From the
Human macrophage metalloelastase (HME) is a recent addition to
the matrix metalloproteinase (MMP) family that was initially found to
be expressed in alveolar macrophages of cigarette smokers. To
understand more about HME expression, analysis of the structure and
location of the gene was performed. The gene for HME is composed of 10
exons and 9 introns, similar to the stromelysins and collagenases, and
HME shares the highly conserved exon size and intron-exon borders with
other MMPs. The 13-kilobase (kb) HME gene has been localized by
fluorescence in situ hybridization to chromosome
11q22.2-22.3, the same location of the interstitial collagenase
and stromelysin genes. We determined that HME and stromelysin 1 genes
are physically linked within 62 kb utilizing pulse-field gel
electrophoresis. The promoter region of the HME gene contains several
features common to other MMP genes including a TATA box 29 bp upstream
to the transcription initiation site, an AP-1 motif, and a PEA3
element. HME mRNA is not detectable in normal adult tissues but is
induced in rapidly remodeling tissues such as the term placenta. In
situ hybridization and immunohistochemistry of placental tissue
demonstrated HME mRNA and protein expression in macrophages and stromal
cells. Cell-specific expression and response to inflammatory stimuli
such as endotoxin is conferred within 2.8 kb of the HME 5`-flanking
sequence as demonstrated by HME promoter-CAT expression constructs.
Knowledge of the genomic organization and chromosomal location of HME
may allow us to further define mechanisms responsible for cell- and
tissue-specific expression of HME.
Matrix metalloproteinases (MMPs)
The MMP
family consists of matrix-degrading proteinases that share sequence
identity (
Macrophage elastase
activity was detected in mouse peritoneal macrophage conditioned media
in 1975(14) , and a 22-kDa protein responsible for this activity
was isolated in 1981(15) . However, only recently have the cDNA
sequences of murine (16) and human macrophage elastase (17) been reported. Cloning and expression of these cDNAs
demonstrated that macrophage elastase is a distinct member of the
metalloproteinase gene family with potent elastolytic activity and a
broad substrate specificity similar to the stromelysins. HME mRNA and
protein are expressed in human alveolar macrophages derived from
cigarette smokers. Given the abundance of macrophages in the lower
airways, we have targeted the HME gene as a potential candidate in the
pathogenesis of pulmonary emphysema.
Similar to other MMPs,
macrophage elastase activity is most likely regulated both at the
transcriptional (1, 18, 19) and
post-transcriptional level(3, 11) . Activation of the
secreted proenzyme within the extracellular space and inhibition by
tissue inhibitors of metalloproteinases represent post-translational
regulation. Macrophage elastase mRNA and protein are up-regulated by
endotoxin and inhibited by glucocorticoids(16, 17) . To
begin to understand the basis for expression of HME in normal and
pathologic states, the aim of this study was to characterize the
structure and function of the gene for human macrophage
metalloelastase.
Immunohistochemical
staining was performed on the term placenta specimens described above
using the immunoperoxidase staining technique (Vectastain ABC Kit;
Vector Laboratories, Inc., Burlingame, CA) according to the
manufacturer's instructions. The primary antibody for HME
represents affinity-purified antisera directed against a peptide
previously demonstrated to be specific for HME (17) used in a
1:1000 dilution. Macrophages were identified using anti-CD68 antiserum
at a dilution of 1:2000. The incubation time for the primary and
secondary antibody was 30 min each at room temperature. Slides were
counterstained with hematoxylin. Controls were performed with nonimmune
rabbit serum.
Murine
macrophage P388D1 cells, cultured in Dulbecco's modified
Eagle's medium, were used to assess expression in macrophages and
LPS responsiveness. These cells express murine macrophage elastase
basally with up-regulation of murine macrophage elastase in response to
LPS, similar to human macrophages and HME. In addition, unlike human
mononuclear phagocyte cell lines, these cells do not require
differentiation with phorbol esters or other agents, which complicate
interpretation of results. Human umbilical vein endothelial cells,
cultured in endothelial basic medium (with 30% human serum), were also
used for transient transfection experiments. Transient transfections
were performed with 10 µg of each plasmid (1 µg of
HSVtk-
In this report we have shown that the gene for human
macrophage metalloelastase (HME) is organized into 10 exons and 9
introns on chromosome 11q22.2-22.3 closely linked to stromelysin
1. Sequencing of the 5`-flanking region demonstrated several features
common to other MMPs including a TATA box ending 29 bp upstream from
the transcriptional start site and potential AP-1 and PEA3 cis-acting elements. The gene does not appear to be
transcribed in normal quiescent adult tissue but is expressed in
macrophage and stromal cells of the actively remodeling term placenta.
