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Identification of a Vitamin D Response Element in the Proximal Promoter of the Chicken Carbonic Anhydrase II Gene

1998; Elsevier BV; Volume: 273; Issue: 17 Linguagem: Inglês

10.1074/jbc.273.17.10638

ISSN

1083-351X

Autores

Isabelle Quélo, Irma Machuca, Pierre Jurdic,

Tópico(s)

Coagulation, Bradykinin, Polyphosphates, and Angioedema

Resumo

The carbonic anhydrase II gene, whose transcription is enhanced by 1,25-dihydroxyvitamin D3(1,25-(OH)2D3), encodes an important enzyme in bone-resorbing cells derived from the fusion of monocytic progenitors. We analyzed the 1,25-(OH)2D3-mediated activation of the avian gene by transient transfection assays with promoter/reporter constructs into HD11 chicken macrophages and by DNA mobility shift assays. Deletion and mobility shift analyses indicated that the −62/−29 region confers 1,25-(OH)2D3responsiveness and forms DNA-protein complexes. The addition of an anti-vitamin D receptor (VDR) antibody inhibited binding to this sequence, whereas anti-retinoid X receptor (RXR) antibody generated a lower mobility complex. Therefore, we concluded that this element binds a VDR·RXR heterodimer, but the addition of extra 1,25-(OH)2D3 had no effect on the formation of this complex. Moreover, the use of nuclear extracts from 1,25-(OH)2D3-treated macrophages led to the formation of an additional high mobility complex also composed of VDR·RXR heterodimer. Mutations provided evidence that the 1,25-(OH)2D3-mediated activation of the carbonic anhydrase II gene is mediated by VDR·RXR heterodimers bound to a DR3-type vitamin D response element with sequence AGGGCAtggAGTTCG. This vitamin D response element is also functional in the ROS 17/2.8 osteoblasts. The carbonic anhydrase II gene, whose transcription is enhanced by 1,25-dihydroxyvitamin D3(1,25-(OH)2D3), encodes an important enzyme in bone-resorbing cells derived from the fusion of monocytic progenitors. We analyzed the 1,25-(OH)2D3-mediated activation of the avian gene by transient transfection assays with promoter/reporter constructs into HD11 chicken macrophages and by DNA mobility shift assays. Deletion and mobility shift analyses indicated that the −62/−29 region confers 1,25-(OH)2D3responsiveness and forms DNA-protein complexes. The addition of an anti-vitamin D receptor (VDR) antibody inhibited binding to this sequence, whereas anti-retinoid X receptor (RXR) antibody generated a lower mobility complex. Therefore, we concluded that this element binds a VDR·RXR heterodimer, but the addition of extra 1,25-(OH)2D3 had no effect on the formation of this complex. Moreover, the use of nuclear extracts from 1,25-(OH)2D3-treated macrophages led to the formation of an additional high mobility complex also composed of VDR·RXR heterodimer. Mutations provided evidence that the 1,25-(OH)2D3-mediated activation of the carbonic anhydrase II gene is mediated by VDR·RXR heterodimers bound to a DR3-type vitamin D response element with sequence AGGGCAtggAGTTCG. This vitamin D response element is also functional in the ROS 17/2.8 osteoblasts. Recent work points to the complexity of the molecular mechanisms involved in the vitamin D3 signaling pathway. It has been known for some time that in addition to the binding of the vitamin D receptor (VDR) 1The abbreviations used are: VDR, vitamin D receptor; VDRE, vitamin D response element; RXR, retinoid X receptor; bp, base pair(s); DR3, direct repeats of two hexameric core binding sites spaced by three nucleotides; CAII, carbonic anhydrase II; 1,25-(OH)2D3, 1,25-dihydroxyvitamin D3; tk, thymidine kinase; CAT or Cat, chloramphenicol acetyltransferase; CMV, cytomegalovirus; EMSA, electrophoretic mobility shift assay; RAR, retinoic acid receptor; TR, thyroid hormone receptor; Mi, microphalmia transcription factor. 1The abbreviations used are: VDR, vitamin D receptor; VDRE, vitamin D response element; RXR, retinoid X receptor; bp, base pair(s); DR3, direct repeats of two hexameric core binding sites spaced by three nucleotides; CAII, carbonic anhydrase II; 1,25-(OH)2D3, 1,25-dihydroxyvitamin D3; tk, thymidine kinase; CAT or Cat, chloramphenicol acetyltransferase; CMV, cytomegalovirus; EMSA, electrophoretic mobility shift assay; RAR, retinoic acid receptor; TR, thyroid hormone receptor; Mi, microphalmia transcription factor. to certain vitamin D response elements (VDREs) as homodimer (3Carlberg C. 