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• W1984231661 abstract "The activation of glycolytic flux is a biochemical characteristic of growing cells. Several reports have demonstrated the role of fructose 2,6-bisphosphate in this process. In this paper we show that the levels of 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase (6PF2K/Fru-2,6-P2ase) mRNA are modulated in response to serum and growth factors and this effect is due to regulation of its transcription rate. The modulation of the expression of this enzyme by growth factors differs according their mitogenic effect; both lysophosphatidic acid and epidermal growth factor, when added alone, increased the mRNA levels, but endothelin had no effect. Furthermore, cAMP, which acts as an antimitogenic signal in Rat-1 fibroblasts, produced a decrease in 6PF2K/Fru-2,6-P2ase mRNA and inhibited the effects of lysophosphatidic acid and epidermal growth factor on 6PF2K/Fru-2,6-P2ase expression. PD 098059, a specific inhibitor of the activation of the mitogen-activated protein kinase, was able to prevent the effect of EGF on 6PF2K/Fru-2,6-P2ase gene expression. These results imply that activation of mitogen-activated protein kinase is required for the stimulation of the transcription of 6PF2K/Fru-2,6-P2ase by EGF. The activation of glycolytic flux is a biochemical characteristic of growing cells. Several reports have demonstrated the role of fructose 2,6-bisphosphate in this process. In this paper we show that the levels of 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase (6PF2K/Fru-2,6-P2ase) mRNA are modulated in response to serum and growth factors and this effect is due to regulation of its transcription rate. The modulation of the expression of this enzyme by growth factors differs according their mitogenic effect; both lysophosphatidic acid and epidermal growth factor, when added alone, increased the mRNA levels, but endothelin had no effect. Furthermore, cAMP, which acts as an antimitogenic signal in Rat-1 fibroblasts, produced a decrease in 6PF2K/Fru-2,6-P2ase mRNA and inhibited the effects of lysophosphatidic acid and epidermal growth factor on 6PF2K/Fru-2,6-P2ase expression. PD 098059, a specific inhibitor of the activation of the mitogen-activated protein kinase, was able to prevent the effect of EGF on 6PF2K/Fru-2,6-P2ase gene expression. These results imply that activation of mitogen-activated protein kinase is required for the stimulation of the transcription of 6PF2K/Fru-2,6-P2ase by EGF. Quiescent cells can be stimulated to recommence DNA synthesis by distinct and interactive signal transduction pathways. These signaling pathways trigger a cascade of molecular events required for progression of DNA replication (1Rozengurt E. Science. 1986; 234: 161-166Crossref PubMed Scopus (843) Google Scholar, 2Rozengurt E. Erusalimsky J. Mehmet H. Morris C. Nanberg E. Sinnet-Smith J. Cold Spring Harbor Symp. Quant. Biol. 1988; 53: 945-954Crossref PubMed Google Scholar). One of the essential processes for this progression is the activation of the glycolytic flux (3Warburg O. Science. 1956; 123: 309-324Crossref PubMed Scopus (9125) Google Scholar, 4Krebs E.G. Belfrage P. Donner J. Stralford P. Mechanism of Insulin Action. Elsevier Science Publishers, Amsterdam1986: 13-32Google Scholar). It is known that glycolysis is stimulated both when growth promoting agents are added to quiescent cells and in many types of transformed cells (5Diamond I. Legg A. Schneider J.A. Rozengurt E. J. Biol. Chem. 1978; 253: 866-871Abstract Full Text PDF PubMed Google Scholar, 6Driedger P.E. Blumberg P.M. Cancer Res. 1977; 37: 3257-3265PubMed Google Scholar, 7Schneider J.A. Diamond I. Rozengurt E. J. Biol. Chem. 1979; 253: 872-877Abstract Full Text PDF Google Scholar). The major regulatory step in the glycolytic pathway is 6-phosphofructo-1-kinase activity. Activation of glycolysis involves the increase of fructose 2,6-bisphosphate (Fru-2,6-P2) 1The abbreviations used are: Fru-2,6-P2fructose 2,6-bisphosphate6PF2K/Fru-2,6-P2ase6-phosphofructo-2-kinase/fructose-2,6-bisphosphataseFCSfetal calf serumCATchloramphenicol acetyltransferaseEGFepidermal growth factorLPAlysophosphatidic acidMAPKmitogen-activated protein kinaseERKextracellular regulated kinaseMEKMAPK or ERK kinasePKCprotein kinase CTPA12-O-tetradecanoylphorbol-13-acetateCTxcholera toxinMEMminimum essential medium. levels, a potent allosteric stimulator of 6-phosphofructo-1-kinase (reviewed in Refs. 8Hue L. Rider K. Biochem. J. 1987; 245: 313-324Crossref PubMed Scopus (331) Google Scholar and 9Pilkis S.J. Claus T.H. Kurland I.J. Lange A.J. Annu. Rev. Biochem. 1995; 64: 799-835Crossref PubMed Scopus (222) Google Scholar). Fru-2,6-P2 content is regulated by the bifunctional enzyme 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase (6PF2K/Fru-2,6-P2ase; EC 2.7.1.105/3.1.3.46), which catalyzes both its synthesis and its degradation (8Hue L. Rider K. Biochem. J. 1987; 245: 313-324Crossref PubMed Scopus (331) Google Scholar, 9Pilkis S.J. Claus T.H. Kurland I.J. Lange A.J. Annu. Rev. Biochem. 