Cloning and Functional Characterization of the 5′-Flanking Region of the Human Monocyte Chemoattractant Protein-1 Receptor (CCR2) Gene
1999; Elsevier BV; Volume: 274; Issue: 8 Linguagem: Inglês
10.1074/jbc.274.8.4646
ISSN1083-351X
AutoresKeizô Yamamoto, Hideo Takeshima, Kazuya Hamada, Mitsuyoshi Nakao, Takeshi Kino, Toru Nishi, Masato Kochi, Jun‐ichi Kuratsu, Teizo Yoshimura, Yukitaka Ushio,
Tópico(s)Chemokine receptors and signaling
ResumoThe human monocyte chemoattractant protein-1 receptor designated hCCR2 is an essential co-receptor in cell entry by the human immunodeficiency virus as well as a receptor for monocyte chemoattractant protein-1, a member of the family of C-C chemokines that mediate monocyte chemotaxis. To elucidate the molecular mechanisms underlying the transcriptional regulation of hCCR2, we cloned and sequenced the hCCR2 gene; it was approximately 8 kilobase pairs in length and consisted of three exons divided by two introns. In the 5′-flanking region, there were the typical mammalian promoter consensus elements, a CAAT box and a TATA box, resulting in a single transcription initiation site. In addition, we found clustered tissue-specific cis-regulatory elements such as GATA consensus sequences, Oct-1 binding sequences, and CAAT/enhancer-binding protein binding sequences. Luciferase assays with various promoter deletions and gel mobility shift assays indicated that threecis-regulatory elements located within the region from −89 to +118 are required for basal activity in THP-1 cells. One element is an octamer sequence 36-base pair upstream from the TATA box; it binds mainly to Oct-1 and is capable of increasing transcriptional activity. The other two elements, which are tandem recognition sites of the CAAT/enhancer-binding protein family, are located in the 5′-untranslated region and account for the transcriptional activation as well as the tissue specificity of hCCR2. The human monocyte chemoattractant protein-1 receptor designated hCCR2 is an essential co-receptor in cell entry by the human immunodeficiency virus as well as a receptor for monocyte chemoattractant protein-1, a member of the family of C-C chemokines that mediate monocyte chemotaxis. To elucidate the molecular mechanisms underlying the transcriptional regulation of hCCR2, we cloned and sequenced the hCCR2 gene; it was approximately 8 kilobase pairs in length and consisted of three exons divided by two introns. In the 5′-flanking region, there were the typical mammalian promoter consensus elements, a CAAT box and a TATA box, resulting in a single transcription initiation site. In addition, we found clustered tissue-specific cis-regulatory elements such as GATA consensus sequences, Oct-1 binding sequences, and CAAT/enhancer-binding protein binding sequences. Luciferase assays with various promoter deletions and gel mobility shift assays indicated that threecis-regulatory elements located within the region from −89 to +118 are required for basal activity in THP-1 cells. One element is an octamer sequence 36-base pair upstream from the TATA box; it binds mainly to Oct-1 and is capable of increasing transcriptional activity. The other two elements, which are tandem recognition sites of the CAAT/enhancer-binding protein family, are located in the 5′-untranslated region and account for the transcriptional activation as well as the tissue specificity of hCCR2. Monocyte chemoattractant protein-1 (MCP-1) 1The abbreviations MCP-1monocyte chemoattractant protein-1CCRC-C chemokine receptorhCCRhuman CCRC/EBPCAAT/enhancer-binding proteinUTRuntranslated regionLucluciferasekbpkilobase pair(s)bpbase pair(s)PCRpolymerase chain reaction 1The abbreviations MCP-1monocyte chemoattractant protein-1CCRC-C chemokine receptorhCCRhuman CCRC/EBPCAAT/enhancer-binding proteinUTRuntranslated regionLucluciferasekbpkilobase pair(s)bpbase pair(s)PCRpolymerase chain reactionis a member of the C-C chemokine family that mediates leukocyte chemotaxis. We initially isolated it from the human malignant glioma cell line U-105 MG as a specific chemoattractant for monocytes (1Kuratsu J. Leonard E.J. Yoshimura T. J. Natl. Cancer Inst. 1989; 81: 347-351Crossref PubMed Scopus (36) Google Scholar, 2Yoshimura T. Robinson E.A. Tanaka S. Appella E. Kuratsu J. Leonard E.J. J. Exp. Med. 1989; 169: 1449-1459Crossref PubMed Scopus (406) Google Scholar). Physiologically, MCP-1 is produced by endothelial cells, smooth muscle cells, and macrophages in response to a variety of mediators including platelet-derived growth factor, tumor necrosis factor α, interleukin 1, epidermal growth factor, and interferon-γ (3Yoshimura T. Leonard E.J. Baggiolini M. Sorg C. Cytokine. 