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Published ahead of print on May 30, 2003, doi:10.1165/rcmb.2003-0028OC
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American Journal of Respiratory Cell and Molecular Biology. Vol. 29, pp. 571-582, 2003
© 2003 American Thoracic Society
DOI: 10.1165/rcmb.2003-0028OC

Human ADAM33 Messenger RNA Expression Profile and Post-Transcriptional Regulation

Shelby P. Umland, Charles G. Garlisi, Himanshu Shah, Yuntao Wan, Jun Zou, Kristine E. Devito, Whei-Mei Huang, Eric L. Gustafson and Robert Ralston

Departments of Allergy and Human Genomics, Schering-Plough Research Institute, Kenilworth, New Jersey; and Canji Inc., San Diego, California

Address correspondence to: Shelby P. Umland, Ph.D., Schering-Plough Research Institute, 2015 Galloping Hill Road, K15-1-1700, Kenilworth, NJ 07033. E-mail: shelby.umland{at}spcorp.com


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
We examined transcript expression and post-transcriptional regulation of human ADAM33, a recently identified asthma gene. A detailed messenger RNA (mRNA) expression profile was obtained using Northern, reverse transcription polymerase chain reaction, and in situ hybridization analyses. ADAM33 mRNA was expressed significantly in smooth muscle–containing organs, minimally in immune organs and hematopoietic cells, and highly in repairing duodenal granulation tissue. Expression was seen in asthmatic subepithelial fibroblasts and smooth muscle but not in respiratory epithelium. In all tissues, transcripts of ~ 5 kb predominated over those of ~ 3.5 kb by 2- to 5-fold. The effect of the 3' untranslated region (UTR) on ADAM33 protein expression and maturation was examined. The presence of the 3'UTR in untagged full-length constructs promoted prodomain removal, detected as mature ~ 100 kD protein by ADAM33-reactive antibodies; in its absence, maturation was 2- to 3-fold less in HEK293 cells. His-tagged and untagged constructs lacking the 3'UTR demonstrated that lack of maturation was not a result of tag-mediated effects. Minimal maturation of ADAM33 occurred in primary lung and MRC5 fibroblasts following adenoviral-mediated expression of ADAM33 lacking the 3'UTR. In contrast, prodomain removal was observed with plasmids and adenovirus encoding only the pro- and catalytic domains. Thus, the 3'UTR of ADAM33 and domains downstream of the catalytic domain regulate potential ADAM33 activity. Mechanisms of regulation of ADAM33, distinct from closely related ADAMs, thus include mRNA localization and processing and protein maturation.

Abbreviations: antibody, Ab • a distintegrin and metalloprotease, ADAM • bronchial smooth muscle cells, BSMC • complimentary DNA, cDNA • Chinese hamster kidney cells, CHO-K1 • cycle threshold, Ct • epidermal growth factor, EGF • human embryonic kidney cells, HEK • a tag of six histidine residues, his • messenger RNA, mRNA • normal human lung fibroblasts, NHLF • in situ hybridization, ISH • polymerase chain reaction, PCR • recombinant adenovirus, rAd • reverse transcription PCR, RT-PCR • Tris-buffered saline, TBS • TBS with 0.1% Tween 20, TBS-T • tumor necrosis factor, TNF • untranslated region, UTR


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Recently, a disintegrin and metalloprotease (ADAM) 33, a newly discovered ADAM family member, was identified as an asthma susceptibility gene through a genetic linkage and association study of families with asthma (1). Defining the role of ADAM33 in the pathophysiology of asthma will require examination at many levels. ADAMs are a family of type I transmembrane zymogen proteins that typically contain an N-terminal secretion signal and prodomain followed by metalloprotease, disintegrin-like, cysteine-rich epidermal growth factor (EGF)-like, transmembrane, and cytoplasmic domains (2). A major function of those ADAMs containing the active site consensus sequence (HEXXHXXGXXH) is the shedding of the ectodomains of membrane proteins (3). However, for very few ADAMs have specific substrates been identified and biologically validated (4). Importantly, post-translational modification of these zymogens, involving removal of the prodomain from proximity to the catalytic site, is required for activation of catalytic activity (2, 5).

The predicted amino acid sequence of human and mouse ADAM33 (1, 6) includes the active site sequence 345HEIGH-SLGLSHD356 in the metalloprotease domain, suggesting catalytic activity. In addition, ADAM33 belongs to a subfamily (1, 6) in which others were shown to be catalytically active. ADAM19 cleaves membrane-anchored neuregulin-ß1, a member of the EGF receptor family (7); ADAM12 sheds heparin-binding EGF(8) and cleaves insulin-like growth factor binding protein–3 and –5 (9). The extracellular matrix component fibronectin is cleaved by ADAM9 (10). The less related ADAM17 (tumor necrosis factor [TNF]-{alpha}–converting enzyme), the most thoroughly studied ADAM, sheds membrane-bound TNF-{alpha}, thereby releasing soluble TNF-{alpha}. Other substrates of ADAM17 include TNF-{alpha} receptor, transforming growth factor-{alpha}, L-selectin, interleukin-1 receptor type II, and amyloid precursor protein (2); the receptor kinase ErbB4/Her4, a member of the EGF receptor family (11), and fractalkine, a CX3C chemokine (12).

Given the types of cell-associated molecules processed proteolytically by ADAMs, it is clear that ADAMs modulate the cellular environment by regulating growth factor, receptor, and adhesion molecule accessibility by converting membrane-bound molecules to soluble forms. In addition, ADAMs interact with major components of the extracellular matrix and thus may be involved in the remodeling of tissue as well as the cell surface. There is evidence that these interactions are facilitated by the disintegrin domains of ADAMs, which interact with integrins on several cell types (2, 3). ADAMs function in diverse cellular processes, such as cell fusion, including muscle cell fusion and fertilization, neurogenesis (2), adipogenesis (13), and inflammation (14). Thus, it is not suprising that several ADAMs have been implicated in pathologic processes. ADAM17 contributes to rheumatoid arthritis (14), due to its processing of TNF-{alpha}, a major mediator of inflammation, from its proform to the active soluble form. A role for ADAM12 in cardiac hypertophy has been demonstrated through its shedding of myocardial heparin-binding EGF (8). Most recently, ADAM33 was implicated as an asthma susceptibility gene (1), the validity of which requires corroboration at the level of function.

A critical part of identifying the role of ADAM33 in the pathophysiology of asthma will be the identification of its biological substrates. Because ADAMs cleave either cell surface or intracellularly located membrane-bound molecules in a juxtacrine not paracrine fashion (15), we undertook as our first objective a detailed analysis of the messenger RNA (mRNA) expression profile of human ADAM33 in many normal tissues and cell types by Northern analysis and reverse transcription polymerase chain reaction (RT-PCR). This was correlated with the spatial distribution of ADAM33 mRNA in intact tissue from normal and diseased tissue, including asthmatic lung, by in situ hybridization (ISH). Previously, a limited analysis of the mRNA expression profile of ADAM33 indicated its presence in many tissue types, including heart, lung, and small intestine (1, 6); cultured lung fibroblasts and bronchial smooth muscle cells (BSMC) (1) expressed significant levels of ADAM33.

