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Targeted truncation of the ADAM17 cytoplasmic domain in mice results in protein destabilization and a hypomorphic phenotype

2021; Elsevier BV; Volume: 296; Linguagem: Inglês

10.1016/j.jbc.2021.100733

ISSN

1083-351X

Autores

José M. Lora, Gisela Weskamp, Thomas M. Li, Thorsten Maretzky, Dorjee T.N. Shola, Sébastien Monette, Stefan F. Lichtenthaler, Theresa Lu, Chingwen Yang, Carl Blobel,

Tópico(s)

Viral Infectious Diseases and Gene Expression in Insects

Resumo

A disintegrin and metalloprotease 17 (ADAM17) is a cell-surface metalloprotease that serves as the principle sheddase for tumor necrosis factor α (TNFα), interleukin-6 receptor (IL-6R), and several ligands of the epidermal growth factor receptor (EGFR), regulating these crucial signaling pathways. ADAM17 activation requires its transmembrane domain, but not its cytoplasmic domain, and little is known about the role of this domain in vivo. To investigate, we used CRISPR-Cas9 to mutate the endogenous Adam17 locus in mice to produce a mutant ADAM17 lacking its cytoplasmic domain (Adam17Δcyto). Homozygous Adam17Δcyto animals were born at a Mendelian ratio and survived into adulthood with slightly wavy hair and curled whiskers, consistent with defects in ADAM17/EGFR signaling. At birth, Adam17Δcyto mice resembled Adam17−/− mice in that they had open eyes and enlarged semilunar heart valves, but they did not have bone growth plate defects. The deletion of the cytoplasmic domain resulted in strongly decreased ADAM17 protein levels in all tissues and cells examined, providing a likely cause for the hypomorphic phenotype. In functional assays, Adam17Δcyto mouse embryonic fibroblasts and bone-marrow-derived macrophages had strongly reduced ADAM17 activity, consistent with the reduced protein levels. Nevertheless, ADAM17Δcyto could be stimulated by PMA, a well-characterized posttranslational activator of ADAM17, corroborating that the cytoplasmic domain of endogenous ADAM17 is not required for its rapid response to PMA. Taken together, these results provide the first evidence that the cytoplasmic domain of ADAM17 plays a pivotal role in vivo in regulating ADAM17 levels and function. A disintegrin and metalloprotease 17 (ADAM17) is a cell-surface metalloprotease that serves as the principle sheddase for tumor necrosis factor α (TNFα), interleukin-6 receptor (IL-6R), and several ligands of the epidermal growth factor receptor (EGFR), regulating these crucial signaling pathways. ADAM17 activation requires its transmembrane domain, but not its cytoplasmic domain, and little is known about the role of this domain in vivo. To investigate, we used CRISPR-Cas9 to mutate the endogenous Adam17 locus in mice to produce a mutant ADAM17 lacking its cytoplasmic domain (Adam17Δcyto). Homozygous Adam17Δcyto animals were born at a Mendelian ratio and survived into adulthood with slightly wavy hair and curled whiskers, consistent with defects in ADAM17/EGFR signaling. At birth, Adam17Δcyto mice resembled Adam17−/− mice in that they had open eyes and enlarged semilunar heart valves, but they did not have bone growth plate defects. The deletion of the cytoplasmic domain resulted in strongly decreased ADAM17 protein levels in all tissues and cells examined, providing a likely cause for the hypomorphic phenotype. In functional assays, Adam17Δcyto mouse embryonic fibroblasts and bone-marrow-derived macrophages had strongly reduced ADAM17 activity, consistent with the reduced protein levels. Nevertheless, ADAM17Δcyto could be stimulated by PMA, a well-characterized posttranslational activator of ADAM17, corroborating that the cytoplasmic domain of endogenous ADAM17 is not required for its rapid response to PMA. Taken together, these results provide the first evidence that the cytoplasmic domain of ADAM17 plays a pivotal role in vivo in regulating ADAM17 levels and function. Cell–cell interactions are crucial for the development and maintenance of multicellular organisms. 