Transcriptional analysis with an HME promoter-CAT reporter construct
demonstrated that cell-specific expression as well as up-regulation of
HME by LPS in macrophages is conferred by the immediate 5`-flanking
sequence of the gene.
The HME gene spans 13 kb. Size of the 10 exons
and intron-exon borders are highly conserved among the MMPs. The
proenzyme domain is encoded within the first 2 exons, the catalytic
domain is derived from the end of exon 2 as well as exons 3-5,
and the C-terminal domain arises from exons 6-10. The
collagenases and stromelysins share this common 10-exon organization,
while the gelatinases have additional exons inserted corresponding to
additional domains that share homology to fibronectin (gelatinases A
and B) and type V collagen (gelatinase B) (34). Matrilysin, which lacks
the C-terminal domain, is composed of similar exons 1-5 with a
unique 6th exon(32) . Similarities in genomic organization
suggest that the MMP gene family may have evolved through gene
duplication and exon shuffling.
The HME gene was localized to
chromosome 11q22.2-22.3 by fluorescent in situ hybridization. Several other MMP genes have been mapped to this
same location (30) including stromelysin 1, stromelysin 2, and
interstitial collagenase. Interestingly, HME shares greatest sequence
identity with these enzymes (49% amino acid sequence identity between
HME with both stromelysin 1 and collagenase). Furthermore, these three
other genes form a cluster within 135 kb of each other oriented:
centromere-stromelysin 2-interstitial collagenase-stromelysin
1(30) . We performed pulsed-field gel electrophoresis on high
molecular weight DNA digested with several rare cutting restriction
enzymes to determine if the HME gene was physically linked to this
cluster. Our results suggest that HME is located within 62 kb of
stromelysin 1, but we were unable to demonstrate close linkage with
interstitial collagenase. Consistent with this finding, stromelysin 1
and HME genes were both found in our
Sequence analysis of the
promoter region of the HME gene demonstrated the presence of a TATA box
and several potential cis-acting regulatory elements. The TATA
box, a feature common to all MMPs with the exception of gelatinase A,
is located at position -37 to -29 from the transcriptional
start site. An AP-1 site is present from position -80 to
-74. This element is present in all MMPs sequenced to date, with
the exception of gelatinase A, between positions -57 and
-79. PEA3 elements are also common among human MMPs (except
gelatinases A and B). However, the location, number, and orientation of
these sites differ somewhat(32) . In general, MMPs contain 1 or
2 PEA3 elements in either or both orientations between positions
-90 and -215. HME contains one PEA3 site in the forward
orientation beginning at position -345, somewhat further upstream
than other MMPs. We have yet to determine if the AP-1 and PEA3 sites
act synergistically to confer responsiveness to phorbol esters, growth
factors, and oncogenes as they do for other genes.
MMPs are not
expressed in normal adult cells under normal, quiescent conditions.
These potentially destructive genes are induced in physiologic
conditions requiring tissue remodeling such as during development,
growth, and tissue repair. Abnormal expression of these potent
proteinases can lead to tissue destruction and impaired organ function.
The HME mRNA tissue survey (Fig. 7) was consistent with this
hypothesis. HME steady-state mRNA was not detected in normal adult
tissues. HME mRNA was abundantly expressed in the term placenta. This
rapidly remodeling/involuting tissue is comprised of endothelial cells,
stromal fibroblast-like mesenchymal cells, and Hofbauer cells of
macrophage origin. In situ hybridization and
immunohistochemistry demonstrated that both stromal cells and
macrophages express HME mRNA and protein, while endothelial cells do
not. Previously, we demonstrated that human alveolar macrophages of
several cigarette smokers express HME mRNA and protein(17) .
