoradiograms (Fugi PhosphoImager, Paris, France) band at 9 h post-stimulation) (Fig. 1B, middle panel). Whereas and are representative of more than two independent experiments. a slight effect was observed with a weakly mitogenic combina- RESULTS tion of epidermal growth factor plus insulin, neither factor MAPK MAPK Potent Mitogens Promote Long-term p42/p44 Activa- alone was able to promote long term p42 activation, cyclin tion, Cyclin D1 Expression, and pRb Phosphorylation—D-type D1 expression, and pRb phosphorylation, suggesting an inter- cyclin expression profile was analyzed in whole cell extracts of dependent relationship between the long lasting activation MAPKs G -arrested Chinese hamster fibroblasts (CCL39), by Western phase of p42/p44 , cyclin D1 expression, and pRb inacti- blot technique using specific antibodies. In G -arrested CCL39 vation in CCL39 fibroblasts. MAPK cells, cyclin D1 protein expression was barely detectable (Fig. The p42/p44 Cascade Positively Regulates Cyclin D1 1). However, serum stimulation of cells led to a dramatic accu- Expression in Fibroblasts—To investigate the role of the p42/ MAPK mulation of cyclin D1 protein that became easily detectable at p44 signaling pathway in the regulation of cyclin D1 ex- 6 h poststimulation and increased until cells entered S-phase pression more directly, we used previously characterized ex- (Fig. 1A). As previously shown in other fibroblastic cell lines pression constructs to modulate either positively or negatively MAPK (30, 49), the protein expression level of cyclin D1 in CCL39 cells the endogenous p42/p44 activity (see “Experimental Pro- was not cell cycle-modulated and only a modest peak of accu- cedures”). Cyclin D1 transcription was monitored by transfect- mulation was observed in late G (12–16 h poststimulation). ing CCL39 cells with a previously cloned fragment of the hu- Cyclin D2 and D3 expression were also analyzed in CCL39 cells man cyclin D1 promoter fused to the luciferase reporter gene using specific monoclonal antibodies. In contrast to cyclin D1, (D1D-944) (39), together with the relevant constructs. The re- MAPK for that expression was strictly dependent on the presence of sults showed that the p42/p44 activity strongly affects mitogens, cyclin D3 was present in resting cells and its level cyclin D1 transcription in CCL39 cells. When a constitutively was poorly increased by growth factors (Fig. 1A). Cyclin D2 was activated form of MAPK kinase (MKK1-SS/DD) was expressed, not detectable in either quiescent or growth factor-stimulated a large increase in luciferase expression (6–10-fold) could be CCL39 cells. The hyperphosphorylation of pRb was analyzed detected in exponentially growing cells, when compared with for each time point following serum stimulation of resting cells. the luciferase expression in control cells transfected with the A specific antibody able to detect the active hypophosphoryl- empty vector (Fig. 2A). This dramatic up-regulation of the ated form of pRb (lower band) as well as the inactive hyper- cyclin D1 reporter expression in cells overexpressing the con- phosphorylated form of the protein (upper band) was used. In stitutively active MKK1 mutant likely resulted from a higher MAPK G -arrested cells, pRb was exclusively found in its active hy- p42/p44 activity in these cells, since co-expression of the pophosphorylated state, suggesting that cyclin D3, although MAPK phosphatase (MKP-1), a dual specificity phosphatase MAPKs expressed does not contribute to pRb phosphorylation in this shown to be able to inactivate p42/p44 (42), totally abol- cell system and thus, is not a limiting factor for progression ished the MKK1-SS/DD-dependent increase in cyclin D1-lucif- through G -phase of the cell cycle. However, pRb inactivation (hyperphosphorylated form) became apparent at 10–12 h post- stimulation, when cyclin D1 expression was maximal (Fig. 1A, G. L’Allemain, J. N. Lavoie, and J. Pouysse´gur, unpublished data. Regulation of Cyclin D1 Expression by MAPKs 20611 MAPK FIG.2. The p42/p44 signal transduction pathway positively regulates cyclin D1 promoter activity and protein expression in CCL39 fibroblasts. Cyclin D1 promoter activity and protein expression were monitored following transfection with the indicated constructs (A and C) in exponentially growing CCL39 cells as well as (B and D) in serum-starved cells stimulated with mitogens. For cyclin D1-luciferase MAPK expression assays, cells were transfected with the appropriate empty vector or with vector containing members of the p42/p44 cascade as indicated, together with the cyclin D1-luciferase reporter gene D1D-944. For experiments performed in exponentially growing cells, luciferase activity