1995; 64: 799-835Crossref PubMed Scopus (222) Google Scholar). Different 6PF2K/Fru-2,6-P2ase isoenzymes have been described depending on the tissues and cell specificity. In rat, the liver, the muscle, and the F-type isoforms are encoded by the same gene arising from distinct promoters (10Dupriez V.J. Darville M.I. Antoine I.V. Gegonne A. Ghysdael J. Rousseau G.G. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 8224-8228Crossref PubMed Scopus (40) Google Scholar, 11Lange A.J. Espinet C. Hall R. El-Maghrabi M.R. Vargas A.M. Miksicek R.J. Granner D.K. Pilkis S.J. J. Biol. Chem. 1992; 267: 15673-15680Abstract Full Text PDF PubMed Google Scholar). These isoenzymes differ with respect to their N-terminal sequence, regulation by phosphorylation, and kinetic properties. It has been shown that the F-type is the isoform expressed in several proliferative tissues and in serum-stimulated Rat-1 fibroblasts (10Dupriez V.J. Darville M.I. Antoine I.V. Gegonne A. Ghysdael J. Rousseau G.G. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 8224-8228Crossref PubMed Scopus (40) Google Scholar). Although extensive studies have been performed on the regulation of liver and muscle isoforms, this is not the case of F-type. fructose 2,6-bisphosphate 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase fetal calf serum chloramphenicol acetyltransferase epidermal growth factor lysophosphatidic acid mitogen-activated protein kinase extracellular regulated kinase MAPK or ERK kinase protein kinase C 12-O-tetradecanoylphorbol-13-acetate cholera toxin minimum essential medium. Different reports have demonstrated that Fru-2,6-P2 levels increase after stimulation of fibroblasts with fetal bovine serum, insulin, epidermal growth factor (EGF), or tumor promoter phorbol esters (12Bruni P. Farnararo M. Vasta V. D'Alessandro A. FEBS Lett. 1983; 159: 39-42Crossref PubMed Scopus (19) Google Scholar, 13Mojena M. Boscà L. Hue H Biochem. J. 1985; 232: 521-527Crossref PubMed Scopus (27) Google Scholar, 14Bruni P. Vasta V. Farnararo M. Biochim. Biophys. Acta. 1986; 887: 23-28Crossref PubMed Scopus (9) Google Scholar, 15Bruni P. Vasta V. Farnararo M. FEBS Lett. 1987; 222: 27-31Crossref PubMed Scopus (12) Google Scholar, 16Rousseau G.G. Fisher Y. Guening M.A. Marchand M.J. Testar X. Hue L. Prog. Cancer Res. Ther. 1988; 35: 150-163Google Scholar, 17Hamer M.J. Dickson A. Biochem. J. 1990; 269: 685-690Crossref PubMed Scopus (21) Google Scholar, 18Taylor D. Evanson J.M. Woolley D.E. Biochem. J. 1990; 269: 573-577Crossref PubMed Scopus (29) Google Scholar, 19Boscà L. Rousseau G.G. Hue H. Proc. Natl. Acad. Sci. U. S. A. 1985; 82: 6440-6444Crossref PubMed Scopus (61) Google Scholar, 20Boscà L. Mojena M. Ghysdael J. Rousseau G.G. Hue L. Biochem. J. 1986; 236: 596-599Crossref Scopus (45) Google Scholar, 21Dalmau M. Bartrons R. Gil J. Exp. Cell. Res. 1994; 212: 93-96Crossref PubMed Scopus (15) Google Scholar). In some cases, the increase in this metabolite has been correlated with an increase in the Vmax of 6-phosphofructo-2-kinase activity (19Boscà L. Rousseau G.G. Hue H. Proc. Natl. Acad. Sci. U. S. A. 1985; 82: 6440-6444Crossref PubMed Scopus (61) Google Scholar, 20Boscà L. Mojena M. Ghysdael J. Rousseau G.G. Hue L. Biochem. J. 1986; 236: 596-599Crossref Scopus (45) Google Scholar, 21Dalmau M. Bartrons R. Gil J. Exp. Cell. Res. 1994; 212: 93-96Crossref PubMed Scopus (15) Google Scholar, 22Cascales M. Martin-Sanz P. Boscà L. Eur. J. Biochem. 1992; 207: 391-397Crossref PubMed Scopus (12) Google Scholar, 23Denis-Pouxviel C. Gauthier T. Daviaud D. Murat J.C. Biochem. J. 1990; 268: 465-470Crossref PubMed Scopus (12) Google Scholar, 24Baulida J. Onetti R. Bassols A. Biochem. Biophys. Res. Commun. 1992; 183: 1216-1223Crossref PubMed Scopus (19) Google Scholar). Although this metabolic effect is clear, the mechanism by which it is achieved is not known. Recently, a study of the regulation of Fru-2,6-P2 metabolism through different signal transduction pathways has been performed using murine Swiss 3T3 fibroblasts as a model (21Dalmau M. Bartrons R. Gil J. Exp. Cell. Res. 1994; 212: 93-96Crossref PubMed Scopus (15) Google Scholar). However, the attempts to measure regulation of 6PF2K/Fru-2,6-P2ase mRNA levels and transcription rate were not successful because the cDNA probes available did not recognize Swiss 3T3 6PF2K/Fru-2,6-P2ase mRNA. The aim of this work is to study the signaling pathways that control the transcription of the 6PF2K/Fru-2,6-P2ase gene during cell proliferation. For this propose, we have investigated the effect of EGF, lysophosphatidic acid (LPA), endothelin, and cholera toxin (CTx) on 6PF2K/Fru-2,6-P2ase gene expression in Rat-1 fibroblasts and the role of