Regulation of ectodomain shedding of ADAM33 in vitro and in vivo
Authors: Elizabeth R. Davies, Laura Denney, Marieke Wandel, Clare M. Lloyd, Donna E. Davies, Hans Michael Haitchi
Journal: Journal of Allergy and Clinical Immunology (2019)

A disintegrin and metalloprotease (ADAM)33 is a susceptibility gene for asthma and bronchial hyperresponsiveness (BHR). ADAM33 is a transmembrane protein, but a soluble protein containing the metalloprotease domain (sADAM33) has previously been identified in bronchoalveolar lavage fluid (BALF) from subjects with asthma and its levels significantly and negatively correlated with FEV1, suggesting a role in the development of airflow obstruction.1Lee J.Y. Park S.W. Chang H.K. Kim H.Y. Rhim T. Lee J.H. et al.A disintegrin and metalloproteinase 33 protein in patients with asthma: relevance to airflow limitation.Am J Respir Crit Care Med. 2006; 173: 729-735Crossref PubMed Scopus (99) Google Scholar We have shown that sADAM33 in asthmatic BALF is enzymatically active and that the sADAM33 metalloprotease causes angiogenesis and myogenesis in vivo or ex vivo.2Davies E.R. Kelly J.F. Howarth P.H. Wilson D.I. Holgate S.T. Davies D.E. et al.Soluble ADAM33 initiates airway remodeling to promote susceptibility for allergic asthma in early life.JCI Insight. 2016; 1 (e87632)Crossref Scopus (29) Google Scholar, 3Puxeddu I. Pang Y.Y. Harvey A. Haitchi H.M. Nicholas B. Yoshisue H. et al.The soluble form of a disintegrin and metalloprotease 33 promotes angiogenesis: implications for airway remodeling in asthma.J Allergy Clin Immunol. 2008; 121 (1406.e1-4): 1400-1406Abstract Full Text Full Text PDF PubMed Scopus (82) Google Scholar Furthermore, in vivo allergen challenge causes shedding of enzymatically active sADAM33 into BALF of wild-type mice, and in a transgenic mouse model, lung-specific sADAM33 expression causes airway remodeling, which enhances eosinophil recruitment with associated BHR following allergen sensitization and challenge.2Davies E.R. Kelly J.F. Howarth P.H. Wilson D.I. Holgate S.T. Davies D.E. et al.Soluble ADAM33 initiates airway remodeling to promote susceptibility for allergic asthma in early life.JCI Insight. 2016; 1 (e87632)Crossref Scopus (29) Google Scholar Although TGF-β is a trigger for sADAM33 release in vitro,3Puxeddu I. Pang Y.Y. Harvey A. Haitchi H.M. Nicholas B. Yoshisue H. et al.The soluble form of a disintegrin and metalloprotease 33 promotes angiogenesis: implications for airway remodeling in asthma.J Allergy Clin Immunol. 2008; 121 (1406.e1-4): 1400-1406Abstract Full Text Full Text PDF PubMed Scopus (82) Google Scholar there is no mechanistic information or evidence of in vivo relevance. Because it is not possible to test directly the importance of TGF-β in ectodomain shedding in human asthma, we characterized the mechanism(s) of the TGF-β–induced ectodomain shedding of murine ADAM33 and determined its importance for shedding of ADAM33 in vivo. Detailed methodology is provided in the Methods section in this article's Online Repository at www.jacionline.org. Initially, we confirmed that murine ADAM33 was similar to human ADAM33 in its sensitivity to TGF-β–induced ectodomain shedding.3Puxeddu I. Pang Y.Y. Harvey A. Haitchi H.M. Nicholas B. Yoshisue H. et al.The soluble form of a disintegrin and metalloprotease 33 promotes angiogenesis: implications for airway remodeling in asthma.J Allergy Clin Immunol. 2008; 121 (1406.e1-4): 1400-1406Abstract Full Text Full Text PDF PubMed Scopus (82) Google Scholar As expected, TGF-β treatment caused a dose-dependent increase in sADAM33 in supernatants of Cos-7 cells expressing murine ADAM33 (see Fig E1, A and B, in this article's Online Repository at www.jacionline.org). The main band had a molecular weight of around 102 kDa, indicating that the entire ectodomain had been shed; however, further processing was also evident, suggesting loss of the inhibitory prodomain. Consistent with this, there was a significant increase in ADAM33 enzymatic activity in cell-free supernatants following TGF-β treatment (Fig E1, C). Pericellular proteolysis is frequently mediated by members of the ADAM or MT-MMP families