Transcriptional Regulation of CHI3L1, a Marker Gene for Late Stages of Macrophage Differentiation

Abstract

The protein product of the CHI3L1 gene, human cartilage 39-kDa glycoprotein (HC-gp39), is a tissue-restricted, chitin-binding lectin and member of glycosyl hydrolase family 18. In contrast to many other monocyte/macrophage markers, its expression is absent in monocytes and strongly induced during late stages of human macrophage differentiation. To gain insights into the molecular mechanisms underlying its cell typerestricted and maturation-associated expression in macrophages, we initiated a detailed study of the proximal HC-gp39 promoter. Deletion analysis of reporter constructs in macrophage-like THP-1 cells localized a region directing high levels of macrophage-specific reporter gene expression to ∼300 bp adjacent to the major transcriptional start site. The promoter sequence contained consensus binding sites for several known factors, and specific binding of nuclear PU.1, Sp1, Sp3, USF, AML-1, and C/EBP proteins was detectable in gel shift assays. In vivo footprinting assays with dimethyl sulfate demonstrate that the protection of corresponding sequences was enhanced in macrophages compared with monocytes. Mutational analysis of transcription factor binding sites indicated a predominant role for a single Sp1 binding site in regulating HC-gp39 promoter activity. In addition, gel shift assays using nuclear extracts of monocytes and macrophages demonstrated that the binding of nuclear Sp1, but not Sp3, markedly increases during macrophage differentiation. Our results further highlight the important role of Sp1 in macrophage gene regulation. The protein product of the CHI3L1 gene, human cartilage 39-kDa glycoprotein (HC-gp39), is a tissue-restricted, chitin-binding lectin and member of glycosyl hydrolase family 18. In contrast to many other monocyte/macrophage markers, its expression is absent in monocytes and strongly induced during late stages of human macrophage differentiation. To gain insights into the molecular mechanisms underlying its cell typerestricted and maturation-associated expression in macrophages, we initiated a detailed study of the proximal HC-gp39 promoter. Deletion analysis of reporter constructs in macrophage-like THP-1 cells localized a region directing high levels of macrophage-specific reporter gene expression to ∼300 bp adjacent to the major transcriptional start site. The promoter sequence contained consensus binding sites for several known factors, and specific binding of nuclear PU.1, Sp1, Sp3, USF, AML-1, and C/EBP proteins was detectable in gel shift assays. In vivo footprinting assays with dimethyl sulfate demonstrate that the protection of corresponding sequences was enhanced in macrophages compared with monocytes. Mutational analysis of transcription factor binding sites indicated a predominant role for a single Sp1 binding site in regulating HC-gp39 promoter activity. In addition, gel shift assays using nuclear extracts of monocytes and macrophages demonstrated that the binding of nuclear Sp1, but not Sp3, markedly increases during macrophage differentiation. Our results further highlight the important role of Sp1 in macrophage gene regulation. Human blood monocytes are able to differentiate into morphologically and functionally heterogeneous effector cells, including macrophages, myeloid dendritic cells, and osteoclasts (1Hume D.A. Ross I.L. Himes S.R. Sasmono R.T. Wells C.A. Ravasi T. J. Leukocyte Biol. 2002; 72: 621-627PubMed Google Scholar). In contrast to the differentiation of precursor cells, this development generally proceeds in the absence of proliferation. In myelomonocytic cell types, several transcription factors have been identified that are important for lineage commitment, subsequent differentiation, and cell type-specific gene regulation. These include members of the Ets, C/EBP, and CBF families and several other transcription factors (2Friedman A.D. Oncogene. 2002; 21: 3377-3390Crossref PubMed Scopus (269) Google Scholar, 3Tenen D.G. Hromas R. Licht J.D. Zhang D.E. Blood. 