Combined ADAMTS10 and ADAMTS17 inactivation exacerbates bone shortening and compromises extracellular matrix formation

Combined ADAMTS10 and ADAMTS17 inactivation exacerbates bone shortening and compromises extracellular matrix formation | bioRxiv /* */ /* */ <!-- <!-- /*! * yepnope1.5.4 * (c) WTFPL, GPLv2 */ (function(a,b,c){function d(a){return"[object Function]"==o.call(a)}function e(a){return"string"==typeof a}function f(){}function g(a){return!a||"loaded"==a||"complete"==a||"uninitialized"==a}function h(){var a=p.shift();q=1,a?a.t?m(function(){("c"==a.t?B.injectCss:B.injectJs)(a.s,0,a.a,a.x,a.e,1)},0):(a(),h()):q=0}function i(a,c,d,e,f,i,j){function k(b){if(!o&&g(l.readyState)&&(u.r=o=1,!q&&h(),l.onload=l.onreadystatechange=null,b)){"img"!=a&&m(function(){t.removeChild(l)},50);for(var d in y[c])y[c].hasOwnProperty(d)&&y[c][d].onload()}}var j=j||B.errorTimeout,l=b.createElement(a),o=0,r=0,u={t:d,s:c,e:f,a:i,x:j};1===y[c]&&(r=1,y[c]=[]),"object"==a?l.data=c:(l.src=c,l.type=a),l.width=l.height="0",l.onerror=l.onload=l.onreadystatechange=function(){k.call(this,r)},p.splice(e,0,u),"img"!=a&&(r||2===y[c]?(t.insertBefore(l,s?null:n),m(k,j)):y[c].push(l))}function j(a,b,c,d,f){return q=0,b=b||"j",e(a)?i("c"==b?v:u,a,b,this.i++,c,d,f):(p.splice(this.i++,0,a),1==p.length&&h()),this}function k(){var a=B;return a.loader={load:j,i:0},a}var l=b.documentElement,m=a.setTimeout,n=b.getElementsByTagName("script")[0],o={}.toString,p=[],q=0,r="MozAppearance"in l.style,s=r&&!!b.createRange().compareNode,t=s?l:n.parentNode,l=a.opera&&"[object Opera]"==o.call(a.opera),l=!!b.attachEvent&&!l,u=r?"object":l?"script":"img",v=l?"script":u,w=Array.isArray||function(a){return"[object Array]"==o.call(a)},x=[],y={},z={timeout:function(a,b){return b.length&&(a.timeout=b[0]),a}},A,B;B=function(a){function b(a){var a=a.split("!"),b=x.length,c=a.pop(),d=a.length,c={url:c,origUrl:c,prefixes:a},e,f,g;for(f=0;f<d;f++)g=a[f].split("="),(e=z[g.shift()])&&(c=e(c,g));for(f=0;f<b;f++)c=x[f](c);return c}function g(a,e,f,g,h){var i=b(a),j=i.autoCallback;i.url.split(".").pop().split("?").shift(),i.bypass||(e&&(e=d(e)?e:e[a]||e[g]||e[a.split("/").pop().split("?")[0]]),i.instead?i.instead(a,e,f,g,h):(y[i.url]?i.noexec=!0:y[i.url]=1,f.load(i.url,i.forceCSS||!i.forceJS&&"css"==i.url.split(".").pop().split("?").shift()?"c":c,i.noexec,i.attrs,i.timeout),(d(e)||d(j))&&f.load(function(){k(),e&&e(i.origUrl,h,g),j&&j(i.origUrl,h,g),y[i.url]=2})))}function h(a,b){function c(a,c){if(a){if(e(a))c||(j=function(){var a=[].slice.call(arguments);k.apply(this,a),l()}),g(a,j,b,0,h);else if(Object(a)===a)for(n in m=function(){var b=0,c;for(c in a)a.hasOwnProperty(c)&&b++;return b}(),a)a.hasOwnProperty(n)&&(!c&&!--m&&(d(j)?j=function(){var a=[].slice.call(arguments);k.apply(this,a),l()}:j[n]=function(a){return function(){var b=[].slice.call(arguments);a&&a.apply(this,b),l()}}(k[n])),g(a[n],j,b,n,h))}else!c&&l()}var h=!!a.test,i=a.load||a.both,j=a.callback||f,k=j,l=a.complete||f,m,n;c(h?a.yep:a.nope,!!i),i&&c(i)}var i,j,l=this.yepnope.loader;if(e(a))g(a,0,l,0);else if(w(a))for(i=0;i (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0];var j=d.createElement(s);var dl=l!='dataLayer'?'&l='+l:'';j.src='//www.googletagmanager.com/gtm.js?id='+i+dl;j.type='text/javascript';j.async=true;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-M677548'); Skip to main content Home About Submit ALERTS / RSS Search for this keyword Advanced Search New Results Combined ADAMTS10 and ADAMTS17 inactivation exacerbates bone shortening and compromises extracellular matrix formation Nandaraj Taye , View ORCID Profile Stylianos Z. Karoulias , Zerina Balic , Lauren W. Wang , Belinda B. Willard , Daniel Martin , Daniel Richard , View ORCID Profile Alexander S. Okamoto , Terence