was measured 48 h posttransfection and normalized using b-galactosidase as an internal control, by cotransfecting the pCH110 reporter construct. For mitogen-stimulated activation of cyclin D1-luciferase, cells were serum-starved 1 day after transfection for 30 h and then stimulated with 10% FCS for 18 h. The experiments were performed in duplicate, and data are representative of at least three independent experiments. The fold increase in luciferase activity was calculated relative to the basal expression level of D1D-944, which was set to 1 unit, and was corrected for empty vector effects. C and D, immunoblots of total cell lysates from transfected cells. Endogenous cyclin D1 protein expression was analyzed in MAPK CCL39 transfected with the marker gene pNHE3, together with expression constructs containing members of the p42/p44 cascade or the appropriate empty vector (EV) as indicated. Forty-eight hours post-transfection, cells were submitted to an acid-load selection and then allowed to recuperate for at least4hin DMEM, 7.5% FCS. C, cells were kept in the exponentially growing phase, in the presence of 7.5% FCS, or D, serum-starved for 24 h and then stimulated with 1 unit/ml thrombin for 9 h. Cells were lysed in SDS-sample buffer and equal amounts of each extract were processed for SDS-PAGE and Western blot analysis using specific antibodies. Cyclin D1 and pRb protein levels were analyzed by scanning of the autoradiograms and are expressed as fold increase in protein levels as compared with the levels measured in cells transfected with the empty vector. C, MKK1-SS/DD: cyclin D1 5 2.1 6 0.18, pRb upper band 5 1.40 6 0.13, pRb lower band 5 1.52 6 0.05; MKP-1: cyclin D1 5 0.52 6 0.09, pRb upper band 5 0.55 6 0.15, pRb lower band 5 0.85 6 0.06. D, fold increase in thrombin-induced cyclin D1 protein levels; MKK1 5 1.1 6 0.12, MKK1-SS/DD 5 1.25 6 0.15, MKP-1 5 0.49 6 0.06. Fold increase in resting MKK1-SS/DD as compared with thrombin-stimulated EV cells: 1.15 6 0.11. Results are the means 6 S.E. of three independent experiments. Endogenous MKK1 expression (lower band) is shown as an internal control. erase expression (Fig. 2A). No significant effects of these plas- MKK1 activity in cells expressing this mutated form of MKK1 mids were observed in the same experiments on the (40). Moreover, the serum-stimulated luciferase expression was b-galactosidase internal control reporter expression. A positive strongly inhibited (80%) in cells expressing the negative regu- effect on the cyclin D1-luciferase expression was also measured latory constructs, as compared with control cells expressing the in G -arrested cells expressing the constitutively active form of reporter gene alone (Fig. 2B). These results thus demonstrate MAPKs MAPK MKK1. In these cells, activation of the p42/p44 in the that the p42/p44 signal transduction pathway positively absence of mitogen led to an increase of cyclin D1-luciferase regulates cyclin D1 transcription in fibroblasts. expression that was even higher than the level measured in Previous studies have shown that an increase in the level of serum-stimulated cells expressing the wild type MKK1 (Fig. cyclin D1 messenger RNA does not always lead to an increase 2B). However, the reporter gene expression in these cells was in cyclin D1 protein levels in transfected cells, suggesting that still inducible by serum to the same extent as in control cells post-transcriptional processes might also play an important MAPK (2–4-fold). Conversely, when the p42/p44 cascade was role in the regulation of cyclin D1 expression (50–52). We thus blocked by expression of inhibitory constructs, cyclin D1-lucif- examined whether the modulation of cyclin D1 transcription by MAPK erase expression was strongly reduced in exponentially grow- the p42/p44 pathway also resulted in modification of cy- MAPK MAPK ing cells. Expression of dominant-negative p44 (p44 - clin D1 protein levels in CCL39 fibroblasts. The effects of the T192A) or MKP-1 produced a 45–80% inhibition of cyclin D1- same constructs (MKK1, MKK1-SS/DD, or MKP-1) on the level luciferase expression, respectively (Fig. 2A). As expected, of endogenous cyclin D1 protein were analyzed in CCL39 cells. expression of the negative regulatory MKK1-S222A construct To do so, the expression plasmids were co-transfected with a 1 1 inhibited cyclin D1-luciferase expression to a lesser extent selection vector that encodes an amiloride-resistant Na /H (30%), correlating with a partial inhibition of endogenous exchanger isoform, NHE3. Nontransfected cells were selec- 20612 Regulation of Cyclin D1 Expression by MAPKs tively eliminated by acid-load selection, as described under “Experimental Procedures,” and