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Nucleic Acids Res. 1994; 22: 2902-2907Crossref PubMed Scopus (75) Google Scholar).The differentiating effects of 1,25-(OH)2D3have been studied extensively in a large number of in vitrosystems using cultures of leukemia cells (28Suda T. Proc. Soc. Exp. Biol. Med. 1989; 191: 214-220Crossref PubMed Scopus (54) Google Scholar, 29Feldman J. Federico M.H.H. Sonohara S. Katayama M.L.H. Koike M.A.A. Roela R.A. Da Silva M.R.P. Brentani M.M. Leukemia Res. 1993; 17: 97-101Crossref PubMed Scopus (11) Google Scholar), keratinocytes (30Pillai S. Bikle D.D. Elias P.M. Skin Pharmacol. 1988; 1: 149-160Crossref PubMed Scopus (36) Google Scholar, 31Regnier M. Darmon M. Differentiation. 1991; 47: 173-188Crossref PubMed Scopus (53) Google Scholar, 32Chen T.C. Persons K. Uskokovic M.R. Horst R.L. Holick M.F. J. Nutr. Biochem. 1993; 1993: 49-57Crossref Scopus (60) Google Scholar), or bone cells (33Roodman G.J. MacDonald B.R. Kuehl T.J. Mundy G.R. Proc. Natl. Acad. Sci. U. S. 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Res. 1992; 7: 405-414Crossref PubMed Scopus (47) Google Scholar), and their differentiation pathway is partly under the control of 1,25-(OH)2D3 (37Bar-Shavit Z. Teitelbaum S.L. Reitsma P. Hall A. Pegg L.E. Trial J. Kahn A.J. Proc. Natl. Acad. Sci. U. S. A. 1983; 80: 5907-5911Crossref PubMed Scopus (342) Google Scholar, 38Woods C. Domenget C. Solari F. Gandrillon O. Lazarides E. Jurdic P. Endocrinology. 1995; 136: 85-95Crossref PubMed Scopus (34) Google Scholar, 39Norman A.W. Hurwitz S. J. Nutr. 1993; 123: 310-316Crossref PubMed Scopus (41) Google Scholar, 40Roodman G.D. Endocr. Rev. 1996; 17: 308-332PubMed Google Scholar). These cells express some characteristic markers such as tartrate-resistant acid phosphatase, vitronectin receptor, calcitonin receptor, and CAII. CAII, expressed at high levels in osteoclasts (41Hillstrom Shapiro L. Venta P.J. Yu Y.S.L. Tashian R. FEBS Lett. 1989; 249: 307-310Crossref PubMed Scopus (26) Google Scholar, 42Zheng M.H. Fan Y. Wysocki S. Wood D.J. Papadimitriou J.M. J. Bone Miner. Res. 1993; 8: 113-118Crossref PubMed Scopus (37) Google Scholar), plays an important role in the extracellular acidification required for bone resorption and therefore bone remodeling. In particular, CAII deficiency is one of the factors responsible for the osteopetrosis characterized in humans by a renal tubular acidosis and a cerebral calcification (43Roth D.E. Venta P.J. Tashian R.E. Sly W.S. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 1804-1808Crossref PubMed Scopus (103) Google Scholar).We have shown previously that 1,25-(OH)2D3enhances the expression of CAII in chicken primary blood-derived macrophages (38Woods C. Domenget C. Solari F. Gandrillon O. Lazarides E. Jurdic P. Endocrinology. 1995; 136: 85-95Crossref PubMed Scopus (34) Google Scholar). Furthermore, 1,25-(OH)2D3activates the CAII gene expression at the transcriptional level in the chicken monocytic BM2 cells induced to differentiate into macrophages by lipopolysaccharides and phorbol 12-myristate 13-acetate (44Lomri A. Baron R. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 4688-4692Crossref PubMed Scopus (37) Google Scholar) as well as in the human promyelocytic leukemia cells HL60 after phorbol 12-myristate 13-acetate stimulation (45Biskobing D.M. Nanes M.S. Rubin J. Endocrinology. 1994; 134: 1493-1498Crossref PubMed Scopus (11) Google Scholar). CAII gene expression is also activated transcriptionally by thyroid hormone in normal erythrocytic cells (46Pain B. Melet F. Samarut J. New Biol. 1990; 2: 284-294PubMed Google Scholar), and several domains in the avian CAII promoter have been shown to control the thyroid hormone regulation of transcription (47Disela C. Glineur C. Bugge T. Sap J. Sengl G. Dodgson J. Stunnenberg J. Beug H. Zenke M. Genes Dev. 1991; 5: 2033-2047Crossref PubMed Scopus (60) Google Scholar,48Rascle A. Ghysdael J. Samarut J. Oncogene. 