4. Karger, Basel, Switzerland1992: 131-152Google Scholar). The expression of MCP-1 is also increased under pathological conditions with monocyte-rich inflammatory processes such as atherosclerosis (4Takeya M. Yoshimura T. Leonard E.J. Takahashi K. Hum. Pathol. 1993; 24: 534-539Crossref PubMed Scopus (245) Google Scholar), rheumatoid arthritis (5Koch A.E. Kunkel S. Harlow S.L. Johnson L.A. Evanoff H.L. Haines G.K. Burdick M.D. Pope R.M. Strieter R.M. J. Clin. Invest. 1992; 90: 772-779Crossref PubMed Scopus (580) Google Scholar), and certain malignant tumors (6Walter S. Bottazzi B. Govoni D. Colotta F. Mantovani A. Int. J. Cancer. 1991; 49: 431-435Crossref PubMed Scopus (62) Google Scholar, 7Takeshima H. Kuratsu J. Takeya M. Yoshimura T. Ushio Y. J. Neurosurg. 1994; 80: 1056-1062Crossref PubMed Scopus (86) Google Scholar).The cDNA of the specific receptor for human MCP-1, which is designated hCCR2, was recently cloned and shown to belong to seven-transmembrane domain receptor families (8Charo I.F. Myers S.J. Herman A. Franci C. Connolly A.J. Coughlin S.R. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 2752-2756Crossref PubMed Scopus (637) Google Scholar) that couple via heterotrimeric G-proteins to affect cellular responses. The activation of hCCR2 is blocked by pertussis toxin, suggesting that hCCR2 couples to Gαi-class G-proteins (9Myers S.J. Wong L.M. Charo I.F. J. Biol. Chem. 1995; 270: 5786-5792Abstract Full Text Full Text PDF PubMed Scopus (162) Google Scholar). The hCCR2 gene has been cloned, and the mechanism of how it produces two alternatively spliced variants that differ only in their carboxyl-terminal tail has been elucidated (10Wong L.-M. Myers S.J. Tsou C.-L. Gosling J. Arai H. Charo I.F. J. Biol. Chem. 1997; 272: 1038-1045Crossref PubMed Scopus (111) Google Scholar). Human CCR5, one of the members of the C-C chemokine receptor family, acts as an essential cell surface co-receptor with CD4 in cell entry by macrophage-tropic human immunodeficiency virus type 1 strains (11Alkhatib G. Combadiere C. Broder C.C. Feng Y. Kennedy P.E. Murphy P.M. Berger E.A. Science. 1996; 272: 1955-1958Crossref PubMed Scopus (2437) Google Scholar, 12Deng H. Liu R. Ellmeier W. Choe S. Unutmaz D. Burkhart M. Di Marzio P. Marmon S. Sutton R.E. Hill C.M. Davis C.B. Peiper S.C. Schall T.J. Littman D.R. Landau N.R. Nature. 1996; 381: 661-666Crossref PubMed Scopus (3189) Google Scholar). In addition, hCCR2 and hCCR3 have been implicated as human immunodeficiency virus type 1 co-receptors in certain cell types (13Doranz B.J. Rucker J. Yi Y. Smyth R.J. Samson M. Peiper S.C. Parmentier M. Collman R.G. Doms R.W. Cell. 1996; 85: 1149-1158Abstract Full Text Full Text PDF PubMed Scopus (1684) Google Scholar, 14He J. Chen Y. Farzan M. Choe H. Ohagen A. Gartner S. Busciglio J. Yang X. Hofmann W. Newman W. Mackay C.R. Sodroski J. Gabuzda D. Nature. 1997; 385: 645-649Crossref PubMed Scopus (811) Google Scholar).We previously reported that tumor-associated macrophages attracted by MCP-1 inhibit the growth of transplanted rat tumors in vivo(15Yamashiro S. Takeya M. Nishi T. Kuratsu J. Yoshimura T. Ushio Y. Takahashi K. Am. J. Pathol. 1994; 145: 856-867PubMed Google Scholar). We posited that if we could stimulate the expression of MCP-1 in tumors and the expression of hCCR2 in monocytes, we would be able to obtain a greater inhibitory effect by a larger number of infiltrated macrophages against the growth of MCP-1-producing tumors such as human malignant glioma. The treatment of malignant tumors by the enhancement of an intrinsic immune system may be possible. The promoter region of the human MCP-1 gene has already been analyzed; it contains a distal nuclear factor κB binding site for induction by interleukin 1, tumor necrosis factor α, and a proximal GC box for basal transcriptional activity that is important for the transcriptional activation of MCP-1 (16Ueda A. Okuda K. Ohno S. Shirai A. Igarashi T. Matsunaga K. Fukushima J. Kawamoto S. Ishigatsubo Y. Okubo T. J. Immunol. 