From the Divisions of ** Endocrinology and Metabolism,
¶ Cellular and Molecular Medicine, and Cardiology,
Department of Medicine, University of California, San Diego,
La Jolla, California 92093-0651
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ABSTRACT |
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Macrosialin is a transmembrane glycoprotein that
is highly expressed in macrophages. In the present studies, macrosialin
mRNA levels are shown to be markedly up-regulated during macrophage differentiation of bone marrow progenitor cells in response to macrophage colony-stimulating factor and granulocyte-macrophage colony-stimulating factor. To investigate the mechanisms
responsible for regulation of macrosialin expression, we have
isolated the macrosialin gene and performed an initial analysis of its
transcriptional regulatory elements. The macrosialin promoter and 7.0 kilobase pairs of 5'-flanking information direct high levels of
reporter gene activity in monocyte/macrophage-like cells, but little or no expression in nonmyeloid cells. This pattern of expression is
dependent on regulatory elements located between 7.0 and
2.5 kilobase pairs from the transcriptional start site that exhibit strong enhancer activity in macrophages and repressor activity in
nonmyeloid cells. Analysis of the proximal macrosialin promoter indicates that combinatorial interactions between at least four classes
of transcriptional activators, including PU.1/Spi-1 and members of the
AP-1 family are required for basal promoter function. PU.1/Spi-1 and
c-Jun act synergistically to activate the macrosialin promoter in
a nonmyeloid cell line, suggesting that combinatorial interactions
between these proteins are involved in regulating macrosialin
expression during macrophage differentiation.
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INTRODUCTION |
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Macrosialin is a heavily glycosylated murine transmembrane protein that belongs to the lysosomal/endosomal-associated membrane glycoprotein (lamp)1 family (1-4). Unlike other members of this family, which are constitutively and ubiquitously expressed in all cell types, macrosialin is specifically expressed in macrophages, and to a lesser extent in dendritic cells (reviewed by Gordon et al. (5)) (6, 7). CD68, the human homologue of macrosialin (8), is expressed very early during granulomonopoietic differentiation, with intracellular staining found in bone marrow progenitor cells that express CD34 and myeloperoxidase (9). During further myeloid differentiation, CD68 remains strongly expressed in myeloperoxidase-positive, lactoferrin-negative, and CD14-negative cells. These cells represent myeloblasts, promyelocytes, and promonocytes. Interestingly, terminal differentiation toward the neutrophil/granulocyte lineage results in a marked decrease in CD68 expression, while continued differentiation toward the monocytic lineage is accompanied by further up-regulation of CD68 (9). In contrast to macrosialin expression, CD68 expression also has been localized, although at much lower levels, in certain lymphocyte subsets, megakaryocytes, and malignant hematopoietic cells (9-11). This somewhat more extended pattern of expression may reflect differences in antibody specificities or slightly different functions between macrosialin and CD68 (7, 12).
The biological functions of macrosialin and CD68 are not known. Although some studies have found CD68 to be exclusively localized in intracellular membrane compartments (13) in resting macrophages, more sensitive methods have detected a small fraction on the cell surface (9). Because macrosialin and CD68 are highly expressed, this small percentage of surface expression may nevertheless be of functional significance. Macrosialin and CD68 possess unique mucin-like extracellular domains located at the N-terminal region. In response to inflammatory stimuli, these regions undergo complex alterations in their patterns of N- and O-linked glycosylation (7, 14) (reviewed by da Silva et al. (15)), and an increased fraction of macrosialin is found on the cell surface (16). The glycosylated regions of macrosialin and CD68 may play a role in protecting these proteins from the harsh hydrolytic environment found in lysosomes and/or may act as ligands for cell adhesion molecules, such as selectins. Saitoh et al. (17) have demonstrated that lamp-1 on leukemic cells can bind to E-selectin (17). Because macrosialin and CD68 are strongly expressed in monocytes and undergo changes in cell surface expression during an inflammatory response, it has been postulated that they might play roles in phagocytosis, and cell-cell and cell-pathogen interactions (1). Recently, macrosialin has been demonstrated to bind oxidized low density lipoproteins and account for 30-50% of its uptake by activated THP-1 cells in vitro (16, 18), suggesting that it may contribute to the development of macrophage foam cells in atherosclerotic lesions.
Because expression of macrosialin is up-regulated during macrophage differentiation, it also provides a model for understanding mechanisms that control macrophage-specific gene expression. The development of macrophages from bone marrow progenitor cells is regulated by a myriad of cytokines and colony-stimulating factors that include M-CSF and GM-CSF (19). Although significant progress has been made in identifying components of the signal transduction pathways that are activated by M-CSF and GM-CSF, the mechanisms by which these factors act to regulate the transcription of specific target genes so as to coordinate the proliferation and development of the monocytic lineage remain poorly understood.
To investigate molecular mechanisms that regulate early events in the
program of macrophage differentiation, we have isolated the macrosialin
gene, defined the exon structure, and performed an initial
characterization of its promoter. The macrosialin promoter directs high
levels of reporter gene expression in several monocyte/macrophage cell
lines. Genomic sequences residing between 2.5 and
7 kb upstream of
the translational start site contain cis-active elements that exhibit enhancer activities in monocyte/macrophage cells and
silencer activities in nonmyeloid cells. Analysis of the proximal promoter suggests that combinatorial interactions between several classes of transcription factors are required for high levels of
activity. Mutations or deletions of binding sites for PU.1/Spi-1 and
AP-1 severely impair macrosialin promoter activity. Conversely, high
levels of macrosialin promoter activity can be established in a
nonmyeloid cell by co-expression of c-Jun and PU.1/Spi-1. These
observations suggest that PU.1 and c-Jun functionally cooperate to
regulate macrosialin expression during macrophage differentiation.