D. Capellini , Suneel S. Apte , View ORCID Profile Dirk Hubmacher doi: https://doi.org/10.1101/2025.01.23.634616 Nandaraj Taye 1 Orthopedic Research Laboratories, Leni & Peter W. May Department of Orthopedics, Icahn School of Medicine at Mount Sinai , New York, NY, 10029, USA Find this author on Google Scholar Find this author on PubMed Search for this author on this site Stylianos Z. Karoulias 1 Orthopedic Research Laboratories, Leni & Peter W. May Department of Orthopedics, Icahn School of Medicine at Mount Sinai , New York, NY, 10029, USA Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Stylianos Z. Karoulias Zerina Balic 1 Orthopedic Research Laboratories, Leni & Peter W. May Department of Orthopedics, Icahn School of Medicine at Mount Sinai , New York, NY, 10029, USA Find this author on Google Scholar Find this author on PubMed Search for this author on this site Lauren W. Wang 3 Department of Biomedical Engineering, Cleveland Clinic Lerner Research Institute , Cleveland, OH, 44195, USA 4 Department of Orthopaedic Surgery, Cleveland Clinic Orthopaedic and Rheumatologic Institute , Cleveland, OH, 44195, USA Find this author on Google Scholar Find this author on PubMed Search for this author on this site Belinda B. Willard 2 Proteomics and Metabolomics Core, Cleveland Clinic Lerner Research Institute , Cleveland, OH, 44195, USA Find this author on Google Scholar Find this author on PubMed Search for this author on this site Daniel Martin 3 Department of Biomedical Engineering, Cleveland Clinic Lerner Research Institute , Cleveland, OH, 44195, USA Find this author on Google Scholar Find this author on PubMed Search for this author on this site Daniel Richard 5 Human Evolutionary Biology, Harvard University , Cambridge, MA, 02138, USA Find this author on Google Scholar Find this author on PubMed Search for this author on this site Alexander S. Okamoto 5 Human Evolutionary Biology, Harvard University , Cambridge, MA, 02138, USA Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Alexander S. Okamoto Terence D. Capellini 5 Human Evolutionary Biology, Harvard University , Cambridge, MA, 02138, USA 6 Broad Institute of MIT and Harvard , Cambridge, MA, 02142, USA Find this author on Google Scholar Find this author on PubMed Search for this author on this site Suneel S. Apte 3 Department of Biomedical Engineering, Cleveland Clinic Lerner Research Institute , Cleveland, OH, 44195, USA 4 Department of Orthopaedic Surgery, Cleveland Clinic Orthopaedic and Rheumatologic Institute , Cleveland, OH, 44195, USA Find this author on Google Scholar Find this author on PubMed Search for this author on this site Dirk Hubmacher 1 Orthopedic Research Laboratories, Leni & Peter W. May Department of Orthopedics, Icahn School of Medicine at Mount Sinai , New York, NY, 10029, USA 7 Mindich Child Health and Development Institute, Icahn School of Medicine at Mount Sinai , New York, NY, 10029, USA Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Dirk Hubmacher For correspondence: dirk.hubmacher{at}mssm.edu Abstract Full Text Info/History Metrics Preview PDF Abstract Weill-Marchesani syndrome (WMS) is characterized by severe short stature, short hands and feet (brachydactyly), joint contractures, tight skin, and heart valve, eye, and skin anomalies. Whereas recessive WMS is caused by mutations in ADAMTS10 , ADAMTS17 , or LTBP2 , dominant WMS is caused by mutations in FBN1 (encoding fibrillin-1). Since bone growth is driven by chondrocyte proliferation and hypertrophy in the growth plates, the genetics of WMS suggests that the affected ECM proteins act within the same pathway to regulate chondrocyte and growth plate function. Here, we investigated the role of the secreted ADAMTS proteases ADAMTS10 and ADAMTS17 in growth