Our second objective was to investigate post-transcriptional regulation of ADAM33. Post-transcriptional events are likely to be major contributors to ADAM33 activity because members of this protease subfamily are initially synthesized as inactive pro-proteins. Untranslated regions (UTRs) of genes, particularly 3'UTR sequences, mediate post-transcriptional and translational control of protein production by affecting mRNA degradation (16), nuclear export, and subcellular localization (17). In addition, alterations in 3'UTR-mediated functions have been associated with disease (18). Because ADAM33 contains several polymorphisms in the 3'UTR that are significantly associated with asthma, we investigated the effect of the 3'UTR on heterologous ADAM33 protein expression and maturation. This was done by both plasmid and recombinant adenovirus-mediated ADAM33 expression in transformed cell lines and primary lung fibroblasts, the latter being identified from our mRNA studies as a major cell type expressing ADAM33 in the normal and asthmatic lung.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Cell Culture
Human embryonic kidney (HEK) cells (HEK293) and Hela cells were obtained from Canji, Inc. (San Diego, CA). HEK293 cells were grown in complete media (Dulbecco's modified eagle's medium [DMEM]), (Gibco Invitrogen, Inc., Grand Island, NY), 10 mM Hepes, 10% FBS, 50 U/ml penicillin, 50 µg/ml streptomycin, and 2 mM L-glutamine). Hela cells were grown in DMEM high glucose with 10% FBS and antibiotics. MRC5 cells, a lung fibroblast line #CCL-171 (ATCC, Manassas, VA) were grown in Earle's Modified Eagle's medium, 10% fetal bovine serum and 2 mM L-glutamine. Normal human lung fibroblasts (NHLF) (BioWhittaker, Walkersville, MD) were grown in the FGM-2 BulletKit medium according to the manufacturer's specifications. Primary cells (T cells, neutrophils, eosinophils) were isolated from the peripheral blood of healthy volunteers. CD4+ T cells were differentiated for the indicated number of days (d12, d13, d20) into T helper type 1 (TH1) or TH2 cells as previously described (19). Neutrophils and eosinophils (20) were isolated from peripheral blood of healthy donors by standard methods. Normal human bronchial epithelial cells (BioWhittaker) were grown according to the accompanying specifications or in air/liquid interface cultures (21). The cell and tissue sources and methods of construction of the complimentary DNA (cDNA) library panel have been described previously (22, 23). Human lung tissue was obtained from the Anatomic Gift Foundation according to Institutional Review Board (IRB) approval; human saphenous vein was obtained, subsequent to IRB approval and patient informed consent, as discard tissue from coronary artery bypass grafts from Hackensack University Medical Center for Biomedical Research (Hackensack, NJ).

mRNA Expression Analysis TaqMan Analysis—Human Tissue Panel
Human tissue autopsy samples were purchased from Zoion Diagnostics (Shrewsbury, MA), subsequent to IRB approval. Postmortem times for tissue collection ranged from 2–6 h. Each tissue type was obtained from three donors. Total RNA was isolated from the tissues using TRI-reagent (MRC, Cincinnati, OH), and tested for quality and quantity using an Agilent 2,100 Bioanalyzer and RNA Labchips (Agilent, Waldbroun, Germany). Real-time quantitative polymerase chain reaction (PCR) was tested by the fluorogenic 5'-nuclease PCR assay (TaqMan) with an ABI Prism 5,700 Sequence Detection System (PE ABI, Foster City, CA). The TaqMan probes were labeled with the dye 6-carboxyfluorescein at the 5'-end of the sequence and with the quencher 6-carboxytetramethylrhodamine at the 3'-end, and all were synthesized by PE ABI. The TaqMan reagents for ADAM33 were designed using the uterus cDNA clone (GenBank accession AF466287), which differs in sequence from AB055891 (6). Probe 4–2403T (Table 1) matches the cDNA sequence encoding the latter but not the former sequence, which differ in the presence of the GCA alanine codon and, consequently, any results obtained with this probe were always confirmed by other TaqMan reagents. The final concentrations of the primers and probe in the PCR reactions were 200 nM and 100 nM, respectively. The PCR reactions were prepared using the components from the TaqMan Gold RT-PCR kit (PE ABI) and assembled according to the manufacturer's instructions. Each 25 µl PCR reaction contained 5.0 µl (50 ng) of total RNA prepared as described above. The RT-PCRs were performed in a single 96-well plate as follows: 48°C, 30 min; 95°C, 20 min; followed by 40 cycles at 95°C for 15 s and 60°C for 1 min. A separate plate of the same RNAs was used to quantitate 18S RNA as an internal quality control. Absolute quantitation was done using a standard curve generated with five 2-fold serial dilutions of the pcDNA3.1(-) (Invitrogen Corp., Carlsbad, CA) plasmid containing full length ADAM33 (GenBank accession number AF466287), starting at 0.5 ng.


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  		TABLE 1 Primer and probe sequences

 
TaqMan Analysis—Primary Cell–Tissue Panel and cDNA Library Panel
Primary cells, cell cultures (resting or activated), and tissues were homogenized in 4 M guanidine isothiocyanate solution (Invitrogen) with 50 mM Tris-hydrochloride (pH 7.5), 25 mM ethylenediamine tetraacetic acid, 0.05% sodium laurylsarcosine, and 0.15% ß-mercaptoethanol (Sigma, St Louis, MO). The homogenate was overlaid on 5.7 M cesium chloride (Roche, Indianapolis, IN) with 10 mM ethylenediamine tetraacetic acid and RNA was collected following ultracentrifugation at 25,000 rpm for 25 h at 25°C using an SW28 rotor in a Beckman XL-80 centrifuge (Beckman Coulter, Fullerton, CA). RNA was quantitated by absorbance at 260/280 nm. cDNA was synthesized using Superscript II reverse transcriptase (Invitrogen) with 5 µM oligo d(T)15 and 1.25 µM oligo p(dN)6 (Roche). These cDNAs or library cDNAs (22, 23) (Table 2), prepared from various primary human cells and cell lines, were tested for expression of ADAM33 by real-time quantitative PCR. Ubiquitin (Table 1) was used as an internal control for quantification of the total amount of cDNA analyzed. A total of 10–20 ng of cDNA per sample was analyzed in a 50 µl reaction mixture. The PCRs were performed in duplicate in 96-well plates. Each 50 µl reaction contained 10 µl (50 ng) of each sample. The cycling conditions were: 50°C, 2 min; 95°C, 10 min; followed by 45 cycles at 95°C for 15 s and 60°C for 1 min. The quantification of the amplicons per reaction was done according to the comparative cycle threshold (Ct) number method as described in the PE ABS User Bulletin No. 2 (Foster City, CA) and expressed as 2(-{Delta}Ct) x 1,000.