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EXPERIMENTAL PROCEDURES |
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Materials--
The murine embryonic stem cell P1 library was
obtained from Genomic Systems. Human recombinant M-CSF, IFN-, murine
recombinant GM-CSF, TNF-
, and IL-3 were from R&D Systems. Murine
recombinant IFN-
was purchased from Genzyme. TPA and LPS were from
Sigma. Plasmids Bluescript SK II and KS II were from Stratagene;
pCDNA-3 was from Invitrogen; and pGEM T and the TNT®-coupled
reticulocyte lysate system were from Promega. DNA sequencing was
performed using the Sequenase 7-diaza-dGTP DNA sequencing kit (U. S. Biochemical Corp.) and the 35S-ATP from Amersham Corp.
[
-32P]dCTP, [
-32P]ATP, and
[
-32P]UTP were obtained from Andotek. Restriction
enzymes were from New England Biolabs, and modifying enzymes and
Lipofectin were from Life Technologies Inc. DOTAP was purchased from
Boehringer Mannheim. Luciferin was purchased from Analytical
Luminesence, and Galacto-Light was purchased from Tropix. Poly(dI·dC)
and Ficoll-Hypaque were purchased from Pharmacia Biotech Inc. c-Jun and
Jun-B antibodies were obtained from Santa Cruz Biotechnology.
Isolation of Macrophages and Bone Marrow Progenitor
Cells--
Thioglycollate-induced mouse peritoneal macrophages were
obtained from B6/D2 mice, and bone marrow cells were isolated and purified on Ficoll-Hypaque gradients as described previously (20). After initial purification, the bone marrow progenitor cells (5.0 × 107 cells) were plated onto 150-mm tissue culture plates
in 50 ml of bone marrow medium (21). The cells were treated with either recombinant human M-CSF (20 ng/ml), recombinant murine GM-CSF (4 ng/ml), or recombinant murine IFN- (1000 units/ml) and assayed 24-72 h later.
RNA Analysis--
Total RNA was isolated by the guanidium
thiocyanate method (22). RNase protection assays were performed as
described previously (23). The antisense RNA probe for macrosialin
corresponded to nucleotides 787-1072. The antisense probe for rat
-actin corresponded to nucleotides 2452-2594. The probes were
hybridized with 20 µg of total RNA or tRNA as a control, digested
with RNase A (30 µg/ml), and analyzed on a 10% denaturing
polyacrylamide gel.
Isolation and Characterization of Genomic Clones-- A mouse embryonic stem cell P1 library was screened using PCR oligonucleotides of sequence 5'-CTGATCTTGCTAGGACCGCT-3' and 5'-GCTGGTCGTAGGGCTG-3' corresponding to nucleotides 110-129 and 222-241 of the macrosialin cDNA, respectively. Three overlapping P1 clones were obtained. The PCR product generated with the two oligonucleotides was used to probe restriction fragments of one of these clones. An 8.5-kb BamHI fragment hybridizing to the PCR product was subcloned into Bluescript SK II, generating the plasmid JC59. Sequence analysis confirmed that this fragment contained the 5' end of the macrosialin cDNA, but lacked the 3' end of the macrosialin-coding sequences. Therefore oligonucleotides corresponding to the 3' end of the macrosialin cDNA (5'-CAGAATTCATCTCTTCGAGAGCTC-3' and 5'-GATGCTCAGAGGGGCTGGT-3' corresponding to nucleotides 785-807 and 1058-1077, respectively) were used to amplify genomic macrosialin sequences. A 500-bp PCR product was generated and subcloned into pGEM T, JC67. Sequence confirmed that it contained the 3' end of the macrosialin gene. A 180-bp fragment from JC67 was used to probe the P1 clone that had been digested with BamHI in a Southern blot. A 3.2-kb fragment was subcloned into Bluescript SK II and confirmed by sequencing to contain the 3' end of genomic macrosialin and the flanking region generating plasmid, JC74. Sequence analysis was performed using an ABI automated DNA sequencer and the MacDNAsis program (Hitachi).
Identification of the Transcriptional Start Site by Primer
Extension and RNase Protection Analysis--
The transcriptional start
site was mapped by primer extension and RNase protection assays. In
primer extension assays, antisense oligonucleotides
(5'-CCAGCTAGGCTACACCAGTTCCTTC-3' and 5'-AGGGAGAAGCTTGGCAGAGATGC-3') corresponding to nucleotides 41 to
17 and
11 to +11, relative to
the translation start site in the macrosialin gene were labeled with
[
-32P]ATP using T4 polynucleotide kinase
and annealed at 30 °C with 10 µg of total murine peritoneal
macrophage RNA. Annealing conditions and subsequent procedures were
carried out as described previously (22). For RNase protection studies
plasmid JC59, containing the genomic fragment of the macrosialin
5'-flanking region, was linearized with NsiI, which is
located 235 bp upstream of the translational start site. A
32P-labeled antisense cRNA probe was generated from this
plasmid using T7 RNA polymerase, and RNase protection
assays were performed using 20 µg of total RNA as described
above.