1994; 153: 2052-2063PubMed Google Scholar). However, the promoter region for thehCCR2 gene remains to be characterized.To elucidate the molecular mechanisms that regulate hCCR2, we cloned the hCCR2 gene, sequenced approximately 1.7 kbp of the 5′-flanking region, and mapped the transcription initiation site. We also assessed the promoter activity of the 5′-flanking region using luciferase (Luc) assays and gel mobility shift assays. We found that the Oct-1 binding sequence located 36 bp upstream from the TATA box and the tandem CAAT/enhancer-binding protein (C/EBP) binding sequences located at +50 to +77 within the 5′-untranslated region (UTR) are essential for the transcriptional activation and the tissue specificity of hCCR2 expression.DISCUSSIONIn the present work, we isolated and functionally characterized the 5′-flanking region of the hCCR2 gene to elucidate its regulatory mechanism. The structural analysis disclosed that the proximal 5′-flanking region of this gene contained a classic TATA box, a CAAT box, and clustered consensus sequences for tissue-specificcis-regulatory elements, i.e. the GATA binding sequence, Oct-1 binding sequence, and C/EBP binding motifs. This is compatible with the fact that the hCCR2 gene is not a housekeeping gene and is regulated in a cell-specific manner.Among the members of the CCR family, only the promoter region for hCCR5 has been isolated and functionally characterized to date (23Mummidi S. Ahuja S.S. McDaniel B.L. Ahuja S.K. J. Biol. Chem. 1997; 272: 30662-30671Crossref PubMed Scopus (153) Google Scholar). The transcription of the hCCR5 gene was initiated by two different promoters, and both proximal and distal promoters lack the canonical TATA or CAAT box. Instead, they contain the consensus sequence for several transcriptional factors such as Oct-1 and multiple motifs for GATA-1, which are also observed in the hCCR2gene. It is interesting that these closely related genes have rather different transcriptional initiations, although they are thought to share the same ancestral gene.The Luc assay using various deletion mutants of the hCCR2promoter region demonstrated that at least four regions are important for the constitutive expression of hCCR2 in THP-1 cells: (a) −429 to −186 for activation, (b) −185 to −123 for repression, (c) −89 to −59 for activation, and (d) +36 to +118 for cell specificity and activation. Among these four regions, we focused on the latter two, because the deletion mutant containing only these regions (−89 to +118) was sufficient for the basal expression and tissue specificity.The gel mobility shift assay and mutagenesis demonstrated that Oct-1 bound mainly to the octamer consensus sequence (ATGCAAAT) located in the third region (from −89 to −59) and was probably responsible for the trans-activation of this region. Although Oct-1 is ubiquitously expressed (24Schaffner W. Trends Genet. 1989; 5: 37-39Abstract Full Text PDF PubMed Scopus (75) Google Scholar), it is reduced or under the detectable level in CCR2-negative cell lines (J-111 and U-251 MG cells) (Fig. 8). This suggests that Oct-1 plays a certain role in the tissue-specific expression of hCCR2.The most important cis-acting element was located at a rather unusual position, the 5′-UTR of the hCCR2 gene (+36 to +118). It has been reported that the 5′-UTR could regulate the expression of genes in two different ways, one of which is that the 5′-UTR can work as a tissue-specific active translational enhancer taking a stable stem and loop structure; examples are the human γ-glutamyl transferase gene (25Diederich M. Wellman M. Visvikis A. Puga A. Siest G. FEBS Lett. 1993; 332: 88-92Crossref PubMed Scopus (21) Google Scholar), the thymidine kinase gene (26Pelletier J. Sonenberg N. Cell. 1985; 40: 515-526Abstract Full Text PDF PubMed Scopus (383) Google Scholar), the glutathione peroxidase gene (27Kurata H. FEBS Lett. 1992; 312: 10-14Crossref PubMed Scopus (17) Google Scholar), and the ornithine decarboxylase gene (28Kashiwagi K. Ito K. Igarashi K. Biochem. Biophys. Res. Commun. 1991; 178: 815-822Crossref PubMed Scopus (22) Google Scholar). Stable secondary structures were predicted between +12 and +118 of the hCCR2 gene, presenting the free energy formation of −43.6 kcal/mol. However, the present Northern blot analysis indicated that the hCCR2 gene is regulated at least at the transcriptional level in various cell lines (Fig. 4). Therefore, we should focus on the other mechanism, the transcriptional activity of the 5′-UTR, which has been demonstrated in the human plasminogen gene (29Meroni G. Buraggi G. Taramelli R. Eur. J. Biochem. 