- Email: [email protected]
Repression of the 5-lipoxygenase gene by c-myb overexpression in differentiated HL-60 cells
Repression of the 5Lipoxygenase Gene by c-myb Overexpression in Differentiated HL-60 Cells An&~ Ponto&, Jean-Paul 2%irionb, and Pierre Siroh”’
Department of Pharmacology’ and Microbiologyb, Faculty of Medicine, University of Sherbrooke, Sherbrooke (P.Q.), Canada, JlH 5N4 This paper reports on the involvement of c-MYB in the regulation of 5lipoxygenase gene expression during differentiation of human HL-60 cells. We demonstrate that c-MYB binds, the 5-lipoxygenase promoter in undifferentiated cells but not in DMSO-‘diffqqtiated cells. Also, we show that overexpression of c-myb cDNA in differentiated HL-60 cells represses the S-lipoxygenase gene expression. Keywords:
5-lipoxygenase; c-myb; gene regulation; repression; differentiation
Introduction Leukotrienes (LTs) are important mediators involved in pathogenesis of various immunologic and inflammatory disorders like asthma, allergic rhinitis, rheumatoid arthritis, inflammatory bowel disease and psoriasis [ 11. The 5lipoxygenase (5-LOi EC1.13.11.34) catalyzes the first two reactions that lead to the biosynthesis of leukotrienes from arachidonic acid [2-51. Due to its key position in the biosynthesis of LTs, a better understanding of the regulation of the 5-LO gene in mammalian cells is essential for the development of new therapeutic agents. ‘Present address: Department of Pathology, Massachusetts General Hospital and Harvard Medical School, 149 Thirteenth Street, Charlestown, MA 02129 USA. ‘To whom correspondence should be addressed: Dr. Pierre Sirois, Department of Pharmacology, Faculty of Medicine, University of Sherbrooke, Sherbrooke [P.Q.) Canada JlH 5N4.
Proaaglandins 53:49-58, 1997 8 1997 by Elsevia Science Inc. 655 Avenue of the Americas, New York, NY 10010
0090-6980/97/$17.00 PII !3090-6980(96~140-2
Repression of the 5-Lipoxygenase Gene by c-myb: Ponton et al. The human promyelocytic leukemia cell line HL-60 is known to express very low levels of 5LO activity [6, 71. However when these cells differentiate into granulocyte or monocyte-like cells upon treatment with dimethyl sulfoxide (DMSO), retinoic acid or 1,25dihydroxyvitamin D3, they express the S-LO gene and synthesize LTs [6, 8-91. Similarly, the 5LO gene is expressed specifically in haematopoietic cells and seems to be regulated during differentiation [lo]. Haematopoietic stem cells do not produce LTs prior to differentiation and this is most likely due to their inability to express the 5-LO gene. Moreover, macrophage differentiation is known to be associated with early up-regulation of the 5-LO pathway that seems to be triggered by down-regulation of the c-myb gene product (c-MYB), a nuclear DNAbinding protein [ 111. It has been reported that undifferentiated normal myeloid precursor cells as well as HL-60 cells, that do not synthesize LTs, express high levels of c-myb [12-141. These results in conjunction with the fact that the promoter of the human 5-LO gene contains a sequence element homologous to the consensus binding site for c-MYB [ 151, led us to speculate that c-myb may act as a repressor of the 5-LO gene in undifferentiated cells by binding to its promoter. Downregulation of c-myb gene as the cells differentiate would thus allow expression of the 5-LO gene which would then be expressed constitutively like a housekeeping gene [ 161. In this paper, we investigate the role of c-MYB in the regulation of the 5-LO gene during differentiation using the HL-60 cells as a test model to show that: 1) there is indeed a good correlation between the expression of the c-myb gene and the repression of the 5-LO gene during different stages of differentiation of the HL-60 cells, 2) c-MYB (or a c-MYB-like protein) binds specifically to the 5-LO promoter in undifferentiated cells and 3) overexpression of c-myb in HL-60 cells differentiated with DMSO inhibits the expression of the 5-LO gene. Materials and Methods Cell Lines HL-60 cells from the ATCC (American Type Culture Collection) were grown at 37°C in RPMI-1640 supplemented with 10% heat-inactivated fetal bovine serum (FBS), 50 units/ml of penicillin and 50 mg/ml of streptomycin in 5% COZ. To induce differentiation, cells were subcultured at 0.2 x 106 cells/ml and exposed to DMSO at a final concentration of 1.3% (v/v) for up to 5 days.