Constructions of Reporter Constructs and Site-directed
Mutagenesis--
Macrosialin-luciferase reporter genes were
constructed by changing the translational start site ATG to a
EcoRI site by PCR mutagenesis using an antisense
oligonucleotide of sequence 5'-AGGGAGAATTCTGGCAGAGATGC-3' and a sense primer of sequence 5'-CAAGCCTTTAATTCCCAGCAT-3'
corresponding to the sequence starting 664 bp upstream from the
macrosialin translational start site in plasmid JC59. The 675-bp PCR
product was sequenced and verified to be correct. The PCR product was digested with NheI and EcoRI and used to replaced
the corresponding sequence within the wild type macrosialin gene and
subcloned into Bluescript KS II (AL9). The 7.0 kb 5'-flanking region
was excised with BamHI and EcoRI and subcloned
into
5'PSV2 luciferase to generate Mac 7.0-luciferase. Deletions of
the 5'-flanking region were created by restriction enzyme digestion
using the following enzymes: NotI (
5.5 kb),
HindIII (
2.5 kb), Spe I (
803 bp), NheI (
614
bp), and PvuII (
250 bp). Additional 5' deletions were
generated by PCR using an antisense primer
5'-AGGGAGAAGCTTGGCAGATGC-3', replacing the ATG site with a
HindIII site, and the following sense primers containing
BamHI restriction sites:
1060,
5'-ATTTGCTGGATCCAATCTACAG-3';
221, 5'-GAGGTAACGGATCCTTTGTAC-3';
203, 5'-CGCCCGGATCCGAACGTCAC3-';
133,
5'-GCTGAGGATCCTGAGTCAGGT-3';
108, 5'-GTGGGATCCTTTTAGTTAAGG-3';
77, 5'-GGCTTTGGATCCCCTCTTCCA-3'. The
31 to
1 construct was generated by annealing two complementary oligonucleotides of sequence 5'-GATCCTGTGTAGCCTAGCTGGTCTGAGCATCTCTGCCA-3' and 5'-
AGCTTGGCAGAGATGCTCAGACCAGCTAGGCTACACAG-3' and subcloning these
into the
5' PSV2-luciferase reporter gene at the BamHI
and HindIII sites. In addition, a 3' deletion was generated,
deleting a putative Ets binding site at
46, using an antisense
oligonucleotide of sequence 5'-CCTCAAGCTTATCCCCTTTGCCTTCTC-3' and the
221 sense oligonucleotide described above for PCR amplification. Similarly, mutations in the AP-1, PU.1/Spi-1, GC region and the CCAAT
binding sites were generated by PCR using the
221 construct as a
template. A general strategy for developing the mutations of these
binding sites using overlapping PCR mutagenesis was employed. For
example, the mutation of the AP-1 site at
132 was constructed by
creating external primers containing BamHI and
HindIII sites at
221 and
1 primers respectively as
described above. The internal primers
5'-TGAGGTGTCCTCGAGAGGTTT-3' (forward) and
5'-AAACCTCTCGAGGACACCTCA-3' (reverse) introduced an
XhoI restriction site into the AP-1 site. The two PCR
products were digested with either BamHI and
XhoI, or XhoI and HindIII, ligated
together and subcloned into the luciferase expression vector at
BamHI and HindIII sites. Mutations of the PU.1/Spi-1, GC region and CCAAT boxes were similarly done introducing a
NotI restriction site into the PU.1/SPi-1 site
(5'-TATTTTAGTGCGGCCGCGTGAGGCTTT-3' (forward) and
5'-AAAGCCTCACGCGGCCGCACTAAAATA-3' (reverse)); an EcoRI restriction site into the GC region
(5'-TAACGGATCCTTTGTAGAATTCACTGA-3' (forward) and
5'TCAGTGAATTCTACAAAGGATCCGTTA-3' (reverse)); and a
BglII restriction site into the second CCAAT box
(5'-TGTGAAAAGATCTGGCTTGAGTGG-3' (forward) and
5'-CCACTCAAGCCAGATCTTTTCACA-3' (reverse)). For the 1st
CCAAT box mutation, a PCR product was made using a primer containing a
XbaI restriction site at the CCAAT box region
(5'-TAACGGATCCTTTGTACCGCCCACTGAGAACGTCACTGTCTAGAACAGCCTAAT-3') and the
1 antisense HindIII oligonucleotide primer. All
constructs generated by PCR were confirmed by dideoxy sequencing and
restriction enzyme digestion.
Cell Culture and Transient Expression Analysis--
Transient
transfections using THP-1 (monocytic leukemia), U937 (histocytic
leukemia), HL-60 (acute promyelocytic leukemia), Jurkat (T cells), and
BaF/3 (murine pro-B cell) and BaF/3 cells expressing the murine GM-CSF
-receptor (gifts from A. D. D'Andrea) were performed by
electroporation as described previously (24) using 5-10 µg of total
plasmid DNA. HeLa (cervical endothelial), P-19 (mouse embryonic
carcinoma), and GC-3 (anterior pituitary) were transfected by the
calcium phosphate method (25) using 2 µg of total plasmid DNA. MCF-7
(breast adenocarcinoma) and RAW 264.7 (murine monocyte) were
transfected with 2 µg of total plasmid DNA using DOTAP and
Lipofectin, respectively, following the manufacturer's instructions.
The myeloid cell lines were harvested for luciferase and
-galactosidase activity 24 h after the time of transfection, whereas the nonmyeloid and the RAW 264.7 cells were harvested 48 h
after transfection. Luciferase activity was measured in a Monolight
2010 luminometer (Analytical Luminesence) as described previously (26).
Luciferase activity was normalized to
-galatosidase activity
directed by a co-transfected plasmid containing the
-actin promoter
linked to the
-galatosidase gene.
-Galactosidase activity was
assayed using Galacto-Light following the manufacturer's instructions and also measured in the Monolight 2010 luminometer. An equimolar amount of the
-actin luciferase construct was used as an external standard. U937 and BaF/3 cell lines were also treated with various cytokines and chemokines for 14-48 h prior to harvesting. After electroporation, the U937 cells were resuspended in 0.5% fetal bovine
serum in RPMI prior to treatment. The BaF/3 cells were maintained in
either RPMI with 10% fetal bovine serum and 5% WEHI-3 conditioned
medium or RPMI with 1% fetal bovine serum and 0.5 ng/ml murine IL-3.
Prior to electroporation, the BaF/3 cells were cytokine-starved for
6 h and resuspended in 0.5-1.0% fetal bovine serum without
cytokines in RPMI after electroporating the cells. The cells were
treated with either TPA, 1 × 10
7 M;
LPS, 10 ng/ml; retinoic acid, 1 × 10
6
M; recombinant human and murine GM-CSF, 4 ng/ml;
recombinant human IFN-
, 1000 units/ml; TNF-
, 100 ng/ml; and
recombinant murine IL-3, 10 ng/ml. A
-actin/
-galatosidase
construct was used as an internal control in the U937 cells and a
SV40-
-galatosidase construct was used in the BaF/3 cells.