1994; 9: 2853-2867PubMed Google Scholar). In previous work, we identified a VDRE between positions −1203 and −1187 of the CAII promoter which mediates 1,25-(OH)2D3 responsiveness to the herpes simplex virus thymidine kinase (tk) minimal promoter in the Drosophila SL3 cell line and in human MCF-7 cells (23Quélo I. Kahlen J.P. Rascle A. Jurdic P. Carlberg C. DNA Cell Biol. 1994; 13: 1181-1187Crossref PubMed Scopus (40) Google Scholar). This VDRE, bound by a VDR·RXR heterodimer, is however not functional in an avian macrophage cell line.In the present study, we have looked for specific 1,25-(OH)2D3 regulation of the CAII gene transcription in macrophages. We have studied the ligand-dependent transactivation of the avian CAII promoter in the chicken HD11 macrophage cell line in which numerous hormonal nuclear receptors and CAII gene are expressed endogenously. 2I. Quélo, and P. Jurdic, manuscript in preparation. 2I. Quélo, and P. Jurdic, manuscript in preparation. We have looked for hormone response elements in this promoter and localized an element conferring 1,25-(OH)2D3-mediated activation (VDRE) to a 34-base pair region located −62/−29 upstream the transcriptional start site. The VDRE was defined precisely after methylation interference assays and by using mutated forms of this sequence. This VDRE, functional in HD11 and in ROS 17/2.8 cell lines, has a DR3 structure with sequence AGGGCA tgg AGTTCG and is specifically bound by a heterodimer formed by VDR and the α or γ isoform of RXR.DISCUSSIONIn this work, we showed that the chicken CAII promoter activity is induced in response to 1,25-(OH)2D3 in an avian macrophage cell line. We identified a domain in the CAII promoter responsible for 1,25-(OH)2D3-mediated transactivation. Transient transfection assays in HD11 macrophages of total or deleted fragments of the CAII promoter allowed us to define a region involved in 1,25-(OH)2D3 responsiveness. In macrophages, most if not all DNA sequences essential for CAII gene basal and 1,25-(OH)2D3-induced expression are within the first 178 bp upstream of the initiation site, referred to as the proximal promoter. It is noteworthy that the level of 1,25-(OH)2D3 activation increases with the 5′-end progressive deletions of the promoter. A repressor domain further upstream may be present and would explain the increasing activation of CAII promoter correlated with deletions. Further deletions and mutational analyses of the CAII proximal promoter allowed us to determine a more precise vitamin D-responsive domain, between residues −53 and −39 of the CAII promoter. We showed that this VDRE is highly functional in macrophages and is bound specifically by a complex formed by a RXR·VDR heterodimer. Lastly, we showed that mutations that abolished protein binding to the VDRE inhibit vitamin D-dependent activation in macrophages.Many have shown that the VDR has a binding preference for the direct repeat composed of AG(G/T)TCA motifs spaced by 3 bp (2Mangelsdorf D.J. Thummel C. Beato M. Herrlich P. Schutz G. Umesono K. Blumberg B. Kastner P. Mark M. Chambon P. Evans R.M. Cell. 1995; 83: 835-839Abstract Full Text PDF PubMed Scopus (6027) Google Scholar, 3Carlberg C. Bendik I. Wyss A. Meier E. Sturzenbecker L.J. Grippo J.F. Hunziker W. Nature. 1993; 361: 657-660Crossref PubMed Scopus (500) Google Scholar, 53Mader S. Leroy P. Chen J.Y. Chambon P. J. Biol. Chem. 1993; 268: 591-600Abstract Full Text PDF PubMed Google Scholar, 54Ebihara K. Masuhiro Y. Kitamoto T. Suzawa M. Uematsu Y. Yoshizawa T. Ono T. Harada H. Matsuda K. Hasegawa T. Masushige S. Kato S. Mol. Cell. Biol. 1996; 16: 3393-3400Crossref PubMed Scopus (76) Google Scholar). The CAII proximal VDRE consists of an imperfect tandem of 6 bases spaced by three nucleotides with the sequence AGGGCA for the 5′-motif and AGTTCG for the downstream motif. Mutational analysis revealed the validity of the DR3 structure for the CAII proximal VDRE. Oligonucleotides containing mutations within the spacer motif of this DR3 (m4 and m8 mutants) were still able to bind VDR·RXR heterodimer and have a very mild effect on transactivation efficiency. In contrast, mutations within the two hexameric motifs of the DR3 element inhibited 1,25-(OH)2D3-mediated transcriptional activation and most of the protein-DNA interactions. Furthermore, our results indicate that the 3′-element integrity of this VDRE is more crucial for binding than the 5′-element. This is evidenced by the ability of a mutated 5′-element VDRE (m3 mutant) to compete still for VDR binding with the native sequence, whereas mutations in the 3′-element (m5 and m6 mutants) resulted in a marked decrease in VDR binding to the mutant element. In contrast with others studies showing the important role of the residues in 5′-position