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  		TABLE 2 ADAM33 mRNA expression in cDNA libraries

 
Northern Analysis
Expression of ADAM33 mRNA was examined using Northern blots obtained from Clontech (Palo Alto, CA). Hybridization was performed using [32P]dCTP random primer-labeled DNA probes at ~ 2 x 106 cpm/ml. A DNA probe in the ADAM33 3'-UTR was generated by PCR using the primer pairs 2561F/3013R (Table 1). The amplified probe was purified by Qiagen Gel extraction columns ProbeQuant G50 Micro columns (Amersham Pharmacia Biotech, Piscataway, NJ). A ß-actin probe (Clontech) was prepared and used according to the manufacturer's specifications. Blots were hybridized in ExpressHyb (Clontech) and washed according to the manufacturer's specifications. All blots were exposed to Kodak Biomax MS film (Eastman Kodak, Rochester, NY) for the indicated time at -70°C. Quantitation of the ~ 5- and 3.5-kb bands observed was done with the Bio-Rad Molecular Imager Fx instrument and Quantity One software (Bio-Rad Laboratories, Hercules, CA).

In Situ Hybridization
ISH was conducted by LifeSpan Biosciences, Inc. (Seattle, WA). A 540-bp fragment corresponding to nucleotides 2,341 to 2,880 of the sequence was amplified using 2341F/2880R primers (Table 1) and the fragment subcloned into pCRII (Invitrogen). In vitro transcription was done using SP6 (antisense) and T7 (sense) RNA polymerases in the presence of 35S-UTP (Perkin-Elmer, Boston, MA). After transcription, the probes were column-purified and separated by electrophoresis on a 5% polyacrylamide gel to confirm size and purity. Serial tissue sections from archival paraffin samples were digested with proteinase K (Roche, Indianapolis, IN) and hybridized with the probes at ~ 3.3–5.8 x 107 dpm/ml for 18 h at 62°C. The slides were treated with RNAse A (Sigma) and washed stringently in 0.1x saline sodium citrate buffer at 67°C for 2 h. The slides were then coated with Kodak NTB-2 emulsion (Eastman Kodak), exposed for 7 or 10 d at 4°C, and developed using Kodak D-10 Developer and Fixer. Slides were stained with hematoxylin and eosin and imaged using a DVC 1310C camera-coupled Nikon microscope (Nikon, Melville, NY). All tissues used were screened initially with a probe for ß-actin mRNA to ensure that RNAs were preserved within the archival paraffin samples. For each sample, adjacent serial sections were also hybridized with the control sense riboprobe. For each tissue type, two donors were examined.

cDNA Constructs
For expression in eukaryotic cells, several constructs were prepared. The full-length cDNA for ADAM33 was isolated from a primary cDNA clone (GenBank accession number AF466287) (1), subcloned into pcDNA3.1(-) (Invitrogen ), and designated (1–812+UTR). Using this plasmid, the active site glutamic acid was mutated to an alanine (1–812E346A+UTR) using oligonucleotide primers and PfuTurbo DNA polymerase as described by the manufacturer (QuickChange Site-Directed Mutagenesis Kit; Stratagene, La Jolla, CA). The single basepair change from GAG to GCG generated a PvuI site that was used for diagnostic purposes. For expression of the full-length gene without the 3' UTR, a PCR fragment corresponding to amino acids 1–812 was generated using primers that incorporated a XhoI site into the 5' end and a NotI site into the 3' end of the amplicon. The EcoRI/NotI or XhoI/NotI fragments from an intermediate vector (pCR4-TOPO; Invitrogen) were purified and ligated into pcDNA3.1/Myc-his(+)B or pcDNA3.1(-), respectively. This resulted in constructs of full-length ADAM33 containing no 3' UTR sequences with (1–812-{Delta}UTR-myc/his) or without (1–812-{Delta}UTR) a 3' Myc-his tag. For construction of a plasmid containing only the ADAM33 leader sequence, prodomain and catalytic domain terminating at aspartic acid 432, PCR primers were designed to add an XhoI site to the 5'end of the gene fragment and a HindIII site to the 3'end. The 3'end oligonucleotide also incorporated sequence encoding six histidines and a stop codon and was ligated into pcDNA3.1(-)(1–432-his). All constructs were verified by restriction endonuclease digestion and DNA sequencing. Plasmids were purified from Escherichia coli extracts using DNA affinity columns (Qiagen GmbH, Hilden, Germany).

Adenovirus Vectors for Expression of ADAM33
Two recombinant adenovirus forms of ADAM33 were constructed: a full-length form, recombinant adenovirus (rAd)-1–812, lacking the 3'UTR and a form, truncated at amino acid 424 (1), rAd-1–424-V5/his, linked to a V5 epitope and his tag. Each was a replication-defective vector containing a CMV driven expression cassette in the E1 region. The Ad5-based viral backbone used for both constructs had deletions of the entire E1 region, coordinates 459–3327, and part of the E3 region, coordinates 25838–32004 (24, 25). rAD-1–424-V5/his was constructed by subcloning a PCR product encoding amino acids 1–424 of human ADAM33 linked to sequences encoding a V5 epitope and his tag and stop codon into the CMV expression cassette within the transfer plasmid, pTRACN'B', which contains the left-hand end of the vector. The sequence-confirmed pTRACN'B' transfer plasmid containing the ADAM 33(1–424)V5–his cDNA was recombined in E. coli strain BJ5183 with plasmid pTG4215 containing the remainder of the A5 vector genome as described previously (26). The resulting infectious plasmid was used to generate rAd-1–424-V5/his virus by transfection of HEK293 cells. rAd-1–812 was constructed similarly using the plasmid pcDNA3.1(+)-1–812{Delta}UTR-myc/his as the template for PCR-based subcloning. The control virus, ZZCB, is a replication-defective adenovirus, analogous to the rAd-ADAM33(1–424)V5/his and rAd-ADAM33(1–812) vectors, in which the E1 region was replaced by an "empty" CMV expression cassette (24). Recombinant adenovirus vectors were purified by column chromatography and particle concentrations were measured as described (27).

Transfections
One day before plasmid transfection, HEK293 cells were cultured in 12-well plates at 1.5 X 105 cells/well in complete media at 37°C in 5% CO2. On the day of transfection, cells were washed with media (serum- and antibiotic-free) and transfected with 0.8 µg plasmid DNA using Lipofectamine and Plus reagent (Invitrogen) as described by the manufacturer. After 5 h at 37°C, cells were rinsed and cultured in complete media containing 1x nonessential amino acids (Gibco, Invitrogen) instead of serum. Transiently transfected cells were maintained in culture for 48 h. For rAd infections, HEK293 and Hela cells were infected with 1 x 109 particles/ml of recombinant virus for 2 h, after which the media was exchanged with DMEM high glucose containing 0.1% bovine serum albumin. MRC5 and NHLF cells at 1.5 x 105 cells/well in 6-well plates were infected with 109 and 108 particles/ml, respectively, and treated as above for HEK293 plasmid transfections. HEK293 cell supernatants and lysates were harvested at 24 h and Hela, NHLF, and MRC5 were harvested at 48 h after infection.