1996; 236: 373-382Crossref PubMed Scopus (25) Google Scholar) and the Aγ-globin gene (30Amrolia P.J. Cunningham J.M. Ney P. Nienhuis A.W. Jane S.M. J. Biol. Chem. 1995; 270: 12892-12898Crossref PubMed Scopus (47) Google Scholar), indicating that the transcriptional factor bound to a specific region within 5′-UTR.We particularly noted the position near +75, where two putative C/EBP binding sequences were located in tandem, because pGL3-0.18KΔE, which partially disrupted the C/EBP binding consensus, reduced the Luc activity by nearly 60%. The gel mobility shift assay using a 73-bp fragment containing tandem C/EBP binding sequences as a probe detected three prominent bands. Competition using the C/EBP consensus sequence and the supershift assay demonstrated that each band was related to C/EBPα, C/EBPβ, or C/EBPδ. Finally, the introduction of mutation in each motif disclosed that site 1 was the most important site for the tissue-specific expression of hCCR2.The C/EBP family of transcriptional factors is involved in tissue-specific gene expression in adipocytes, hepatocytes, and monocyte/macrophages (31Wedel A. Ziegler-Heitbrock H.W.L. Immunobiology. 1995; 193: 171-185Crossref PubMed Scopus (198) Google Scholar). In particular, target genes for C/EBP include acute phase response genes in liver cells and cytokine genes in monocytes/macrophages (31Wedel A. Ziegler-Heitbrock H.W.L. Immunobiology. 1995; 193: 171-185Crossref PubMed Scopus (198) Google Scholar). Our present results demonstrate that CCR2-negative cells (J-111 and U-251 cells) had considerably reduced expression of the C/EBP family (Fig. 8). Therefore, it is reasonable to speculate that the C/EBP binding sequence located in the 5′-UTR can regulate the expression of hCCR2 in a monocyte-specific manner.It is interesting that C/EBPβ and C/EBPδ contribute to the lipopolysaccharide response of MCP-1, the ligand of CCR2 (32Bretz J.D. Williams S.C. Baer M. Johnson P.F. Schwartz R.C. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 7306-7310Crossref PubMed Scopus (74) Google Scholar). MCP-1 and CCR2 are probably coordinately regulated at the transcriptional level by the C/EBP family for effective immune responses. The tumor cytotoxicity of macrophages from C/EBPβ knockout mice was severely impaired (33Tanaka T. Akira S. Yoshida K. Umemoto M. Yoneda Y. Shirafuji N. Fujiwara H. Suematsu S. Yoshida N. Kishimoto T. Cell. 1995; 80: 353-361Abstract Full Text PDF PubMed Scopus (464) Google Scholar). Our previous study showed that the growth of MCP-1-producing tumors was inhibited by the infiltration of tumor-associated macrophages (15Yamashiro S. Takeya M. Nishi T. Kuratsu J. Yoshimura T. Ushio Y. Takahashi K. Am. J. Pathol. 1994; 145: 856-867PubMed Google Scholar), indicating that MCP-1 and CCR2 may play an important role in the cytotoxicity of macrophages in cancer. Although we were unable to identify the stimulation that effectively induces the expression of hCCR2, our present findings provide a useful foundation for further studies. Monocyte chemoattractant protein-1 (MCP-1) 1The abbreviations MCP-1monocyte chemoattractant protein-1CCRC-C chemokine receptorhCCRhuman CCRC/EBPCAAT/enhancer-binding proteinUTRuntranslated regionLucluciferasekbpkilobase pair(s)bpbase pair(s)PCRpolymerase chain reaction 1The abbreviations MCP-1monocyte chemoattractant protein-1CCRC-C chemokine receptorhCCRhuman CCRC/EBPCAAT/enhancer-binding proteinUTRuntranslated regionLucluciferasekbpkilobase pair(s)bpbase pair(s)PCRpolymerase chain reactionis a member of the C-C chemokine family that mediates leukocyte chemotaxis. We initially isolated it from the human malignant glioma cell line U-105 MG as a specific chemoattractant for monocytes (1Kuratsu J. Leonard E.J. Yoshimura T. J. Natl. Cancer Inst. 1989; 81: 347-351Crossref PubMed Scopus (36) Google Scholar, 2Yoshimura T. Robinson E.A. Tanaka S. Appella E. Kuratsu J. Leonard E.J. J. Exp. Med. 1989; 169: 1449-1459Crossref PubMed Scopus (406) Google Scholar). Physiologically, MCP-1 is produced by endothelial cells, smooth muscle cells, and macrophages in response to a variety of mediators including platelet-derived growth factor, tumor necrosis factor α, interleukin 1, epidermal growth factor, and interferon-γ (3Yoshimura T. Leonard E.J. Baggiolini M. Sorg C. Cytokine. 4. Karger, Basel, Switzerland1992: 131-152Google Scholar). The expression of MCP-1 is also increased under pathological conditions with monocyte-rich inflammatory processes such as atherosclerosis (4Takeya M. Yoshimura T. Leonard E.J. Takahashi K. Hum. Pathol. 1993; 24: 534-539Crossref PubMed Scopus (245) Google Scholar), rheumatoid arthritis (5Koch A.E. Kunkel S. Harlow S.L. Johnson L.A. Evanoff H.L. Haines G.K. Burdick M.D. Pope R.M. Strieter R.M. J. Clin. Invest. 1992; 90: 772-779Crossref PubMed Scopus (580) Google Scholar), and certain malignant tumors (6Walter S. Bottazzi B. Govoni D. Colotta F. Mantovani A. Int. J. Cancer. 1991; 49: 431-435Crossref PubMed Scopus (62) Google Scholar, 7Takeshima H. Kuratsu J. Takeya M. Yoshimura T. Ushio Y. J. Neurosurg. 