outside the VDRE, we have shown here that the nucleotides located outside the VDRE were not essential either for the binding of the protein complex on DNA or for the transactivation activity.The sequences of numerous known natural positive VDREs, generally of the DR3 type, identified within the promoter regions of different genes, have been aligned. It is of interest to note that the hexameric core binding sites are rather degenerated, although they all can specifically bind VDR complexes and confer transactivation upon 1,25-(OH)2D3 stimulation. We have observed that the upstream motif of the consensus VDRE is more conserved than the downstream one. The difference between the two half-sites may indicate preferential binding of each receptor of the complex bound to DNA. Previous studies have shown that RXRs bind preferentially to the 5′-core binding site in retinoic acid and thyroid hormone response elements as well as in VDREs (1Umesono K. Murakami K.K. Thompson C.C. Evans R.M. Cell. 1991; 65: 1255-1266Abstract Full Text PDF PubMed Scopus (1488) Google Scholar, 7Freedman L.P. Arce V. Fernandez R.P. Mol. Endocrinol. 1994; 8: 265-273Crossref PubMed Scopus (79) Google Scholar, 25Colnot S. Lambert M. Blin C. Thomasset M. Perret C. Mol. Cell. Endocrinol. 1995; 113: 89-98Crossref PubMed Scopus (39) Google Scholar, 26Towers T.L. Luisi B.F. Asianov A. Freedman L.P. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 6310-6314Crossref PubMed Scopus (107) Google Scholar, 27Nishikawa J.I. Kitaura M. Matsumoto M. Imagawa M. Nishihara T. 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Matsumoto M. Sakoda K. Kitaura M. Imagawa M. Nishihara T. J. Biol. Chem. 1993; 268: 19739-19743Abstract Full Text PDF PubMed Google Scholar).Consistent with the results obtained with gel retardation experiments, we have demonstrated by transient transfection experiments that the vitamin D stimulation of the CAII gene expression acts, in vivo, through VDR and RXR dimerization.2 Furthermore, we have also shown that, in vivo, addition of RXR ligand does not interfere with the 1,25-(OH)2D3response and so, does not affect the stability of VDR·RXR heterodimers in the cells nor the transactivation efficiency (49Maurer R.A. Notides A.C. Mol. Cell. Biol. 1987; 7: 4247-4254Crossref PubMed Scopus (202) Google Scholar).Surprisingly, two specific complexes, C1 and C2, were formed on the CAII proximal VDRE with nuclear extracts from 1,25-(OH)2D3-treated HD11 cells, whereas only one complex (C1) was formed with vehicle-treated extracts or recombinant proteins. However, C1 and C2 complexes bound to this VDRE are both composed at least of VDR and RXR heterodimers. We have speculated that in vivo, 1,25-(OH)2D3 induces a specific conformational change of the VDR·RXR heterodimer leading to the formation of the C2 complex, whereas in vitro, only the C1 complex is observed. This C2 higher mobility complex could be caused by the binding of 1,25-(OH)2D3 to the VDR, although the addition of extra 1,25-(OH)2D3 to the EMSA reaction mixture had no effect on the DNA binding affinity of the protein complex. In such conditions no change in mobility or complex composition was observed. This suggests that in vitro, binding of the ligand is not necessary for the binding of proteins to DNA. In contrast, in vivo, cell exposure to 1,25-(OH)2D3 could change the complex conformation on DNA and in doing so could either recruit or displace the binding of either a coactivator or a corepressor. The two complexes, C1 and C2, obtained by EMSAs using nuclear extracts, may reflect the possible involvement of known or unknown factors in addition to the VDR·RXR heterodimer. In fact, the transcription factor TFIIB, which was shown to interact directly with VDR (61MacDonald P.N. Sherman D.R. Dowd D.R. Jefcoat Jr., S.C. DeLisle R.K. J. Biol. Chem. 1995; 270: 4748-4752Abstract Full Text Full Text PDF PubMed Scopus (152) Google Scholar), has been described as forming part of in these complexes and appears to be required for the VDR to bind the VDREs (62Haussler M. Jurutka P. Haussler C. Hsieh J.C. Thompson P. Remus L. Selznick S. Encinas C. Whitfield G.K. Norman A.W. Bouillon R. Thomasset L. Vitamin D Chemistry, Biology and Clinical Applications of the Steroid Hormone. University of California, Riverside, CA1997: 210-217Google Scholar). 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