plate function and ECM formation. We generated Adamts10 ; Adamts17 double knockout (DKO) mice, which showed significant postnatal lethality compared to single Adamts10 or Adamts17 KO mice. Importantly, we observed severe bone shortening DKO mice, which correlated with a narrower hypertrophic zone in their growth plates. ADAMTS17 substrates identified by N-terminomics and yeast two-hybrid screening identified the ECM proteins fibronectin and collagen VI (COL6). However, validation experiments did not reveal direct proteolysis of either fibronectin or COL6 by ADAMTS17. We then investigated ECM formation in primary ADAMTS10- and ADAMTS17-deficient skin fibroblasts and observed compromised fibronectin deposition concomitant with aberrant intracellular accumulation of fibrillin-1. These findings support a role for ADAMTS17 in ECM protein secretion and assembly. Collectively, our data suggest that ADAMTS10 and ADAMTS17 regulate bone growth by regulating chondrocyte hypertrophy or hypertrophic chondrocyte turnover. Mechanistically, ADAMTS17 appears to be a critical regulator of ECM protein secretion or pericellular matrix assembly, whereas ADAMTS10 likely modulates ECM formation at later stages, possibly regulating the spatio-temporal deposition of fibrillin isoforms. Introduction Acromelic dysplasias are a group of genetic conditions resulting in severe short stature and shortening of distal limb elements 1 , 2 . Among them, Weill-Marchesani syndrome (WMS) has been characterized genetically resulting from mutations in distinct, but functionally related genes, encoding extracellular matrix (ECM) proteins 1 , 3 . Recessive pathogenic variants in secreted ADAMTS10 cause Weill-Marchesani syndrome 1 (WMS1) 4 – 6 . Distinct WMS sub-types can also be caused by dominant mutations in fibrillin-1 ( FBN1 , WMS2) or recessive mutations in LTBP2 (WMS3) or ADAMTS17 (WMS4) suggesting that these four genes may act together in pathways that regulate development and homeostasis of affected tissues 4 , 7 – 11 . WMS is characterized by short stature, lens dislocation, microspherophakia and other eye anomalies, progressive joint stiffness, and tight skin 3 , 12 . Lens dislocation and/or changes in the outflow track can block the drainage of aqueous humor from the anterior chamber of the eye and cause glaucoma in WMS. WMS1 also has cardiovascular manifestations such as patent ductus arteriosus, pulmonary valve dysplasia, which can lead to pulmonary stenosis, and in rare cases aortic aneurysms 6 , 8 , 12 . In dogs, mutations in ADAMTS10 and ADAMTS17 were associated with primary open angle glaucoma 13 – 17 . Homozygosity of a glaucoma-causing canine ADAMTS17 variants was also associated with short stature in several dog breeds 18 . WMS-causing ADAMTS10 and ADAMTS17 mutations are distributed over the entire molecule and result in loss-of-function or haploinsufficiency due to impaired secretion 1 , 3 , 10 , 11 . The fact that mutations in ADAMTS10 and ADAMTS17 each cause WMS suggests that these genes cooperate, have superimposed mechanisms, or act in the same pathways that regulate the development or homeostasis of affected tissues, including the growth plate, which drives bone growth, the skin, and the eye 19 , 20 . ADAMTS10 and ADAMTS17 each bind to fibrillin-1, which may provide a scaffold for their tissue-specific deposition or functional regulation 21 , 22 . In addition, ADAMTS10 was shown to promote the assembly of fibrillin-1 in cell culture 21 . The 19 ADAMTS proteases are involved in diverse biological processes including tissue morphogenesis and homeostasis 23 , 24 . Mutations in several ADAMTS proteases cause birth defects and inherited connective tissue disorders in humans and other species 25 . In addition, ADAMTS