Northern analysis of of mRNA from several other cell types including
human vein endothelial cells and adult skin fibroblasts failed to
demonstrate HME expression. Transcription analysis using an HME
promoter-CAT reporter construct demonstrated that cell-specific
expression of HME was conferred by the immediate 5`-flanking region of
the HME gene. As observed in placenta, the HME promoter region was
sufficient to drive expression in macrophages but not endothelial
cells. The HME promoter also did not drive CAT expression in adult skin
fibroblasts. We did, however, observe HME mRNA in placental
stromal/fibroblast-like cells by in situ hybridization. This
discrepancy may be related to differences in the microenvironment
within these tissues or related perhaps to fibroblast differentiation.
Further studies are needed to characterize HME expression in
fibroblasts.
Furthermore, we have previously shown that HME mRNA is
up-regulated by LPS in alveolar macrophages. In this study we found
that CAT expression linked to the HME promoter was also enhanced by
LPS. Nuclear factor
Exon
and intron sequences are shown in uppercase and lowercase,
respectively. The 5` donor GT and the 3` acceptor AG are underlined.
Genomic DNA was cut to completion with rare
cutting restriction enzymes above. Pulsed field gel electrophoresis was
performed, and Southern hybridization was performed using MMP cDNA
probes. The size of the hybridizing fragments in kilobases are shown
above.
(
)comprise a family of structurally related matrix-degrading
enzymes that play a major role in tissue remodeling and repair
associated with development and
inflammation(1, 2, 3) . Abnormal expression of
MMPs may contribute to a variety of destructive diseases including
tumor invasiveness(4, 5) ,
arthritis(6, 7) , atherosclerosis(8) , aneurysm
formation(9) , and pulmonary emphysema(10) .
32-49% at the amino acid level) and common
structural domains. In general, these enzymes are secreted as zymogens
which are activated within the extracellular space. Catalytic activity
is dependent on coordination of zinc and is specifically inhibited by
the tissue inhibitors of metalloproteinases(3, 11) .
Family members can be loosely divided into groups based on matrix
degrading capacity. As a whole, they are able to cleave all
extracellular matrix components. Fibroblast-derived interstitial
collagenase, neutrophil collagenase, and a recently described
collagenase (12) expressed in breast carcinoma cells, which is
similar to rodent collagenase, are unique in their capacity to degrade
interstitial collagens. There are two gelatinase/type IV collagenases
of 72 kDa (gelatinase A) and 92 kDa (gelatinase B). The stromelysins
and matrilysin have a broad spectrum of susceptible substrates
including all basement membrane components. Recently, a 66-kDa
membrane-type metalloproteinase has been described that has the
capacity to activate other MMPs(13) .
Molecular Cloning and Sequencing of HME Genomic
DNA
250,000 plaques from a human genomic (leukocyte) library
constructed in EMBL3 were screened using full-length MME cDNA as the
probe. We isolated the most strongly hybridizing duplicate-positive
clone to homogeneity. Phage DNA was purified, and the insert was
released with XhoI and cut to completion with several
restriction enzymes. Digested DNA was transferred to GeneScreen Plus
(Dupont NEN), and Southern hybridization was performed using MME cDNA
as the probe. Two strongly hybridizing EcoRI fragments of 6 kb
and 2.4 kb were subcloned into pUC 9 and sequenced on both strands
using the dideoxy chain termination method of Sanger and cloned T7 DNA
polymerase (Sequenase 2, U. S. Biochemical Corp.). DNA sequences were
compiled with the Geneworks program (IntelliGenetics, Inc.). These
fragments contained 3 kb of the 5`-flanking region as well as exons 1
through 5. To obtain the remainder of the HME gene as well as closely
related genes, a human genomic library constructed in the P1
bacteriophage was screened (Genomes Systems Inc., St. Louis, MO). This
library, which contains inserts averaging 85 kb in length, was screened
by the polymerase chain reaction (PCR) using oligonucleotide primers
between exon 3 and exon 4. A single clone containing the entire HME
gene was isolated and DNA was prepared(20) . PCR products
between consecutive exons were subcloned and intron-exon borders were
obtained by sequencing. Southern hybridization was performed to map
linked genes as described below.