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The goal of this study was to generate mice carrying a mutant form of endogenous ADAM17 that lacks its cytoplasmic domain, including all previously described signaling motifs, in order to learn more about the role of the cytoplasmic domain in regulating the function of ADAM17 in vivo. In order to generate mice that express endogenous ADAM17 with a truncated cytoplasmic domain, we used CRISPR-Cas9 to introduce a targeted knock-in (KI) mutation, in which an HA-tag with a stop codon was attached immediately C-terminal to the transmembrane domain of ADAM17 (following DKKLD699, Fig. 1A, see Experimental procedures and Fig. S1 for details). The resulting mutant ADAM17Δcyto lacks almost all of the cytoplasmic domain of ADAM17 (cytoplasmic amino acid residues 700–827) and corresponds to an ADAM17Δcyto mutant that fully rescues ADAM17-dependent shedding when overexpressed in Adam17−/− mouse embryonic fibroblasts (mEFs) (27Le Gall S.M. Maretzky T. Issuree P.D.A. Niu X.-D. Reiss K. Saftig P. Khokha R. Lundell D. Blobel C.P. ADAM17 is regulated by a rapid and reversible mechanism that controls access to its catalytic site.J. Cell Sci. 2010; 123: 3913-3922Crossref PubMed Scopus (143) Google Scholar). Founder mice carrying the Adam17Δcyto KI mutation were generated following standard protocols (see Experimental procedures for details) and bred to homozygosity. The presence of the Adam17Δcyto mutation was verified by sequencing PCR fragments from genomic DNA of mutant mice compared with wild-type controls (Fig. 1, B and C). Offspring of heterozygous matings (Adam17Δcyto/+ x Adam17Δcyto/+) were genotyped by genomic PCR (Fig. 1D) and were born at the expected Mendelian ratio (Fig. 1E). Homozygous mutant Adam17Δcyto animals appeared grossly normal (Fig. 2A) and did not display behavioral abnormalities during routine handling compared with wild-type controls. However, closer inspection revealed that Adam17Δcyto mice had slightly wavy fur (Fig. 2B) and curly whiskers (Fig. 2C, red arrows), which are characteristic phenotypes for mice carrying mutations affecting ADAM17/EGFR signaling (16Chalaris A. Adam N. Sina C. Rosenstiel P. Lehmann-Koch J. Schirmacher P. Hartmann D. Cichy J. Gavrilova O. Schreiber S. Jostock T. Matthews V. Hasler R. Becker C. Neurath M.F. et al.Critical role of the disintegrin metalloprotease ADAM17 for intestinal inflammation and regeneration in mice.J. Exp. Med. 2010; 207: 1617-1624Crossref PubMed Scopus (207) Google Scholar, 44Brandl K. Sun L. Neppl C. Siggs O.M. Le Gall S.M. Tomisato W. Li X. Du X. Maennel D.N. Blobel C.P. Beutler B. MyD88 signaling in nonhematopoietic cells protects mice against induced colitis by regulating specific EGF receptor ligands.Proc. Natl. Acad. Sci. U. S. A. 2010; 107: 19967-19972Crossref PubMed Scopus (119) Google Scholar, 45Hassemer E.L. Le Gall S.M. Liegel R. McNally M. Chang B. Zeiss C.J. Dubielzig R.D. Horiuchi K. Kimura T. Okada Y. Blobel C.P. Sidjanin D.J. The waved with open eyelids (woe) locus is a hypomorphic mouse mutation in Adam17.Genetics. 