mitogen-activated kinase (MAPK) in this regulation. [ä-32P]dCTP (3000 Ci/mmol), [ä-32P]UTP (3000 Ci/mmol), [6-3H]thymidine (5 Ci/mmol), ECL Western kit, and Hybond-N membranes were from Amersham International (Amersham, Bucks., United Kingdom (UK)). The random-primer DNA-labeling kit was from Boehringer Mannheim. Fetal calf serum, minimum essential medium, and penicillin/streptomycin were from Bio-Whittaker. EGF, LPA, insulin, and endothelin-1 were from Sigma. Anti-phosphotyrosine monoclonal antibody PY20 was from Affiniti Research Products Ltd. (Nottingham, UK), and anti-MAPK monoclonal antibody was from Zymed Laboratories, Inc. (San Francisco, CA). PD 098059 was a gift from Dr. A. Saltiel, Parke-Davis Pharmaceutical Division (Detroit, MI). Other materials and chemicals were of the highest quality available. Rat-1 fibroblasts were routinely grown in minimum essential medium (MEM) supplemented with 10% (v/v) fetal calf serum (FCS), 100 units of penicillin/ml, and 100 μg of streptomycin/ml at 37°C in a 5% CO2 atmosphere. For experimental purposes, cells were subcultured in six-well plates or 100-mm dishes and their quiescence was induced by maintaining the cells in serum-free medium during 24 h. After this, cells were stimulated with either FCS or different growth factors. Quiescent Rat-1 cells were activated with 10% FCS and different growth factors. At the indicated times, cells were exposed to [6-3H]thymidine (0.5 μCi/ml) for the last 1 h of stimulation. Trichloroacetic acid-precipitable material was dissolved in 0.1 M NaOH and quantified by liquid scintillation counting. To measure Fru-2,6-P2 levels, Rat-1 cells were homogenized in 0.1 M NaOH, heated at 80°C for 15 min, and centrifuged at 12,000 x g for 5 min. Fru-2,6-P2 was determined in supernatants by its ability to activate pyrophosphate-dependent 6-phosphofructo-1-kinase from potato tubers as described by Van Schaftingen et al. (25Van Schaftingen E. Lederer B. Bartrons R. Hers H.G. Eur. J. Biochem. 1982; 129: 191-195Crossref PubMed Scopus (529) Google Scholar). Immunoblot analysis were performed essentially as described by Burnette (26Burnette W.N. Anal. Biochem. 1981; 112: 195-203Crossref PubMed Scopus (5844) Google Scholar). For 6PF2K/Fru-2,6-P2ase, we used a 1:1000 dilution of a polyclonal antibody raised against the rat liver protein (27El-Maghrabi M.R. Correia J.J. Heil P.J. Pate T. Coob C. Pilkis S.J. Proc. Natl. Acad. Sci. U. S. A. 1986; 83: 5005-5009Crossref PubMed Scopus (48) Google Scholar). Western blot analysis of MAPK phosphorylation was performed as described previously (28Sun H. Charles C.H. Lau L.F. Tonks N.K. Cell. 1993; 75: 487-493Abstract Full Text PDF PubMed Scopus (1020) Google Scholar) using anti-phosphotyrosine or anti-MAPK monoclonal antibodies. Bound antibodies were detected by enhanced chemiluminescence method. The amount of protein was determined by densitometric scanning of the autoradiograms. Total RNA was extracted from Rat-1 cells by the LiCl/urea method (29Auffray C. Rougeon F. Eur. J. Biochem. 1980; 107: 303-314Crossref PubMed Scopus (2084) Google Scholar). Northern blot analyses were carried out by standard procedures (30Sambrook J. Fritsch E.F. Maniatis T. Molecular Cloning: A Laboratory Manual. 2nd Ed. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1989Google Scholar). To detect 6PF2K/Fru-2,6-P2ase mRNA, a 1.4-kilobase EcoRI fragment from pPKB plasmid was used as a probe (31Tauler A. Rosenberg A.H. Colosia A. Studier W.F. Pilkis S.J. Proc. Natl. Acad. Sci. U. S. A. 1988; 85: 6642-6646Crossref PubMed Scopus (27) Google Scholar). The integrity of the RNA was verified by observing the rRNA bands in the ethidium bromide gel under UV irradiation. The level of mRNA was evaluated by densitometric scanning of the autoradiograms and corrected for the amount of 18 S rRNA using a ribosomic cDNA probe (32Gerlach W.L. Bedbrook J.R. Nucleic Acids Res. 1979; 7: 1869-1885Crossref PubMed Scopus (1262) Google Scholar). Stable transfections were performed by the CaPO4 method as described by Sambrook et al. (30Sambrook J. Fritsch E.F. Maniatis T. Molecular Cloning: A Laboratory Manual. 2nd Ed. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1989Google Scholar). Rat-1 cells were plated at 106 cells/100-mm culture dish 24 h before transfection. The DNA precipitate (50 μg of the HindIII/BspmI 5′ flanking region of the F isoform of 6PF2K/Fru-2,6-P2ase cloned into pBLCAT3 plasmid as described by Lange (11Lange A.J. Espinet C. Hall R. El-Maghrabi M.R. Vargas A.M. Miksicek R.J. Granner D.K. Pilkis S.J. J. Biol. Chem. 1992; 267: 15673-15680Abstract Full Text PDF PubMed Google Scholar) and 5 μg of pSV2Neo) were left overnight. After 24 h, the cells were trypsinized and replated at 1:4 dilution. G418 (400 μg/ml) was added 24 h later. Transfections containing resistant colonies were pooled for experimentation. CAT activity determinations were carried out as described by Ausebel et al. (33Ausebel F.M. Brent R. Kingston R.E. Moore D.D. Seidman J.G. Smith J.A. Struhl K. Current Protocols in Molecular Biology. John Wiley & Sons Inc., New York1994Google Scholar). Protein was determined as described by Bradford (34Bradford M. Anal. Biochem. 