Antibody Production
Peptides containing amino-terminal cysteines were synthesized (Zymed Laboratories, Inc., South San Francisco, CA) corresponding to regions within the prodomain (amino acids 44 – 61, NH2-(C)VLDGQPWRTVSLEEPVSK-COOH), catalytic domain (amino acids 303 to 319; NH2-RAFQGATVGLAPVEGMC-COOH), and cytoplasmic domain (amino acids 777 to 790; NH2-(C) DPENSHEPSSHPEK-COOH) of ADAM33 (1). Antibodies were made, purified, and characterized as previously described (28). The antibody designations are as follows: Pro1—reactive to the prodomain peptide 44 - 61; ASP2—reactive to the catalytic domain peptide 303—319; Cyt2—reactive to the cytoplasmic domain peptide 777–790.

Western Blotting
After transfection, the cell supernatants were collected after centrifugation and cell extracts were prepared from HEK293 and Hela cells as described (29). Lysis buffer contained 1% NP-40, 10 mM 1,10-phenanthroline and 1x Complete Protease Inhibitor Cocktail (Roche) in Tris-buffered saline (TBS; 20 mM Tris-hydrochloride, 500 mM sodium chloride, pH 7.5). Cell culture media were concentrated 5- to 10-fold using Centriprep YM10 filter devices (Millipore, Bedford, MA) and lysates were used directly. Where indicated, samples were enriched for glycoproteins by ConA-sepharose (Amersham Pharmacia Biotech) as described (29). Samples were heat-denatured with SDS, reduced with dithiothreitol (DTT) and equivalent protein separated by electrophoresis through 7.5%, 10%, or 4–20% SDS-polyacrylamide gels (Bio-Rad). Molecular mass was estimated by comparison to Kaleidoscope (Bio-Rad) or BenchMark (Invitrogen) prestained standards included on every gel. Proteins were transferred to polyvinylidene fluoride membranes by electroblotting. For immunodetection, membranes were blocked with 5% ECL blocking reagent (Amersham) in TBS containing 0.1% Tween 20 (TBS-T) for 1 h at room temperature. After rinsing three times with TBS-T, membranes were incubated with antibodies (1–2 ug/ml affinity-purified antipeptide antibodies and 1:5,000 horseradish peroxidase–conjugated antihistidine antibody (Invitrogen) for 1 h. Unbound antibodies were removed by washing with TBS-T and, if required, membranes were incubated with goat anti-rabbit immunoglobulin G conjugated to horseradish peroxidase (1:25,000; Jackson ImmunoResearch Laboratories, Inc., West Grove, PA) for 30 min. Unbound antibodies were removed by washing and immobilized antigen–antibody complexes were detected with ECL chemiluminescence detecting reagents (Amersham) as described by the manufacturer. The amount of the pro and mature forms of ADAM33 as a percentage of total was calculated by digital scanning of developed films and analysis with Quantity One software (Bio-Rad). Multiple films were acquired to assure bands did not saturate the film.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Human ADAM33 mRNA Expression Profile
To expand upon previous studies of the distribution of ADAM33 mRNA (1, 6), real-time quantitative PCR was performed on a panel of human tissues to quantitate ADAM33 mRNA levels. For these analyses, each tissue type was represented by three healthy human donors. Expression was examined using primers that amplified a fragment detectable by a probe within the cytoplasmic and 3'UTR region of the transcript (4–2384F/4–2467R, Table 1). No expression was observed in kidney, liver, pancreas, bone marrow, or spleen (Figure 1). No ADAM33 expression was detected in cell lines derived from colon (n = 4), ovary (n = 4), breast (n = 5), and prostate (n = 3) tumors, as well as gliomas (n = 7) and melanomas (n = 4) (data not shown). These cell lines were of epithelial origin. The highest expression observed occurred in diverse tissues with a significant smooth muscle component, such as the urethra and bladder and in the vasculature (saphenous vein, aorta, and coronary artery). Approximately 10-fold lower levels of ADAM33 mRNA were found in the lung, trachea, and bronchus. ADAM33 mRNA was observed in all brain regions examined at a level ~ 100-fold below that seen in the vasculature or smooth muscle–containing organs. Of note, for 70% of the 62 tissues shown, the expression level for each of the 3 donors did not differ by more than 10-fold. Similar results were obtained with the primer set 3–2090F/3–2290R (Table 1) amplifying a fragment between the transmembrane and cytoplasmic region.



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  		Figure 1. TaqMan analysis of ADAM33 mRNA in human tissues. ADAM33 mRNA levels were quantitated by TaqMan using the methods for the human tissue Taqman array described in MATERIALS AND METHODS. The primers and probe set used was 4–2384F/4–2467R/4–2403T (Table 1). Similar results were obtained with 3–2090F/3–2290R/3–2237T set. Values are expressed as RNA copies per 50 ng RNA input (mean ± SD of three individual samples per tissue).

 
To identify specific cell types in which transcription of ADAM33 occurred, cDNAs derived from RNA isolated from cells of hematopoeitic origin (T cells, neutrophils, eosinophils), as well as bronchial epithelial and lung fibroblast cells, were tested by quantitative PCR. Expression was examined using 3–2090F/3–2290R and 4–2384F/4–2467R (Table 1) with similar results obtained. No expression was observed in differentiated TH0 (B21 cells), TH1, or TH2 cells (data not shown). In addition, no expression was detected in these hematopoietic cells with stimuli relevant to each cell type (T cells stimulated with anti-CD3 or costimulated with anti-CD28 antibody [19], lipopolysaccharide stimulation of neutrophils, and interleukin-5 stimulation of eosinophils [30] data not shown). Lastly, normal human bronchial epithelial cells grown under standard liquid conditions or in air/liquid interface cultures, in which differentiation of ciliated and secretory cells occurs (21), did not express human ADAM33. In the same experiments, the absence of expression in these cell types contrasted with the significant expression of ADAM33 seen in lung tissue (35 ± 29, n = 13, 2(-{Delta}Ct) x 1,000) and that detected in saphenous vein (600 ± 20, n = 2, 2(-{Delta}Ct) x 1,000).

The results above suggested that ADAM33 mRNA detected in the lung was unlikely to originate in hematopoeitic cells. To confirm this and identify cell types contributing to ADAM33 expression in the lung, a screen of cDNA libraries derived from other resting and activated primary cells, cell lines, and fetal tissues was done using 3–2090F/3–2290R primers (Table 2). Primary cells of hematopoeitic origin including T and B cells, monocytes, mast, dendritic, and natural killer (NK) cells did not express ADAM33 mRNA. Consistent with the lack of expression in primary bronchial epithelial cells (Figure 2), A549 cells (Table 2, samples 48, 49), an epithelial cell line, and CHA cells (sample 51), a kidney epithelial cell line, did not express ADAM33. Interestingly, significant expression was seen in MRC5 cells (sample 50), a lung fibroblast cell line, and in Jurkat cells (sample 14, 15), a transformed T cell line. ADAM33 was expressed ubiquitously in human fetal tissue (samples 52–62), with the exception of fetal brain (sample 56). The highest expression in fetal tissue was seen in uterus (sample 60).