50
Prostaglandins 1997:53, January
Repression of the 5-Lipoxygenase Gene by c-myb: Ponton et al. Plasmid Construction Plasmid pcDNAI/NEO-myb/s was constructed from pBCM8 that has the entire c-myb cDNA 1171.The pBCM8 plasmid was cut with Wet I and Xho I, and the c-myb fragment was subcloned in the polylinker of pcDNAI/NEO (Invitrogen, CA) at the Not I and Xho I sites. All the plasmids were prepared by alkaline lysis and purified with Qiagen plasmid purification procedures. DNA Transfection The plasmid pcDNAI/NEO-myb/s or the pcDNAI/NEO (7.5 ug) was diluted in Hepes buffer (20 mM Hepes, 150 mM NaCl) to a final volume of 75 ~1. In a separate reaction tube, the DOTAP (70 ~1, 1 mgJm1; (Boehringer Mannheim, Laval, Canada), was diluted in Hepes buffer to a final volume of 180 ~1. The DOTAP and DNA solutions were mixed and incubated for 10 min at room temperature. Then, the DOTAP/DNA mixture was added to the HL-60 cells suspension (10 ml of 1 x lo6 cells/ml) with had been previously treated for 72 h with DMSO (1.3% v/v). The transfected cells were incubated at 37°C and after 24 h, the medium was replaced with fresh RPMI 1640. Incubation was continued for another 24 h before RNA extraction. RNA Extraction and Northern Blot Analysis Total cellular RNA was isolated using the TRIzolTMReagent solution (GIBCO BRL) as described by the manufacturer. Northern blotting was performed as described [18]. The human 5-LO cDNA was from Dr J. Evans (Merck Frosst Lab., Montreal). The c-myb and the GAPDH cDNAs were provided by Dr R. Dalla-Favera (Colombia University, N. Y.) and by Dr. Richard Leduc (University of Sherbrooke, Canada) respectively. The cDNAs (5-LO, c-myb and GAPDH) were labeled with [a-“91 dCTP (3000 Ci/mmol) by random priming and the probes purified over Sepharose G-50 spun columns. Oligonucleotides All the oligonucleotides were obtained from General Synthesis and Diagnostics (Toronto, Canada) and were all gel purified. DNA-gel Mobility Shift Assays Nuclear protein extracts from undifferentiated or differentiated HL-60 cells were prepared as described [ 191.The nuclear protein extracts ( 10 or 50 pg) were preincubated for 20 min at 25°C with poly(dI-dC)*poly(dIdC) (40pg/ml) alone or with an excess of cold double-stranded oligonuProstaglandins 1997:53, January
51
Repression of the 5-Lipoxygenase Gene by c-myb: Ponton et al. cleotide (as indicated in figure legend) in buffer containing 25 mM of HEPES-KOH (pH 719);40mM NaCl; 1mM EDTA; 5% glycerol and 1mM DTT. After preincubation, radiolabelled double-stranded oligonucleotide (10000 cpm) was added in each reaction and incubated for 20 min at 25°C. Reaction volumes of 25 d were immediately loaded onto 5% TGE (50 mM Tris, pH 7.5, 380 mM glycine, 2mM EDTA) native polyacrylamide gel and the electrophoresis was done at 4°C for 1 h. The probe was prepared by annealing a 5’-radiolabelled single-stranded oligonucleotide with a 3 fold molar excess of the complementary oligonucleotide in 0.15 M NaCl. The annealing mixture was heated to 65°C for 2 min and cooled to room temperature within 30 min. The double-stranded oligonucleotide was purified on a 15% native polyacrylamide gel, eluted overnight in water and passed on a Sepharose GlO column [20]. Cold competitor oligonucleotides were annealed in equimolar ratios, purified on a Sepharose GlO column and used in molar excess relative to the probe as indicated in figure legend. The sequence of the oligonucleotides that corresponds to the c-MYB binding site from the human 5-LO promoter was: (forward), dGAAATAAACCGTTATGTTTATj (reverse), dATAAACA TAACGGTTTATTTC. Semiquantitative Analysis of RNA by PCR Total RNA was extracted from differentiated HL-60 cells transfected with pcDNAI/NEO-myb/s or pcDNAI/NEO as control. About 6 pg of each RNA were reverse-transcribed in 3 mM MgCl2, 75 mM KCI, 50 mM Tris-HC1 (pH 8.3), 2 ng/@ of random primers (Pharmacia), 0.5 mM each dNTP, 2.0 U/p1 of RNase inhibitor and 10 U/p1 of Superscript II reverse transcriptase (BRL). The reactions were carried out at 37°C for 90 min in a final volume of 60 pl. The enzyme was then inactivated at 94°C for 10 min. Aliquots (20 ~1) of the reverse transcription reactions were amplified by polymerase chain reaction (PCR) using sets of primers specific for 5-LO, c-myb or (3-actin under the following reaction conditions: 4.0 mM MgCl2, 50 mM KCl, 10 mM Tris-HCl pH 8.3, 0.2 mM each dNTP, 25 U/ml AmpliTaq DNA polymerase (PerkinElmer) and 0.45 uM of each primer in a final volume of 100 ~1. The sequence of each primer was as follows: 5-LO (forward), dGCAGTCCTGCTCTGTGTAGAATGGG; 5-LO (reverse), dACCATTGAGCAGATCGTGGACACGC; c-myb (forward), dCGGCACA GCATATATAGC; c-myb (reverse), dGGCTTTTGAAGACTCCTG, (3actin (forward), dGGCACCACACCTTCTACAATGA; B-actin (reverse), dTCGTGATGGACTCCGGTGAC.
52
Prostaglandins 1997:53, January
Repression of the SLipoxygenase
Gene by c-myb: Ponton et al.
Results and Discussion Expression of the Human 5LO and c-myb Genes in Undifferentia ted and Differentiated HL-60 Cells The expression of the 5-LO and c-myb genes was determined by Northern blot analysis. The Fig. 1, lane 2, shows that the 5-LO gene is expressed in HL-60 cells treated with DMSO for five days as determined by the mRNA accumulation (Fig. 1, lane 2), but the amount of mRNA is barely detectable in undifferentiated cells (lane 1). These results are in agreement with the results of other investigators (6, 71.These results indicate that the HL-60 cells used in our experiments have not undergone any clonal modifications and can be used to study the expression of the &LO gene. The same blot was then rehybridized with c-myb c-DNA as a probe and as expected the level of mRNA accumulation was very high in the undifferentiated cells and low in differentiated cells as demonstrated in other myeloid cell lines [ 12-14, 21 j. A band is also present just below the c-myb specific signal which may result from the crosshybridization of the probe with another member of the myb gene family
-
S-LO
-
1
GAPDB
2
FIGURE1. Expressionof the human 5-LO and c-myb genes in HL-60 and differentiated HL-60 calls. Total mRNA was extracted from HL-60 (lane 1) and DMSO treated HL-SOcells (lane 2). Each line contains 10 pg of RNA. The blot was hybridizedwith the 32P-labeIed 5LO cbNA insert.After autoradiiraphy, the 5-LO probe was eluted and the filter was rehybridiredtih a “P-labeled plasmid containing the c-myb cDNA and rehybridized again after elution with a GAPDH probe.