Co-transfections of expression vectors into P-19 cells used 1.0 µg of
reporter plasmid and either or both 100 ng of a CMV-PU.1 expression
vector and 100 ng of a CMV-c-Jun expression vector. 50 ng of
-actin/
-galatosidase construct was also co-transfected and used
as an internal standard. 100-200 ng of the empty vector, pcDNA-3,
were used as a control. Salmon sperm DNA was used as a carrier to
equalize the total amount of transfected DNA. All transfections were
performed with triplicate points, at least three times.
Preparation of Nuclear Extracts and Electrophoretic Mobility
Shift Assays--
Nuclear extracts were prepared from TPA-treated
THP-1 cells as described previously (27). To determine the binding of
nuclear factors to regions within the macrosialin promoter,
double-stranded oligonucleotides with 5' overhangs were synthesized.
The oligonucleotides were labeled either with
[-32P]dCTP using the Klenow fragment of DNA polymerase
or with [
-32P]dATP using T4 polynucleotide
kinase. The sequences of the oligonucleotides corresponding to the
macrosialin AP-1 element were 5'-gatccAGGTGTCTGAGTCAGGTTTGG-3' (sense)
and 5'-gatctCCAAACCTGACTCAGACACCT-3' (antisense), where lowercase
letters denote non-native sequences added to facilitate cloning. The
sequences of the sense and antisense oligonucleotide corresponding to
the macrosialin PU.1/Spi-1 element were 5'-gatccGTTAAGGGAAGTGA-3' and
5'-gatctTCACTTCCCTTAAC-3', respectively. Five micrograms of nuclear
extract were incubated with 0.5 µg of poly(dI·dC) and 5-50-fold
molar excess of specific or mutant unlabeled competitor (5'-
TGAGGTGTCCTCGAGAGGTTT-3' for the AP-1 mutation and
5'-TATTTTAGTGCGGCCGCGTGAGGCTTT-3' for the PU.1/Spi-1
mutation) in 150 mM KCl, 10 mM Tris, pH 8.0, 0.1 mM EDTA, 50 mM dithiothreitol, 5 µg of
bovine serum albumin, and 5% glycerol for 30 min on ice. One
microliter of probe (100,000 cpm) was added to the reaction mixture and
incubated for another 30 min on ice. For supershift assay, 1-2 µl of
antibody were added to the mixture and preincubated on ice for 1 h. The reaction mixtures were spun down at 4 °C and run on a 6%
nondenaturing polyacrylamide gel at 300 V. Anti c-Jun monoclonal IgG
raised against a peptide corresponding to amino acids 56-69 of the
human c-Jun and anti-Jun-B polyclonal IgG raised against amino acids
45-61 of mouse Jun-B were used. Guinea pig anti-PU.1 antiserum was
raised against a recombinant peptide, corresponding to amino acids
157-272, that is specific to the DNA binding domain. In
vitro translated products of c-Jun and PU.1 were synthesized using
the TNT®-coupled reticulocyte lysate system following the
manufacturer's instructions.
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RESULTS |
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Macrosialin mRNA Is Up-regulated during Macrophage
Differentiation--
To assess macrosialin expression during
macrophage differentiation, RNase protection assays were performed.
Bone marrow progenitor cells were enriched by purification
through Ficoll gradients and cultured in the presence of M-CSF or
GM-CSF to induce proliferation and differentiation. Macrosialin
mRNA was detected in the bone marrow progenitor cells, consistent
with the observation that CD68 is expressed in CD34+ cells
(9). In addition, macrosialin mRNA levels were markedly up-regulated in response to both M-CSF and GM-CSF within 24 h, reaching maximal levels by 72 h in M-CSF-treated cells.
Intriguingly IFN-, a potent regulator of numerous inflammatory
responses in macrophages (28), inhibited the induction of macrosialin
mRNA in response to M-CSF by more than 50%.
Thioglycollate-elicited peritoneal macrophages demonstrated the highest
levels of macrosialin mRNA, which were approximately half as
abundant as
-actin mRNA when corrected for the relative specific
activities of the macrosialin and
-actin probes (Fig.
1).
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Cloning and Characterization of the Macrosialin Gene-- To investigate molecular mechanisms controlling macrosialin gene expression at the transcriptional level, genomic clones containing macrosialin regulatory elements and coding sequences were isolated from a mouse P1 clone (Fig. 2A). Two adjacent BamHI fragments were subcloned that together contain 7.0 kb of 5'-flanking information, the macrosialin structural gene, and 1.35 kb of 3'-flanking information. Comparison of the genomic and cDNA sequences indicate that the primary macrosialin transcript contains six exons. With the exception of the junction between exon 4 and exon 5, all of the assigned splice donor and acceptor sites exhibit close matches to consensus splice donor and acceptor sequences. Exon 1, which is of variable length due to multiple transcriptional start sites, as described below, contains the translational start site and encodes the first 14 amino acids of the signal peptide. Exon 2, which is the largest exon (439 bp), encodes the remaining 7 amino acids of the signal peptide and the majority of serine and threonine residues thought to be substrates for O-linked glycosylation. In addition, exon 2 encodes five potential N-linked glycosylation sites (Asn-X-Ser/Thr), the so-called proline hinge, and the first of four cysteine residues that are conserved in members of the lamp family and are involved in intramolecular disulfide bonds. Exon 3 contains the second conserved cysteine residue and one potential N-linked glycosylation site. Exon 4 contains a single potential N-linked glycosylation site, while exon 5 contains two potential N-linked glycosylation sites and the third conserved cysteine residue. Exon 6 contains the final conserved cysteine residue and encodes the transmembrane domain and cytoplasmic tail. The predicted cytoplasmic tail sequence is RRRQSTYQPL, which is similar to other members of the lamp family in containing three basic amino acids and a conserved tyrosine. Previous studies have suggested that an alternative form of macrosialin might be expressed with a truncated C-terminal tail of sequence RR*, due to either a second, highly related gene or an alternative exon (1). No evidence was found for an alternative exon that would encode a truncated cytoplasmic domain in the 1.35 kb of the further 3'-flanking sequence. As only one genomic P1 clone was characterized, the possibility of a second highly related gene cannot be excluded. However, Southern blotting experiments using probes to the 3' end of the cloned macrosialin gene have thus far been consistent with the presence of a single gene. These observations suggest that versions of the macrosialin cDNA that encode truncated proteins may have arisen as artifacts during PCR amplification or cDNA library construction.