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  		Figure 2. Analysis of human ADAM33 expression by Northern analysis. Northern blotting was done using a [32P]dCTP probe corresponding to the ADAM33 3'UTR (2561F/3013R, Table 1). Temperatures for prehybridization, hybridization, and washes were 65, 65, and 50°C, respectively. Blots were exposed to film for 72 h. For each tissue, the ratio of signal intensity of the ~ 5 to 3.5 kb bands is shown in parentheses. (A) Clontech Human MTN Blot III: Lane 1, stomach (2.9); 2, thyroid (2.0); 3, spinal cord (2.3); 4, lymph node (2.6); 5, trachea (2.7); 6, adrenal gland (2.3); 7, bone marrow (1.8). (B) Clontech Human Muscle MTN Blot: Lane 1, skeletal muscle (2.8); 2, uterus (3.0); 3, colon (4.6); 4, small lintestine (3.9); 5, bladder (2.6); 6, heart (2.3); 7, stomach (3.6); 8, prostate (4.1). (C) Clontech Human Immune II Blot: Lane 1, spleen (2.6); 2, lymph node (2.8); 3, thymus (3.7); 4, peripheral blood leukocytes (2.3); 5, bone marrow (2.8); 6, fetal liver (3.4). (D) Human Brain MTN Blot II: Lane 1, cerebellum (3.2); 2, cerebral cortex (3.5); 3, medulla (2.9); 4, spinal cord (2.6); 5, occipital pole (4.2); 6, frontal lobe (4.2); 7, temporal lobe (3.2); 8, putamen (2.9). Blots were stripped, rehybridized with a ß-actin probe, and exposed to film for 2 h. For each blot, A–D, the upper panel represents ADAM33 and the lower panel represents ß-actin hybridization. Size markers are in kb.

 
It has been shown previously by limited Northern analysis that ADAM33 transcripts comprise two major bands of ~ 5 and 3.5 kb (1). To determine if this was characteristic of ADAM33, additional tissue samples were analyzed by Northern blotting using Clontech blots (Figure 2). Two predominant bands of ~ 5 and 3.5 kb were seen in all tissues. The highest levels of expression by Northern analysis were observed in tissue with a significant contribution of smooth muscle, such as stomach (Figure 2A, lane 1; Figure 2B, lane 7), uterus, colon, small intestine, heart, and prostate (Figure 2B, lanes 2–6, 8). Skeletal muscle expressed much lower levels of ADAM33 mRNA (Figure 2B, lane 1). ADAM33 was expressed at moderately high levels in the trachea, which also contains smooth muscle (Figure 2A, lane 5). Variable levels of ADAM33 mRNA were observed in tissues of hematopoietic origin: negligible levels were seen in spleen (Figure 2C, lane 1), bone marrow (Figure 2A, lane 7; and 3C, lane 5) and peripheral blood leukocytes (Figure 2C, lane 4), while lymph node (Figure 2A, lane 4; and 2C, lane 2) and thymus (Figure 2C, lane 3) expressed higher levels. (ß-actin levels for the blot in Figure 2C were the highest of the four blots.) Relative to smooth muscle–containing tissue, expression of ADAM33 mRNA in various brain regions was much lower (Figure 2D). A high correlation of rank order of expression levels of ADAM33 in the same human tissue types was observed between the two methods, Northern analysis (Figure 2) and quantitative RT-PCR (Figure 1).

Two additional points can be made regarding the expression of ADAM33 by Northern analysis. First, higher levels of the larger band were observed consistently in all tissues. Given that the known full-length cDNAs encode only the 3.5 kb transcript and the nature of the larger forms is unknown, the intensities of the two bands were quantitated to determine whether particular tissues expressed different ratios of the different size transcripts (Figure 2). Despite a broad range of expression levels (differing by more than 100-fold) in the different tissues, the ratios of the signal intensity of the two bands occurred in a narrow range of 1.8 to 4.5. Second, in several tissues with the highest expression levels (Figure 2B:, uterus, lane 2; small intestine, lane 4; and heart, lane 6), a transcript of 7–8 kb was observed.

The results above indicated a unique expression pattern of human ADAM33 with a presence in cells of mesenchymal origin, such as fibroblasts and smooth muscle cells, and an absence in nonmesenchymal hematopoietic cells and epithelial cells. To further define this pattern, examination of ADAM33 by ISH was performed in intact tissue from normal and disease states, including asthma. Based upon the distribution of ADAM33 mRNA by quantitative PCR (Figure 1) and the origin of an ADAM33 cDNA clone (1), uterus was selected as a positive control tissue. Within the uterine tissue, stromal cells (Figure 3A) and myometrial and vascular smooth muscle (Figure 3B) showed significant hybridization to the antisense probe, with little hybridization to the sense probe (Figure 3C). In the normal lung bronchus, ADAM33 mRNA was not detected in respiratory epithelium (Figure 3D) where ß-actin mRNA was highly expressed (Figure 3E), demonstrating the integrity of the RNA. However, ADAM33 mRNA was detected in the submucosal smooth muscle of the bronchus (Figure 3F). Importantly, in asthmatic tissue, where the constricted airway and mucus plug (Figure 3G) was indicative of asthmatic airway disease, ADAM33 mRNA was present in submucosal smooth muscle cells (Figure 3I) underlying the respiratory epithelium, within which no expression was detected (Figure 3H). Lastly, granulation tissue, obtained from an ulcerous duodenum and undergoing a repair response to injury, was examined. Significant hybridization was seen in smooth muscle cells (Figure 3J) and in reactive fibroblasts along the ulcer base (Figure 3K); these cells displayed the highest level of expression within the ISH study. Interestingly, tissue macrophages showed moderate positivity (Figure 3K); these cells were negative when hybridized to the sense control probe (Figure 3L). ADAM33-expressing macrophages were also observed in soft connective tissue of the hand undergoing a chronic inflammatory response and in the edematous submucosal tissue of a nasal polyp (data not shown).




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  		Figure 3. ADAM33 mRNA expression in intact tissue by in situ hybridization. Archival tissues were screened for ß-actin mRNA to ensure intact RNA. Adjacent serial sections were hybridized to ADAM33 antisense and sense probes and subsequently stained with hematoxylin and eosin. The probes recognized a 540-bp fragment corresponding to nucleotides 2,341 to 2,880 in the 3'UTR of ADAM33 (Table 1). (A) Normal uterus (surgical) showing stromal cells, ADAM33 antisense probe, 60x. (B) Uterus showing myometrial smooth muscle, ADAM33 antisense probe, 60x. (C) Uterus showing stroma, ADAM33 sense probe, 60x. (D) Normal bronchus from 58-yr-old male, showing epithelium, ADAM33 antisense probe, 60x. (E) ß-actin antisense probe, 40x. (F) Normal bronchus from 60-yr-old male showing submucosal smooth muscle, ADAM33 antisense probe, 60x. Arrowhead indicates representative smooth muscle cell with ADAM33-specific hybridization. (G) Asthma lung from an 18-yr-old female with asthma who died of respiratory failure, hematoxylin and eosin stained, 10x. (H) Tissue as in (G), showing submucosal smooth muscle and epithelial layer, ADAM33 antisense probe, 60x. (I) Asthma lung from a 13-yr-old male with asthma who died of respiratory failure showing submucosal smooth muscle, ADAM33 antisense probe, 60x. Arrowhead indicates representative smooth muscle cell with ADAM33 specific hybridization. (J) Duodenum, ulcer granulation tissue (surgical) from a 62-yr-old male showing smooth muscle cells, ADAM33 antisense probe, 60x; Arrowheads indicate representative smooth muscle cells with ADAM33 specific hybridization. (K) Tissue as in (J), showing ulcer base containing macrophages (yellow arrowhead) and reactive fibroblasts (green arrowhead), ADAM33 antisense probe, 60x; (L) tissue as in (K), ADAM33 sense probe, 60x; macrophages are indicated by yellow arrowhead. Scale is indicated on each panel.