Localization of the HME Gene by in Situ Hybridization and
Digital Imaging
To localize the chromosomal locus encoding the
HME gene, a genomic probe (6 kb) was labeled by nick translation with
biotin-11-dUTP and used for fluorescence in situ hybridization
as described by Zimonjic et al.(21) and Popescu et
al.(22) . Chromosomes obtained from
methotrexate-synchronized normal peripheral leukocyte cultures were
pretreated with RNase, denatured for 2 min at 70 °C in 2
SSC, 70% (v/v) formamide, and hybridized with the DNA probe (200 ng) in
2
SSC, 50% (v/v) formamide, 10% (w/v) dextran sulfate, 2
Denhardt's solution, 1% Tween 20 (v/v), and 50 µg of
human Cot-1 DNA (Life Technologies, Inc.) probe for 18 h at 37 °C.
Posthybridization washing was in 2
SSC, 50% formamide at 42
°C (3
6 min each) followed by 0.1
SSC at 60 °C
(3
6 min each). Biotin-labeled DNA was detected by fluorescein
isothiocyanate-conjugated avidin DCS and antiavidin antibodies (Vector
Laboratories). Chromosomes were counterstained with propidium iodide
and examined with an Olympus BH2 epifluorescence microscope. Digital
image acquisition and analysis as well as posthybridization chromosome
banding have been described in detail
elsewhere(21, 22) .
DNA Analysis by Pulsed-field Gel
Electrophoresis
High molecular weight genomic DNA samples from
four different lymphoblastoid cell lines (CGM, MKH, KH18, and JB18)
were prepared in low melting point agarose as
described(23, 24) . Restriction digests were performed
by incubating a cell plug with 100 units (10 units/µg of DNA)
of the following restriction enzymes; BssHII, EagI, SacII, SfiI, SmaI, XhoI, NaeI, MluI, and ClaI. Restriction fragments
were separated in a 1% agarose gel using a contour-clamped, homogeneous
electric field (CHEF DR2, Bio-Rad) apparatus(24) .
Electrophoresis conditions included a 6 V/cm (160 V) field strength,
0.5
TBE buffer at 12 °C, and 20-24-h running times. A
linear ramped switch interval that started at 20 s and ended at 40 s
resolved fragments between 50 and 200 kb, and intervals between 35 and
70 s resolved bands between 400 and 800 kb. DNA fragments were
depurinated in 0.25 N HCl and transferred to reinforced
nitrocellulose (Nitroplus 2000, 0.45 µm; Micron Separations,
Westboro, MA) using an ammmonium acetate procedure (25) and
routine blotting techniques(26) . Filters were baked (80 °C
for 2 h) and then hybridized to
P-labeled probes
(macrophage metalloelastase, stromelysin-1, and interstitial
collagenase full-length cDNA probes) in a hybridization mixture
containing 40% formamide, 10% dextran sulfate, 4
SSC, 1
Denhardt's solution, and 10 µg/ml sheared salmon sperm DNA.
Blots were washed twice in 2
SSC, 0.1% SDS at room temperature
and twice in 0.2
SSC, 0.1% SDS at 50 °C. Autoradiograms
were developed between 1 and 7 days. Filters were stripped of a probe
by incubation in 0.1% SDS at 80 °C. Fragment sizes were determined
by comparison to concatemers of
phage DNA and the electrophoretic
karyotype of Saccharomyces cerevisiae strain
AB1380(23) .
Determination of Intron-Exon Junctions and Intron
Sizes
The junctions between exons 1-5 and introns
1-4 were determined by sequencing the entire region using the
clone derived from the EMBL3 genomic library. To determine the
organization of exons 6-10 and introns 5-9, we amplified
fragments spanning consecutive exons by the polymerase chain reaction
and sequenced the exons and intron-exon junctions. Oligonucleotides
used for PCR were selected based on HME cDNA sequence. PCR conditions
included 100 ng of P1 DNA, 250 µM dNTP, 20 pmol of
each oligonucleotide primer, and 2.5 units of KlenTaq-LA in a buffer of
50 mM Tris-HCl, pH 9.2, 2.5 mM MgCl
, 16
mM (NH
)
SO
, 150 µg/ml
bovine serum albumin. The PCR amplification reactions consisted of a
20-s denaturation at 99 °C, followed by 35 cycles of denaturation
at 99 °C for 1 s, annealing at 55 °C for 30 s, and extension at
72 °C for 1 min 30 s. Amplified introns were sized by agarose gel
electrophoresis that included DNA (base pair) standards. Each PCR
fragment was gel-purified (Bioclean kit, U.S. Biochemical Corp.),
subcloned in pNOT (5`Prime-3`Prime, Boulder, CO), and sequenced. These
sequences were compared with human HME cDNA sequences to identify
exonic sequences and intron-exon boundaries.