proteases contribute to the progression of acquired disease, such as the aggrecanase ADAMTS5 in osteoarthritis 26 . These diverse roles are attributed to the actions of ADAMTS proteases on distinct substrates. Recognized ECM substrates for ADAMTS proteases include proteoglycans such as aggrecan and versican, the ECM scaffolding proteins fibrillin-1, fibrillin-2, and fibronectin, and several collagens 23 . Most ADAMTS proteases are secreted as inactive zymogens whose activation requires proteolytic removal of their prodomains by furin or other proprotein convertases 27 – 30 . ADAMTS10, however, lacks a canonical furin recognition site and is poorly processed by furin. Consistent with poor furin-processing, ADAMTS10 appears to be an inefficient protease, although, once activated by mutagenesis to restore a canonical furin-processing site, ADAMTS10 could cleave fibrillin-1 and fibrillin-2 in vitro 21 , 31 . However, alternative furin-independent mechanisms of ADAMTS10 activation in vitro or in vivo remain elusive. ADAMTS17 is secreted as an active protease, but fragments itself extensively and efficiently, including within the catalytic domain, prior to its release from the surface of HEK293 cells 22 . These findings suggested that ADAMTS17 may act as a protease in the secretory pathway or at the cell surface. ADAMTS17 substrates other than itself have not been identified. We showed previously that ADAMTS17 interacted with fibrillin-1 and fibrillin-2 but did not cleave either 22 , 32 . Adamts10 and Adamts17 inactivation in mice was previously reported with differential phenotypes. While Adamts10 knockout (KO) mice did not result in short stature, Adamts17 KO mice had shorter bones and growth plate abnormalities 31 , 33 . Interestingly, the knock-in of a WMS mutation into the mouse Adamts10 locus resulted in short stature and growth plate abnormalities 34 . In addition, knock-in of an ADAMTS10 mutation that causes glaucoma in dogs into the mouse Adamts10 locus also resulted in short stature 35 . Together, these findings support a role for ADAMTS10 and ADAMTS17 in regulating bone growth and thus height. If and how ADAMTS10 and ADAMTS17 cooperate in regulating bone growth and in the formation and maintenance of other tissues affected in WMS is not known. Here, we investigated the genetic interactions of ADAMTS10 and ADAMTS17 by analyzing the bone and skin phenotypes of Adamts10 ; Adamts17 double KO (DKO) mice. For insights on molecular mechanisms, we evaluated potential ADAMTS17 substrates and binding partners identified by N-terminomics and yeast two-hybrid screening, respectively. The findings of our studies, taken together with prior work strongly suggest a cooperative role for these proteases in skeletal growth and provide a putative molecular basis for their ECM-regulatory activities. Results Combined Adamts10 and Adamts17 inactivation resulted in early postnatal mortality and reduced body size To generate Adamts10 ; Adamts17 DKO mice, we combined a previously published Adamts10 KO allele with a novel Adamts17 KO allele. The Adamts10 KO allele was generated by replacing 41 bp of Adamts10 exon 5 with an IRES- lacZ - neo cassette 31 . The Adamts17 KO allele was generated using CRISPR/Cas9-induced non-homologous end-joining with guide RNAs targeting Adamts17 exon 3 ( Fig. 1A ). The resulting dinucleotide insertion (AT) in exon 3 of Adamts17 ( Adamts17 670_671insAT, NM_001033877.4) caused a reading frame shift (p.I190fsX12), which resulted in a premature termination codon presumed to trigger nonsense-mediated mRNA decay ( Fig. 1A ). The AT insertion in Adamts17 was verified by Sanger sequencing of a PCR product amplified from template DNA isolated from toe tissue with Adamts17 -specific primers