 
The significant expression of ADAM33 mRNA in lymph node and thymus by Northern analysis was seemingly at odds with its lack of expression in many hematopoietic cells tested by the more sensitive method of RT-PCR. To reconcile these differences, ISH was done on lymph node, thymus, and spleen tissue (data not shown). In these lymphatic tissues, the strongest hybridization signals obtained with the antisense probe to ADAM33 occurred in vascular endothelial cells, smooth muscle cells, and subcapsular fibroblasts. Within the lymph nodes and spleen, plasma cells, neutrophils, and eosinophils were negative. Occasional lymphocytes were positive in interfollicular and paracortex regions of the lymph node and red pulp of the spleen. Thymic lymphocytes did not demonstrate specific hybridization to the ADAM33 antisense probe.

Requirement for the 3'UTR in ADAM33 Protein Maturation
The studies described above indicated that ADAM33 is constitutively transcribed at a low level in many cell types and tissues. Little evidence of further inducibility of ADAM33 mRNA was obtained in those cells and tissues or in epithelial or hematopoietic cells. Thus, we investigated post-transcriptional regulation. ADAM33 is highly polymorphic, with several polymorphisms in the 3'UTR that are significantly associated with asthma. Because 3'UTRs are known to regulate mRNA stability (16), cellular localization (17), and translational efficiency, we investigated its effect on the expression and maturation of ADAM33 protein. HEK293 cells were transfected with various ADAM33 cDNA constructs, each containing the native signal and prodomain sequences to allow for proper targeting, folding, and processing. The following constructs were compared: full-length ADAM33 without the 3'UTR (1–812{Delta}UTR), full-length ADAM33 with a myc/his tag without the 3'UTR (1–812{Delta}UTR-myc/his), full-length ADAM33 with its 3'UTR (1–812+UTR), and the same with a mutation from 346E to 346A to inactivate the catalytic site (1–812E346A+UTR). Also included was a construct of ADAM33, which included the prodomain and catalytic domains only, with a carboxyl terminus at aspartic acid 432 and a 6-his tag (1–432-his). Following transfection of HEK293 cells, the cell supernatants and lysates were immunoblotted. For detection, antibodies to ADAM33 were used because there is evidence of loss of C-terminal tags through autocatalysis (31). Only those constructs of full-length ADAM33 containing the 3'UTR showed significant processing of the immature, ~ 120 kD form of ADAM33 protein (Figure 4A, lanes 2 and 3) to a form displaying higher mobility of ~ 100 kD. This was also observed for the construct 1–812E346A+UTR, with the catalytic site-inactivating mutation (lane 3), demonstrating that prodomain removal is not due to autocatalysis. Both the ~ 120- and 100-kD forms were detectable in cell lysates only with Cyt2 antibody and thus represent membrane-bound forms. Only the larger ~ 120-kD protein was also recognized by the antiprodomain antibody, Pro1, and thus represents the latent, unprocessed form of ADAM33 (data not shown). The proportion of mature form to total ADAM33 protein expressed from the 1–812+UTR and 1–812E346A+UTR constructs was 21 ± 8% (n = 6) and 27 ± 7% (n = 5), respectively. Both constructs without the 3'UTR, 1–812{Delta}UTR and 1–812{Delta}UTR-myc/his (Figure 4A, lanes 4 and 5, respectively) expressed mainly the latent form of ADAM33 with minimal mature form. The latent, unprocessed form but not the processed form of ADAM33 expressed from the 1–812{Delta}UTR-myc/his construct was detected with an anti–his antibody (Figure 4A, lane 7), demonstrating the presence of the C-terminal tag. The proportion of mature form to total ADAM33 protein expressed from 1–812{Delta}UTR and 1–812{Delta}UTR-myc/his constructs was 12 ± 2% (n = 4) and 8 ± 5% (n = 5), respectively. Thus, in the presence of the 3'UTR, 2- to 3-fold more mature ADAM33 protein was detected.



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  		Figure 4. Effect of 3'UTR on ADAM33 expression and maturation. (A) HEK293 cells were transfected with full-length ADAM33 constructs and 48 h cell lysates were analyzed by Western blotting with antibodies that recognize the cytoplasmic domain (Cyt2, lanes 1–5), or anti–his antibody (lanes 6–7). Lanes 1 and 6, control vector; lane 2, 1–812+UTR; lane 3, 1–812E346A+UTR; lane 4, 1–812{Delta}UTR; lanes 5 and 7, 1–812{Delta}UTR-myc/his. (B–C) HEK293 cells were transfected with 1–432-his construct and 48 h cell lysates (B), or concentrated culture medium (C), analyzed by Western blotting with antibodies that recognize the catalytic domain (ASP2, lanes 1, 2, 7, and 8); the prodomain (Pro1, lanes 3, 4, 9, and 10); or anti–his (lanes 5, 6, 11, and 12). P = proform, m = mature form.

 
In HEK293 cells, the plasmid construct 1–432-his, lacking domains distal to the catalytic domain as well as the 3'UTR, was efficiently processed from a form of ~ 70 kD in cell lysates (Figure 4B), detectable by both Pro1 (lane 4), ASP2 (lanes 2) and anti–his antibodies (lane 6) to a secreted processed form of ~ 40 kD (C). The doublet clearly observed (Figure 4B, lanes 2, 6) is due to differential gylcosylation (data not shown; Ref. 28). It has been similarly found for ADAM12-S (9) that the cleaved prodomain remains associated with the mature protein. The soluble secreted form of ADAM33 was detectable by both ASP2 (C, lane 8), reactive to the catalytic domain, and anti–his antibody (C, lane 12) but not Pro1 (C, lane 10), although a smaller protein (~ 30 kD), the cleaved prodomain free of the catalytic domain, in the cell culture medium was detected (C, lane 10). In contrast to the culture medium where there is an accumulation of secreted catalytic domain, in the cell lysate no processed catalytic domain was detected, probably due to rapid secretion. Lastly, a comparison of the full length and pro-catalytic domain constructs with N-terminal tags, demonstrates that the presence of this tag does not, in and of itself, disrupt prodomain removal from ADAM33.