1994; 80: 1056-1062Crossref PubMed Scopus (86) Google Scholar). monocyte chemoattractant protein-1 C-C chemokine receptor human CCR CAAT/enhancer-binding protein untranslated region luciferase kilobase pair(s) base pair(s) polymerase chain reaction monocyte chemoattractant protein-1 C-C chemokine receptor human CCR CAAT/enhancer-binding protein untranslated region luciferase kilobase pair(s) base pair(s) polymerase chain reaction The cDNA of the specific receptor for human MCP-1, which is designated hCCR2, was recently cloned and shown to belong to seven-transmembrane domain receptor families (8Charo I.F. Myers S.J. Herman A. Franci C. Connolly A.J. Coughlin S.R. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 2752-2756Crossref PubMed Scopus (637) Google Scholar) that couple via heterotrimeric G-proteins to affect cellular responses. The activation of hCCR2 is blocked by pertussis toxin, suggesting that hCCR2 couples to Gαi-class G-proteins (9Myers S.J. Wong L.M. Charo I.F. J. Biol. Chem. 1995; 270: 5786-5792Abstract Full Text Full Text PDF PubMed Scopus (162) Google Scholar). The hCCR2 gene has been cloned, and the mechanism of how it produces two alternatively spliced variants that differ only in their carboxyl-terminal tail has been elucidated (10Wong L.-M. Myers S.J. Tsou C.-L. Gosling J. Arai H. Charo I.F. J. Biol. Chem. 1997; 272: 1038-1045Crossref PubMed Scopus (111) Google Scholar). Human CCR5, one of the members of the C-C chemokine receptor family, acts as an essential cell surface co-receptor with CD4 in cell entry by macrophage-tropic human immunodeficiency virus type 1 strains (11Alkhatib G. Combadiere C. Broder C.C. Feng Y. Kennedy P.E. Murphy P.M. Berger E.A. Science. 1996; 272: 1955-1958Crossref PubMed Scopus (2437) Google Scholar, 12Deng H. Liu R. Ellmeier W. Choe S. Unutmaz D. Burkhart M. Di Marzio P. Marmon S. Sutton R.E. Hill C.M. Davis C.B. Peiper S.C. Schall T.J. Littman D.R. Landau N.R. Nature. 1996; 381: 661-666Crossref PubMed Scopus (3189) Google Scholar). In addition, hCCR2 and hCCR3 have been implicated as human immunodeficiency virus type 1 co-receptors in certain cell types (13Doranz B.J. Rucker J. Yi Y. Smyth R.J. Samson M. Peiper S.C. Parmentier M. Collman R.G. Doms R.W. Cell. 1996; 85: 1149-1158Abstract Full Text Full Text PDF PubMed Scopus (1684) Google Scholar, 14He J. Chen Y. Farzan M. Choe H. Ohagen A. Gartner S. Busciglio J. Yang X. Hofmann W. Newman W. Mackay C.R. Sodroski J. Gabuzda D. Nature. 1997; 385: 645-649Crossref PubMed Scopus (811) Google Scholar). We previously reported that tumor-associated macrophages attracted by MCP-1 inhibit the growth of transplanted rat tumors in vivo(15Yamashiro S. Takeya M. Nishi T. Kuratsu J. Yoshimura T. Ushio Y. Takahashi K. Am. J. Pathol. 1994; 145: 856-867PubMed Google Scholar). We posited that if we could stimulate the expression of MCP-1 in tumors and the expression of hCCR2 in monocytes, we would be able to obtain a greater inhibitory effect by a larger number of infiltrated macrophages against the growth of MCP-1-producing tumors such as human malignant glioma. The treatment of malignant tumors by the enhancement of an intrinsic immune system may be possible. The promoter region of the human MCP-1 gene has already been analyzed; it contains a distal nuclear factor κB binding site for induction by interleukin 1, tumor necrosis factor α, and a proximal GC box for basal transcriptional activity that is important for the transcriptional activation of MCP-1 (16Ueda A. Okuda K. Ohno S. Shirai A. Igarashi T. Matsunaga K. Fukushima J. Kawamoto S. Ishigatsubo Y. Okubo T. J. Immunol. 1994; 153: 2052-2063PubMed Google Scholar). However, the promoter region for thehCCR2 gene remains to be characterized. To elucidate the molecular mechanisms that regulate hCCR2, we cloned the hCCR2 gene, sequenced approximately 1.7 kbp of the 5′-flanking region, and mapped the transcription initiation site. We also assessed the promoter activity of the 5′-flanking region using luciferase (Luc) assays and gel mobility shift assays. We found that the Oct-1 binding sequence located 36 bp upstream from the TATA box and the tandem CAAT/enhancer-binding protein (C/EBP) binding sequences located at +50 to +77 within the 5′-untranslated region (UTR) are essential for the transcriptional activation and the tissue specificity of hCCR2 expression. DISCUSSIONIn the present work, we isolated and functionally characterized the 5′-flanking region of the hCCR2 gene to elucidate its regulatory mechanism. The