cyclin D1 protein levels were analyzed in whole cell extracts of resistant cells by Western blots using a specific antibody. In exponentially growing cells expressing the constitutively active MKK1 mutant, a higher level of cyclin D1 could be detected as compared with cyclin D1 expression in control cells transfected with either the empty vector or the wild type MKK1 (Fig. 2C). This increase of cyclin D1 protein level in MKK1-expressing cells kept in serum-sup- plemented medium was associated with an overall increase in the amount of the hyperphosphorylated form of pRb; however, FIG.3. Time course of cyclin D1 accumulation and pRb phos- the relative total amount of pRb expression was also increased, phorylation in estradiol or serum-stimulated CCL39-DRaf-1:ER keeping constant the ratio between the hyper- and hypophos- cells. CCL39-DRaf-1:ER cells were serum-starved for 24 h and stimu- MAPKs phorylated forms. Conversely, inactivation of p42/p44 by lated with either 10 mM estradiol or 10% FCS for varying periods as expression of the MKP-1 markedly reduced cyclin D1 protein indicated (hours). Cells were then lysed in SDS sample buffer, and equal amounts of whole cell lysates were separated by SDS-PAGE in expression below the level detected in control cells (Fig. 2C). 10% gels (cyclin D1) or in 7.5% gels (pRb). Cyclin D1 and pRb protein The significant inhibition of cyclin D1 protein expression in levels were analyzed by Western blotting using specific antibodies, MKP1-overexpressing cells also correlated with a 45% decrease following electrotransfer of total proteins onto nitrocellulose. in the amount of the hyperphosphorylated form of pRb, sug- gesting that the resulting low level of cyclin D1 in these cells cells, indicating that activation of the Raf pathway alone (lead- was limiting for cyclin D1-associated kinase activity. When MAPK ing to p42/44 activation) initiated positive regulatory sig- similar experiments were performed in resting cells, a strong nals responsible for cyclin D1 protein synthesis. Nevertheless, positive effect on cyclin D1 expression could be detected upon the Raf pathway alone was not sufficient to promote activation MAPK activation of the p42/p44 pathway. As shown in Fig. 2D, of the D1-associated kinase activity, since neither pRb phos- cyclin D1 was barely detectable in serum-starved control cells, phorylation (Fig. 3, lower panels) nor in vitro cdk4 kinase whereas in serum-starved MKK1-SS/DD expressing cells, a activity (data not shown) could be detected in estradiol-stimu- growth factor-independent expression of cyclin D1 was de- lated CCL39-DRaf-1:ER cells. tected. Furthermore, this increase of cyclin D1 protein level MAPK The p38/HOG Cascade Antagonizes Mitogen-induced mediated by expression of the constitutively active MKK1 mu- Expression of Cyclin D1 in Fibroblasts—In addition to the tant was comparable with the level measured in thrombin- MAPK p42/p44 pathway, at least two other MAPK cascades are stimulated control cells (see quantitations in the legend of Fig. implicated in the transduction of external stimuli in mamma- MAPK 2D). Conversely, when the p42/p44 activity was blocked by MAPK lian cells, the p38/HOG and the JNKs (2). We next exam- the presence of an elevated level of MKP-1, thrombin-induced ined whether other MAPK pathways could have some regula- accumulation of cyclin D1 was severely impaired, being inhibited tory effects on cyclin D1 expression. Previous studies indicated by as much as 51%, as compared with the level measured in cells that the proto-oncogene c-jun can increase expression of a transfected with the corresponding empty vector (Fig. 2D). Alto- cyclin D1 promoter-controlled luciferase reporter in co-trans- gether, these results strongly suggest an important contribu- fection experiments, suggesting a positive regulatory effect of MAPK tion of the p42/p44 signal transduction pathway in the the JNK pathway on cyclin D1 transcription (39, 53). However, regulation of cyclin D1 expression in response to growth sig- the effect on protein expression has not been shown. The role of nals, both at the level of transcription and protein synthesis. MAPK the p38 cascade in gene expression is less clear. We thus MAPK Activation of the p42/p44 Cascade Is Sufficient to Pro- MAPK focused on the putative effect of the p38 cascade on cyclin mote Cyclin D1 Accumulation in CCL39-DRaf-1:ER Cells—The D1 transcription and protein expression in CCL39 cells. Co- MAPK contribution of the p42/p44 cascade on mitogen-induced transfection experiments were performed, using plasmids en- MAPK cyclin D1 protein synthesis was next examined, using a CCL39- coding different members of this cascade: the p38 kinase derived