that are sensitive to the broad-spectrum hydroxamic acid–based inhibitor, GM6001; however, we found that it did not affect the shedding (see Fig E2, A and B, in this article's Online Repository at www.jacionline.org) or activity (Fig E2, C) of ADAM33. We also confirmed that GM6001 had no effect on the activity of purified recombinant ADAM33 (Fig E2, D). Because ADAM33 has a unique substrate-binding site and its catalytic activity is insensitive to GM6001,4Orth P. Reichert P. Wang W. Prosise W.W. Yarosh-Tomaine T. Hammond G. et al.Crystal structure of the catalytic domain of human ADAM33.J Mol Biol. 2004; 335: 129-137Crossref PubMed Scopus (74) Google Scholar we postulated that ADAM33 ectodomain shedding was autocatalytic. Consistent with this, mutation of E347A in the catalytic site suppressed shedding of sADAM33 both at baseline and in response to TGF-β (Fig 1, A) and this was accompanied by a significant reduction in enzymatic activity in cell-free supernatants (Fig 1, B). These data suggest that a substantial component of the shedding of sADAM33 is autocatalytic. TGF-β activates multiple signals including mothers against decapentaplegic homolog (SMAD) and non-SMAD pathways. Inhibition of SMAD signaling using SB431452 significantly suppressed TGF-β1–induced shedding of immunoreactive and enzymatically active ADAM33 (Fig 1, C and D). In contrast, the MAP2K/mitogen-activated protein kinase kinase inhibitor PD98059 dose-dependently increased shedding of immunoreactive and enzymatically active forms of sADAM33 (Fig 1, E and F). These data suggest a complex effect of TGF-β signaling, with SMAD activation being stimulatory for ADAM33 shedding, whereas mitogen-activated protein kinase (MAPK) activation has a negative regulatory effect. The latter effect may be mediated via TIMP-3, a known inhibitor of ADAM33 enzymatic activity,5Zou J. Zhu F. Liu J. Wang W. Zhang R. Garlisi C.G. et al.Catalytic activity of human ADAM33.J Biol Chem. 2004; 279: 9818-9830Crossref PubMed Scopus (94) Google Scholar which is reported to be induced by TGF-β via MAPK signaling.6Qureshi H.Y. Sylvester J. El Mabrouk M. Zafarullah M. TGF-beta-induced expression of tissue inhibitor of metalloproteinases-3 gene in chondrocytes is mediated by extracellular signal-regulated kinase pathway and Sp1 transcription factor.J Cell Physiol. 2005; 203: 345-352Crossref PubMed Scopus (71) Google Scholar Thus, inhibiting MAPK activation with PD98059 may inhibit TIMP3 expression and release ADAM33 from the effects of this natural inhibitor leading to increased autocatalytic shedding and augmented enzyme activity. Although ectodomain shedding can be regulated by natural protein inhibitors, other mechanisms including membrane trafficking, protein maturation, and substrate presentation have also been shown to be important for the regulation of other sheddases.7Lichtenthaler S.F. Lemberg M.K. Fluhrer R. Proteolytic ectodomain shedding of membrane proteins in mammals—hardware, concepts, and recent developments.EMBO J. 2018; 37 (e99456)Crossref Scopus (138) Google Scholar Whether release of ADAM33 is preceded by its membrane trafficking or maturation and/or by assembly into higher order complexes requiring additional protein interactions to exosites located either on the ADAM33 or adapter proteins remains to be determined. Epithelial injury leads to release of TGF-β, and we have postulated that this is the source of the TGF-β that drives the increase in sADAM33 observed in response to allergen challenge in vivo.2Davies E.R. Kelly J.F. Howarth P.H. Wilson D.I. Holgate S.T. Davies D.E. et al.Soluble ADAM33 initiates airway remodeling to promote susceptibility for allergic asthma in early life.JCI Insight. 2016; 1 (e87632)Crossref Scopus (29) Google Scholar To test this hypothesis, we used BALF from mice in which lung epithelial Tgfb1 was conditionally deleted in bronchial epithelial club cells before intranasal administration of either 25 μg house dust mite extract or recombinant murine IL-33.8Denney L. Byrne A.J. Shea T.J. Buckley J.S. Pease J.E. Herledan G.M. et al.Pulmonary epithelial cell-derived cytokine TGF-beta1 is a critical cofactor for enhanced innate lymphoid cell function.Immunity. 