1976; 112: 195-203Google Scholar) with bovine serum albumin as a standard. Statistical significance of difference was assessed by Student's unpaired t test. Confluent Rat-1 fibroblasts were made quiescent by maintaining them in serum-free medium for 24 h and then shifted to medium supplemented with 10% fetal-calf serum. After serum addition, the cells were harvested and samples were taken to be analyzed at different times. The data reported in Table I show that, after being exposed to serum, an increase of the intracellular Fru-2,6-P2 content was detected. During this process, the amount of 6PF2K/Fru-2,6-P2ase protein also increased. This was measured by immunobloting with an antibody obtained against the liver 6PF2K/Fru-2,6-P2ase (Fig. 1). To determine whether the abundance of 6PF2K/Fru-2,6-P2ase protein was correlated with the amount of 6PF2K/Fru-2,6-P2ase mRNA, Northern blot analysis was done using a 1.4-kilobase cDNA probe (31Tauler A. Rosenberg A.H. Colosia A. Studier W.F. Pilkis S.J. Proc. Natl. Acad. Sci. U. S. A. 1988; 85: 6642-6646Crossref PubMed Scopus (27) Google Scholar). As shown in Fig. 2, there was a transient increase in the level of 6PF2K/Fru-2,6-P2ase mRNA with a maximum at 12-15 h (5-fold increase over serum deprivation mRNA levels) and a gradual decline to serum-free mRNA levels by 30 h. The analysis of the incorporation of [6-3H]thymidine into DNA during the course of this experiment revealed that the peak in mRNA accumulation was coincident with the onset of DNA synthesis (Fig. 2).Table IEffect of serum addition on fructose 2,6-bisphosphate levelsFru-2,6-P2 content6 h12 h24 hpmol/mg of proteinControl97 ± 13119 ± 1781 ± 7+ FCS154 ± 17*211 ± 15**233 ± 16** Open table in a new tab Fig. 2Expression of 6PF2K/Fru-2,6-P2ase mRNA during the cell cycle of Rat-1 fibroblasts. Quiescent Rat-1 fibroblasts were stimulated with MEM supplemented with 10% FCS (v/v). At the indicated incubation times, total RNA (20 μg/lane) was extracted from cultured cells in the presence (▪) or absence (□) of FCS and analyzed by Northern blot. Upper, a representative Northern blot obtained with preparations including FCS is shown. Lower, the intensity of the autoradiographic signal was quantified by densitometric scanning and corrected for the amount of RNA obtained with the 18 S rRNA probe. Values are expressed relative to the value of control cells, which was considered as 1 arbitrary unit. Data are means ± S.E. of three independent experiments. To measure thymidine incorporation, at the indicated incubation times, serum-stimulated cells were exposed to [6-3H]thymidine (0.5 μCi) during the last 1 h of stimulation, and incorporation was assayed by the trichloroacetic acid standard procedure. Each point (○) represents the mean ± S.E. of three independent experiments, each one performed by triplicate.View Large Image Figure ViewerDownload Hi-res image Download (PPT) To determine whether the regulation of 6PF2K/Fru-2,6-P2ase mRNA levels was due to a regulation of its transcription rate, Rat-1 fibroblasts were stable transfected with a CAT reporter gene under transcriptional control of a 1.7-kilobase genomic DNA fragment that contain the F-type promoter (11Lange A.J. Espinet C. Hall R. El-Maghrabi M.R. Vargas A.M. Miksicek R.J. Granner D.K. Pilkis S.J. J. Biol. Chem. 1992; 267: 15673-15680Abstract Full Text PDF PubMed Google Scholar). In preliminary RNase protection experiments, we observed that the F-type isoform is the only one expressed in these cells. Quiescent transfected cells were stimulated with 10% of serum, and CAT activity was measured at different times. As shown in Fig. 3, up to 4-fold induction of CAT activity was found at 4 and 8 h after addition of serum. In contrast, CAT activity did not change in cells incubated in the absence of serum during this period. In order to study the signal transduction pathways that control the expression of 6PF2K/Fru-2,6-P2ase in Rat-1 fibroblasts during proliferation, we determined the effect of EGF, LPA, endothelin, and cholera toxin (CTx) on 6PF2K/Fru-2,6-P2ase mRNA levels. As shown in Table II, EGF at 10 ng/ml and LPA at 70 μM increased the levels of 6PF2K/Fru-2,6-P2ase mRNA 4- and 7-fold, respectively. No significant change in 6PF2K/Fru-2,6-P2ase mRNA levels was detected after endothelin addition. Furthermore, 12-O-tetradecanoylphorbol-13-acetate (TPA), an activator of protein kinase C (PKC) also failed to increase 6PF2K/Fru-2,6-P2ase mRNA. In contrast, CTx at 10 ng/ml decreased the 6PF2K/Fru-2,6-P2ase mRNA levels by approximately 50%. In agreement with the results from measurement