cell line (CCL39-DRaf-1:ER) expressing an estradiol- (MKK3), the constitutively active MKK3 mutant (MKK3- MAPK dependent human Raf-1 protein kinase (44). In this cell line, S189G/T193G called MKK3-Glu), the p38 , and the domi- the DRaf-1:ER chimera is activated in response to estradiol, MAPK MAPK nant-negative p38 (p38 -TY/AF), together with the MAPKs thereby activating MKK1 and then p42/44 . Previous cyclin D1-luciferase reporter construct. Surprisingly, overex- characterization of CCL39-DRaf-1:ER cells has shown that ad- pression of MKK3, which resulted in a higher basal level of MAPK dition of estradiol to serum-starved cells stimulates p42/ p38 activity in CCL39 cells, did not activate cyclin D1 MAPKs MAPK p44 within minutes. The p42/44 activity increases promoter; rather cyclin D1-luciferase expression was reduced for up to 1 h, thus reaching a level comparable to the maximal by 33% in exponentially growing cells, when compared with MAPK p42/44 activation measured in serum-stimulated CCL39 cells transfected with the empty vector (Fig. 4A). A stronger control cells, and remains elevated in the presence of estradiol. inhibitory effect was detected when a wild type form of the MAPK We used this cell system to directly measure the specific con- p38 was expressed together with MKK3, as if endogenous MAPK MAPK tribution of Raf . MKK1 . p42/p44 cascade on cyclin D1 p38 was limiting. In contrast, expression of dominant- MAPK protein expression. CCL39-DRaf-1:ER cells were serum- negative p38 -TY/AF together with MKK3 totally abol- starved for 24 h and then stimulated with either 10 mM estra- ished the MKK3-mediated inhibition of cyclin D1 luciferase diol or 10% FCS for varying periods. Whole cell extracts were expression, indicating that the negative effect on cyclin D1 analyzed for cyclin D1 protein expression and the pRb phos- transcription results from increased MKK3 activity. Expres- phorylation state by Western blots using specific antibodies. sion of the constitutively active MKK3 mutant (MKK3-Glu) The results showed that estradiol-treated CCL39-DRaf-1:ER elicited a more pronounced inhibition of cyclin D1-luciferase cells accumulated cyclin D1 protein to a level comparable with expression (62%) than the wild type MKK3, a result further that stimulated in the same cells by 10% serum (Fig. 3). More- indicating that the inhibitory effect was dependent on MKK3 over, cyclin D1 accumulation followed a similar time course in activity. When assays were performed in G -arrested cells, estradiol-treated cells when compared with serum-stimulated basal luciferase expression of the cyclin D1 promoter-controlled Regulation of Cyclin D1 Expression by MAPKs 20613 MAPK MAPK FIG.4. Activation of the p38/HOG cascade interferes with cyclin D1 expression. A and B, the effects of p38 activation on cyclin D1-luciferase expression were measured in exponentially growing cells kept in serum-supplemented medium (A) or in serum-starved cells (B). MAPK CCL39 were co-transfected with D1D-944 and expression vectors containing members of the p38 cascade, as indicated. A, luciferase activity was measured 48 h after the transfection and was normalized to the b-galactosidase activity by co-transfecting the pCH110 reporter. B, transfected cells were serum-starved for 30 h and stimulated with 10% FCS for 18 h. The fold increase in luciferase activity was calculated relative to the basal level of cyclin D1-luciferase, which was set to 1 unit and corrected for empty vector effects. Data are representative of at least three independent experiments. C and D, immunoblots of whole cell lysates from transfected cells. CCL39 were co-transfected with pNHE3 and expression constructs MAPK containing members of the p38 cascade or the appropriate empty vector (EV) as indicated. Transfected cells were selected 48 h after the transfection by applying an acid-load selection. C, cells were kept in the exponential growing phase in 7.5% serum-supplemented medium. D, cells were allowed to recover after the acid-load selection treatment in DMEM 7.5% FCS for at least 4 h before being starved for 24 h and stimulated in the presence of 1 unit/ml thrombin for 9 h. Equal amounts of whole cell lysates were separated by SDS-PAGE and proteins were detected by Western blots using specific antibodies against either cyclin D1 or pRb. Protein levels were quantitated by scanning of the autoradiograms. C, the amount of cyclin D1, upper and lower bands of pRb in MKK3 expressing cells as compared with the levels measured in EV cells were 0.55 6 0.055, 0.74 6 0.08, 1.4 6 0.06, respectively. D, thrombin-induced cyclin D1 protein levels in transfected cells expressing the various constructs as MAPK MAPK compared