Prostaglandins 1997:53, January
53
Repression of the 5-Lipoxygenase Gene by c-myb: Ponton et al. [22]. As an appropriate control, we have determined the expression of the 5-LO and c-myb genes in the human monocyte cell line THP-1, which can differentiate in macrophages and in this regard should behave like the HL-60 cells. As expected, we find that prior to differentiation, these cells do not express the &LO gene but do show a high level of expression for the c-myb gene (data not shown) as with the HL-60 cells. Specific Binding of MYB-like Protein to the 5-LO Promoter To explain the results above we hypothesized that the c-myb gene product (c-MYB) may be a repressor of the 5-LO gene expression. It has been shown that the promoter of the 5-LO gene contains a sequence element (TAACGG) [ 151 which corresponds to the c-MYB consensus DNAbinding site (T/CAACG/TG) [23]. To test our hypothesis, the sequence (dATAAACA TAACGGTTTATTTC) from the 5-LO promoter was used as a probe in DNA-gel mobility shift analysis in order to determine if the binding of MYB occurs in undifferentiated HL-60 cells only. Nuclear protein extracts were made with either differentiated or undifferentiated cells and mixed with the probe as described in the materials and methods section. Fig. 2 A shows that DNA-proteins complexes were detectable with nuclear extracts from undifferentiated HL-60 cells but not from differentiated cells (compare lanes 2 and 3 with lanes 4 and 5). Two specific complexes (Cl and C2) are formed, one of them could result from the binding of c-MYB to its specific site, and the second complex from the binding of another member of the MYB family or dimerization of MYB with itself or with another protein [24]. The retarded complexes are sequence-specific because they are titered out by molar excesses of cold probe (Fig. 2 B, lane 2 and 3), but not by an unrelated probe of the same length (data not shown). We cannot rule out the possibility that another protein might have recognized the same binding site. This is, however, unlikely because the band-shift analysis strongly suggests that c-MYB or a c-MYB-like protein which is present only in undifferentiated HL-60 cells binds the c-MYB binding site present on the human 5-LO promoter. Overexpression of c-myb cDNA in DMSO Treated HL-60 Cells In order to verify whether or not down-regulation of the 5-LO gene could be achieved by overexpressing the c-myb gene, an expression vector containing the full length cDNA for c-myb was transfected in HL-60 cells treated with DMSO for 72 h. The level of mRNA expression was determined by RT-PCR as described in materials and methods. As shown in Fig. 3, lane 2, the cells transfected with the vector alone express the 5-LO gene but not the c-myb gene, which confirms the results obtained by Northern blot. Interestingly, the mRNA levels for the 5-LO 54
Prostaglandins 1997:53, January
Repression of the 5-Lipoxygenase Gene by c-myb: Ponton et al. B.
A.
1
2
3
4
5
1
2
3
FIGURE2. The 5-LO promoter contains sequences that bind C-MYB related proteins. A 32Plab&d double-stranded oliionudeotide that corresponds to the C-MB binding ebmer(t from the !&LO promotar was used in DNA mobltity gel-shift assays with (in A) 1Qcg and 5gpg of nuclear extracts from HL80 (lane 2 and 3) and fmm DMSO traatad HL-60 calIs (lane 4 end 5). Lane 1 contains no nuclear extracts. B. Lanes 1,2 and 3 contain 5Omg of nudaar axtracts from HL-60 cells. Lane 2 and 3 contain 10X and 50X molar excess of cold otigonudeoWa that was added before the incubation with the probe. In all cases the proba was 10,000 cpm par reaction.
gene, as measured by RT-PCR, are greatly reduced when the cells are transfected with the plasmid containing the c-myb cDNA (Fig. 3, lane 1) This indicates that the transcription for the 5-LO gene is inhibited by overexpression of the c-myb gene which is now expressed as shown by RT-PCR. In conclusion, the data reported here strongly suggests that c-MYB acts as a repressor of the S-LO gene in undifferentiated HL-60 cells. As the c-myb gene is expressed only in haematopoietic stem cells, this may explain why the &LO is not expressed in these cells. However the 5-LO gene is known not to be expressed in other cell types such as fibroblasts or epithelial cells that do not express the c-myb gene either [14). In these cases, c-MYB carmot be held responsible for the non-expression of the S-LO gene which suggests that thus another mechanism of control for gene expression exist. Moreover, it has been reported that B-MYB, another member of the MYB family, binds the same DNA sequence as c-MYB, but to the contrary of c-MYB, B-MYB is expressed in nonhaematopoietic cells [25]. Therefore, B-MYB may play the same role as c-MYB in non-haematopoitic cells. To test this hypothesis we carried Prostaglandins 1997:53, January
55
Repression of the SLipoxygenase
Gene by c-myb: Ponton et al.
c-myb -
FIGURE 3. Overexpression of c-r?@ cDNA in DMSO treated HL-60 cells. DMSO treated HL-80 cells were transfected with an expression vector (pcDNAI/NEO) containing the full length cDNA for c-myb (lane 1) or with the expression vector alone (lane 2). After 48 h, the RNA was extracted and submitted to RT-PCR analysis with oligonucleotides corresponding to the 5-LO, c-myb and p-actin gene.
out RT-PCR analysis with mRNA from HeLa cells, which do not express the c-myb and the 5LO genes as demonstrated by Northern blot analysis (data not shown). We found that B-myb is expressed in these cells (data not shown). Thus, if B-MYB can really repress the 5LO gene in non-haematopoietic cells, this finding may aid in the development of new potential therapeutic agents. Acknowledgments We thank Solange Cloutier for technical assistance. This work was supported by the Medical Research Council of Canada. AP is in receipt of a Fellowship from Fonds de la Recherche en Sante du Quebec. References Lewis, R.A., Austen, K.F. and Soberman, R.J. Leukotrienes and other products of the 5lipoxygenase pathway. N. Engl. J. Med. 323,645-655, 1990. Samuelsson, B. Leukotrienes: mediators of immediate hypersensitivity reactions and inflammation. Science 220, 568-575, 1983. Rouzer, C.A., Matsumoto, T. and Samuelsson, B. Single protein from human leukocytes possesses 5-lipoxygenase and leukotriene A4 synthase activities. Proc Nat1 Acad Sci USA 83, 857-861, 1986. Shimizu, T., Izumi, T., Seyama, Y., Tadokoro, K., Rgdmark, 0. and Samuelsson, B. Characterization of leukotriene A4 synthase from murine
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Repression of the SLipoxygenase
5.