To confirm the lack of processing in the absence of the 3'UTR using a different expression system, a recombinant adenovirus containing amino acids 1–812 of ADAM33 (rAd-1–812) was used. In addition, an rAd containing only the pro- and catalytic domains of ADAM33, with a carboxyl terminus at glutamic acid 424, and containing a V5/his tag (rAd-1–424-V5/his), was evaluated. HEK293 and Hela cells were infected and cell lysates and cell culture medium evaluated by immunoblotting (Figure 5). Only one protein form of ~ 120 kD was detectable by ASP2 in rAd-1–812-infected HEK293 (Figure 5A, lane 2) and Hela (Figure 5A, lane 5) cell lysates. This ~ 120-kD protein was also detectable by Pro1 in HEK293 (lane 8) and Hela (lane 11) cells, indicating that this form represented latent, unprocessed ADAM33. Neither of these specific bands was observed in lysates infected with control virus (lanes 1, 7 and 4, 10). Expression of ADAM33 was not observed in the culture medium of rAd-1–812–infected cells, indicating it is only membrane-bound. rAd-1–424-V5/his expressed an unprocessed form of ~ 60 kD, detectable by both antibodies, in both HEK293 (lanes 3, 9) and Hela (lanes 6, 12) cell lysates. No processed catalytic domain was detected in the cell lysates. Pro1 also detected a smaller protein (~ 26 kD) in lysates of each cell (lanes 9, 12), again indicating the presence of prodomain not physically associated with the catalytic domain. In the corresponding cell medium (Figure 5B), a soluble processed form of ~ 40 kD was detectable by ASP2 (lanes 2, 4) in both cell types. In addition, a protein of ~ 26 kD was detected by Pro1 in the medium (lanes 6, 8).



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  		Figure 5. rAd-mediated ADAM33 expression and maturation. HEK293 (A, lanes 1–3, 7–9; B, lanes 1–2, 5–6) and Hela cells (A, lanes 4–6, 10–11; B, lanes 3–4, 7–8) were infected with rAd ADAM33 constructs and 24 or 48 h cell lysates (A) or culture medium (B) were analyzed by Western blotting with ASP2 antibody (A, lanes 1–6; B, lanes 1–4) or Pro1 (A, lanes 7–12; B, lanes 5–8). Control rAd vector, (A) lanes 1, 4, 7, 10; (B) lanes 1, 3, 5, and 7; rAd-1–812, (A) lanes 2, 5, 8, and 11; rAd-1–424-V5/his, (A) lanes 3, 6, 9, and 12; B, lanes 2, 4, 6, and 8. *indicates nonspecific band.

 
HEK293 and Hela cells are transformed, nonprimary cell lines of epithelial origin; in our mRNA studies, we found no evidence of epithelial expression of ADAM33. To evaluate the efficiency of ADAM33 maturation in the absence of the 3'UTR in cells that endogenously express ADAM33 mRNA, rAd-1–812 was used to infect primary NHLF and MRC5 fibroblasts. Ectopic expression of ADAM33 in fibroblasts was less efficient than in HEK293 or Hela cells and required enrichment for detection. In ConA-sepharose–selected cell lysates from rAd-1–812 infected NHLF (Figure 6, lane 2), only the unprocessed ~ 120-kD form was detected by Cyt2 (lane 2) and only a minor amount of the processed form of ADAM33 was detected in infected MRC5 cells by Cyt2 antibody (lane 6). The proportion of the processed to mature form of ADAM33 in fibroblasts was ~ 17% (n = 2). Interestingly, no endogenous ADAM33 protein was detected in control NHLF or MRC5 cells (Figure 6, lanes 1, 3, and 5) under the conditions used. Infection of NHLF and MRC5 cells with rAd-1–424-V5/his produced a pattern of unprocessed and processed ADAM33 protein in cell lysates and culture medium (data not shown) that was identical to that observed in HEK293 and Hela cells (Figure 5A and 5B).



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  		Figure 6. rAd-mediated ADAM33 expression and maturation in fibroblasts. NHLF (lanes 1, 2) and MRC5 (lanes 3–6) cells were infected with rAd-1–812 and 48 h cell lysates analyzed by Western blotting following ConA-sepharose enrichment of glycoproteins. ADAM33 protein was detected using Cyt2 (lanes 1–2, 5–6) or Pro1 (lanes 3–4) antibodies. Control rAd vector, lanes 1, 3, and 5; rAd-1–812, lanes 2, 4, and 6.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
To begin to elucidate the role of ADAM33 in the pathophysiology of asthma, the objectives of the current study were 2-fold. The first was to thoroughly define the mRNA expression pattern of ADAM33 and to identify the cell types in the normal and asthmatic lung that express ADAM33. A detailed expression profile of ADAM33 will facilitate the identification of its substrates with which it colocalizes. Our second objective was to examine post-transcriptional regulation. Because ADAMs undergo significant post-transcriptional modification, events that can be regulated by the 3'UTR—and the 3'UTR of ADAM33 is highly polymorphic—we examined the effect of the 3'UTR on protein expression and maturation.

The unique tissue and cell distribution profile of ADAM33 provides insight into pathophysiologic processes in which ADAM33 may be involved. The highest ADAM33 mRNA expression observed occurred in diverse tissues with a significant smooth muscle component, such as the urethra, bladder, uterus and prostate and in the vasculature (saphenous vein, aorta, and coronary artery). Approximately 10-fold lower levels of ADAM33 mRNA were found in the trachea and bronchus, which also contain smooth muscle. This tissue distribution pattern was supported at the cellular level in isolated BSMC (1) and lung fibroblasts, findings confirmed by ISH in intact tissue. In the asthmatic lung, ADAM33 mRNA was observed in fibroblasts and smooth muscle cells underlying the respiratory epithelium of the bronchus and trachea. Expression within the structural cells of the airway wall is consistent with the hypothesis that ADAM33 is involved in airways hyperresponsiveness, a phenotype with which this gene is strongly associated in genetic linkage and association studies (1). Interestingly, the highest level of ADAM33 mRNA observed in the ISH studies was seen in the granulation areas of a duodenal ulcer. Because levels of ADAM33 in healthy duodenal samples were not among the highest observed in a broad set of human tissues (Figure 1), the high levels seen in the ulcer base perhaps indicate a strong upregulation of ADAM33 within injured/repairing tissue. Additional, quantitative ISH studies of asthmatic lung samples are required to document upregulation of ADAM33 mRNA in airways undergoing tissue remodeling. Our studies have not successfully incorporated immunohistochemistry because our anti-ADAM33 antibodies were raised to peptides, and endogenous ADAM33 is a low-level protein (Figure 6). However, using concentrated protein from large quantities of cells and tissues, confirmation of translation of endogenous ADAM33 mRNA to protein has been obtained in primary BSMC, MRC5 fibroblasts and human bronchus by immunoblotting (28).