with the level measured in EV cells: MKK3 5 0.72 6 0.095, p38 5 0.83 6 0.06, MKK3 1 p38 5 0.45 6 0.16. Results are the MAPK means 6 S.E. for three independent experiments. Endogenous MKK1 or p38 expression are shown as internal controls. reporter was also reduced (Fig. 4B). Furthermore, increasing ated form, suggesting that the MKK3-mediated interfering MAPK p38 activity by expression of MKK3 resulted in a marked effect on cyclin D1 expression was affecting cyclin D1/cdk4–6 inhibition of the serum-induced cyclin D1-luciferase expression activity (Fig. 4C). Even though this modification of the pRb MAPK in resting cells. This result suggests that the p38 cascade phosphorylation state was not pronounced, we could reproduc- could be involved in the negative regulation of cyclin D1 tran- ibly quantitate 25% decrease of the slow-migrating form. Sim- scription and thus antagonize the mitogen-dependent stimula- ilarly, the thrombin-induced accumulation of endogenous cy- tion of cyclin D1 transcription mediated, at least in part, by the clin D1 was inhibited by 55% upon co-expression of the MAPK MAPK p42/p44 cascade. p38 together with its activator, MKK3, when compared MAPK This opposing effect of the p38 cascade, as compared with the level of thrombin-induced cyclin D1 in cells trans- MAPK with the positive regulatory effect of the p42/p44 pathway, fected with the empty vectors (Fig. 4D). MAPK was also detected on endogenous cyclin D1 protein expression As an independent approach to probe the role of the p38 MAPK in CCL39. The effects of MKK3 and p38 were monitored cascade, we used a chemical compound, the SB203580, which MAPK following co-transfection of the relevant constructs together has been described recently as a specific inhibitor for p38 , MAPKs with the selection vector encoding an amiloride-resistant but without inhibitory action on p42/p44 and JNKs (54, 1 1 MAPK Na /H exchanger isoform, which allowed us to monitor cyclin 55). When the endogenous p38 activity was inhibited by D1 protein levels in a population of cells enriched for the pretreatment of CCL39 with SB203580, the cyclin D1-lucifer- transfected genes. Cyclin D1 expression was measured in ase expression was reproducibly enhanced by 2–3-fold (Fig. whole cell lysates of resistant cells by immunoblotting using a 5A). Furthermore, treatment of MKK3-expressing cells with specific antibody. The results showed that cyclin D1 protein SB203580 reversed the inhibition of cyclin D1-luciferase ex- levels were reproducibly decreased by 40–50% in cells express- pression, a result that indicates that the MKK3-mediated in- ing the MKK3 construct when compared with control cells hibition of cyclin D1 transcription results from an increased MAPK transfected with the corresponding empty vector (Fig. 4C). This p38 activity. A 2–3-fold enhancing effect of SB203580 on inhibition of cyclin D1 expression was associated with a slight the level of endogenous cyclin D1 protein expression was also decrease in the amount of the slow-migrating hyperphospho- observed in exponentially growing cells as well as in resting rylated form of pRb in the same cell extracts, together with a cells in absence of mitogens (Fig. 5B). Altogether, these results MAPK corresponding increase in the amount of the hypophosphoryl- strongly suggest that the p38 cascade may exert a nega- 20614 Regulation of Cyclin D1 Expression by MAPKs FIG.6. IL-1b attenuates the thrombin-induced expression of cyclin D1 in CCL39 cells. CCL39 cells were serum-starved for 24 h and then stimulated in the presence of either 0.1 unit/ml thrombin or 0.1 unit/ml thrombin together with 20 ng/ml IL-1b for 9 h. Equal amounts of whole cell lysates were separated by SDS-PAGE in 10% gels, and cyclin D1 protein levels were determined on Western blots and quan- titated by scanning of the autoradiograms. The thrombin-induced cyclin D1 expression was inhibited by 26.5 6 2.06% in the presence of IL-1b. Percent IL-1b inhibition is the mean 6 S.E. for at least two independent experiments and was calculated relative to the level measured in cells stimulated with a-thrombin only. Cyclin D1 immunoblot is shown. these two signaling pathways. Therefore, this result strongly indicates that the differential regulation of cyclin D1 by the MAPK MAPK and the p38 cascades could have some phys- p42/p44 iological relevance. DISCUSSION Entry into the cell cycle upon growth factor stimulation requires fine coordination of events from the membrane to the nucleus. To pass the restriction point, mammalian cells must be continuously “fired” by signals during the first 6–8 h that precede the onset of DNA replication (56). At least two types of Ser/Thr protein kinases play a determinant role