6.
7.
8.
9.
10.
11.
12.
13.
14.
15.
16. 17.
18.
Gene by c-myb: Ponton et al.
mast cells: evidence for its identity to arachidonate 5lipoxygenase. Proc Nat1 Acad Sci USA 83,4175-4179, 1986. Samuelsson, B., Dahlen, S.-E., Lindgren, J.A., Rouzer, C.A. and Serhan, C.N. Leukotrienes and lipoxins; structures, biosynthesis, and biological effects. Science 237, 1171-1176, 1987. Dixon, R.A.F., Jones, R.E., Diehl, R.E., Bennett, CD., Kargman, S. and Rouzer, C.A. Cloning of the cDNA for human 5lipoxygenase. Proc Nat1 Acad Sci USA 85,416-420, 1988. Piechele, G., Colli, S., Tremoli, E. and Maggi, A. 5-lipoxpygenase gene expression in HL60 cells during differentiation with DMSO. Pharmacological Res.27, 53-60, 1993. Harris, P. and Ralph, P. Human leukemic models of myelomonocytic development: a review of the HL-60 and U937 cell lines. J. Leukocyte Biol. 37, 407-422, 1985. Anthes, J.C., Bryant, R.W., Musch, M.W., Ng, K. and Siegel, M.I. Calcium ionophore and chemotactic peptide stimulation of peptidoleukotriene synthesis in DMSO-differentiated HL60 cells. Inflammation, 10, 145-156, 1986. Balcarek, J.M., Theisen, T.W., Cook, M.N., Varrichio, A., Hwang, S.-M., Strohsacker, M.W. and Crooke, S.T. Isolation and characterization of a cDNA clone encoding rat 5lipoxygenase. J. Biol. Chem. 263, 13937-13941, 1988. Habenicht, A.J.R., Goerig, M., Rothe, D.E.R., Specht, E., Ziegler, R., Glomset, J.A. and Graf, T. Early reversible induction of leukotriene synthesis in chicken myelomonocytic cells transformed by a temperature-sensitive mutant of avian leukemia virus E26. Proc. Natl. Acad. Sci. USA 86, 921-924, 1989. Yanagisawa, H., Nagasawa, T., Kuramochi, S., Abe, T., Ikawa, Y. and Todokoro, K. Constitutive expression of exogenous c-myb gene causes maturation block in monocyte-macrophage differentiation. Bioch. Bioph. Acta, 1088,380-384, 1991. Clarke, M., Kukowska-Latollo, J.F., Westin, E., Smith, M. and Prochownik, E. Constitutive expession of a c-myb cDNA blocks friend murine erythroleukemia cell differentiation. Mol. Cell Biol. 8, 884-892, 1988. Duprey, S.P. and Boettiger, D. Developmental regulation of c-myb in normal myeloid progenitor cells. Proc. Natl. Acad. Sci. USA 82, 6937-6941, 1985. Hoshiko, S., Radmark, 0. and Samuelsson, B. Characterization of the human 5lipoxygenase gene promoter. Proc. Natl. Acad. Sci. USA 87, 90739077, 1990. Bird, A.P. CpG islands as gene markers in the vertebrate nucleus. Trends Genet., 3:,342-347, 1987. Majello, B., Kenyon, L.C. and Dalla-Favera, R. Human c-myb protooncogene: nucleotide sequence of cDNA and oganization of the genomic locus. Proc. Natl. Acad. Sci. USA 83, 9636-9640, 1986. Ponton, A., Coulombe, B. and Skup, D. Decreased expression of tissue inhibitor of metallopoteinases in metastatic tumor cells leading to increased levels of collagenase activity. Cancer Res. 51,2138-2143, 1991.
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Repression of the 5-Lipoxygenase Gene by c-myb: Ponton et al, 19. Ponton, A., Coulombe, B., Steyaert, A., Williams, B.R.G. and Skup, D. Basal expression of the gene (TIMP) encoding the murine tissue inhibitor of metalloproteinases is mediated through APl- and CCAAT-binding factors. Gene 116,187-194, 1992. 20. Levine, M. and Manley, J.L. Transcriptional repression of eukaryotic promotors. Cell 59, 405408, 1989. 21. Gonda, T.J. and Metcalf, D. Expression of myb, myc and fos protooncogenes during the differentiation of a murine myeloid leukaemia. Nature 310,249-251, 1984.. 22. Nomura, N., Takahashi, M., Matsui, M., Ishii, S., Date, T., Sasamoto, S. and Ishizaki, R. Isolation of human cDNA clones of myb-related genes, A-myb and B-myb. Nucleic Acids Res. 16, 11075-l 1088, 1988. 23. Ness, S.A., Marknell, A. and Graf, T. The v-myb oncogene product binds to and activates the promyelocyte-specific mim-1 gene. Cell 59, 1115-1125 , 1989. 24. Ramsay, R.G., Ishii, S. and Gonda, T.J. Interaction of the Myb protein with specific DNA binding sites. J. Biol. Chem. 267, 5656-5662, 1992. 25. Reiss, K., Travali, S., Calabretta, B. and Baserga, R. Growth regulated expression of B-myb in fibroblasts and hematopoietic cells. J. Cell. Phys. 148, 338-343, 1991.