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Transcription of the Macrosialin Gene Is Initiated at Multiple
Start Sites--
The transcription start sites of the macrosialin
gene were determined by primer extension and RNase protection
analysis using total RNA from murine peritoneal macrophages that
were thioglycollate-elicited to maximize the macrosialin mRNA
levels. Two oligonucleotides that are complementary to the macrosialin
mRNA from 17 to
41 and +11 to
11 bp from the translational
start site were used. Primer extension experiments utilizing the
antisense oligonucleotide corresponding to nucleotides
17 to
41 of
the macrosialin cDNA relative to the translational start site
resulted in several extension products, with the majority ending 55-79
nucleotides upstream of the initiator methionine (Fig.
3A). Extension products were not detected for the +11 to
11 oligonucleotide, possibly due to
secondary structure of the macrosialin mRNA. To confirm the presence of multiple start sites, RNase protection assays were performed using the 5'-flanking region of the genomic sequence as a
template for generation of an antisense RNA transcript. The RNase
protection assay also demonstrated multiple sized fragments (Fig.
3B) corresponding to the start sites identified in the
primer extension analysis using the
17 to
41 primer. These
observations are consistent with the presence of sequences that match
the consensus for Inr elements (YYANT/AYY) (29) at
83 and
111, with
the sequence at
83 closely associated with several transcriptional start sites. Intriguingly, the Inr element at
111 is only 1 bp removed from a consensus TATA box and is favorably positioned with
respect to the Inr element at
83. Because there was no predominant start site, we have utilized a convention in which upstream regulatory elements are numbered relative to the translational start site, with
the majority of transcription initiated at clusters of start sites
between
55 and
79 bp upstream (Figs. 2 and 3). The 5' end of the
previously published macrosialin cDNA ends at
93, indicating that
it was generated from a relatively rare mRNA initiated upstream of
the major start sites (1).
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The Macrosialin Promoter Is Preferentially Expressed in Myeloid
Cells--
To begin to characterize transcriptional regulatory
elements that control macrosialin expression, a 7.0-kb fragment of the 5'-flanking region of the macrosialin gene containing the
transcriptional start sites was linked to a luciferase reporter gene
and transiently transfected into myeloid and nonmyeloid cells (Fig.
4A). The activity of the
macrosialin promoter was normalized to the activity of the -actin
promoter to correct for different transfection efficiencies observed in
the various cell types. As shown in Fig. 4A, the macrosialin promoter displayed a preferential activity in myeloid cell lines versus nonmyeloid cells lines. RAW cells, which represent a
macrophage-like cell, demonstrated the highest levels of basal promoter
activity. High levels of expression were also observed in the monocytic THP-1 cells and U937 which, represent monoblast-like cells. The macrosialin promoter was also expressed in HL-60 cells, which are
capable of differentiating into either monocyte, macrophage, or
granulocyte-like cells in response to vitamin D, TPA, or retinoic acid,
respectively (30, 31). Jurkat cells exhibited the lowest level of
promoter activity among the hematopoietic cell lines. Treatment of
HL60, U937, and THP-1 cells with TPA to induce macrophage differentiation increased the level of promoter activity in each of
these cell lines by 3-5-fold. The results are consistent with the
effect of TPA on CD68 protein levels observed in THP-1 cells (16). TPA
did not have any significant effect on the more fully differentiated
RAW cell line. Interestingly, HeLa but not other nonmyeloid cells
examined, showed significant levels of promoter activity that was also
inducible by TPA. The 7.0-kb macrosialin regulatory elements exhibited
a level of promoter activity in TPA-treated THP-1 cells that was
approximately one-fourth the promoter activity directed by the
-actin promoter. This observation is consistent with the relative
levels of macrosialin and
-actin mRNA determined by RNase
protection assays in primary macrophages (Fig. 1) and suggests that the
macrosialin promoter is one of the most highly active
macrophage-specific promoters yet identified. A direct comparison with
the scavenger receptor A gene regulatory elements, which also direct
macrophage-specific expression (20, 25, 32), indicated that the
macrosialin promoter is approximately 100-fold more active with respect
to both basal and TPA-stimulated activity (Fig. 4B).
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Regulation of Macrosialin Promoter Activity--
To determine
whether or not the macrosialin promoter responds to cytokines and other
signaling molecules that affect the proliferation and maturation of
macrophages, U937 cells were transiently transfected with the Mac-7.0
luciferase reporter gene and treated with combinations of TPA, GM-CSF,
IFN-, retinoic acid, TNF-
, and LPS. GM-CSF treatment alone
increased promoter activity slightly above basal activity, and a more
than additive effect was observed when cells were treated with both TPA
and GM-CSF (Fig. 4C). IFN-
and retinoic acid had no
effect on basal promoter activity, but inhibited
TPA-dependent transcription by approximately 50%. TNF-
and LPS inhibited both basal and TPA-dependent promoter
activity to a similar extent (Fig. 4C).