during this critical period of G /G progression: (i) the MAP kinase iso- 0 1 MAPK MAPKs FIG.5. Inhibition of the p38 cascade enhances cyclin D1 forms p42/p44 (ERK2/ERK1) and (ii) the G cyclin-cdk expression. A, CCL39 cells were co-transfected with D1D-944 and complexes, particularly cyclin D1/cdk4. Specific inactivation of MKK3, or the empty vector (EV). One day after transfection, cells were either of these two protein kinase signaling systems results in exposed to 10 mM of SB203580 for 16 h, and luciferase activity was specific growth arrest in G (1, 12, 27, 30, 31). measured. Data are representative of at least three independent exper- iments. The fold increase in luciferase activity was calculated relative to We previously demonstrated that persistent activation of MAPKs the basal expression level of D1D-944, which was set to 1 unit and p42/p44 is critical for the commitment of cell cycle entry corrected for empty vector effects. D1-luc, D1-luciferase. B, cyclin D1 in fibroblasts (5, 11). Only potent mitogens are capable of immunoblots of CCL39 whole cell lysates. Exponentially growing (up- MAPKs stimulating a sustained kinase activation of p42/p44 and per panel) or thrombin-stimulated resting CCL39 cells (lower panel) were exposed to 10 mM SB203580 for 9 h, and whole cell lysates were the concomitant nuclear translocation of these enzymes (5). separated by SDS-PAGE in 10% gels. Cyclin D1 protein levels were The long-term activation (that represents 10–30% of the peak detected by Western blotting and quantitated by scanning of the auto- activity measured 5 min following mitogen addition) usually radiograms. Data are the means 6 S.E. for at least three independent persists for several hours before declining to barely detectable experiments and are expressed as fold increase of cyclin D1 protein levels in cells incubated with SB203580 as compared with the level levels when cells enter and progress through S-phase (5, 11). MAPKs measured in untreated control cells. Exponentially growing cells, In marked contrast to p42/p44 , cyclin D1/cdk4 activity in 2.03 6 0.15; G -arrested cells, 2.6 6 0.38; thrombin-stimulated cells, CCL39 fibroblasts emerges rather late in G -phase and in- 1.15 6 0.09. creases progressively as the cells approach and pass through S-phase. Based on this temporal difference in activation and tive control on cyclin D1 expression. on the mitogenic regulatory roles of these two protein kinase A single external stimulus can lead to the simultaneous MAPKs signaling systems, we postulated that the p42/p44 control activation of multiple MAPK signal transduction pathways (2). the activation of cyclin D1/cdk4-cdk6, the first complex to be However, the magnitude and time course of the response ob- activated in early G . However, mechanisms that govern G - 1 1 served for each pathway is greatly dependent upon the nature cyclin/cdk activation are complex. First, de novo synthesis of of the stimulus. In order to determine if the opposing effects of MAPK MAPK the regulatory cyclin subunit is required, and this step is cer- p42/p44 and p38 activation on cyclin D1 expression tainly the most limiting step in G -arrested cells. Second, ap- were physiologically relevant, we designed experiments to propriate sites on the catalytic subunit must be phosphorylated measure the differential effects on cyclin D1 in response to by the activating kinase CAK and dephosphorylated by the appropriate stimuli. a-Thrombin mainly activates p42/ MAPKs MAPK specific cdc25 phosphatase (57). Third, multiple G -cdk inhib- p44 , whereas IL-1b drives a strong p38 activation MAPKs itors (CKIs) must be blunted for the kinase activity to emerge with little effect on p42/p44 . When these two MAPK MAPKs (58). It is possible that p42/p44 control many of these pathways were simultaneously activated by the co-addition of steps acting on the cyclin D1/cdk4-cdk6 activation. The present a-thrombin and IL-1b to resting CCL39 cells, the cyclin D1 study focused only on the first level of regulation that concerns protein level obtained after a 9-h stimulation was attenuated the growth factor-sensitive cyclin D1 induction. by 25–30% as compared with the level measured in cells ex- We found that among the D-type cyclins, cyclin D1 is the only posed to thrombin alone (Fig. 6). Based on co-transfection ex- detectable early G -cyclin to be regulated by growth factors in periments shown above, the reduced expression of cyclin D1 MAPKs obtained upon simultaneous activation of p42/p44 and MAPK 6 p38 likely resulted from opposite regulatory effects of A. Brunet, J.