Editor: Dr. W. Smith
Received: 09-04-96
Accepted : 09-26-96
Prostaglandins 1997~53, January
Department of Pharmacology’ and Microbiologyb, Faculty of Medicine, University of Sherbrooke, Sherbrooke (P.Q.), Canada, JlH 5N4 This paper reports on the involvement of c-MYB in the regulation of 5lipoxygenase gene expression during differentiation of human HL-60 cells. We demonstrate that c-MYB binds, the 5-lipoxygenase promoter in undifferentiated cells but not in DMSO-‘diffqqtiated cells. Also, we show that overexpression of c-myb cDNA in differentiated HL-60 cells represses the S-lipoxygenase gene expression. Keywords:
5-lipoxygenase; c-myb; gene regulation; repression; differentiation
Introduction Leukotrienes (LTs) are important mediators involved in pathogenesis of various immunologic and inflammatory disorders like asthma, allergic rhinitis, rheumatoid arthritis, inflammatory bowel disease and psoriasis [ 11. The 5lipoxygenase (5-LOi EC1.13.11.34) catalyzes the first two reactions that lead to the biosynthesis of leukotrienes from arachidonic acid [2-51. Due to its key position in the biosynthesis of LTs, a better understanding of the regulation of the 5-LO gene in mammalian cells is essential for the development of new therapeutic agents. ‘Present address: Department of Pathology, Massachusetts General Hospital and Harvard Medical School, 149 Thirteenth Street, Charlestown, MA 02129 USA. ‘To whom correspondence should be addressed: Dr. Pierre Sirois, Department of Pharmacology, Faculty of Medicine, University of Sherbrooke, Sherbrooke [P.Q.) Canada JlH 5N4.
Proaaglandins 53:49-58, 1997 8 1997 by Elsevia Science Inc. 655 Avenue of the Americas, New York, NY 10010
0090-6980/97/$17.00 PII !3090-6980(96~140-2
Repression of the 5-Lipoxygenase Gene by c-myb: Ponton et al. The human promyelocytic leukemia cell line HL-60 is known to express very low levels of 5LO activity [6, 71. However when these cells differentiate into granulocyte or monocyte-like cells upon treatment with dimethyl sulfoxide (DMSO), retinoic acid or 1,25dihydroxyvitamin D3, they express the S-LO gene and synthesize LTs [6, 8-91. Similarly, the 5LO gene is expressed specifically in haematopoietic cells and seems to be regulated during differentiation [lo]. Haematopoietic stem cells do not produce LTs prior to differentiation and this is most likely due to their inability to express the 5-LO gene. Moreover, macrophage differentiation is known to be associated with early up-regulation of the 5-LO pathway that seems to be triggered by down-regulation of the c-myb gene product (c-MYB), a nuclear DNAbinding protein [ 111. It has been reported that undifferentiated normal myeloid precursor cells as well as HL-60 cells, that do not synthesize LTs, express high levels of c-myb [12-141. These results in conjunction with the fact that the promoter of the human 5-LO gene contains a sequence element homologous to the consensus binding site for c-MYB [ 151, led us to speculate that c-myb may act as a repressor of the 5-LO gene in undifferentiated cells by binding to its promoter. Downregulation of c-myb gene as the cells differentiate would thus allow expression of the 5-LO gene which would then be expressed constitutively like a housekeeping gene [ 161. In this paper, we investigate the role of c-MYB in the regulation of the 5-LO gene during differentiation using the HL-60 cells as a test model to show that: 1) there is indeed a good correlation between the expression of the c-myb gene and the repression of the 5-LO gene during different stages of differentiation of the HL-60 cells, 2) c-MYB (or a c-MYB-like protein) binds specifically to the 5-LO promoter in undifferentiated cells and 3) overexpression of c-myb in HL-60 cells differentiated with DMSO inhibits the expression of the 5-LO gene. Materials and Methods Cell Lines HL-60 cells from the ATCC (American Type Culture Collection) were grown at 37°C in RPMI-1640 supplemented with 10% heat-inactivated fetal bovine serum (FBS), 50 units/ml of penicillin and 50 mg/ml of streptomycin in 5% COZ. To induce differentiation, cells were subcultured at 0.2 x 106 cells/ml and exposed to DMSO at a final concentration of 1.3% (v/v) for up to 5 days.