structural analysis disclosed that the proximal 5′-flanking region of this gene contained a classic TATA box, a CAAT box, and clustered consensus sequences for tissue-specificcis-regulatory elements, i.e. the GATA binding sequence, Oct-1 binding sequence, and C/EBP binding motifs. This is compatible with the fact that the hCCR2 gene is not a housekeeping gene and is regulated in a cell-specific manner.Among the members of the CCR family, only the promoter region for hCCR5 has been isolated and functionally characterized to date (23Mummidi S. Ahuja S.S. McDaniel B.L. Ahuja S.K. J. Biol. Chem. 1997; 272: 30662-30671Crossref PubMed Scopus (153) Google Scholar). The transcription of the hCCR5 gene was initiated by two different promoters, and both proximal and distal promoters lack the canonical TATA or CAAT box. Instead, they contain the consensus sequence for several transcriptional factors such as Oct-1 and multiple motifs for GATA-1, which are also observed in the hCCR2gene. It is interesting that these closely related genes have rather different transcriptional initiations, although they are thought to share the same ancestral gene.The Luc assay using various deletion mutants of the hCCR2promoter region demonstrated that at least four regions are important for the constitutive expression of hCCR2 in THP-1 cells: (a) −429 to −186 for activation, (b) −185 to −123 for repression, (c) −89 to −59 for activation, and (d) +36 to +118 for cell specificity and activation. Among these four regions, we focused on the latter two, because the deletion mutant containing only these regions (−89 to +118) was sufficient for the basal expression and tissue specificity.The gel mobility shift assay and mutagenesis demonstrated that Oct-1 bound mainly to the octamer consensus sequence (ATGCAAAT) located in the third region (from −89 to −59) and was probably responsible for the trans-activation of this region. Although Oct-1 is ubiquitously expressed (24Schaffner W. Trends Genet. 1989; 5: 37-39Abstract Full Text PDF PubMed Scopus (75) Google Scholar), it is reduced or under the detectable level in CCR2-negative cell lines (J-111 and U-251 MG cells) (Fig. 8). This suggests that Oct-1 plays a certain role in the tissue-specific expression of hCCR2.The most important cis-acting element was located at a rather unusual position, the 5′-UTR of the hCCR2 gene (+36 to +118). It has been reported that the 5′-UTR could regulate the expression of genes in two different ways, one of which is that the 5′-UTR can work as a tissue-specific active translational enhancer taking a stable stem and loop structure; examples are the human γ-glutamyl transferase gene (25Diederich M. Wellman M. Visvikis A. Puga A. Siest G. FEBS Lett. 1993; 332: 88-92Crossref PubMed Scopus (21) Google Scholar), the thymidine kinase gene (26Pelletier J. Sonenberg N. Cell. 1985; 40: 515-526Abstract Full Text PDF PubMed Scopus (383) Google Scholar), the glutathione peroxidase gene (27Kurata H. FEBS Lett. 1992; 312: 10-14Crossref PubMed Scopus (17) Google Scholar), and the ornithine decarboxylase gene (28Kashiwagi K. Ito K. Igarashi K. Biochem. Biophys. Res. Commun. 1991; 178: 815-822Crossref PubMed Scopus (22) Google Scholar). Stable secondary structures were predicted between +12 and +118 of the hCCR2 gene, presenting the free energy formation of −43.6 kcal/mol. However, the present Northern blot analysis indicated that the hCCR2 gene is regulated at least at the transcriptional level in various cell lines (Fig. 4). Therefore, we should focus on the other mechanism, the transcriptional activity of the 5′-UTR, which has been demonstrated in the human plasminogen gene (29Meroni G. Buraggi G. Taramelli R. Eur. J. Biochem. 1996; 236: 373-382Crossref PubMed Scopus (25) Google Scholar) and the Aγ-globin gene (30Amrolia P.J. Cunningham J.M. Ney P. Nienhuis A.W. Jane S.M. J. Biol. Chem. 1995; 270: 12892-12898Crossref PubMed Scopus (47) Google Scholar), indicating that the transcriptional factor bound to a specific region within 5′-UTR.We particularly noted the position near +75, where two putative C/EBP binding sequences were located in tandem, because pGL3-0.18KΔE, which partially disrupted the C/EBP binding consensus, reduced the Luc activity by nearly 60%. The gel mobility shift assay using a 73-bp fragment containing tandem C/EBP binding sequences as a probe detected three prominent bands. Competition using the C/EBP consensus sequence and the supershift assay demonstrated that each band was related to C/EBPα, C/EBPβ, or C/EBPδ. Finally, the introduction of mutation in each motif disclosed that site 1 was the most important site for the tissue-specific expression of hCCR2.The C/EBP family of transcriptional factors is involved in tissue-specific gene expression in adipocytes, hepatocytes, and monocyte/macrophages (31Wedel A. Ziegler-Heitbrock H.W.L. Immunobiology. 