-M. Brondello, and J. Pouysse´gur, unpublished results. Regulation of Cyclin D1 Expression by MAPKs 20615 CCL39 cells. Furthermore, we demonstrated that the p42/ result in agreement with our previous finding in cells expressing MAPK p44 cascade controls cyclin D1 expression in response to the constitutively active MKK1 mutant (13). Interestingly, a growth signals thus contributing to the regulation of S-phase CCL39-derived cell line expressing high levels of human cyclin entry. Such a modulation of cyclin D1-associated kinase activ- D1 exhibited a similar higher sensitivity to growth factors. MAPK ity will participate in pRb inactivation (pRb hyperphosphoryl- In marked contrast to the positive action of p42/p44 MAPK ation). As a result, E2F-regulated gene expression is expected activation, the p38 signaling pathway exerted a negative MAPK MAPK to be suppressed in cells where p42/p44 activity is inhib- effect on cyclin D1 expression. Activation of the p38 cas- ited. In fact, not only cyclin D1 and its associated kinase activ- cade led to a significant decrease in cyclin D1 transcription ity were blocked in cells expressing either the dominant-nega- and, conversely, inhibition of this signaling pathway by the MAPK tive p44 or the MKP-1, but also the cdk2 activity was specific inhibitor SB203580 had an opposite enhancing effect. MAPK inhibited (data not shown). This result is consistent with the Although less pronounced, this inhibitory effect of the p38 fact that cdk2 activation in late G /early S-phase is dependent on cyclin D1 expression was also visualized at the level of upon transcription and synthesis of its regulatory subunits, protein synthesis and reproducibly detected using different MAPK cyclin E and cyclin A, which in turn require E2F release for strategies (either expression of MKK3, p38 , or pretreat- their expression, as many other inducible genes at the G /S ment with SB203580). In addition, IL-1b, a physiological in- MAPK boundary (59–62). Athough preliminary data suggest that ducer of p38 activation, showed a similar antagonizing MKP-1 could have the ability to inactivate other members of effect on thrombin-induced cyclin D1 accumulation in CCL39 the MAPK family, in particular the JNKs, a similar inhibition cells. However, treatment of CCL39 fibroblasts with IL-1b also of cyclin D1 expression, pRb hyperphosphorylation and cdk2 increases the JNK activity. The fact that the JNK cascade MAPK activity were observed using the dominant-negative p44 has been shown to promote cyclin D1 transcription and cell mutant, a more specific interfering construct. Considering pre- proliferation in fibroblasts (21, 53) may explain the poor inhib- vious studies that have shown that inhibition of the p42/ itory effect of IL-1b on DNA synthesis in CCL39 cells. How- MAPK p44 cascade blocks DNA synthesis and cell proliferation ever, the results presented here strongly suggest that the MAPK (1), we conclude that the requirement of this signaling pathway p38 -mediated inhibition of cyclin D1 expression may have for S-phase entry may thus rely in part on its essential function more dramatic effects in cell systems where IL-1b negatively as a positive regulator of cyclin D1 expression. regulates cell division. Interestingly, it has been recently MAPK This study revealed that not only the p42/p44 cascade is shown that hypophosphorylation of pRb could mediate the required for cyclin D1 transcription and protein synthesis, but G /G growth arrest induced by IL-1b in human A375-C6 mel- 0 1 it is sufficient by itself. Expression of a constitutively active anoma cells (63). MAP kinase kinase produced an increase in cyclin D1 protein The molecular mechanisms underlying the negative regula- MAPK level in absence of any other growth signal, which was equiv- tion of cyclin D1 expression by p38 is not clear. The tran- MAPK alent to the expression level measured in cells stimulated with scription factor ATF2 is substrate for p38 and, therefore, a strong mitogen. More importantly, the use of a CCL39-de- may account for the effect of this signaling pathway. The cyclin rived cell line expressing an estrogen-dependent human Raf-1 D1 promoter contains multiple regulatory elements (TRE, E2F, protein kinase (CCL39-DRaf-1:ER) demonstrated that the ex- Oct, SP1, CRE) and some uncharacterized elements that may MAPK clusive activation of the Raf . MKK1 . p42/p44 cascade also play a role in transcription of the gene (39). Thus cyclin D1 was able to induce cyclin D1 protein expression to the same expression may be responsive to a large set of transcription magnitude and with an identical time course as that induced by factors. In addition, multiple MAPK cascades appears to be serum in these cells. Even though stimulation of the Raf path- implicated in the regulation of the promoter activity (this way