50
Prostaglandins 1997:53, January
Repression of the 5-Lipoxygenase Gene by c-myb: Ponton et al. Plasmid Construction Plasmid pcDNAI/NEO-myb/s was constructed from pBCM8 that has the entire c-myb cDNA 1171.The pBCM8 plasmid was cut with Wet I and Xho I, and the c-myb fragment was subcloned in the polylinker of pcDNAI/NEO (Invitrogen, CA) at the Not I and Xho I sites. All the plasmids were prepared by alkaline lysis and purified with Qiagen plasmid purification procedures. DNA Transfection The plasmid pcDNAI/NEO-myb/s or the pcDNAI/NEO (7.5 ug) was diluted in Hepes buffer (20 mM Hepes, 150 mM NaCl) to a final volume of 75 ~1. In a separate reaction tube, the DOTAP (70 ~1, 1 mgJm1; (Boehringer Mannheim, Laval, Canada), was diluted in Hepes buffer to a final volume of 180 ~1. The DOTAP and DNA solutions were mixed and incubated for 10 min at room temperature. Then, the DOTAP/DNA mixture was added to the HL-60 cells suspension (10 ml of 1 x lo6 cells/ml) with had been previously treated for 72 h with DMSO (1.3% v/v). The transfected cells were incubated at 37°C and after 24 h, the medium was replaced with fresh RPMI 1640. Incubation was continued for another 24 h before RNA extraction. RNA Extraction and Northern Blot Analysis Total cellular RNA was isolated using the TRIzolTMReagent solution (GIBCO BRL) as described by the manufacturer. Northern blotting was performed as described [18]. The human 5-LO cDNA was from Dr J. Evans (Merck Frosst Lab., Montreal). The c-myb and the GAPDH cDNAs were provided by Dr R. Dalla-Favera (Colombia University, N. Y.) and by Dr. Richard Leduc (University of Sherbrooke, Canada) respectively. The cDNAs (5-LO, c-myb and GAPDH) were labeled with [a-“91 dCTP (3000 Ci/mmol) by random priming and the probes purified over Sepharose G-50 spun columns. Oligonucleotides All the oligonucleotides were obtained from General Synthesis and Diagnostics (Toronto, Canada) and were all gel purified. DNA-gel Mobility Shift Assays Nuclear protein extracts from undifferentiated or differentiated HL-60 cells were prepared as described [ 191.The nuclear protein extracts ( 10 or 50 pg) were preincubated for 20 min at 25°C with poly(dI-dC)*poly(dIdC) (40pg/ml) alone or with an excess of cold double-stranded oligonuProstaglandins 1997:53, January
51
Repression of the 5-Lipoxygenase Gene by c-myb: Ponton et al. cleotide (as indicated in figure legend) in buffer containing 25 mM of HEPES-KOH (pH 719);40mM NaCl; 1mM EDTA; 5% glycerol and 1mM DTT. After preincubation, radiolabelled double-stranded oligonucleotide (10000 cpm) was added in each reaction and incubated for 20 min at 25°C. Reaction volumes of 25 d were immediately loaded onto 5% TGE (50 mM Tris, pH 7.5, 380 mM glycine, 2mM EDTA) native polyacrylamide gel and the electrophoresis was done at 4°C for 1 h. The probe was prepared by annealing a 5’-radiolabelled single-stranded oligonucleotide with a 3 fold molar excess of the complementary oligonucleotide in 0.15 M NaCl. The annealing mixture was heated to 65°C for 2 min and cooled to room temperature within 30 min. The double-stranded oligonucleotide was purified on a 15% native polyacrylamide gel, eluted overnight in water and passed on a Sepharose GlO column [20]. Cold competitor oligonucleotides were annealed in equimolar ratios, purified on a Sepharose GlO column and used in molar excess relative to the probe as indicated in figure legend. The sequence of the oligonucleotides that corresponds to the c-MYB binding site from the human 5-LO promoter was: (forward), dGAAATAAACCGTTATGTTTATj (reverse), dATAAACA TAACGGTTTATTTC. Semiquantitative Analysis of RNA by PCR Total RNA was extracted from differentiated HL-60 cells transfected with pcDNAI/NEO-myb/s or pcDNAI/NEO as control. About 6 pg of each RNA were reverse-transcribed in 3 mM MgCl2, 75 mM KCI, 50 mM Tris-HC1 (pH 8.3), 2 ng/@ of random primers (Pharmacia), 0.5 mM each dNTP, 2.0 U/p1 of RNase inhibitor and 10 U/p1 of Superscript II reverse transcriptase (BRL). The reactions were carried out at 37°C for 90 min in a final volume of 60 pl. The enzyme was then inactivated at 94°C for 10 min. Aliquots (20 ~1) of the reverse transcription reactions were amplified by polymerase chain reaction (PCR) using sets of primers specific for 5-LO, c-myb or (3-actin under the following reaction conditions: 4.0 mM MgCl2, 50 mM KCl, 10 mM Tris-HCl pH 8.3, 0.2 mM each dNTP, 25 U/ml AmpliTaq DNA polymerase (PerkinElmer) and 0.45 uM of each primer in a final volume of 100 ~1. The sequence of each primer was as follows: 5-LO (forward), dGCAGTCCTGCTCTGTGTAGAATGGG; 5-LO (reverse), dACCATTGAGCAGATCGTGGACACGC; c-myb (forward), dCGGCACA GCATATATAGC; c-myb (reverse), dGGCTTTTGAAGACTCCTG, (3actin (forward), dGGCACCACACCTTCTACAATGA; B-actin (reverse), dTCGTGATGGACTCCGGTGAC.
52
Prostaglandins 1997:53, January
Repression of the SLipoxygenase
Gene by c-myb: Ponton et al.
Results and Discussion Expression of the Human 5LO and c-myb Genes in Undifferentia ted and Differentiated HL-60 Cells The expression of the 5-LO and c-myb genes was determined by Northern blot analysis. The Fig. 1, lane 2, shows that the 5-LO gene is expressed in HL-60 cells treated with DMSO for five days as determined by the mRNA accumulation (Fig. 1, lane 2), but the amount of mRNA is barely detectable in undifferentiated cells (lane 1). These results are in agreement with the results of other investigators (6, 71.These results indicate that the HL-60 cells used in our experiments have not undergone any clonal modifications and can be used to study the expression of the &LO gene. The same blot was then rehybridized with c-myb c-DNA as a probe and as expected the level of mRNA accumulation was very high in the undifferentiated cells and low in differentiated cells as demonstrated in other myeloid cell lines [ 12-14, 21 j. A band is also present just below the c-myb specific signal which may result from the crosshybridization of the probe with another member of the myb gene family
-
S-LO
-
1
GAPDB
2
FIGURE1. Expressionof the human 5-LO and c-myb genes in HL-60 and differentiated HL-60 calls. Total mRNA was extracted from HL-60 (lane 1) and DMSO treated HL-SOcells (lane 2). Each line contains 10 pg of RNA. The blot was hybridizedwith the 32P-labeIed 5LO cbNA insert.After autoradiiraphy, the 5-LO probe was eluted and the filter was rehybridiredtih a “P-labeled plasmid containing the c-myb cDNA and rehybridized again after elution with a GAPDH probe.