1997; 90: 489-519Crossref PubMed Google Scholar). In particular the Ets family transcription factor PU.1 is essential for normal monocyte/macrophage differentiation and cell type-specific gene expression (4McKercher S.R. Torbett B.E. Anderson K.L. Henkel G.W. Vestal D.J. Baribault H. Klemsz M. Feeney A.J. Wu G.E. Paige C.J. Maki R.A. EMBO J. 1996; 15: 5647-5658Crossref PubMed Scopus (930) Google Scholar, 5Scott E.W. Simon M.C. Anastasi J. Singh H. Science. 1994; 265: 1573-1577Crossref PubMed Scopus (1279) Google Scholar, 6Tondravi M.M. McKercher S.R. Anderson K. Erdmann J.M. Quiroz M. Maki R. Teitelbaum S.L. Nature. 1997; 386: 81-84Crossref PubMed Scopus (452) Google Scholar). The defect of PU.1–/–hematopoietic progenitors may be attributed to their failure to express the receptors for macrophage colony stimulating factor, granulocyte colony stimulating factor, and granulocyte macrophage colony stimulating factor, which are critical for progenitor cell survival and proliferation (7Anderson K.L. Smith K.A. Conners K. McKercher S.R. Maki R.A. Torbett B.E. Blood. 1998; 91: 3702-3710Crossref PubMed Google Scholar). PU.1 is also directly involved in the regulation of a large number of genes that are associated with myeloid, and particularly with monocytic, differentiation including for instance, PU.1 itself (8Chen H. Ray-Gallet D. Zhang P. Hetherington C.J. Gonzalez D.A. Zhang D.E. Moreau-Gachelin F. Tenen D.G. Oncogene. 1995; 11: 1549-1560PubMed Google Scholar), CD11b (9Pahl H.L. Scheibe R.J. Zhang D.E. Chen H.M. Galson D.L. Maki R.A. Tenen D.G. J. Biol. Chem. 1993; 268: 5014-5020Abstract Full Text PDF PubMed Google Scholar), CD18 (10Rosmarin A.G. Caprio D. Levy R. Simkevich C. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 801-805Crossref PubMed Scopus (99) Google Scholar), FcγRI (11Eichbaum Q.G. Iyer R. Raveh D.P. Mathieu C. Ezekowitz R.A. J. Exp. Med. 1994; 179: 1985-1996Crossref PubMed Scopus (102) Google Scholar), FcγRIII (12Feinman R. Qiu W.Q. Pearse R.N. Nikolajczyk B.S. Sen R. Sheffery M. Ravetch J.V. EMBO J. 1994; 13: 3852-3860Crossref PubMed Scopus (81) Google Scholar), c-fes (13Heydemann A. Juang G. Hennessy K. Parmacek M.S. Simon M.C. Mol. Cell Biol. 1996; 16: 1676-1686Crossref PubMed Scopus (75) Google Scholar), macrophage scavenger receptor (14Moulton K.S. Semple K. Wu H. Glass C.K. Mol. Cell Biol. 1994; 14: 4408-4418Crossref PubMed Scopus (160) Google Scholar), TFEC (15Rehli M. Lichanska A. Cassady A.I. Ostrowski M.C. Hume D.A. J. Immunol. 1999; 162: 1559-1565PubMed Google Scholar), or Toll-like receptor 4 (16Rehli M. Poltorak A. Schwarzfischer L. Krause S.W. Andreesen R. Beutler B. J. Biol. Chem. 2000; 275: 9773-9781Abstract Full Text Full Text PDF PubMed Scopus (213) Google Scholar). The acquisition of a mature macrophage phenotype is clearly distinct from monocytic lineage commitment and requires expression of a new set of genes. Also, most “macrophage-specific” marker genes that have been studied previously are already expressed in peripheral blood monocytes. To gain insight into the molecular mechanisms of macrophage differentiation, we identified a number of genes that are specifically up-regulated during the differentiation of human blood monocytes into macrophages (17Krause S.W. Rehli M. Kreutz M. Schwarzfischer L. Paulauskis J.D. Andreesen R. J. Leukocyte Biol. 1996; 60: 540-545Crossref PubMed Scopus (168) Google Scholar). Because of its unique cell type-restricted and maturation-associated expression, the gene CHI3L1 was chosen as a model to investigate late transcriptional events during macrophage differentiation. The CHI3L1 gene product, human cartilage 39-kDa glycoprotein (HC-gp39, 1The abbreviations used are: HC-gp39, human cartilage 39-kDa glycoprotein; MO, monocytes; PMA, phorbol 12-myristate 13-acetate; PBS, phosphate-buffered saline; EMSA, electrophoretic mobility shift assay; DMS, dimethyl sulfate; LM-PCR, ligation-mediated PCR. also known as YKL-40), is a mammalian member of glycosyl hydrolase family 18 (18Hakala