Primer Extension
Primer extension was used to
determine the transcription start site of the human macrophage elastase
gene. A synthetic antisense oligonucleotide primer
(CTCCAGAAGCAGTGGCCTGCAGGAGCAGTA) located in exon 1 downstream of the
ATG was end-labeled with [-
P] ATP (Amersham
International, Buckinghamshire, United Kingdom). Human placenta
poly(A
) RNA (10 µg) (Clontech) was primed for 2 h
at 55 °C and reverse-transcribed with avian myeloblastosis virus
reverse transcriptase (10 units) (Promega) for 90 min at 42 °C. The
primer-extended product was then digested with 500 ng of DNase-free
RNase A for 30 min at 37 °C. Ammonium acetate was added to a final
concentration of 2 M, and the sample was extracted with
phenol-chloroform-isoamyl alcohol (25:24:1) and precipitated with
ethanol. Following lyophilization, the sample was dissolved in sterile
water, and the primer-extended product was analyzed on a 6%
polyacrylamide, 8 M urea gel in parallel with an HME genomic
subclone sequenced with the same primer and a
P-labeled
DNA ladder obtained by digestion of plasmid
X174 with HinfI.
RNA Analysis by Northern Hybridization
A Northern
blot containing equal amounts (2 µg) of poly(A) RNA from various normal human tissues was obtained from Clontech.
Northern blot analysis of RNA was performed under high stringency
conditions as described previously (16, 17) using
full-length HME cDNA as the probe.
In Situ Hybridization and Immunohistochemistry of HME in
Human Placenta
In situ hybridization was performed as
described in detail by Prosser et al.(27, 28) .
cRNA probes were generated using a 400-bp HME cDNA fragment (bp
1-400) subcloned into pGEM7(+) and linearized to generate
both sense and antisense strands. The cDNAs were then transcribed in
the presence of [-
S]UTP with T7 or SP6
polymerase (Promega). Tissue sections of term human placenta (5 µm)
were fixed in 4% paraformaldehyde, rinsed in phosphate-buffered saline,
and digested with proteinase K. Sections were then rinsed in 0.1 M triethanolamine, acetylated in 0.25% acetic anhydride, and
dehydrated in a graded series of ethanol solutions. The sections were
covered with 25-50 µl of hybridization buffer containing 2
10
cpm/µl of
S-labeled RNA probe.
Sections were incubated at 55 °C for 18 h in a humidified chamber.
After hybridization, unhybridized cRNA probe was removed by digestion
with RNase A, and slides were washed under stringent conditions. Slides
were hand-dipped in Kodak NT/B2 emulsion, followed by autoradiographic
exposure for 2 weeks, after which time the photographic emulsion was
developed and stained with hematoxylin-eosin.
Transient Transfection Analysis with an HME Promoter-CAT
Reporter Construct
The HME 5`flanking sequence (HindIII-XhoI fragment, -2,750 to +41) was
subcloned upstream of the chloramphenicol acetyltransferase (CAT)
reporter gene in the pCAT vector. Control constructs included pBLCAT2,
a CAT construct driven by the HSV thymidine kinase promoter, and a
promoter-less CAT construct, pSKCAT. In addition, cells were
cotransfected with the HSVtk--galactosidase plasmid which was used
as an internal control for transfection efficiency.
-galactosidase) on subconfluent cultures using lipofectamine
as described by the manufacturer (Life Technologies, Inc.). The
transfection medium was changed after 6 h of incubation and replaced
with fresh medium ± LPS. After a total of 24 h, the cell
extracts were normalized for
-galactosidase activity, and CAT
activity was determined by incubation of cell extracts with
[
C]chloramphenicol. Acetylated and nonacetylated
forms were separated by thin layer chromatography followed by
autoradiography and scintillation counting.
Genomic Organization of HME
To characterize the
HME gene, two EcoRI fragments obtained from screening a human
genomic DNA library constructed in EMBL3 were subcloned and sequenced
on both strands in their entirety. This allowed us to identify exons
1-5 of HME. To complete the 3` portion of the gene, a P1 clone of
85 kb containing the entire HME gene was isolated. The polymerase
chain reaction was used to amplify fragments between consecutive exons.