Prostaglandins 1997:53, January
53
Repression of the 5-Lipoxygenase Gene by c-myb: Ponton et al. [22]. As an appropriate control, we have determined the expression of the 5-LO and c-myb genes in the human monocyte cell line THP-1, which can differentiate in macrophages and in this regard should behave like the HL-60 cells. As expected, we find that prior to differentiation, these cells do not express the &LO gene but do show a high level of expression for the c-myb gene (data not shown) as with the HL-60 cells. Specific Binding of MYB-like Protein to the 5-LO Promoter To explain the results above we hypothesized that the c-myb gene product (c-MYB) may be a repressor of the 5-LO gene expression. It has been shown that the promoter of the 5-LO gene contains a sequence element (TAACGG) [ 151 which corresponds to the c-MYB consensus DNAbinding site (T/CAACG/TG) [23]. To test our hypothesis, the sequence (dATAAACA TAACGGTTTATTTC) from the 5-LO promoter was used as a probe in DNA-gel mobility shift analysis in order to determine if the binding of MYB occurs in undifferentiated HL-60 cells only. Nuclear protein extracts were made with either differentiated or undifferentiated cells and mixed with the probe as described in the materials and methods section. Fig. 2 A shows that DNA-proteins complexes were detectable with nuclear extracts from undifferentiated HL-60 cells but not from differentiated cells (compare lanes 2 and 3 with lanes 4 and 5). Two specific complexes (Cl and C2) are formed, one of them could result from the binding of c-MYB to its specific site, and the second complex from the binding of another member of the MYB family or dimerization of MYB with itself or with another protein [24]. The retarded complexes are sequence-specific because they are titered out by molar excesses of cold probe (Fig. 2 B, lane 2 and 3), but not by an unrelated probe of the same length (data not shown). We cannot rule out the possibility that another protein might have recognized the same binding site. This is, however, unlikely because the band-shift analysis strongly suggests that c-MYB or a c-MYB-like protein which is present only in undifferentiated HL-60 cells binds the c-MYB binding site present on the human 5-LO promoter. Overexpression of c-myb cDNA in DMSO Treated HL-60 Cells In order to verify whether or not down-regulation of the 5-LO gene could be achieved by overexpressing the c-myb gene, an expression vector containing the full length cDNA for c-myb was transfected in HL-60 cells treated with DMSO for 72 h. The level of mRNA expression was determined by RT-PCR as described in materials and methods. As shown in Fig. 3, lane 2, the cells transfected with the vector alone express the 5-LO gene but not the c-myb gene, which confirms the results obtained by Northern blot. Interestingly, the mRNA levels for the 5-LO 54
Prostaglandins 1997:53, January
Repression of the 5-Lipoxygenase Gene by c-myb: Ponton et al. B.
A.
1
2
3
4
5
1
2
3
FIGURE2. The 5-LO promoter contains sequences that bind C-MYB related proteins. A 32Plab&d double-stranded oliionudeotide that corresponds to the C-MB binding ebmer(t from the !&LO promotar was used in DNA mobltity gel-shift assays with (in A) 1Qcg and 5gpg of nuclear extracts from HL80 (lane 2 and 3) and fmm DMSO traatad HL-60 calIs (lane 4 end 5). Lane 1 contains no nuclear extracts. B. Lanes 1,2 and 3 contain 5Omg of nudaar axtracts from HL-60 cells. Lane 2 and 3 contain 10X and 50X molar excess of cold otigonudeoWa that was added before the incubation with the probe. In all cases the proba was 10,000 cpm par reaction.
gene, as measured by RT-PCR, are greatly reduced when the cells are transfected with the plasmid containing the c-myb cDNA (Fig. 3, lane 1) This indicates that the transcription for the 5-LO gene is inhibited by overexpression of the c-myb gene which is now expressed as shown by RT-PCR. In conclusion, the data reported here strongly suggests that c-MYB acts as a repressor of the S-LO gene in undifferentiated HL-60 cells. As the c-myb gene is expressed only in haematopoietic stem cells, this may explain why the &LO is not expressed in these cells. However the 5-LO gene is known not to be expressed in other cell types such as fibroblasts or epithelial cells that do not express the c-myb gene either [14). In these cases, c-MYB carmot be held responsible for the non-expression of the S-LO gene which suggests that thus another mechanism of control for gene expression exist. Moreover, it has been reported that B-MYB, another member of the MYB family, binds the same DNA sequence as c-MYB, but to the contrary of c-MYB, B-MYB is expressed in nonhaematopoietic cells [25]. Therefore, B-MYB may play the same role as c-MYB in non-haematopoitic cells. To test this hypothesis we carried Prostaglandins 1997:53, January
55
Repression of the SLipoxygenase
Gene by c-myb: Ponton et al.
c-myb -
FIGURE 3. Overexpression of c-r?@ cDNA in DMSO treated HL-60 cells. DMSO treated HL-80 cells were transfected with an expression vector (pcDNAI/NEO) containing the full length cDNA for c-myb (lane 1) or with the expression vector alone (lane 2). After 48 h, the RNA was extracted and submitted to RT-PCR analysis with oligonucleotides corresponding to the 5-LO, c-myb and p-actin gene.
out RT-PCR analysis with mRNA from HeLa cells, which do not express the c-myb and the 5LO genes as demonstrated by Northern blot analysis (data not shown). We found that B-myb is expressed in these cells (data not shown). Thus, if B-MYB can really repress the 5LO gene in non-haematopoietic cells, this finding may aid in the development of new potential therapeutic agents. Acknowledgments We thank Solange Cloutier for technical assistance. This work was supported by the Medical Research Council of Canada. AP is in receipt of a Fellowship from Fonds de la Recherche en Sante du Quebec. References Lewis, R.A., Austen, K.F. and Soberman, R.J. Leukotrienes and other products of the 5lipoxygenase pathway. N. Engl. J. Med. 323,645-655, 1990. Samuelsson, B. Leukotrienes: mediators of immediate hypersensitivity reactions and inflammation. Science 220, 568-575, 1983. Rouzer, C.A., Matsumoto, T. and Samuelsson, B. Single protein from human leukocytes possesses 5-lipoxygenase and leukotriene A4 synthase activities. Proc Nat1 Acad Sci USA 83, 857-861, 1986. Shimizu, T., Izumi, T., Seyama, Y., Tadokoro, K., Rgdmark, 0. and Samuelsson, B. Characterization of leukotriene A4 synthase from murine
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5.