B.E. White C. Recklies A.D. J. Biol. Chem. 1993; 268: 25803-25810Abstract Full Text PDF PubMed Google Scholar). In contrast to chitotriosidase, a related active chitinase that is also expressed during late stages of macrophage differentiation (19Boot R.G. Renkema G.H. Strijland A. van Zonneveld A.J. Aerts J.M. J. Biol. Chem. 1995; 270: 26252-26256Abstract Full Text Full Text PDF PubMed Scopus (339) Google Scholar), HC-gp39 lacks hydrolytic activity, probably because of a single amino acid substitution in the catalytic center region and acts as a chitin-specific lectin (20Renkema G.H. Boot R.G. Au F.L. Donker-Koopman W.E. Strijland A. Muijsers A.O. Hrebicek M. Aerts J.M. Eur. J. Biochem. 1998; 251: 504-509Crossref PubMed Scopus (334) Google Scholar). In vivo, HC-gp39 levels are increased in serum, synovial fluid, and cartilage of rheumatoid arthritis patients (18Hakala B.E. White C. Recklies A.D. J. Biol. Chem. 1993; 268: 25803-25810Abstract Full Text PDF PubMed Google Scholar, 21Johansen J.S. Jensen H.S. Price P.A. Br. J. Rheumatol. 1993; 32: 949-955Crossref PubMed Scopus (214) Google Scholar), in various inflammatory disorders (22Vos K. Steenbakkers P. Miltenburg A.M. Bos E. van Den Heuvel M.W. van Hogezand R.A. de Vries R.R. Breedveld F.C. Boots A.M. Ann. Rheum. Dis. 2000; 59: 544-548Crossref PubMed Scopus (103) Google Scholar), in the liver of patients with alcoholic cirrhosis (23Johansen J.S. Moller S. Price P.A. Bendtsen F. Junge J. Garbarsch C. Henriksen J.H. Scand. J. 1997; 32: PubMed Scopus Google Scholar), in of patients with J.S. C. M. C. Price P.A. Eur. J. 1995; Full Text PDF PubMed Scopus Google and C. J.S. Price P.A. S. Br. J. 1999; PubMed Scopus Google Scholar), and in macrophages in the R.G. van van B.E. Renkema G.H. Aerts J.M. de Vries C.J. Biol. 1999; PubMed Scopus Google Scholar). The expression a role in and HC-gp39 was to proliferation of human cells and to and protein a role as a factor A.D. White C. H. Biochem. J. 2002; PubMed Scopus Google Scholar). The expression of HC-gp39 is to a number of cell (18Hakala B.E. White C. Recklies A.D. J. Biol. Chem. 1993; 268: 25803-25810Abstract Full Text PDF PubMed Google Scholar), synovial cells (18Hakala B.E. White C. Recklies A.D. J. Biol. Chem. 1993; 268: 25803-25810Abstract Full Text PDF PubMed Google Scholar), B. Price P.A. J.S. J. Proc. 1998; Google Scholar), and macrophages (17Krause S.W. Rehli M. Kreutz M. Schwarzfischer L. Paulauskis J.D. Andreesen R. J. Leukocyte Biol. 1996; 60: 540-545Crossref PubMed Scopus (168) Google and a with late macrophage differentiation in expression is in monocytes and detectable in dendritic cells (17Krause S.W. Rehli M. Kreutz M. Schwarzfischer L. Paulauskis J.D. Andreesen R. J. Leukocyte Biol. 1996; 60: 540-545Crossref PubMed Scopus (168) Google Scholar). To the molecular mechanisms of gene expression during late stages of macrophage differentiation, we the expression of the gene for HC-gp39 the transcriptional and the CHI3L1 gene M. Krause S.W. Andreesen R. 1997; PubMed Scopus Google and initiated a detailed of its macrophage-specific promoter. important for HC-gp39 promoter in macrophages and to Sp1, C/EBP, and CBF family transcription factors in binding sites for transcription factors for promoter reporter in THP-1 a role for Sp1 family factors was in regulating HC-gp39 promoter activity. used from are from for from blood cells of from in a as previously (17Krause S.W. Rehli M. Kreutz M. Schwarzfischer L. Paulauskis J.D. Andreesen R. J. Leukocyte Biol. 1996; 60: 540-545Crossref PubMed Scopus (168) Google Scholar). as and expression of in with amino from human for a of The human monocytic cell THP-1 and in with The human cell and the human cell from A. in cells with or cells from in and was from cell the P. Biochem. 