©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Human Macrophage Metalloelastase
GENOMIC ORGANIZATION, CHROMOSOMAL LOCATION, GENE LINKAGE, AND TISSUE-SPECIFIC EXPRESSION (*)

Azzaq Belaaouaj , J. Michael Shipley , Dale K. Kobayashi , Drazen B. Zimonjic (1), Nicholas Popescu (1), Gary A. Silverman (2), Steven D. Shapiro (§)

From the (1)Respiratory and Critical Care Division, Department of Medicine and Department of Cell Biology and Physiology, Jewish Hospital at the Washington University Medical Center, St. Louis, Missouri 63110, the Laboratory of Biology, Division of Cancer Etiology, NCI, National Institutes of Health, Bethesda, Maryland 20892, and the (2)Joint Program in Neonatology, Harvard Medical School, Boston, Massachusetts 02115

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
REFERENCES

ABSTRACT

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.


INTRODUCTION

Matrix metalloproteinases (MMPs)()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) .

The MMP family consists of matrix-degrading proteinases that share sequence identity (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) .

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.


MATERIALS AND METHODS

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.

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.

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.

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--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.


RESULTS

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.


DISCUSSION

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 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.

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 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

Exon and intron sequences are shown in uppercase and lowercase, respectively. The 5` donor GT and the 3` acceptor AG are underlined.


  
Table: Size of hybridizing fragments with pulsed field gel electrophoresis

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.



FOOTNOTES

*
This work was supported by United States Public Health Service Grants HL2401 and PO-1 HL29594, by an American Lung Association fellowship (to J. M. S.) and Career Investigator award (to S. D. S.), and the Smokeless Tobacco Council for Research Grant 0477 (to G. A. S.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence and reprint requests should be addressed. Tel.: 314-454-7524; Fax: 314-454-8605.

The abbreviations used are: MMPs, matrix metalloproteinases; HME, human macrophage metalloelastase; kb, kilobase(s); bp, base pair(s); PCR, polymerase chain reaction; CAT, chloramphenicol acetyltransferase; LPS, lipopolysaccharide; ATA, ataxia telangiectasia; HSV, herpes simplex virus.


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