2010; 185: 245-255Crossref PubMed Scopus (34) Google Scholar). Gross and histopathological analysis of newborn mice revealed that Adam17Δcyto animals resemble Adam17−/− animals in that their eyes are open at birth (Fig. 3A, top two rows of panels) (7Peschon J.J. Slack J.L. Reddy P. Stocking K.L. Sunnarborg S.W. Lee D.C. Russel W.E. Castner B.J. Johnson R.S. Fitzner J.N. Boyce R.W. Nelson N. Kozlosky C.J. Wolfson M.F. Rauch C.T. et al.An essential role for ectodomain shedding in mammalian development.Science. 1998; 282: 1281-1284Crossref PubMed Google Scholar, 46Horiuchi K. Kimura T. Miyamoto T. Takaishi H. Okada Y. Toyama Y. Blobel C.P. Cutting edge: TNF-{alpha}-converting enzyme (TACE/ADAM17) inactivation in mouse myeloid cells prevents lethality from endotoxin shock.J. Immunol. 2007; 179: 2686-2689Crossref PubMed Google Scholar). Moreover, Adam17Δcyto mice show enlarged and thickened pulmonic, aortic, and tricuspid heart valves (Fig. 3A, middle and lower middle panels, tricuspid valve not shown), similar to Adam17−/− mice (12Jackson L.F. Qiu T.H. Sunnarborg S.W. Chang A. Zhang C. Patterson C. Lee D.C. Defective valvulogenesis in HB-EGF and TACE-null mice is associated with aberrant BMP signaling.EMBO J. 2003; 22: 2704-2716Crossref PubMed Scopus (330) Google Scholar, 46Horiuchi K. Kimura T. Miyamoto T. Takaishi H. Okada Y. Toyama Y. Blobel C.P. Cutting edge: TNF-{alpha}-converting enzyme (TACE/ADAM17) inactivation in mouse myeloid cells prevents lethality from endotoxin shock.J. Immunol. 2007; 179: 2686-2689Crossref PubMed Google Scholar). However, unlike Adam17−/− mice (14Hall K.C. Hill D. Otero M. Plumb D.A. Froemel D. Dragomir C.L. Maretzky T. Boskey A. Crawford H.C. Selleri L. Goldring M.B. Blobel C.P. ADAM17 controls endochondral ossification by regulating terminal differentiation of chondrocytes.Mol. Cell. Biol. 2013; 33: 3077-3090Crossref PubMed Scopus (34) Google Scholar, 15Saito K. Horiuchi K. Kimura T. Mizuno S. Yoda M. Morioka H. Akiyama H. Threadgill D. Okada Y. Toyama Y. Sato K. Conditional inactivation of TNFalpha-converting enzyme in chondrocytes results in an elongated growth plate and shorter long bones.PLoS One. 2013; 8e54853Crossref PubMed Scopus (17) Google Scholar), Adam17Δcyto mice did not have significantly expanded zones of hypertrophic cells in their long bone growth plate (Fig. 3A, femur shown in lower panel).Figure 3Adam17Δcyto mice closely resemble Adam17−/− mice at birth. A, representative images of the heads of newborn wild-type and Adam17Δcyto (littermates) and Adam17−/− mice show open eyes at birth (OEB) in Adam17Δcyto mutants that are similar to the OEB in Adam17−/− mice (top row, white arrows). Corresponding H&E-stained sections of the eye are shown in row 2, with an asterisk marking the open eyelid. Sections of a representative pulmonic valve (middle panel/row 3) and aortic valve (row 4) show similarly enlarged and misshapen tricuspid valves in Adam17Δcyto mutants compared with Adam17−/− mice. However, the growth plate in the Adam17Δcyto mutant appeared normal in size and comparable to the wild-type control and did not display the enlarged zone of hypertrophic cells seen in Adam17−/− mice (bottom row). Scale bars: sections of eyes and femurs, 100 μm, sections of heart valves, 50 μm. B, representative images of the aortic and pulmonic valves in adult Adam17Δcyto mutants are indistinguishable from wild-type controls (left panels). The hair follicles and skin of Adam17Δcyto mutants showed pyogranulomatous inflammation, which also affected the meibomian glands in the eyelids and the zymbal glands near the ear canal (Fig. 3B, right panels). Scale bars: sections of adult heart valves, 100 μm, sections of skin and glands, 50 μm. All images are representative for sections from at least three mice per genotype.View Large Image Figure ViewerDownload Hi-res image Download (PPT) Analysis