162: PubMed Scopus Google Scholar). and as previously (17Krause S.W. Rehli M. Kreutz M. Schwarzfischer L. Paulauskis J.D. Andreesen R. J. Leukocyte Biol. 1996; 60: 540-545Crossref PubMed Scopus (168) Google Scholar). of the region was used as and in was with a corresponding to the proximal promoter region of HC-gp39 that was previously using M. Krause S.W. Andreesen R. 1997; PubMed Scopus Google Scholar). the of the CHI3L1 gene identified analysis and PCR. of a of the proximal promoter was using the and the and sites for and used for into the reporter to the reporter of the HC-gp39 promoter from with and and into the the and reporter of the using the in of and used in reporter The was using the and of binding sites and of constructs and constructs with specific was using in with into the corresponding expression for human Sp1 family proteins a from G. and using the from in footprinting was as G. R. H. J. Biol. Chem. 1995; 270: Full Text Full Text PDF PubMed Scopus Google Scholar). cells with phosphate-buffered in of PBS, and for with of dimethyl sulfate The was the of of and in in and with cells in of and was to A.M. PubMed Scopus Google Scholar). of ligation-mediated of or as P.A. Proc. Natl. Acad. Sci. U. S. A. PubMed Scopus Google Scholar, B. Science. PubMed Scopus Google with G. R. H. J. Biol. Chem. 1995; 270: Full Text Full Text PDF PubMed Scopus Google Scholar). The for are in The was in of of and of for and of the the was to and of as in and B. Science. PubMed Scopus Google Scholar), of of and of 4 the was with and in The was in of of set and the and of for using and the was to of of in a of not the was to for to of and and for and in of was a and the and for with was to the the cells using as (16Rehli M. Poltorak A. Schwarzfischer L. Krause S.W. Andreesen R. Beutler B. J. Biol. Chem. 2000; 275: 9773-9781Abstract Full Text Full Text PDF PubMed Scopus (213) Google Scholar). The cell and using to the 4 cells using of and of of and for the of cells using of and of of and for the of The was and with The cell for and cell for and using the reporter a of was activity. cells using to the 4 cells cells using of and of of reporter of expression and cell for using the reporter of was protein using a and extracts from cells, or in human macrophages using as (15Rehli M. Lichanska A. Cassady A.I. Ostrowski M.C. Hume D.A. J. Immunol. 1999; 162: 1559-1565PubMed Google Scholar). extracts from human monocytes using that with a in a used contained and a of with using or with using The binding contained of nuclear protein or of the in of and of in a of used in and for a of and used have been (15Rehli M. Lichanska A. Cassady A.I. Ostrowski M.C. Hume D.A. J. Immunol. 1999; 162: 1559-1565PubMed Google Scholar). in and of HC-gp39 in Human and Cell identified HC-gp39 as a marker for late stages of macrophage (17Krause S.W. Rehli M. Kreutz M. Schwarzfischer L. Paulauskis J.D. Andreesen R. J. Leukocyte Biol. 1996; 60: 540-545Crossref PubMed Scopus (168) Google Scholar). To of the HC-gp39 several myeloid cell for HC-gp39 The myeloid cell or induced to differentiate into macrophage-like cells with or a analysis of the myeloid cell and as compared with human blood monocytes and in HC-gp39 expression was detectable in or HC-gp39 was also in but was markedly induced THP-1 cells expressed HC-gp39 and expression was further increased the of or HC-gp39 expression in cells and cell also with protein expression analysis not of the HC-gp39 the HC-gp39 promoter cell type-specific in reporter we a of the promoter region of the human CHI3L1 gene into a reporter analysis was in the myeloid cell THP-1 and and cell and which not express HC-gp39 not for with a and results for cell compared to the of a in reporter was detectable in THP-1 cells, which is with HC-gp39 expression in this cell results using a of the proximal promoter of the proximal HC-gp39 are to a and are from are to a and are from in a new Deletion of the HC-gp39 that are important for HC-gp39 promoter in macrophages, constructs the promoter of the human CHI3L1 gene of the proximal region into the and of the promoter or analysis was with reporter constructs of in the monocytic cell THP-1 to sequences in the HC-gp39 in the promoter constructs a reporter activity, the of transcriptional of the transcriptional start site. reporter