These fragments were subcloned and used to determine exon sequence,
intron-exon borders, and intron sizes. Exons 6-10 and introns
5-9 were identified by this strategy. Overall, the HME gene spans
13 kb and contains 10 exons and 9 introns ( Fig. 1and ). Exon sizes range from 92 bp (exon 9) to 247 bp (exon 2),
and introns range from 100 bp (intron 3) to 2,600 bp (intron 8). The
intron-exon junctions, determined by comparing HME genomic and HME cDNA
sequences, conformed to the GT/AG rule for splice junctions ()(29) . Exon nucleotide sequences did not differ
from the previously reported cDNA sequence(17) . The Gene for HME Is Located on Chromosome 11q22.2-22.3 by
Fluorescent in Situ Hybridization (FISH)-In normal human
prometaphase and metaphase chromosomes hybridized with a biotinylated
HME probe, a specific fluorescent signal was detected in 127 (84.67%)
of 150 randomly selected spreads with low nonspecific fluorescein
isothiocyanate background. Hybridization signal consisting of two
symmetrical fluorescent spots on both chromatids at the long arm of at
least one chromosome 11 was observed in 93 (62%) of the analyzed
chromosome spreads (Fig. 2A). In 33 metaphases (22%),
both chromosomes 11 displayed doublets. Such a signal was not observed
on other chromosomes. The position of symmetrical doublets was
determined by on-screen analysis of enlarged digital images of 28
chromosomes from 25 well-banded metaphases (Fig. 2B)
with minimal chromosome overlapping. Direct image comparisons of
labeled and G-banded spreads as well as of pter-signal-qter ratios for
each of the labeled chromosomes, allowed the localization of the HME
gene locus to chromosome region 11q22.2-22.3.
Figure 1:
Structure of the human macrophage
metalloelastase gene. HME gene structure was determined by sequencing a
phage subclone containing exons 1-5 on both strands and by
amplifying fragments between consecutive exons and sequencing exons and
intron-exon junctions from a P1 clone for exons 6-10 and introns
5-9. The HME gene spans
13 kb with introns ranging from 100
bp to 2.7 kb. Also shown are the domains of the HME proenzyme that are
encoded by each exon. Note, exon 1 and most of exon 2 encodes the
prodomain (white), the catalytic domain (hatched) is
encoded by exons 3-5, and the C-terminal domain (black and stippled) is encoded by exons
6-10.
Figure 2:
Localization by fluorescence in situ hybridization of the HME gene to human chromosome
11q22.2-22.3. A, metaphase chromosome spreads after
fluorescence in situ hybridization with a biotin-labeled HME
genomic DNA probe exhibiting specific double fluorescent signals on
chromatids of a single chromosome 11. B, the same metaphase
spread after trypsin-induced G-banding and Wright stain allowing
localization of the signal at chromosome band
11q22.2-22.3.
The HME Gene Is Physically Linked to Stromelysin
1
To determine if the HME gene is physically associated with
other MMP genes located on chromosome 11q22.2-22.3(30) ,
we performed pulsed-field gel electrophoresis on samples of high
molecular weight genomic DNA. Genomic DNA was derived from four
different lymphoblastoid cell lines digested with rare cutting
restriction enzymes. Restriction fragments were transferred to
Nitroplus and hybridized with full-length cDNAs of HME, stromelysin 1,
and interstitial collagenase. Results, summarized in ,
show interstitial collagenase hybridized to different fragments for all
restriction digests except for MluI, where all probes
hybridized to a large nonresolved fragment located in the compression
zone of >1,000 kb. HME and stromelysin 1, on the other hand,
hybridized with the same fragments for nearly all of the digests
performed with the exception of ClaI. The smallest band that
both HME and stromelysin 1 hybridized with was 62 kb, suggesting that
these genes are located within 62 kb of each other. To confirm linkage
of HME to stromelysin-1, we performed Southern hybridization on
restriction digests of the P1 genomic clone containing HME (data not
shown). Both HME and stromelysin 1 probes hybridized to the P1 clone,
but to different restriction fragments within the resolved area of the
gel. Interstitial collagenase and stromelysin 2-specific probes did not
hybridize with the P1 DNA restriction digests.