Identification of Cell-specific Distal Enhancer Elements--
To
identify regulatory elements within the macrosialin gene necessary for
high levels of promoter activity, a 5'-deletion analysis was performed
(Fig. 5). Deletion of the region from
7.0 to
5.5 kb resulted in a 50% reduction in basal promoter
activity and an 85% reduction in TPA-dependent activity in
U937 cells (Fig. 5A). Deletion to
2.5 kb led to a further
reduction in basal promoter activity in U937 cells to 20% of that
observed for the Mac 7.0 reporter, and a reduction of
TPA-dependent activity to approximately 10% of that
observed for Mac 7.0. In contrast, deletions of the macrosialin
promoter to
5.5 and 2.5 kb resulted in progressive increases in basal
and TPA-dependent transcriptional activity in HeLa cells
(Fig. 5B). In P19 cells, which express the Mac 7.0 reporter
gene at very low levels, deletions to
5.5 kb also led to a 3-fold
increase in reporter gene activity (Fig. 5C). In concert, these observations suggest the existence of complex regulatory elements
residing between
2.5 and
7.5 kb upstream of the major transcriptional response site that confer enhancer activities in
myeloid cells and silencer activities in nonmyeloid cells.
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Multiple cis-Active Elements Are Required for Activity of the
Proximal Macrosialin Promoter--
Computer-assisted analysis of the
macrosialin promoter revealed several potential binding sites for
sequence-specific transcription factors. Four putative binding sites
for AP-1 transcription factors were identified at 925,
899,
257,
and
131 bp from the translational start site. Three putative Ets
binding sites, were identified at
919,
332, and
43. A sequence
motif located between
104 to
89 is similar to the PU.1/Spi-1
binding site identified in the macrophage scavenger receptor A gene
(32). Three CCAAT boxes were identified at
338,
191, and
167. A
GC-box was noted at
213. To determine whether these or other elements
were required for function of the macrosialin promoter, an extensive
series of 5'-deletion mutants was evaluated in U937 cells (Fig.
6). Progressive deletions from 1.06 kb to
250 bp resulted in progressive decreases in basal promoter activity
(Fig. 6A). Deletion to
221 resulted in a marked increase
in activity, suggesting that the region between
250 and
221
contains a silencer element (Fig. 6A). Further deletion to
77 abolished promoter activity. Based on these results, a more
detailed analysis was performed of the
221 promoter (Fig. 6C). Mutations were made in the putative GC box, the CCAAT
boxes, the AP-1, and PU.1/Spi-1 binding sites to determine their
potential roles. Mutating each of these regions substantially decreased the basal promoter activity and all but the first CCAAT box had a
decrease in TPA response. Deletion of the Ets binding site did not have
any effect in promoter activity. In concert, these studies establish
the region from
221 to
1 of the macrosialin gene as a proximal
promoter that is capable of conferring a transcriptional response to
TPA in monocyte-like cells. At least four classes of
cis-active elements appear to be required for proximal
promoter activity in these cells; a GC-rich sequence at
213, two
CCAAT boxes at
191 and
167, a putative binding site for PU.1/Spi-1 at
104 and a binding site for AP-1 at
131.
|
The Proximal Macrosialin Promoter Mediates Transcriptional
Activation by GM-CSF--
Based on the regulation of the endogenous
macrosialin mRNA levels by GM-CSF (Fig. 1), we wished to determine
whether macrosialin regulatory elements could confer a transcriptional
response to GM-CSF in a model cell line. BaF/3 cells containing the
murine GM-CSF -receptor were transfected with the macrosialin
deletion constructs (
7.0 to
221) (Fig.
7A). Each construct exhibited a transcriptional response to GM-CSF with the fold of induction being
similar for the
7.0 kb and
221 bp. The results demonstrated that
the proximal promoter is alone sufficient to confer a transcriptional response to GM-CSF. To determine if the same cis-acting
elements that directed the TPA response in the U937 also are
responsible for the GM-CSF response, the mutated versions of the
proximal promoter constructs were transfected into the BaF/3-GM cells
(Fig. 7B). As was observed in the U937 cells, these regions
also affected the basal activity of the proximal promoter in the
BaF/3-GM cells. However, only mutations in the GC-rich region exhibited
a decrease in fold induction when corrected to the new basal level. The
results suggest that additional sequences are primarily responsible for the GM-CSF response within proximal promoter.
|
Interaction of c-Jun and PU.1 with Macrosialin Regulatory Elements-- To identify nuclear proteins that bind to the putative AP-1 and PU.1/Spi-1 elements present in the macrosialin promoter, electrophoretic mobility shift assays were performed. Incubation of nuclear proteins obtained from THP-1 cells with an oligonucleotide probe containing the putative PU.1/Spi-1 element resulted in several complexes, labeled Ia, Ib, and Ic in Fig. 8A. All complexes were effectively competed by the unlabeled macrosialin probe, while Ia and Ic were effectively competed by an oligonucleotide corresponding to a consensus PU.1/Spi-1 binding site. None of these binding activities was competed for by an oligonucleotide in which the PU.1/Spi-1 site was mutated. PU.1 and Spi-1 are the murine and human orthologues of a B cell and macrophage-specific transcription factor belonging to the ets domain gene family (35, 36). PU.1 and Spi-1 are equivalent in size and can be recognized by a specific antiserum raised against PU.1 binding DNA domain (32). To determine whether these complexes contained Spi-1, THP-1 nuclear extracts were incubated with this antiserum prior to addition of the radiolabeled PU.1/Spi-1 probe. As illustrated in Fig. 8B, the PU.1/Spi-1 antibody almost completely abolished complexes Ia and Ic, and a faint supershifted band was observed (lane 3). Complex Ib did not change, consistent with its failure to be competed effectively by the consensus PU.1/Spi-1 binding site. As a control, in vitro translated PU.1 was incubated with the probe and antibody was also added. The in vitro translated product of PU.1 also bound with high affinity to the macrosialin PU.1/Spi-1 site (Fig. 8B, lane 4). The major complex migrated at the same position as complex Ic, which results from partial proteolysis of full-length PU.1. These results indicate that complexes Ia and Ic contain Spi-1 and suggest that complex Ia may represent either multimers of Spi-1 or a ternary complex containing Spi-1 and other nuclear proteins.
|
Functional Cooperation between c-Jun and PU.1 on the Macrosialin
Promoter--
To directly assess whether PU.1 and c-Jun can
functionally cooperate to stimulate macrosialin promoter activity,
experiments were performed in P19 cells, which lack c-Jun and PU.1. The
Mac 221-luciferase construct was co-transfected into P19 cells with expression vectors containing cDNAs for either c-Jun or PU.1 (Fig. 9). Cells were treated with and without
TPA for 14-16 h and harvested 48 h after transfection. Cells that
were transfected with the 221 construct and an empty expression
vector (pcDNA-3) demonstrated very low levels of promoter activity
in the presence or absence of TPA (Fig. 9). Co-transfection of the PU.1
expression vector did not significantly alter promoter activity (Fig.