Several aspects of the expression profile of ADAM33 are of interest. ADAM33 mRNA expression is regulated during development, with expression seen in many fetal tissues at 25–28 wk of gestation, with the exception of fetal brain. The latter contrasts with the ubiquitous though moderate expression observed in the adult brain and suggests a tight regulation of ADAM33 transcription during brain development that is subsequently relaxed. Further insights into the role of ADAM33 in development will be gained by studies of embryogenesis in ADAM33 transgenic mice and knockout mice. Noteworthy is the absence of ADAM33 in several cell types as well as an expression pattern that is distinct from close family members, ADAM12 and ADAM19. The lack of ADAM33 expression in epithelial cells was first observed in numerous cell lines of epithelial origin derived from transformed tissue. ISH studies confirmed the lack of ADAM33 expression in epithelium including respiratory epithelium in the healthy or asthmatic lung. This suggests that effects of ADAM33 on the repairing epithelium in the asthmatic lung would be indirect via its expression and activity within the underlying submucosal smooth muscle layer. Although ADAM12 also displays a preferred expression in smooth muscle, it differs from ADAM33 in its absence in brain and lung by Northern analysis (32).

Of importance to the role of ADAM33 in asthma is the limited expression seen within the immune system. Although we found moderate levels of ADAM33 mRNA in the lymph node and thymus, little expression was seen in the spleen and bone marrow from multiple individuals by RT-PCR and Northern blotting. In contrast, ADAM19 is highly expressed in spleen, leukocytes, and bone marrow (33). Consistent with our observations of minimal ADAM33 expression in spleen and bone marrow is the lack of expression in many hematopoietic cell types isolated and cultured from peripheral blood, including B and T lymphocytes and NK cells, eosinophils, neutrophils, monocytes, and dendritic cells. Moreover, cell type–specific stimuli, known to induce the transcription of genes involved in the regulation of the immune system, failed to induce ADAM33 expression in those hematopoietic cells isolated from the peripheral circulation. Our only evidence of expression of ADAM33 mRNA in hematopoietic cells was that seen by ISH in macrophages localized within the base of a duodenal ulcer and other inflamed tissue. This suggests that mediators generated during or in response to injury may be sufficient to activate ADAM33 transcription in tissue macrophages, but this remains to be confirmed by other methods in additional tissues. The source of the signal in lymph node and thymus seen by Northern or RT-PCR is likely the vascular endothelial and smooth muscle cells and subcapsular fibroblasts because these cells, and not the majority of lymphocytes, demonstrated ADAM33 specific signals by ISH. The absence of ADAM33 in the immune system together with the lack of increase in significance of the genetic association with asthma when the immunologic parameter of immunoglobulin E level was added to the asthma phenotype (1) indicates that ADAM33 is not directly involved in the allergic component of asthma.

The extensive ADAM33 mRNA profile generated here using multiple methods indicates that ADAM33 is constitutively transcribed in many cell types and tissues at a low level. Little evidence of additional inducibility by various stimuli was obtained, particularly in epithelial and hematopoietic cells, where ADAM33 was absent. Thus, our subsequent studies investigated post-transcriptional regulation of ADAM33, which focused on the 3'UTR. This region of the ADAM33 sequence is highly polymorphic, with seven single nucleotide polymorphisms identified, of which one (V4) is alone, or in combination with others within the 3'UTR (V1/V4,V2/V4, and V4/V5) significantly associated with asthma (1). 3'UTR sequences control nuclear export, subcellular localization (17), translational efficiency, and mRNA degradation (16), and alterations in 3'UTR-mediated functions have been associated with disease (18). We investigated the effect of the 3'UTR on ADAM33 protein expression and maturation from an inactive zymogen to a potentially catalytically active protease. The presence of the 3'UTR facilitated removal of the prodomain of ADAM33; in the absence of the 3'UTR, maturation was 2- to 3-fold lower than in the presence of the 3'UTR in HEK293 cells. The use of tagged and untagged constructs both lacking the 3'UTR demonstrated that the lack of maturation was not solely due to interference by the tag with proper intracellular trafficking. The inefficient processing of full-length ADAM33 in the absence of the 3'UTR was observed in transfected HEK293 and Hela cells by plasmid transfection and rAd infection and in monkey cells (COS7) and Chinese hamster kidney cells (CHO-K1) (data not shown). Each of these is a transformed, nonprimary cell line of epithelial origin; in our mRNA studies, we found no evidence of epithelial expression of ADAM33 mRNA. Importantly, only a trace or minor amount of mature ADAM33 protein was detected in primary lung fibroblasts or the MRC5 fibroblast line, respectively, which endogenously express ADAM33 after infection with rAd-1–812, while the proform was readily detected. In addition, the poor processing of the full-length ADAM33 construct in the absence of the 3'UTR contrasted with the efficient processing of the nonmembrane-anchored forms. These findings further suggest that domains downstream of the catalytic domain influence the efficiency of prodomain removal. The 3'UTR affected protein maturation but not the overall level of ADAM33 expression. This suggests that the 3'UTR may affect subcellular localization rather than translational efficiency and mRNA degradation, because the process of prodomain removal from ADAMs occurs within the trans-Golgi network by furin-type pro-protein convertases as the molecule traffics intracellularly (29).

An investigation of sequence motifs in the 3'UTR of ADAM33 that may confer function and be affected by polymorphisms did not reveal any clear candidates. 3'UTR AU-rich elements that regulate mRNA stability (34) are not present. In contrast, two copies (nucleotides 2,594 to 2,608 and 2,955 to 2,973) of a motif, differentiation control element, which mediates translational silencing, are present. However, it is likely that function conferred by this motif requires tandem repeats of this element (35). Also, the asthma-associated polymorphisms did not occur within the differentiation control elements. Lastly, we have evaluated several constructs containing various combinations of the 3'UTR polymorphisms to identify those that play a role in promoting prodomain removal. To date, this evaluation has been unsuccessful, perhaps due to the overriding effect of the regulatory sequences contained in the plasmid.

Finally, our studies point to several mechanisms through which the activity of ADAM33 is regulated. First, mRNA species of 4–5 kb predominate over the the 3–4 kb species by 2- to 5-fold in all tissues examined and may represent nuclear intermediate forms (1). Second, the majority of detectable ADAM33 protein is in the immature form. This contrasts with ADAM17 (31), ADAM12-S (5), and ADAM19 (7), where the vast majority of the total amount of protein expressed is the mature form. Moreover, processing of ADAM33 to the mature form is promoted by the 3'UTR. These regulatory mechanisms are undescribed for other ADAMs. Lastly, localization of ADAM33 mRNA is distinct from its closest relatives, ADAM12 and ADAM19, indicating potential differences in function and substrate utilization. Defining the role of ADAM33 in the pathophysiology of asthma will require understanding each of these regulatory mechanisms.


   Acknowledgments
 
The authors thank Terri McClanahan and Erin Murphy (DNAX Research Institute) for the cDNA library panel used in the TaqMan expression studies, Maureen Laverty (SPRI) for performing the TaqMan expression studies on the human tissue panel, Mike Minnicozzi (SPRI) for discussion of the in situ hybridization slides, and Luquan Wang (SPRI) for sequence analysis of the 3'UTR. The authors also thank Richard Del Mastro, Karen Braunschweiger, and Randall Little (Genome Therapeutics Corporation), and lastly, Carl P. Blobel (Sloan-Kettering), Motasim Billah, and Robert Egan (SPRI) for their support and many useful discussions.

Received in original form January 27, 2003

Received in final form May 2, 2003


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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