2015; 43: 945-958Abstract Full Text Full Text PDF PubMed Google Scholar After house dust mite challenge, lower levels of sADAM33 could be detected in the BALF of Tgfb1−/− mice compared with littermate controls (Fig 2, A) and it also contained less sADAM33 enzymatic activity (Fig 2, B). In the same way, when mice were challenged with IL-33, BALF from Tgfb1−/− mice had a lower level of sADAM33 immunoreactive protein (Fig 2, C) and enzymatic activity (Fig 2, D). Of note, exogenous TGF-β alone was ineffective at stimulating shedding of ADAM33 in vivo. This might be explained either by the fact that TGF-β could not pass through the intact epithelium of unchallenged mice and/or by the low dose and short half-life of the growth factor, which did not allow sufficient time for it to pass through the epithelium to reach the subepithelial mesenchymal cells where ADAM33 is expressed.9Haitchi H.M. Powell R.M. Shaw T.J. Howarth P.H. Wilson S.J. Wilson D.I. et al.ADAM33 expression in asthmatic airways and human embryonic lungs.Am J Respir Crit Care Med. 2005; 171: 958-965Crossref PubMed Scopus (91) Google Scholar Epithelial-derived IL-33 induces rapid release of TGF-β into the airways to enhance migration of innate lymphoid cells (ILCs) and development of a robust ILC2 response that initiates allergic immunity.9Haitchi H.M. Powell R.M. Shaw T.J. Howarth P.H. Wilson S.J. Wilson D.I. et al.ADAM33 expression in asthmatic airways and human embryonic lungs.Am J Respir Crit Care Med. 2005; 171: 958-965Crossref PubMed Scopus (91) Google Scholar Because we found that epithelial-derived TGF-β is also required for ectodomain shedding of ADAM33, it may be speculated that sADAM33 can contribute to the recruitment or activation of ILCs or other innate immune cells, either by affecting matrix turnover or by promoting growth factor or chemokine shedding. Of note, polymorphisms in TGFB, IL33, and ADAM33 genes have each been associated with asthma susceptibility, yet each has a small overall effect on disease development. The involvement of 3 susceptibility gene products in epithelial responses to allergens highlights how they may cooperate to amplify the downstream asthmatic responses. Identification of the involvement of TGF-β in ectodomain shedding of ADAM33 in an in vivo model strengthens the case for exploring how human polymorphic variation in the ADAM33 gene is linked to asthma pathogenesis. Four single nucleotide polymorphisms (S1, S2, T1, and T2) encode amino acid substitutions in the transmembrane and cytoplasmic domain of ADAM33 and have been associated with asthma.4Orth P. Reichert P. Wang W. Prosise W.W. Yarosh-Tomaine T. Hammond G. et al.Crystal structure of the catalytic domain of human ADAM33.J Mol Biol. 2004; 335: 129-137Crossref PubMed Scopus (74) Google Scholar Although the intracellular domain of ADAM33 is relatively short, it is very rich in prolines, having a putative SH3 binding site where the T2 SNP is located, a casein kinase I/II phosphorylation site, and an MAPK consensus sequence that is likely to be important for regulation of ADAM33 function, especially as we have identified a negative regulatory role for MAPK in our current studies. Further work is required to determine whether this effect is direct and involves ADAM33 phosphorylation or indirect via inhibitors such as TIMP3. Alternatively, one mutation Ala395Val is located within the catalytic domain,4Orth P. Reichert P. Wang W. Prosise W.W. Yarosh-Tomaine T. Hammond G. et al.Crystal structure of the catalytic domain of human ADAM33.J Mol Biol. 2004; 335: 129-137Crossref PubMed Scopus (74) Google Scholar which may directly affect catalytic activity. In summary, we have provided direct evidence that epithelial-derived TGF-β is an important regulator of ectodomain shedding of enzymatically active ADAM33 from the mesenchyme. This process appears largely to be autocatalytic and involves SMAD signaling, but is negatively regulated by MAPK signaling. These findings highlight the importance of epithelial-mesenchymal cross-talk in asthma pathogenesis and underscore the potential for co-operation