6.
7.
8.
9.
10.
11.
12.
13.
14.
15.
16. 17.
18.
Gene by c-myb: Ponton et al.
mast cells: evidence for its identity to arachidonate 5lipoxygenase. Proc Nat1 Acad Sci USA 83,4175-4179, 1986. Samuelsson, B., Dahlen, S.-E., Lindgren, J.A., Rouzer, C.A. and Serhan, C.N. Leukotrienes and lipoxins; structures, biosynthesis, and biological effects. Science 237, 1171-1176, 1987. Dixon, R.A.F., Jones, R.E., Diehl, R.E., Bennett, CD., Kargman, S. and Rouzer, C.A. Cloning of the cDNA for human 5lipoxygenase. Proc Nat1 Acad Sci USA 85,416-420, 1988. Piechele, G., Colli, S., Tremoli, E. and Maggi, A. 5-lipoxpygenase gene expression in HL60 cells during differentiation with DMSO. Pharmacological Res.27, 53-60, 1993. Harris, P. and Ralph, P. Human leukemic models of myelomonocytic development: a review of the HL-60 and U937 cell lines. J. Leukocyte Biol. 37, 407-422, 1985. Anthes, J.C., Bryant, R.W., Musch, M.W., Ng, K. and Siegel, M.I. Calcium ionophore and chemotactic peptide stimulation of peptidoleukotriene synthesis in DMSO-differentiated HL60 cells. Inflammation, 10, 145-156, 1986. Balcarek, J.M., Theisen, T.W., Cook, M.N., Varrichio, A., Hwang, S.-M., Strohsacker, M.W. and Crooke, S.T. Isolation and characterization of a cDNA clone encoding rat 5lipoxygenase. J. Biol. Chem. 263, 13937-13941, 1988. Habenicht, A.J.R., Goerig, M., Rothe, D.E.R., Specht, E., Ziegler, R., Glomset, J.A. and Graf, T. Early reversible induction of leukotriene synthesis in chicken myelomonocytic cells transformed by a temperature-sensitive mutant of avian leukemia virus E26. Proc. Natl. Acad. Sci. USA 86, 921-924, 1989. Yanagisawa, H., Nagasawa, T., Kuramochi, S., Abe, T., Ikawa, Y. and Todokoro, K. Constitutive expression of exogenous c-myb gene causes maturation block in monocyte-macrophage differentiation. Bioch. Bioph. Acta, 1088,380-384, 1991. Clarke, M., Kukowska-Latollo, J.F., Westin, E., Smith, M. and Prochownik, E. Constitutive expession of a c-myb cDNA blocks friend murine erythroleukemia cell differentiation. Mol. Cell Biol. 8, 884-892, 1988. Duprey, S.P. and Boettiger, D. Developmental regulation of c-myb in normal myeloid progenitor cells. Proc. Natl. Acad. Sci. USA 82, 6937-6941, 1985. Hoshiko, S., Radmark, 0. and Samuelsson, B. Characterization of the human 5lipoxygenase gene promoter. Proc. Natl. Acad. Sci. USA 87, 90739077, 1990. Bird, A.P. CpG islands as gene markers in the vertebrate nucleus. Trends Genet., 3:,342-347, 1987. Majello, B., Kenyon, L.C. and Dalla-Favera, R. Human c-myb protooncogene: nucleotide sequence of cDNA and oganization of the genomic locus. Proc. Natl. Acad. Sci. USA 83, 9636-9640, 1986. Ponton, A., Coulombe, B. and Skup, D. Decreased expression of tissue inhibitor of metallopoteinases in metastatic tumor cells leading to increased levels of collagenase activity. Cancer Res. 51,2138-2143, 1991.
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Repression of the 5-Lipoxygenase Gene by c-myb: Ponton et al, 19. Ponton, A., Coulombe, B., Steyaert, A., Williams, B.R.G. and Skup, D. Basal expression of the gene (TIMP) encoding the murine tissue inhibitor of metalloproteinases is mediated through APl- and CCAAT-binding factors. Gene 116,187-194, 1992. 20. Levine, M. and Manley, J.L. Transcriptional repression of eukaryotic promotors. Cell 59, 405408, 1989. 21. Gonda, T.J. and Metcalf, D. Expression of myb, myc and fos protooncogenes during the differentiation of a murine myeloid leukaemia. Nature 310,249-251, 1984.. 22. Nomura, N., Takahashi, M., Matsui, M., Ishii, S., Date, T., Sasamoto, S. and Ishizaki, R. Isolation of human cDNA clones of myb-related genes, A-myb and B-myb. Nucleic Acids Res. 16, 11075-l 1088, 1988. 23. Ness, S.A., Marknell, A. and Graf, T. The v-myb oncogene product binds to and activates the promyelocyte-specific mim-1 gene. Cell 59, 1115-1125 , 1989. 24. Ramsay, R.G., Ishii, S. and Gonda, T.J. Interaction of the Myb protein with specific DNA binding sites. J. Biol. Chem. 267, 5656-5662, 1992. 25. Reiss, K., Travali, S., Calabretta, B. and Baserga, R. Growth regulated expression of B-myb in fibroblasts and hematopoietic cells. J. Cell. Phys. 148, 338-343, 1991.
Editor: Dr. W. Smith
Received: 09-04-96
Accepted : 09-26-96
Prostaglandins 1997~53, January