of mRNA levels, addition of EGF and LPA to quiescent Rat-1 fibroblasts induced an increase in Fru-2,6-P2 levels. In contrast, TPA or endothelin did not modify the concentration of this metabolite (Table III).Table IIEffect of growth factors on 6PF2K/Fru-2,6-P2ase mRNA levelsRelative amount of 6PF2K/Fru-2,6-P2ase mRNAArbitrary unitsControl1.0+ FCS6.2 ± 0.1+ EGF3.8 ± 0.3+ LPA7.1 ± 0.8+ TPA1.5 ± 0.1+ ET-11.4 ± 0.2+ CTx0.5 ± 0.1 Open table in a new tab Table IIIEffect of growth factors on fructose 2,6-bisphosphate levelsFructose 2,6-P2 contentpmol/mg of proteinControl119 ± 17+ EGF167 ± 3*+ LPA188 ± 17*+ TPA111 ± 25+ CTx110 ± 11 Open table in a new tab Both EGF and LPA induce the activation of the mitogen-activated protein kinases ERK1 and ERK2 (MAPKs) through the Ras-Raf-MEK pathway (35Cook S.J. Rubinfeld B. Albert I. McCormick F. EMBO J. 1993; 12: 3475-3485Crossref PubMed Scopus (331) Google Scholar, 36Hordijk P.L. Verlaan I. van Corven E.J. Moolenaar W.H. J. Biol. Chem. 1994; 269: 645-651Abstract Full Text PDF PubMed Google Scholar). In Rat-1 fibroblasts, cAMP can inhibit the activation of MAPKs by inhibiting Raf activation (37Cook S.J. McCormick F. Science. 1993; 262: 1069-1071Crossref PubMed Scopus (860) Google Scholar). To examine the correlation between MAPK activation and the increase in 6PF2K/Fru-2,6-P2ase mRNA found after mitogenic responses, quiescent Rat-1 cells were preincubated with CTx for 90 min before stimulation with EGF and the 6PF2K/Fru-2,6-P2ase mRNA levels were then measured. The phosphorylation state of ERK2 is shown in Fig. 4. As we expected, a phosphotyrosine protein that comigrated with ERK2 was detected after EGF and serum addition, but it was almost undetectable with TPA. Preincubation with CTx blocked the effect of EGF on ERK2 phosphorylation (panel B). Under these experimental conditions, EGF produced a 4-fold increase on the levels of 6PF2K/Fru-2,6-P2ase mRNA and preincubation with CTx completely inhibited this effect (panel C). The same results were obtained with LPA (data not shown). Recently, PD 098059 has been identified as a specific cell-permeant inhibitor of MEK (38Dudley D.T. Pang L. Decker S.J. Bridges A.J. Saltiel A.R. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 7686-7689Crossref PubMed Scopus (2581) Google Scholar). To prove that MAPK activation is involved in mediating the effect of EGF on 6PF2K/Fru-2,6-P2ase expression, we examined the effect of this compound on 6PF2K/Fru-2,6-P2ase mRNA levels. As shown in Fig. 5, PD 098059 is able to prevent the increase on 6PF2K/Fru-2,6-P2ase mRNA by EGF. The concentration of inhibitor required to avoid this activation was similar to the one reported to inhibit MAPK (38Dudley D.T. Pang L. Decker S.J. Bridges A.J. Saltiel A.R. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 7686-7689Crossref PubMed Scopus (2581) Google Scholar). The ability of PD 098059 to inhibit the effect EGF on 6PF2K/Fru-2,6-P2ase mRNA levels was correlated with its ability to prevent the activation of 6PF2K/Fru-2,6-P2ase transcription. Cells stably transfected with a CAT reporter gene under transcriptional control of 1.7-kilobase F-type promoter, were incubated at different concentrations of PD 098059 and CAT activity was assayed 8 h after addition of EGF. As expected, PD 098059 suppressed the increase of CAT activity induced by EGF and the activity remaining at basal levels at high concentration of the inhibitor (Fig. 6). Several reports have pointed out the essential role of Fru-2,6-P2 in the activation of glycolytic flux in growing cells (3Warburg O. Science. 1956; 123: 309-324Crossref PubMed Scopus (9125) Google Scholar, 4Krebs E.G. Belfrage P. Donner J. Stralford P. Mechanism of Insulin Action. Elsevier Science Publishers, Amsterdam1986: 13-32Google Scholar). The levels of this metabolite increase in different proliferative states (12Bruni P. Farnararo M. Vasta V. D'Alessandro A. FEBS Lett. 1983; 159: 39-42Crossref PubMed Scopus (19) Google Scholar, 13Mojena M. Boscà L. Hue H Biochem. J. 1985; 232: 521-527Crossref PubMed Scopus (27) Google Scholar, 14Bruni P. Vasta V. Farnararo M. Biochim. Biophys. Acta. 1986; 887: 23-28Crossref PubMed Scopus (9) Google Scholar, 15Bruni P. Vasta V. Farnararo M. FEBS Lett. 1987; 222: 27-31Crossref PubMed Scopus (12) Google Scholar, 16Rousseau G.G. Fisher Y. Guening M.A. Marchand M.J. Testar X. Hue L. Prog. Cancer Res. Ther. 1988; 35: 150-163Google Scholar, 17Hamer M.J. Dickson A. Biochem. J. 1990; 269: 685-690Crossref PubMed Scopus (21) Google Scholar, 18Taylor D. Evanson J.M. Woolley D.E. Biochem. J. 1990; 269: 573-577Crossref PubMed Scopus (29) Google Scholar, 19Boscà L. Rousseau G.G. Hue H. Proc. Natl. Acad. Sci. U. S. A. 1985; 82: 6440-6444Crossref PubMed Scopus (61) Google Scholar, 20Boscà L. Mojena M. Ghysdael J. Rousseau G.G. Hue L. Biochem. J. 1986; 236: 596-599Crossref Scopus (45) Google Scholar, 21Dalmau M. Bartrons R. Gil J. Exp. Cell. Res. 1994; 212: 93-96Crossref PubMed Scopus (15) Google Scholar, 22Cascales M. Martin-Sanz P. Boscà L. Eur. J. Biochem. 