1995; 193: 171-185Crossref PubMed Scopus (198) Google Scholar). In particular, target genes for C/EBP include acute phase response genes in liver cells and cytokine genes in monocytes/macrophages (31Wedel A. Ziegler-Heitbrock H.W.L. Immunobiology. 1995; 193: 171-185Crossref PubMed Scopus (198) Google Scholar). Our present results demonstrate that CCR2-negative cells (J-111 and U-251 cells) had considerably reduced expression of the C/EBP family (Fig. 8). Therefore, it is reasonable to speculate that the C/EBP binding sequence located in the 5′-UTR can regulate the expression of hCCR2 in a monocyte-specific manner.It is interesting that C/EBPβ and C/EBPδ contribute to the lipopolysaccharide response of MCP-1, the ligand of CCR2 (32Bretz J.D. Williams S.C. Baer M. Johnson P.F. Schwartz R.C. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 7306-7310Crossref PubMed Scopus (74) Google Scholar). MCP-1 and CCR2 are probably coordinately regulated at the transcriptional level by the C/EBP family for effective immune responses. The tumor cytotoxicity of macrophages from C/EBPβ knockout mice was severely impaired (33Tanaka T. Akira S. Yoshida K. Umemoto M. Yoneda Y. Shirafuji N. Fujiwara H. Suematsu S. Yoshida N. Kishimoto T. Cell. 1995; 80: 353-361Abstract Full Text PDF PubMed Scopus (464) Google Scholar). Our previous study showed that the growth of MCP-1-producing tumors was inhibited by the infiltration of tumor-associated macrophages (15Yamashiro S. Takeya M. Nishi T. Kuratsu J. Yoshimura T. Ushio Y. Takahashi K. Am. J. Pathol. 1994; 145: 856-867PubMed Google Scholar), indicating that MCP-1 and CCR2 may play an important role in the cytotoxicity of macrophages in cancer. Although we were unable to identify the stimulation that effectively induces the expression of hCCR2, our present findings provide a useful foundation for further studies. In the present work, we isolated and functionally characterized the 5′-flanking region of the hCCR2 gene to elucidate its regulatory mechanism. The structural analysis disclosed that the proximal 5′-flanking region of this gene contained a classic TATA box, a CAAT box, and clustered consensus sequences for tissue-specificcis-regulatory elements, i.e. the GATA binding sequence, Oct-1 binding sequence, and C/EBP binding motifs. This is compatible with the fact that the hCCR2 gene is not a housekeeping gene and is regulated in a cell-specific manner. Among the members of the CCR family, only the promoter region for hCCR5 has been isolated and functionally characterized to date (23Mummidi S. Ahuja S.S. McDaniel B.L. Ahuja S.K. J. Biol. Chem. 1997; 272: 30662-30671Crossref PubMed Scopus (153) Google Scholar). The transcription of the hCCR5 gene was initiated by two different promoters, and both proximal and distal promoters lack the canonical TATA or CAAT box. Instead, they contain the consensus sequence for several transcriptional factors such as Oct-1 and multiple motifs for GATA-1, which are also observed in the hCCR2gene. It is interesting that these closely related genes have rather different transcriptional initiations, although they are thought to share the same ancestral gene. The Luc assay using various deletion mutants of the hCCR2promoter region demonstrated that at least four regions are important for the constitutive expression of hCCR2 in THP-1 cells: (a) −429 to −186 for activation, (b) −185 to −123 for repression, (c) −89 to −59 for activation, and (d) +36 to +118 for cell specificity and activation. Among these four regions, we focused on the latter two, because the deletion mutant containing only these regions (−89 to +118) was sufficient for the basal expression and tissue specificity. The gel mobility shift assay and mutagenesis demonstrated that Oct-1 bound mainly to the octamer consensus sequence (ATGCAAAT) located in the third region (from −89 to −59) and was probably responsible for the trans-activation of this region. Although Oct-1 is ubiquitously expressed (24Schaffner W. Trends Genet. 