of adult Adam17Δcyto animals showed no evident abnormalities in heart weight and gross and histologic morphology of the heart, including the heart valves (Fig. 3B, left panels), suggesting that the heart valve defects are remodeled and return to normal as these animals grow into adults. In addition, we found pyogranulomatous inflammation of hair follicles in the skin and in the meibomian glands in eyelids and zymbal glands near the ear canal, which are both specialized sebaceous glands (Fig. 3B, right panels). There were no other major evident pathological phenotypes in the adult Adam17Δcyto mice compared with their littermate controls. The hypomorphic phenotype of Adam17Δcyto mice raised questions about the underlying cause for the apparently reduced activity of ADAM17. We therefore performed western blots on Concanavalin A-enriched glycoproteins isolated from wild-type, Adam17Δcyto, and Adam17−/− mEFs with rabbit polyclonal antibodies raised against the extracellular domain of mouse ADAM17 (anti-A17-ecto). The samples were run under denaturing, but nonreducing conditions, since the anti-A17-ecto antibodies only recognize nonreduced ADAM17 in western blots (see Fig. S2 and Experimental procedures for details). Under these conditions, the anti-A17-ecto antibody detected the wild-type proform of ADAM17 (Fig. 4A, indicated by an open arrowhead) as well as the mature form (indicted by a black arrowhead) in mEFs. By comparison, the levels of pro-ADAM17Δcyto (open arrowhead) and mature ADAM17Δcyto (black arrowhead) were strongly reduced. When wild-type cells were lysed in the absence of metalloprotease inhibitors (marimastat and 1,10 Phenanthroline, see Experimental procedures for details), the mature form of wild-type ADAM17 underwent postlysis autocatalytic removal of the cytoplasmic domain, as reported previously (47Schlöndorff J. Becherer J.D. Blobel C.P. Intracellular maturation and localization of the tumour necrosis factor alpha convertase (TACE).Biochem. J. 2000; 347 Pt 1: 131-138Crossref PubMed Google Scholar, 48McIlwain D.R. Lang P.A. Maretzky T. Hamada K. Ohishi K. Maney S.K. Berger T. Murthy A. Duncan G. Xu H.C. Lang K.S. Haussinger D. Wakeham A. Itie-Youten A. Khokha R. et al.iRhom2 regulation of TACE controls TNF-mediated protection against Listeria and responses to LPS.Science. 2012; 335: 229-232Crossref PubMed Scopus (219) Google Scholar), which generates a faster migrating form of mature ADAM17 (Fig. 4A, indicated by an asterisk), whereas pro-ADAM17 was not affected. A similar postlysis autocatalytic removal of the cytoplasmic domain was also observed in the related ADAM10 and is thought to have no biological significance in ADAM17 or ADAM10 (49Brummer T. Pigoni M. Rossello A. Wang H. Noy P.J. Tomlinson M.G. Blobel C.P. Lichtenthaler S.F. The metalloprotease ADAM10 (a disintegrin and metalloprotease 10) undergoes rapid, postlysis autocatalytic degradation.FASEB J. 2018; 32: 3560-3573Crossref PubMed Scopus (14) Google Scholar). The proform of ADAM17Δcyto (open arrowhead in Fig. 4A, darker exposure shown on the right) migrated faster than the proform of wild-type ADAM17, consistent with the predicted reduction in molecular weight caused by deletion of the cytoplasmic domain. The mature form of ADAM17Δcyto (black arrowhead in Fig. 4A) comigrated approximately with the autodegraded form of mature wild-type ADAM17 (asterisk). However, the presence or absence of the metalloprotease inhibitors during cell lysis did not affect the migration of mature ADAM17Δcyto. This suggests that ADAM17Δcyto was not detectably subjected to postlysis processing

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