1992; 207: 391-397Crossref PubMed Scopus (12) Google Scholar, 23Denis-Pouxviel C. Gauthier T. Daviaud D. Murat J.C. Biochem. J. 1990; 268: 465-470Crossref PubMed Scopus (12) Google Scholar, 24Baulida J. Onetti R. Bassols A. Biochem. Biophys. Res. Commun. 1992; 183: 1216-1223Crossref PubMed Scopus (19) Google Scholar, 39Rosa J.L. Ventura F. Carreras J. Bartrons R Biochem. J. 1990; 270: 645-649Crossref PubMed Scopus (19) Google Scholar), and this change has been correlated with an increase in 6-phosphofructo-2-kinase activity (18Taylor D. Evanson J.M. Woolley D.E. Biochem. J. 1990; 269: 573-577Crossref PubMed Scopus (29) Google Scholar, 19Boscà L. Rousseau G.G. Hue H. Proc. Natl. Acad. Sci. U. S. A. 1985; 82: 6440-6444Crossref PubMed Scopus (61) Google Scholar, 20Boscà L. Mojena M. Ghysdael J. Rousseau G.G. Hue L. Biochem. J. 1986; 236: 596-599Crossref Scopus (45) Google Scholar, 21Dalmau M. Bartrons R. Gil J. Exp. Cell. Res. 1994; 212: 93-96Crossref PubMed Scopus (15) Google Scholar, 22Cascales M. Martin-Sanz P. Boscà L. Eur. J. Biochem. 1992; 207: 391-397Crossref PubMed Scopus (12) Google Scholar, 23Denis-Pouxviel C. Gauthier T. Daviaud D. Murat J.C. Biochem. J. 1990; 268: 465-470Crossref PubMed Scopus (12) Google Scholar, 24Baulida J. Onetti R. Bassols A. Biochem. Biophys. Res. Commun. 1992; 183: 1216-1223Crossref PubMed Scopus (19) Google Scholar) and recently with an increase in mRNA levels (40Joaquin M. Rosa J.L. Salvado C. Lopez S. Nakamura T. Bartrons R. Gil J. Tauler A. Biochem. J. 1996; 314: 235-240Crossref PubMed Scopus (5) Google Scholar, 41Darville M.I. Antoine I.V. Mertens-Strijhagen J.R. Dupriez V.J. Rousseau G.G. Oncogene. 1995; 11: 1509-1517PubMed Google Scholar). However, the mechanisms by which growth factors regulate 6PF2K/Fru-2,6-P2ase are still unclear. The results presented here demonstrate that MAPK is involved in the regulation of 6PF2K/Fru-2,6-P2ase gene transcription in response to mitogenic signals. The modulation of the expression of 6PF2K/Fru-2,6-P2ase by growth factors is concomitant with their mitogenic response. Serum, LPA, and EGF, complete mitogens for Rat-1 fibroblasts (42van Corven E.J. Groenink A. Jalink K. Eichholtz T. Moolenaar W.H. Cell. 1989; 59: 45-54Abstract Full Text PDF PubMed Scopus (674) Google Scholar), were able to increase the mRNA levels of 6PF2K/Fru-2,6-P2ase and Fru-2,6-P2 content. In contrast, endothelin, which has a low mitogenic activity (43Van der Bend R.L. De Widt J. Van Corven E.J. Moolenaar W.H. Van Blitterswijk W.J. Biochem. J. 1992; 285: 235-240Crossref PubMed Scopus (85) Google Scholar), had no significant effect on the expression of this enzyme. Indeed, cAMP produced a decrease in 6PF2K/Fru-2,6-P2ase mRNA levels and inhibited the effect of LPA and EGF. In agreement with these results, Darville et al. (41Darville M.I. Antoine I.V. Mertens-Strijhagen J.R. Dupriez V.J. Rousseau G.G. Oncogene. 1995; 11: 1509-1517PubMed Google Scholar) have demonstrated that cAMP was also able to block the effect of serum on 6PF2K/Fru-2,6-P2ase transcription. Previous studies reported a different effect of cAMP on Fru-2,6-P2 levels in fibroblasts. In Swiss 3T3 (21Dalmau M. Bartrons R. Gil J. Exp. Cell. Res. 1994; 212: 93-96Crossref PubMed Scopus (15) Google Scholar) and human fibroblasts (16Rousseau G.G. Fisher Y. Guening M.A. Marchand M.J. Testar X. Hue L. Prog. Cancer Res. Ther. 1988; 35: 150-163Google Scholar), cAMP induces an increase in the concentration of Fru-2,6-P2, while in chick-embryo fibroblast (19Boscà L. Rousseau G.G. Hue H. Proc. Natl. Acad. Sci. U. S. A. 1985; 82: 6440-6444Crossref PubMed Scopus (61) Google Scholar), the presence of dibutyryl cAMP did not change the levels of this metabolite. It is very likely that the discrepancy between these results is due to the different mitogenic role of cAMP in these cells; cAMP is a mitogenic signal in Swiss 3T3 cells (44Rozengurt E. Legg A. Strang G. Courtenay-Luck N. Proc. Natl. Acad. Sci. U. S. A. 1981; 78: 4392-4396Crossref PubMed Scopus (113) Google Scholar), but it is a strong antimitogen in Rat-1 fibroblasts (37Cook S.J. McCormick F. Science. 