was with the promoter of the promoter to bp a in promoter activity. The was the of a further in this Deletion to bp in a of activity. These results that important for the macrophage-specific of the proximal promoter and of the HC-gp39 analysis localized a region directing macrophage-specific reporter gene expression to ∼300 bp proximal to the major transcriptional start site that was in a study M. Krause S.W. Andreesen R. 1997; PubMed Scopus Google Scholar). In contrast to many other the HC-gp39 promoter a and a bp of the transcriptional sites is in the proximal promoter several binding sites for transcription factors and factors C/EBP, identified analysis using the In we identified several that be members of the Ets including the myeloid and transcription factor To human and the corresponding promoter region of the was with the human HC-gp39 promoter a of the and Deletion of the HC-gp39 further in the proximal HC-gp39 a detailed analysis was of the constructs in THP-1 cells is in The region bp and was for reporter gene this region was from the in reporter of the Sp1 sequence and a binding site for transcription the that of binding Deletion of the region in the of reporter of the region reporter activity, and of the region the and the reporter in the HC-gp39 for protein binding sites the proximal HC-gp39 in vivo footprinting with was using in macrophages, and THP-1 The results for and are in macrophages and THP-1 cells sites as as several sites the Sp1 the and of the site in monocytes. sites to the Sp1 and the as as the C/EBP In protection of binding sites was detectable several the region the was in cell of THP-1 cells and macrophages the the of THP-1 as a model for late macrophage expression of to the HC-gp39 shift to transcription factors that sequence in vivo footprinting or consensus site Because the of PU.1 for expression of myeloid genes been demonstrated in a number of we the of PU.1 to the human HC-gp39 promoter. was with corresponding to sequences a sequence and and using in PU.1 or nuclear extracts of THP-1 cells that specifically to In was the of of but not with a In addition, of was in the of a In contrast to the that PU.1, several specific using the binding proteins and as as used for of the not The region the Sp1 and factor binding sites was also using in using and Sp1, Sp3, and including high mobility factors from and also a region for gene regulation. and with a corresponding identified specific in THP-1 nuclear extracts or factors binding sites for C/EBP factors are and of the site. To which to C/EBP factors, used as the with the that C/EBP proteins the site. Mutational of transcription factor binding sites into the to the of sites for promoter in assays. in the of the proximal promoter in THP-1 cells was not of single binding which is with the results with constructs of the Sp1 binding site the of reporter activity, to The C/EBP site a of reporter activity, several other The of site was not for reporter in THP-1 In constructs or the of of the Sp1 and the C/EBP of the reporter was The of sequences as as binding sites probably of transcription factor in reporter in THP-1 constructs the region the and and sequences of the the proximal promoter to binding sites and to in of the was to the of the Sp1 site a markedly reporter activity, further the of this for HC-gp39 promoter activity. of for of the HC-gp39 in HC-gp39 expression is markedly up-regulated during differentiation of THP-1 To the was of the proximal the of constructs in THP-1 cells or with in the was induced Deletion of the region reporter activity, and of the region the and in a of to the Deletion of the a of that in this region are for the of was also with constructs in the Sp1 its for the of of to the Sp1 strongly important role of the Sp1 in the of HC-gp39 promoter in to macrophage differentiation and differentiation of THP-1 To the levels of nuclear Sp1 family proteins in cells of gel shift assays using a corresponding to the Sp1 in nuclear levels of Sp1 are markedly increased in mature macrophages as compared