9). Similar results were obtained over a wide range of PU.1 expression
vector concentrations (data not shown). When the c-Jun expression
vector was co-transfected with the
221 construct, the basal level of promoter activity also did not significantly change; however, a
10-fold induction was seen when TPA was added. Significantly, co-expression of PU.1 and c-Jun resulted in synergistic increases in
both basal and TPA-dependent transcription.
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DISCUSSION |
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Macrosialin is a transmembrane glycoprotein of uncertain function that has been shown to be highly expressed in macrophages and dendritic cells (9). Consistent with these observations, macrosialin mRNA was observed in murine bone marrow progenitor cells and was markedly up-regulated by M-CSF and GM-CSF, factors that promote the proliferation and differentiation of the monocyte-macrophage lineage. These observations suggest that the macrosialin gene will be a useful model for the investigation of molecular mechanisms that control early events in macrophage differentiation.
In the present studies, we have cloned the macrosialin gene and have
performed an initial characterization of its transcriptional regulatory
elements. A fragment of the macrosialin gene containing the promoter
and 7.0 kb of 5'-flanking information was demonstrated to direct high
levels of reporter gene activity in several monocyte-like cell lines.
Maximal levels of promoter activity in U937 cells required the presence
of enhancer elements located between 7.0 and
2.5 kb of the
translational start site. This region also contained regulatory
elements that inhibited promoter activity in nonmyeloid cells. It will
thus be of interest to identify the proteins that bind to these
regulatory elements and determine their roles in controlling
macrosialin expression in vivo.
In comparison to several other myeloid-specific promoters that we have evaluated, including the scavenger receptor A promoter (32), the macrosialin promoter is very active, directing levels of reporter gene activity that are approximately 100 times higher than the scavenger receptor A promoter. In addition to facilitating the analysis of transcriptional regulatory elements, the relative strength and specificity of the macrosialin promoter is likely to have practical applications in transgenic animal experiments in which the macrosialin promoter is used to overexpress genes of interest in macrophages and dendritic cells.
Consistent with the finding that GM-CSF up-regulates macrosialin
mRNA in bone marrow progenitor cells, the macrosialin promoter and
7.0 kb of 5'- flanking information conferred a strong positive transcriptional response to GM-CSF in BaF/3 cells transfected with the
subunit of the GM-CSF receptor. Furthermore, we have determined
that the proximal 221 bp of promoter information are sufficient to
mediate the GM-CSF response, indicating that the proximal promoter is
likely to play an important role in the GM-CSF-dependent induction of macrosialin transcription, but do not appear to correspond to elements required for phorbal ester responsiveness.
The macrosialin promoter and 5'-flanking sequences also conferred
positive transcriptional responses to the phorbol ester, TPA, which
induces macrophage differentiation of THP-1, U937, and HL60 cells. TPA
regulates the activities of several classes of transcription factors,
including AP-1 proteins, as a consequence of stimulating protein kinase
C (37). Among other events, protein kinase C activates the
Raf/mitogen-activated protein kinase pathway, which has been suggested
to play an important role in directing the proliferation and
differentiation of macrophage progenitor cells in response to M-CSF
(37-42). TPA-dependent induction of the macrosialin
promoter was inhibited by several cytokines and regulatory molecules
that influence macrophage development and function. Inhibition of
TPA-dependent expression of macrosialin by retinoic acid is
of interest because of the potent effects of retinoic acid as an
inducer of granulocyte differentiation in several myeloid leukemic cell
lines (31, 43), which would be anticipated to lead to repression of the
endogenous macrosialin gene. The inhibitory effects of IFN-,
bacterial LPS, and TNF-
on macrosialin promoter activity are very
similar to the inhibitory effects of these substances on the
SR-A gene (44), suggesting that macrosialin and SR-A are
coordinately regulated in terminally differentiated macrophages. Mice
lacking the TNF receptor R1 (p55) develop more atherosclerosis on a
high fat diet than did control animals (45). Thus, the effects of
TNF-
on macrosialin and SR-A expression may be relevant to the
development of atherosclerosis if these proteins play important roles
in the uptake of oxidatively modified lipoproteins. Hsu et.
al. (46) demonstrated that down-regulation of the macrophage
scavenger receptor was not due to a transcriptional decrease, but was
mainly due to the destabilization of the macrophage scavenger receptor
mRNA (46).
Characterization of the proximal macrosialin promoter indicates that at
least four classes of cis-active elements are required for
full activity. Mutation of a GC-box at 213 bp reduces promoter activity by approximately 10-fold. This element is recognized by a DNA
binding activity that is present in many cell types and confers
enhancer activity to a heterologous minimal
promoter.2
Antibody-perturbated gel shift studies, competition experiments with
consensus binding sites, and electrophoretic mobility shift assays with
recombinant proteins have thus far excluded NF-
B, SP-1, SP-3, AML-1,
and Egr-1 as potential factors binding to these elements in U937 cells.
Two putative CCAAT boxes have also been identified within the
macrosialin promoter. Mutation of these regions also reduced the
activity of the
221 promoter by 10-fold. We are currently
investigating whether the two CCAAT bases between
191 and
162 are
binding sites for CCAAT binding proteins such as C/EBP or CREB-binding
protein (CBP).