1993; 262: 1069-1071Crossref PubMed Scopus (860) Google Scholar). With regard to the signaling pathways that control the transcription of the 6PF2K/Fru-2,6-P2ase gene during cell proliferation, our results suggest that agents or growth factors (LPA, EGF) that activate the Ras-Raf-MEK-ERK pathway also increase the levels of 6PF2K/Fru-2,6-P2ase mRNA. In Rat-1 fibroblasts, EGF promotes the activation of ERK1 and ERK2 proteins through a Ras-dependent pathway without affecting inositol phosphate levels (42van Corven E.J. Groenink A. Jalink K. Eichholtz T. Moolenaar W.H. Cell. 1989; 59: 45-54Abstract Full Text PDF PubMed Scopus (674) Google Scholar, 45Van Corven E.J. van Rijswijk A. Jalink K. van der Bend R.L. van Blitterswijk W.J. Moolenaar W.H Biochem. J. 1992; 281: 163-169Crossref PubMed Scopus (236) Google Scholar). In the same cells, LPA activates at least three independent signaling cascades by coupling its receptor to Gi and Gq proteins: stimulation of phospholipases C and D via a pertussis toxin-insensitive G protein (Gq), inhibition of adenylate cyclase via a pertussis toxin-sensitive G protein (Giä), and activation of Ras via a pertussis toxin-sensitive Gi protein (Gβγ) (46Moolenaar W.H. J. Biol. Chem. 1995; 270: 12949-12952Abstract Full Text Full Text PDF PubMed Scopus (566) Google Scholar). The activation of the Ras cascade by LPA produces the stimulation of MAPK (36Hordijk P.L. Verlaan I. van Corven E.J. Moolenaar W.H. J. Biol. Chem. 1994; 269: 645-651Abstract Full Text PDF PubMed Google Scholar). In Rat-1 fibroblasts, Ras is a point of convergence of EGF and LPA signaling pathways in the activation of MAPK. It has been shown that raising the cAMP levels in the cell induces the hyperphosphorylation of Raf-1 by PKA, reducing the binding affinity of Raf-1 to Ras and consequently inhibiting the Ras-Raf-MEK-ERK pathway (47Häfner S. Adler H. Mischak H. Janosch P. Heidecker G. Wolfman A. Pippig S. Lohse M. Ueffing M. Kolch W. Mol. Cell. Biol. 1994; 14: 6696-6703Crossref PubMed Scopus (288) Google Scholar). Our results show that the inhibition of this pathway by cAMP also leads to an inhibition of the stimulatory effect of EGF and LPA on 6PF2K/Fru-2,6-P2ase expression. The effect of cAMP on 6PF2K/Fru-2,6-P2ase expression is probably due to inhibition of MAPK activation. The implication of MAPK cascade on the expression of this enzyme has been suggested previously (41Darville M.I. Antoine I.V. Mertens-Strijhagen J.R. Dupriez V.J. Rousseau G.G. Oncogene. 1995; 11: 1509-1517PubMed Google Scholar). However, definitive experiments were necessary to support this tentative hypothesis. Our finding that a specific inhibitor of MEK can prevent the effect of EGF on 6PF2K/Fru-2,6-P2ase mRNA levels and on the transcriptional levels provides further evidence to confirm the idea that MAPK is involved in regulating 6PF2K/Fru-2,6-P2ase expression during proliferative processes. Activation of PKC increases Fru-2,6-P2 levels in different cells (16Rousseau G.G. Fisher Y. Guening M.A. Marchand M.J. Testar X. Hue L. Prog. Cancer Res. Ther. 1988; 35: 150-163Google Scholar, 17Hamer M.J. Dickson A. Biochem. J. 1990; 269: 685-690Crossref PubMed Scopus (21) Google Scholar, 18Taylor D. Evanson J.M. Woolley D.E. Biochem. J. 1990; 269: 573-577Crossref PubMed Scopus (29) Google Scholar, 19Boscà L. Rousseau G.G. Hue H. Proc. Natl. Acad. Sci. U. S. A. 1985; 82: 6440-6444Crossref PubMed Scopus (61) Google Scholar, 20Boscà L. Mojena M. Ghysdael J. Rousseau G.G. Hue L. Biochem. J. 1986; 236: 596-599Crossref Scopus (45) Google Scholar, 21Dalmau M. Bartrons R. Gil J. Exp. Cell. Res. 1994; 212: 93-96Crossref PubMed Scopus (15) Google Scholar, 22Cascales M. Martin-Sanz P. Boscà L. Eur. J. Biochem. 1992; 207: 391-397Crossref PubMed Scopus (12) Google Scholar, 23Denis-Pouxviel C. Gauthier T. Daviaud D. Murat J.C. Biochem. J. 1990; 268: 465-470Crossref PubMed Scopus (12) Google Scholar, 24Baulida J. Onetti R. Bassols A. Biochem. Biophys. Res. Commun. 1992; 183: 1216-1223Crossref PubMed Scopus (19) Google Scholar). Although there is no direct evidence, several reports suggest that PKC may regulate 6PF2K/Fru-2,6-P2ase gene expression. In chick embryo fibroblasts, incubation with TPA produced an increase in 6PF2K/Fru-2,6-P2ase activity and this effect was abolished in cells incubated with actinomycin D, cycloheximide, or puromycin (19Boscà L. Rousseau G.G. Hue H. Proc. Natl. Acad. Sci. U. S. A. 1985; 82: 6440-6444Crossref PubMed Scopus (61) Google Scholar). In fetal hepatocytes (22Cascales M. Martin-Sanz P. Boscà L. Eur. J. Biochem. 1992; 207: 391-397Crossref PubMed Scopus (12) Google Scholar) and Swiss 3T3 cells (21Dalmau M. Bartrons R. Gil J. Exp. Cell. Res. 1994; 212: 93-96Crossref PubMed Scopus (15) Google Scholar), the rise in 6PF2K/Fru-2,6-P2ase activity produced by phorbol esters was inhibited by cycloheximide, suggesting that it is due to enhanced expression of the protein. Our results demonstrate that, in Rat-1 fibroblasts, endothelin, a strong stimulator of PKC, and TPA, had no detectable effect on 6PF2K/Fru-2,6-P2ase mRNA levels and Fru-2,6-P2, suggesting that activation of PKC pathway alone is not enough to stimulate 6PF2K/Fru-2,6-P2ase expression in these cells. It has been reported that, in Rat-1 fibroblasts, endothelin fails to stimulate ERK phosphorylation, despite its potent coupling to the phospholipase C-PKC second messenger cascade (36Hordijk P.L. Verlaan I. van Corven E.J. Moolenaar W.H. J. Biol. Chem. 1994; 269: 645-651Abstract Full Text PDF