with human blood monocytes. of in and nuclear Sp1 also that the of that was not with family with and was specific for mature differentiation of THP-1 cells also with increased binding of nuclear Sp1 and a of nuclear binding The in macrophages was absent in or THP-1 cells or of the HC-gp39 in further the role of Sp1 family transcription factors as critical of HC-gp39 promoter activity, we in cells Sp1 activity. The was into cells with expression for Sp1 family members in Sp1 and the of able to the HC-gp39 proximal promoter. expression for PU.1 a from was also or in with Sp1 or of the HC-gp39 proximal promoter was detectable with PU.1 and or was detectable with Sp1 or not In the we the transcriptional regulation of human cartilage during macrophage differentiation. a analysis of the proximal HC-gp39 promoter region and the of and factors that are important for the specific expression of HC-gp39 in In contrast to the differentiation of myeloid precursor cells, differentiation of human blood monocytes in and in vivo is not proliferation. Because a number of mechanisms differentiation are to cell other mechanisms during the differentiation of monocytes to the and of the cells, which include the various of macrophages, and myeloid dendritic not is known the factors and mechanisms gene expression during differentiation. To gain insight into the mechanisms during this is to study model genes. have previously a number of genes that are strongly up-regulated during macrophage differentiation (17Krause S.W. Rehli M. Kreutz M. Schwarzfischer L. Paulauskis J.D. Andreesen R. J. Leukocyte Biol. 1996; 60: 540-545Crossref PubMed Scopus (168) Google Scholar). the CHI3L1 gene the glycoprotein HC-gp39 to be model gene to study transcription regulation during differentiation. HC-gp39 is in peripheral blood monocytes and induced during in differentiation of macrophages (17Krause S.W. Rehli M. Kreutz M. Schwarzfischer L. Paulauskis J.D. Andreesen R. J. Leukocyte Biol. 1996; 60: 540-545Crossref PubMed Scopus (168) Google Scholar). a to the molecular mechanisms its expression we and the proximal promoter of the CHI3L1 The proximal promoter was to specific reporter in a human macrophage cell which was also to express high levels of HC-gp39 of HC-gp39 further induced of THP-1 cells with PMA, that the differentiation of THP-1 into macrophage-like cell assays be in human monocytes or we used footprinting analysis to sequence that are nuclear proteins in cell as compared with THP-1 in macrophages and THP-1 cells that the promoter and mechanisms in cell in monocytes or as compared with the strongly cell types, that the promoter in monocytes may in be for transcription gel shift with nuclear extracts from THP-1 cells identified a number of transcription factors binding the sequences that in the footprinting the most in in a region that transcription factors of the Sp1 and or transcription factors C/EBP, AML-1, or The proteins have been in or macrophage-specific gene regulation (2Friedman A.D. Oncogene. 2002; 21: 3377-3390Crossref PubMed Scopus (269) Google Scholar, 3Tenen D.G. Hromas R. Licht J.D. Zhang D.E. Blood. 1997; 90: 489-519Crossref PubMed Google and for important role in the macrophage-specific regulation of Deletion and the predominant of the Sp1 binding or of the Sp1 site the most the promoter and in of reporter activity. because of the of transcription start sites as as the of transcription factor binding the HC-gp39 promoter was other binding sites of of the transcription start sites in the Sp1 a predominant role for Sp1 family factors in HC-gp39 promoter activity. These are in with a number of Sp1 family proteins in the of genes during to macrophage differentiation D. Raveh D. M. M. J. Ezekowitz R.A. Blood. 1997; 90: PubMed Google Scholar, T. C. S. A. C. M. M. K. de G. J. 1999; Full Text Full Text PDF PubMed Google Scholar, S. C. T. P. C. G. J. 1998; Full Text Full Text PDF PubMed Google Scholar, S. T. J. Biol. Chem. Google Scholar, D.E. Hetherington C.J. 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