between different asthma susceptibility genes to drive disease pathogenesis. The Cos-7 cell line, a fibroblast-like cell line, was grown in Dulbecco modified Eagle medium supplemented with 10% FBS, 50 units/mL penicillin, 50 μg/mL streptomycin, 2 mM l-glutamine, 1 mM sodium pyruvate, and 1× nonessential amino acids (Dulbecco modified Eagle medium/FBS) (all from Life Technologies, Paisley, UK). For transfection, cells were placed in non-supplemented Opti-MEM (Life Technologies). Cos-7 cells were transfected with a plasmid encoding full-length murine ADAM33 (MR217277 clone, NM_033615; OriGene, Rockville, Md) or green fluorescence protein using X-treme Gene 9 reagent (Roche, Southampton, UK). After 24 hours, cells were treated with TGF-β1 (Peprotech, London, UK) for 8 hours to assess ectodomain shedding of ADAM33. The TGF-β1 isoform was chosen, as this is the major isoform in adult mice.E1Pelton R.W. Johnson M.D. Perkett E.A. Gold L.I. Moses H.L. Expression of transforming growth factor-beta 1, -beta 2, and -beta 3 mRNA and protein in the murine lung.Am J Respir Cell Mol Biol. 1991; 5: 522-530Crossref PubMed Scopus (108) Google Scholar Where indicated, cells were preincubated for 1 hour with the ALK5/SMAD inhibitor SB431542 (10 μM; Stratech, Ely, UK), the mitogen-activated protein kinase kinase (MAP2K, MEK) inhibitor PD98059 (10 or 25 μM, Sigma, Poole, UK), the broad-spectrum metalloprotease inhibitor GM6001 (10 μM; Merck, Millipore, Watford, UK), or vehicle control before addition of 5 ng/mL TGF-β1. Cell-free supernatants were harvested and cells were lysed into RIPA buffer. Concanavalin A-Sepharose 4B beads (ConA) (Sigma) were used to pull down glycosylated ADAM33 from the supernatants of the transfected Cos-7 cells, as previously described.E2Puxeddu I. Pang Y.Y. Harvey A. Haitchi H.M. Nicholas B. Yoshisue H. et al.The soluble form of a disintegrin and metalloprotease 33 promotes angiogenesis: implications for airway remodeling in asthma.J Allergy Clin Immunol. 2008; 121 (1406.e1-4): 1400-1406Abstract Full Text Full Text PDF PubMed Scopus (90) Google Scholar In brief, 1 mL supernatant was adjusted to contain 20 mM Tris HCl, pH 7.4, 0.5 M NaCl, and protease inhibitors (Sigma) before addition of activated ConA beads (20 μL beads per mL sample). After binding overnight at 4°C, the beads were washed twice in 20 mM Tris HCl, pH 7.4, containing 0.5 M NaCl and then twice in 20 mM Tris HCl, pH 7.4, to remove excess salt. The bound protein was then solubilized into SDS sample buffer containing 0.5 M methyl α-d-mannopyranoside for SDS-PAGE and Western blotting. An ADAM33 enzymatic assay was performed using a fluorescence resonance energy transfer peptide cleavage assay as previously described.E3Zou J. Zhang R. Zhu F. Liu J. Madison V. Umland S.P. ADAM33 enzyme properties and substrate specificity.Biochemistry. 2005; 44: 4247-4256Crossref PubMed Scopus (26) Google Scholar All assay measurements were made at 37°C in real time using a Bio-Rad CFX96 machine for detection of carboxyfluorescein reporter dye (FAM) (450-490 nm excitation and 510-530 nm detection), allowing determination of initial rates. Each reaction contained 7 μL neat BALF or cell supernatant mixed with 2 μL 5x-assay buffer (100 mM HEPES, pH 7, 2.5 M NaCl, 50 mM CaCl2, and 1 mg/mL BSA); the assay was initiated by addition of 44 pmol fluorescence resonance energy transfer peptide (DABCYL-YRVAFQKLAE(FAM)K-NH2) (Severn Biotech, Kidderminster, UK) and 100 pmol ZnCl2 to make a final reaction volume of 10 μL. Enzymatic activity was determined by plotting relative fluorescence units against time after the background had been subtracted. The rate of the reaction (relative fluorescence units/min) was determined from the line of best fit in the linear phase of the assay. Control assays used human recombinant soluble active ADAM33 (positive control) and mutant ADAM33 (E365A) (negative control) as previously described.E2Puxeddu I. Pang Y.Y. Harvey A. Haitchi H.M. Nicholas B. Yoshisue H. et al.The soluble form of a disintegrin and metalloprotease 33 promotes angiogenesis: implications for airway remodeling in asthma.J Allergy Clin Immunol. 