PubMed Google Scholar). The different contribution of PKC to MAPK activation could explain the fact that, in Swiss 3T3 cells, bombesin, another phospholipase C-activating agonist, was able to increase 6PF2K/Fru-2,6-P2ase activity (21Dalmau M. Bartrons R. Gil J. Exp. Cell. Res. 1994; 212: 93-96Crossref PubMed Scopus (15) Google Scholar). In these cells, bombesin stimulates MAPK activity via a pathway that is almost entirely dependent on PKC (48Pang L. Decker J. Saltiel A.R. Biochem. J. 1993; 289: 283-287Crossref PubMed Scopus (71) Google Scholar). Sequence analysis of the F promoter of 6PF2K/Fru-2,6-P2ase reveals the presence of several DNA-binding sites that could directly or indirectly regulate the transcription of the 6PF2K/Fru-2,6-P2ase gene by MAPK activation. A region containing three binding motifs for transcription factors of the ets oncogene family has been found in the F promoter (10Dupriez V.J. Darville M.I. Antoine I.V. Gegonne A. Ghysdael J. Rousseau G.G. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 8224-8228Crossref PubMed Scopus (40) Google Scholar). The transcriptional activity of various Ets-related transcription factors is regulated by MAPK. Thus, c-Ets-2 is both transcriptionally activated and phosphorylated by MAPK (49Coffer P. de Jonge M. Mettouchi A. Binetruy B. Ghysdeal J. Kruijer W. Oncogene. 1994; 9: 911-922PubMed Google Scholar). Furthermore, phosphorylation of the Ets-related transcription factor p62TCF/Elk-1 by MAPK stimulates ternary complex formation at c-fos promoter, thus activating the fos transcription and consequently, the AP-1 DNA binding activity (50Gille H. Sharrocks A.D. Shaw P. Nature. 1992; 358: 414-417Crossref PubMed Scopus (811) Google Scholar, 51Marais R. Wynne J. Treisman R. Cell. 1993; 73: 381-393Abstract Full Text PDF PubMed Scopus (1102) Google Scholar, 52Hill C.S. Marais R. John S. Wynne J. Dalton S. Treisman R. Cell. 1993; 73: 395-406Abstract Full Text PDF PubMed Scopus (329) Google Scholar). Darville et al. (53Darville M.I. Crepin K.M. Hue L. Rousseau G.G. Proc. Natl. Acad. Sci. U. S. A. 1988; 86: 6543Crossref Scopus (56) Google Scholar) reported an AP-1 DNA-binding site about 250 bp downstream of the F promoter, and several reports show that an intronic AP-1 site could regulate gene expression (54Hengerer B. Lindholm D. Heumann R. Rüther U. Wagner E.F. Thoenen H. Proc. Natl. Acad. Sci. U. S. A. 1990; 87: 3899-3903Crossref PubMed Scopus (311) Google Scholar, 55Van Dijck P. Schoonjans K. Sassone Corsi P. Auwerx J. Verhoeven G. Mol. Cell. Biochem. 1993; 125: 127-136Crossref PubMed Scopus (7) Google Scholar, 56Bergers G. Graninger P. Braselmann S. Wrighton C. Busslinger M. Mol. Cell. Biol. 1995; 15: 3748-3758Crossref PubMed Scopus (160) Google Scholar). Indeed, in the same 6PF2K/Fru-2,6-P2ase gene, a potent glucocorticoid response element was identified in the first intron of liver transcript (11Lange A.J. Espinet C. Hall R. El-Maghrabi M.R. Vargas A.M. Miksicek R.J. Granner D.K. Pilkis S.J. J. Biol. Chem. 1992; 267: 15673-15680Abstract Full Text PDF PubMed Google Scholar). Recently, an E2F binding site localized at the 5′ end of the first exon has been suggested to be involved in the serum regulation of F promoter (41Darville M.I. Antoine I.V. Mertens-Strijhagen J.R. Dupriez V.J. Rousseau G.G. Oncogene. 1995; 11: 1509-1517PubMed Google Scholar). E2F transcriptional activity is blocked by its binding to hypophosphorylated retinoblastoma protein (pRB). G1 cyclins, including cyclin D1, forms complexes with cyclin-dependent kinases which can phosphorylate pRB, causing to release E2F (57Weinberg R.A. Cell. 1995; 81: 323-330Abstract Full Text PDF PubMed Scopus (4274) Google Scholar). Although there is no evidence, MAPK pathway could regulate the dissociation of E2F from retinoblastoma protein (pRB) leading to activation of E2F target genes. In this sense, EGF induction of cyclin D1 transcription was antagonized by either dominant negative MAPK or dominant negatives of Ets-2 activation, suggesting that MAPK and Ets-2 function downstream of EGF in the context of cyclin D1 induction (58Albanese C. Johnson J. Watanabe G. Eklund N. Vu D. Arnold A. Pestell R.G. J. Biol. Chem. 1995; 270: 23589-23597Abstract Full Text Full Text PDF PubMed Scopus (759) Google Scholar). It would be very interesting to study whether this putative MAPK-Ets-cyclin D1-pRB-E2F pathway controls 6PF2K/Fru-2,6-P2ase expression. We thank Dr. R. Bartrons for continued interest in this work. We also thank Dr. A. Saltiel at Parke-Davis for generously supplying PD 098059. We thank Dr. J. Hesketh for correcting the English of the manuscript." @default.
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• W1984231661 title "Effect of Growth Factors on the Expression of 6-Phosphofructo2-kinase/Fructose-2,6-bisphosphatase in Rat-1 Fibroblasts" @default.
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