was sufficient to induce cyclin D1 accumulation, it could study) (39, 53), some of that modulating the activity of a com- not promote pRb phosphorylation or cdk2 kinase activation mon substrate. This is the case for Elk-1, a substrate for the (data not shown). As a result, no significant estradiol-induced three distinct mammalian MAPKs, which can therefore inte- DNA synthesis could be detected in this system. The apparent grate signals from multiple MAPK cascades in response to increase in the overall pRb hyperphosphorylation in cells ex- extracellular stimuli (64). The transcription factor ATF2 is also MAPK pressing the constitutively active MKK1 mutant in the pres- a common substrate for p38 and JNKs (17, 18, 20). How- ence of serum may thus likely result from a cooperative effect ever, only the JNK cascade can activate c-Jun (16, 65–67). MAPK between the p42/p44 cascade and other signaling path- Therefore, activation of a specific MAPK cascade is likely to ways. Interestingly, we could detect a similar increase in the produce a differential effect on gene expression. Finally, there overall amount of pRb expression (and thus in the pRb hyper- are many mechanisms for the regulation of the respective phosphorylated form), associated with a marked increase in the MAPK signal transduction pathways and both positive and level of cyclin D1 expression in stable-CCL39 transfectants negative cross-talk between these MAPK cascades probably MAPK expressing a high constitutive level of the MKK1-SS/DD mu- exists, which could explain the interfering effect of the p38 tant. The use of CCL39-DRaf-1:ER-derived cell line allowed us cascade on cyclin D1 expression. to discriminate between secondary versus immediate effects of Although the data obtained here strongly suggest that the MAPK MAPK MAPK p42/p44 activation in a synchronized population of cells. p42/p44 and the p38 exert their regulatory action on MAPK We thus conclude that although activation of the p42/p44 cyclin D1 expression at the level of the transcriptional machin- cascade is necessary and sufficient for cyclin D1 expression, ery, they do not exclude a possible additional post-transcrip- MAPK other signals are required to promote the activation of cyclin tional regulation. It has been suggested that the p42/p44 D1/cdk4–6 complex and thus the hyperphosphorylation of pRb cascade could up-regulate translation initiation of specific to ensure passage through S-phase. However, activation of the genes in response to insulin (68). A recent study has shown that MAPK Raf . MKK1 . p42/p44 by estradiol treatment led to a overexpression of eIF-4E, which is released upon phosphoryl- MAPKs decrease in growth factor requirement for DNA synthesis, a ation of PHAS-1 protein by p42/p44 , increased both cyclin D1 mRNA and protein in resting fibroblasts (52). There is also J.-M. Brondello, F. McKenzie, and J. Pouysse´gur, unpublished results. J.-M. Brondello, A. Brunet, and J. Pouysse´gur, unpublished P. Lenormand and J. Pouysse´gur, unpublished results. results. 9 11 A. Brunet, J. N. Lavoie, and J. Pouysse´gur, unpublished results. J. N. Lavoie, A. Brunet, and J. Pouysse´gur, unpublished results. 20616 Regulation of Cyclin D1 Expression by MAPKs MAPK 19. Van Dam, H., Wilhelm, D., Herr, I., Steffen, A., Herrlich, P., and Angel, P. evidence suggesting a role for p38 in the control of gene (1995) EMBO J. 14, 1798–1811 expression by post-transcriptional regulation of specific gene 20. Raingeaud, J., Gupta, S., Rogers, J. S., Dickens, M., Han, J., Ulevitch, R. 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Han, J., Lee, J.-D., Bibbs, L., and Ulevitch, R. J. (1994) Science 265, 808–811 data were presented showing, in agreement with the results 42. Sun, H., Charles, C. H., Lau, L. F., and Tonks, N. K. (1993) Cell 75, 487–493 presented here, a positive modulation of cyclin D1 transcription 43. Noe¨l, J., Roux, D., and Pouysse´gur, J. (1996) J. Cell Sci., 109, 929–939 MAPK by the p42 (53). This positive effect has been shown to be 44. Samuels, M. L., Weber, M. J., Bishop, J. M., and McMahon, M. (1993) Mol. Cell. Biol. 13, 6241–6252 mediated by the transcription factor c-Ets-2 and dependent on 45. Pouysse´gur, J., Sardet, C., Franchi, A., L’Allemain, G., and Paris, S. (1984) the presence of a putative Ets-like binding domain on the Proc. Natl. Acad. Sci. U. S. A. 81, 4833–4837 46. Counillon, L., Scholz, W., Lang, H. J., and Pouysse´gur, J. (1993) Mol. Phar- proximal region of the human cyclin D1 promoter. macol. 44, 1041–1045 Acknowledgments—We warmly thank Dr. V. Baldin for providing us 47. 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Journal
Journal of Biological Chemistry – Unpaywall
Published: Aug 1, 1996