2008; 121 (1406.e1-4): 1400-1406Abstract Full Text Full Text PDF PubMed Scopus (90) Google Scholar Solubilized samples from ConA pull down or murine BALF were run on 12.5% acrylamide or 8% to 20% gradient gels (Bio-Rad, Watford, UK) and transferred onto polyvinylidene fluoride (PVDF) membranes. The transferred protein was assessed by Ponceau staining to confirm equivalent protein loading before blocking using 2.5% BSA in TBS/Tween and Western blotting. Membranes were probed with a polyclonal goat antibody against the ectodomain of mouse ADAM33 (AF2434, 1:1000, R&D Systems, Abingdon, UK). Secondary antibody was rabbit antigoat IgG horseradish peroxidase antibody (Merck; 1:5000). The blots were visualized using enhanced chemiluminescence (Clarity ECL; Bio-Rad) with an Amersham Imager 600 (GE Healthcare, Buckinghamshire, UK); densitometric images were semiquantified using ImageJ software (National Institutes of Health, Wis) and data plotted as relative intensity. Mice conditionally lacking lung epithelial Tgfb expression (Ccsp-creTgfb1−/−) were generated as previously described.E4Denney L. Byrne A.J. Shea T.J. Buckley J.S. Pease J.E. Herledan G.M. et al.Pulmonary epithelial cell-derived cytokine TGF-beta1 is a critical cofactor for enhanced innate lymphoid cell function.Immunity. 2015; 43: 945-958Abstract Full Text Full Text PDF PubMed Scopus (109) Google Scholar Mice were pretreated with doxycycline (Sigma) (or vehicle as control) to delete Tgfb1 from the bronchiolar epithelial cells 72 hours before intranasal administration of either 25 μg HDM extract (1 mg/mL protein solution dissolved in PBS) or 25 μL of PBS 5 times a week for 3 weeks. Mice were culled 4 hours after the final challenge. In other experiments, carrier-free recombinant murine IL-33 (1 μg/dose in 25 μL PBS) (eBioscience, Thermo Fisher Scientific, Hemel Hempstead, UK) was administered 3 times a week for 1 week and mice culled 18 hours after the final dose. Where required, 50 ng recombinant TGF-β1 in PBS (R&D Systems) was administered intranasally without or with IL-33. At the point of killing, BALF was collected by washing the airways 3 times with 400 μL PBS. After centrifugation to remove cells, supernatants were stored at −80°C. The protein content of the BALF was quantified (BCA Assay, Thermofisher, Hemel Hempstead, UK) and normalized before use in ADAM33 enzyme assays or solubilization in 2x Laemmli sample buffer (Bio-Rad) for analysis of sADAM33 protein by Western blotting. Mice of both sexes were used between age 7 and 12 weeks, housed in specific-pathogen-free conditions, and given food and water ad libitum. All procedures were conducted in accordance with the Animals (Scientific Procedures) Act 1986. Normal distribution of the numeric data was evaluated, and appropriate parametric or nonparametric statistical tests applied. All data are parametric and plotted as mean with 1 SD. Statistical significance was assessed by using Student t test (unpaired data) with Welch correction if SDs were not equal for comparisons between 2 groups. For comparison of 3 or more groups, a 1-way ANOVA with Dunn's multiple comparison test was used. For comparison of 2 or more groups with 2 independent variables, a 2-way ANOVA with Tukey multiple comparison test was used (*P < .05, **P < .01, ***P < .001).Fig E2The broad-spectrum MMP inhibitor GM6001 does not inhibit ADAM33 shedding. Cos-7 cells transiently transfected with full-length murine ADAM33 were preincubated with GM6001 (0-10 μM) for 1 hour before addition of 5 ng/mL TGF-β for 8 hours. Cell-free supernatants were assessed for soluble ADAM33 by Western blotting (A) with densitometry (arbitrary units, AU) (B) and by fluorescence resonance energy transfer peptide cleavage assay (C). Purified recombinant ADAM33 protein was incubated with GM6001 and tested for activity in a fluorescence resonance energy transfer peptide cleavage assay (D). Activity is presented as a percent of that measured in the absence of GM6001. MW, Molecular weight. Data are expressed as mean + SD and are representative of 4 independent experiments.View Large Image Figure ViewerDownload Hi-res image Download (PPT)