Transcriptional Start Site and 5`-Flanking Sequence of
HME
Primer extension analysis was performed to determine the
initiation site of transcription. Human placenta was used as the source
of poly (A) RNA, and a 30-bp antisense oligonucleotide
complementary to nucleotides 59 to 88 in the first exon was
radiolabeled and used to prime cDNA synthesis (Fig. 3). Alignment
of the extended product with HME genomic DNA sequenced with the same
primer was performed on a denaturing gel. Length of the extended
product indicated that transcription of HME RNA initiates 41 bases
upstream from the translation initiation site. The transcription
initiation site is in a pyrimidine-rich region, a common feature of the
cap site sequence(29) .
Figure 3:
Determination of the transcription
initiation site by primer extension analysis. A 30-bp antisense
oligonucleotide complementary to the sequence within exon 1 of HME was
radiolabeled, annealed to human placenta poly (A) RNA,
and reverse-transcribed. The 90-bp product was separated on a
sequencing gel (Sample) applied with a radiolabeled DNA ladder
and a genomic HME sequence with the same primer used for reverse
transcription. The transcription initiation site corresponds to an
adenosine nucleotide 41 bp upstream from the translation initiation
site. A TATA box is present 29 bp upstream from the transcription
initiation site. The inset schematically demonstrates the 5`
region of HME mRNA, the oligonucleotide probe used for primer
extension, and the 90-bp reverse-transcribed
fragment.
1.2 kb of the 5`-flanking region of
the HME gene was sequenced on both strands, and potential cis-acting regulatory elements were identified (Fig. 4).
A putative TATA box was identified, ending 29 bp upstream from the
transcription initiation site. This is within the predicted distance
relative to the site of transcription initiation (31) and
provides confirmation that our cap site is correctly identified at
position +1. Of note, TATA boxes have been found in several other
MMP genes(32) , including mouse macrophage elastase (MME) (not
shown). No canonical CAAT element was found for HME as is the case for
all MMP genes to date.
Figure 4:
Nucleotide sequence of the 5`-flanking
region and first exon of the HME gene. Nucleotides are numbered
relative to the transcription initiation site (adenosine +1). The
TATA box is indicated by lines above and below the sequence,
an AP-1 consensus sequence is indicated within the solid box,
and the PEA3 element is depicted within the dotted box. LBP-1
and TRF elements are underlined. The translation initiation
ATG is indicated (double-underlined). The sequences
complementary to the oligonucleotides used for primer extension (Fig.
3) are underlined with an arrow.
Major potential cis-acting elements
identified within the 5`-flanking region include an AP-1 site
(-74 bp) (18) and a PEA3 binding site (-335
bp)(33) . These elements have also been identified in other MMP
genes(32) . The AP-1 site recognizes a protein complex encoded
by members of the fos and jun oncogene family. The
PEA3 binding site interacts with the products of the ets gene
family. Both AP-1 and PEA3 have been found in other genes to act
synergistically and confer responsiveness to phorbol esters and a
variety of cytokines, growth factors, and oncogenes. Other potential cis-acting elements include LBP-1 and TRF. These elements have
previously been found in viral promoters. The functional significance
of these sites in the HME promoter is uncertain.
HME mRNA Has Limited Expression in Normal Human
Tissues
Northern blot analysis was performed with mRNA from
various normal human tissues including heart, brain, placenta, lung,
liver, skeletal muscle, kidney, and pancreas using HME cDNA as the
probe (Fig. 5). The 1.8-kb HME mRNA was only detected in the
placenta. HME mRNA was not detected in the other normal human tissues
assayed.
Figure 5:
Northern blot analysis of mRNA derived
from adult human tissues. Equal amounts (2 µg) of
poly(A) RNA were harvested from the indicated normal
adult human tissues and subjected to Northern blot analysis under
stringent conditions with random primer-labeled HME cDNA as the probe.
Note, the abundant 1.8-kb HME mRNA detected in the term placenta
represents the only actively remodeling
tissue.
In situ hybridization of term placenta tissue
sections was performed to determine which cell types express HME mRNA (Fig. 6). The antisense HME riboprobe hybridized distinctly to
cells within the villi. Endothelial cells did not express HME mRNA,
while the two other cell types within the villi, the macrophage-like
Hofbauer cells and the stromal-mesenchymal cells, had a strong
hybridization signal (Fig. 6, A and B). The
sense riboprobe did not hybridize to the placenta tissue (Fig. 6, C and D). To verify that both of these cell types
express HME, sequential sections were immunostained with an
affinity-purified antibody directed against HME and a
macrophage-specific anti-CD68 antibody. Comparison of CD68 immunostain (Fig. 6E) with HME immunostain (Fig. 6F)
demonstrates that HME is expressed in all macrophages in addition to
many CD68-negative, spindle-shaped stromal cells.