1989; 5: 37-39Abstract Full Text PDF PubMed Scopus (75) Google Scholar), it is reduced or under the detectable level in CCR2-negative cell lines (J-111 and U-251 MG cells) (Fig. 8). This suggests that Oct-1 plays a certain role in the tissue-specific expression of hCCR2. The most important cis-acting element was located at a rather unusual position, the 5′-UTR of the hCCR2 gene (+36 to +118). It has been reported that the 5′-UTR could regulate the expression of genes in two different ways, one of which is that the 5′-UTR can work as a tissue-specific active translational enhancer taking a stable stem and loop structure; examples are the human γ-glutamyl transferase gene (25Diederich M. Wellman M. Visvikis A. Puga A. Siest G. FEBS Lett. 1993; 332: 88-92Crossref PubMed Scopus (21) Google Scholar), the thymidine kinase gene (26Pelletier J. Sonenberg N. Cell. 1985; 40: 515-526Abstract Full Text PDF PubMed Scopus (383) Google Scholar), the glutathione peroxidase gene (27Kurata H. FEBS Lett. 1992; 312: 10-14Crossref PubMed Scopus (17) Google Scholar), and the ornithine decarboxylase gene (28Kashiwagi K. Ito K. Igarashi K. Biochem. Biophys. Res. Commun. 1991; 178: 815-822Crossref PubMed Scopus (22) Google Scholar). Stable secondary structures were predicted between +12 and +118 of the hCCR2 gene, presenting the free energy formation of −43.6 kcal/mol. However, the present Northern blot analysis indicated that the hCCR2 gene is regulated at least at the transcriptional level in various cell lines (Fig. 4). Therefore, we should focus on the other mechanism, the transcriptional activity of the 5′-UTR, which has been demonstrated in the human plasminogen gene (29Meroni G. Buraggi G. Taramelli R. Eur. J. Biochem. 1996; 236: 373-382Crossref PubMed Scopus (25) Google Scholar) and the Aγ-globin gene (30Amrolia P.J. Cunningham J.M. Ney P. Nienhuis A.W. Jane S.M. J. Biol. Chem. 1995; 270: 12892-12898Crossref PubMed Scopus (47) Google Scholar), indicating that the transcriptional factor bound to a specific region within 5′-UTR. We particularly noted the position near +75, where two putative C/EBP binding sequences were located in tandem, because pGL3-0.18KΔE, which partially disrupted the C/EBP binding consensus, reduced the Luc activity by nearly 60%. The gel mobility shift assay using a 73-bp fragment containing tandem C/EBP binding sequences as a probe detected three prominent bands. Competition using the C/EBP consensus sequence and the supershift assay demonstrated that each band was related to C/EBPα, C/EBPβ, or C/EBPδ. Finally, the introduction of mutation in each motif disclosed that site 1 was the most important site for the tissue-specific expression of hCCR2. The C/EBP family of transcriptional factors is involved in tissue-specific gene expression in adipocytes, hepatocytes, and monocyte/macrophages (31Wedel A. Ziegler-Heitbrock H.W.L. Immunobiology. 1995; 193: 171-185Crossref PubMed Scopus (198) Google Scholar). In particular, target genes for C/EBP include acute phase response genes in liver cells and cytokine genes in monocytes/macrophages (31Wedel A. Ziegler-Heitbrock H.W.L. Immunobiology. 1995; 193: 171-185Crossref PubMed Scopus (198) Google Scholar). Our present results demonstrate that CCR2-negative cells (J-111 and U-251 cells) had considerably reduced expression of the C/EBP family (Fig. 8). Therefore, it is reasonable to speculate that the C/EBP binding sequence located in the 5′-UTR can regulate the expression of hCCR2 in a monocyte-specific manner. It is interesting that C/EBPβ and C/EBPδ contribute to the lipopolysaccharide response of MCP-1, the ligand of CCR2 (32Bretz J.D. Williams S.C. Baer M. Johnson P.F. Schwartz R.C. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 7306-7310Crossref PubMed Scopus (74) Google Scholar). MCP-1 and CCR2 are probably coordinately regulated at the transcriptional level by the C/EBP family for effective immune responses. The tumor cytotoxicity of macrophages from C/EBPβ knockout mice was severely impaired (33Tanaka T. Akira S. Yoshida K. Umemoto M. Yoneda Y. Shirafuji N. Fujiwara H. Suematsu S. Yoshida N. Kishimoto T. Cell. 1995; 80: 353-361Abstract Full Text PDF PubMed Scopus (464) Google Scholar). Our previous study showed that the growth of MCP-1-producing tumors was inhibited by the infiltration of tumor-associated macrophages (15Yamashiro S. Takeya M. Nishi T. Kuratsu J. Yoshimura T. Ushio Y. Takahashi K. Am. J. Pathol. 1994; 145: 856-867PubMed Google Scholar), indicating that MCP-1 and CCR2 may play an important role in the cytotoxicity of macrophages in cancer. Although we were unable to identify the stimulation that effectively induces the expression of hCCR2, our present findings provide a useful foundation for further studies. We thank Ursula Petralia for editorial assistance.
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