Several lines of evidence indicate that AP-1 and PU.1/Spi-1 cooperate
to activate macrosialin transcription via regulatory elements in the
proximal macrosialin promoter. Mutations in either the PU.1/Spi-1 or
AP-1 binding sites reduced activity of the 221 bp promoter by nearly
a factor of 10. Antibody-perturbated gel shift experiments confirmed
that PU.1/Spi-1 and c-Jun are indeed components of protein complexes
that bind to the PU.1 and AP-1 elements, respectively. Furthermore,
coexpression of PU.1 and c-Jun led to synergistic transcriptional
activation of the
221 macrosialin promoter in P19 cells.
These observations are consistent with the proposed roles of PU.1/Spi-1 and c-Jun in regulating critical aspects of macrophage development and function. PU.1/Spi-1 is a B cell and macrophage-specific transcription factor (35) that has been demonstrated to activate several genes that are selectively expressed in these cell types (32, 47, 48). PU.1/Spi-1 has been found in CD34+ cells, indicating that it is present when macrosialin first becomes expressed (49, 50). Disruption of the PU.1 gene results in complete absence of B cells and macrophages, indicating its requirement for the development of these lineages (51). Intriguingly, PU.1 has been found to be located at or near the transcriptional start site of several TATA-less promoters and to interact with TATA binding protein (52, 53). The unusual structure of the macrosialin promoter, in which the PU.1 site lies between a downstream consensus Inr element and an upstream Inr element that is only 1 bp removed from a consensus TATA box, may permit several options for transcriptional initiation and account in part for the relative strength of the macrosialin promoter.
In the present studies, high and low mobility protein DNA complexes
were abolished by the anti-PU.1 antibody. These observations raise the
possibility that the low mobility complex consists of a ternary complex
between PU.1 and another factor. PU.1 has previously been demonstrated
to form a ternary complex with the lymphocyte-specific factor
NF-EM5/Pip or regulatory elements present in the immunoglobulin 3'
enhancer (54, 55). It will be of interest to determine whether
analogous factors exist in macrophages that function to enhance PU.1
activity on macrophage-specific genes.
Recent studies have also implicated c-Jun as an important factor in
mediating at least some of the actions of M-CSF in macrophages (reviewed in Roussel (56)). c-Jun binds as a homodimer or a heterodimer
with other basic leucine zipper proteins to AP-1 elements in a large
number of genes that are activated in macrophages in response to M-CSF,
and has been found to activate a number of these genes in
cotransfection assays. In the case of the SR-A gene,
mutation of the AP-1 binding sites abolishes the transcriptional response of the promoter to M-CSF (57). In addition to mediating the
positive transcriptional effects of M-CSF, AP-1 factors have also been
proposed to be targets of negative regulation by IFN- and retinoic
acid. Recent studies suggest that transcriptional activation by c-Jun,
STAT1
, and retinoic acid receptor requires the recruitment of
coactivator complexes that contain CBP or p300 (57, 58). CBP and p300
appear to be present in rate-limiting amounts in cells, suggesting that
competition for these complexes may account for antagonistic
interaction between activators of AP-1, IFN-
, and the retinoic acid
receptor. These observation also raise the possibility that cooperative
recruitment of CBP·p300 complexes by several locally bound
transcription factors could potentially account for synergistic
interaction between pathways. It will therefore be of interest to
determine the mechanistic basis for synergy between PU.1 and AP-1.
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ACKNOWLEDGEMENTS |
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We thank Dr. Oswald Quehenberger for the macrosialin cDNA clone and Dr. Alan D'Andrea for the BaF/3 wild-type and BaF/3-GM-CSF receptor cell lines. We thank Dr. Daniel Steinberg for critical review of the manuscript. We also thank Dr. Mylène Oglisatro for helpful discussions, Sally Ngo for isolation of the bone marrow progenitor cells, and Tanya Schneiderman for preparation of the manuscript.
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FOOTNOTES |
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* These studies were supported in part by National Institutes of Health Specialized Center of Research on Atherosclerosis Grant HL14197.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EMBL Data Bank with accession number(s) AF039399.
§ Supported by a National Institutes of Health Physician Scientist Award 2K12AG00353-09 to the University of California at San Diego, a Clinician Scientist Award 96004400 from the American Heart Association, and a National Institutes of Health, NHLBI Individual Mentored Clinical Scientist Development Award 1K08HL03625-01.
Current address: The Institute of Cancer Research, Royal
Cancer Hospital, Chester Beatty Laboratories, 237 Fulham Rd., London, SW3 6JB, United Kingdom.
Established Investigator of the American Heart Association. To
whom correspondence should be addressed: Division Endocrinology and
Metabolism, Dept. of Medicine, University of California, San Diego,
9500 Gilman Dr., La Jolla, CA 92093-0651. Tel.: 619-534-6011; Fax:
619-534-8549; E-mail: cglass{at}ucsd.edu.
1
The abbreviations used are: lamp,
lysosomal/endosomal-associated membrane glycoprotein; M-CSF, macrophage
colony-stimulating factor; GM-CSF, granulocyte-macrophage stimulating
factor; IFN-, interferon-
; TNF-
, tumor necrosis factor-
;
IL, interleukin; TPA, phorbol 12-myristate 13-acetate; LPS,
lipopolysaccharide; PCR, polymerase chain reaction; kb, kilobase(s);
bp, base pair(s); Mac, macrosialin; CMV, cytomegalovirus; Inr,
initiator; STAT, signal transducer and activator of transcription;
SR-A, scavenger receptor A; C/EBP, CCAAT enhancer binding protein;
CREB, cAMP response element-binding protein; CBP, CREB binding protein;
DOTAP, N-[1-(2,3-dioleoyloxy)propyl-N,N,N-trimethylammonium
methylsulfate.
2 A. C. Li and C. K. Glass, unpublished results.
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REFERENCES |
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