Figure 6:
Expression of HME mRNA in term placenta by in situ hybridization and immunohistochemistry. Serial
sections of term placenta were pro-cessed for in situ hybridization using S-labeled HME sense or antisense
riboprobes. A and B, photomicrographs of sections
hybridized with antisense HME probe. A, bright field
photomicrograph counterstained with hematoxylin and eosin demonstrate
the architecture of the villi (arrow). B, dark field
illumination demonstrates the presence of silver grains within cells of
the villi. C and D represent photomicrographs of
bright and dark-field, respectively, using a sense control probe.
Immunohistochemistry was performed on serial sections to determine cell
types expressing HME. E, anti-CD68 staining identified
Hofbauer macrophage cells. F, immunostaining with anti-HME on
a serial section verified that both CD68-positive macrophages as well
as CD68-negative stromal cells, but not endothelial cells, express HME.
(
400 magnification)
The Human Macrophage Metalloelastase Promoter Confers
Tissue-specific Expression and LPS Regulation
We analyzed the
promoter activity of HME by transient transfection to determine if
tissue-specific expression and LPS induction of HME were mediated by
the immediate 5`-flanking sequence of the gene. A reporter plasmid
containing the CAT gene under the control of the HME 5`-flanking
sequence was transfected into various cell types. As shown in Fig. 7, 2.8 kb of the HME promoter was sufficient to confer
tissue-specific expression. Similar to cellular expression of HME in
placenta (Fig. 6) and other tissues(17) , the HME promoter
drives expression of CAT in macrophage cells (P388D1 cell line), but
not endothelial cells (human umbilical vein endothelial cells).
Promoterless CAT was devoid of activity in both cell types (data not
shown). A similar lack of activity was observed in keratinocytes and
fibroblasts (data not shown).
Figure 7:
Tissue-specific expression and regulation
of the human macrophage metalloelastase promoter. Constructs containing
2.8 kb of the HME immediate 5`-flanking region linked to CAT and
control constructs pBLCAT2 (and promoterless CAT not shown) were
cotransfected with HSVtk--galactosidase, which was used as an
internal control for transfection efficiency, in P388D1 macrophages and
human umbilical vein endothelial cells. Six h following transient
transfections, fresh medium was added with the absence or addition of
LPS. After a total of 24 h, the cell extracts were normalized for
-galactosidase activity, and CAT activity was determined by
incubation of cell extracts with
[
C]chloramphenicol. Acetylated and nonacetylated
forms were separated by thin layer chromatography followed by
autoradiography.
The immediate promoter region is not
only sufficient to direct cell-specific expression of HME, but this
sequence also mediates LPS-induced up-regulation of HME in macrophages.
As shown in Fig. 7, addition of LPS following transient
transfection enhances HME-driven CAT activity. Measurement of C on amounts of lysate within the linear range
demonstrated a 4-fold increase in transcription in response to LPS.
This response is similar to increases in HME steady-state mRNA levels
observed in response to LPS in human alveolar macrophages(17) .
LPS did not influence CAT activity driven by the HSV promoter in
macrophages (not shown) nor was LPS responsiveness observed for HME-CAT
in human umbilical vein endothelial cells (Fig. 7), suggesting
that LPS induction of the HME promoter is specific for macrophages.
85-kb genomic clone, while
interstitial collagenase and stromelysin 2 were not. At region
11q22-23, the gene for ataxia telangiectasia (complementation
group, ATA) was mapped(35) . Linkage between HME, stromelysin,
and ATA genes would be of interest.
B (NF-
B) has been shown to play a role in
LPS-mediated induction of several genes in macrophages(36) . HME
does not contain an NF-
B site. Further evaluation of the HME
promoter will be required to delineate the important cis elements and trans-acting factors responsible for this
response. Knowledge gained from the genomic organization and
5`-flanking region of the HME gene will help in further delineating the
expression of HME.
Table: Exon and intron organization of HME gene
Table: Size of hybridizing fragments with pulsed field
gel electrophoresis
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.