flanking exon 3 ( Fig. 1B ). Mice homozygous for the Adamts17 AT insertion are referred to as Adamts17 KO. Loss of ADAMTS17 protein was validated by immunostaining tissue and primary skin fibroblasts isolated from wild type (WT) and Adamts17 KO mice using a monoclonal ADAMTS17 antibody. In sections through WT skin and the tibial growth plate, ADAMTS17 immunoreactivity was apparent around hair follicles and in hypertrophic chondrocytes, respectively. This signal was absent or strongly reduced in comparable sections from Adamts17 KO or Adamts10 ; Adamts17 DKO mice ( Fig. 1C ). In primary WT skin fibroblasts, the ADAMTS17 signal was most intense in perinuclear regions, where the endoplasmic reticulum and Golgi are located, and in patches between cells ( Fig. 1C , right). Similar to tissue sections, the ADAMTS17 signal was absent in DKO fibroblasts. Thus, genetic inactivation of ADAMTS17 eliminated ADAMTS17 protein in relevant tissues and primary cells and at the same time validated the specificity of the monoclonal ADAMTS17 antibody. Download figure Open in new tab Figure 1. Generation of Adamts17 KO and Adamts10;Adamts17 DKO mice. A) Domain organization of ADAMTS17 shows location and targeting of exon 3 by CRISPR/Cas9 gRNA to induce non-homologous end joining. The nucleotide and amino acid sequence of the ADAMTS17 WT allele (green) and after AT insertion (red) are indicated. The dinucleotide insertion induced a frameshift, which resulted in a premature stop codon after 12 amino acids. B) Sanger sequencing traces of a PCR product generated with primers flanking exon 3 showing the AT insertion (underlined) in the Adamts17 KO. C) Micrographs of ADAMTS17 immunostaining of sections through WT and Adamts17 KO skin (left), DKO growth plates (middle), and of primary DKO mouse skin fibroblasts (right). The signal in the dermis around hair follicles, in growth plate chondrocytes, and in fibroblasts and their ECM originating from the monoclonal ADAMTS17 antibody was strongly reduced in KO and DKO tissues and cells, indicating lack of ADAMTS17 protein in Adamts17 KO mice. D) Pie chart showing Mendelian distribution of genotypes recovered from Adamts17 Het intercrosses at the time of genotyping (P7-P10) (n=94 mice). E) Breeding scheme to generate WT, Adamts10 KO (10KO), Adamts17 KO (17KO), and DKO mice. F) Pie chart showing distribution of genotypes recovered from Adamts10 Het;Adamts17 Het intercrosses at P7-P10 (n=180 mice). Statistical analysis was performed using Chi square calculation. G) Kaplan-Meier survival analysis of DKO mice. The numbers of observed dead/total mice for the individual genotypes are indicated in brackets. Statistical significance was determined using a log-rank test. H) Whole mount images of WT, 10KO, 10KO;17Het mice at 4 weeks of age shows progressive reduction in body size. I) Bar graphs showing body weights of 4-week-old mice of the indicated genotypes. The number of mice is indicated below the genotypes. J) Bar graphs showing body weight normalized to average femur length for the genotypes that were significantly different in I. In I, J floating bars indicate the 25 th – 75 th percentile range, lines the mean value, and whiskers the standard deviation. Statistical differences in I, J were determined using a one-way ANOVA with post-hoc Tukey test. a, p<0.05 compared to WT. Adamts17 KO mice were born at the expected Mendelian ratio and were viable ( Fig. 1D ). To generate DKO mice, we first crossbred Adamts10 Het and Adamts17 Het mice to generate Adamts10;Adamts17 double-heterozygous mice ( Fig. 1E ). Adamts10 Het; Adamts17 Het mice were then intercrossed to generate offspring with all allelic combinations, among which WT, Adamts10 KO; Adamts17 KO, and Adamts10 ; Adamts17 DKO mice were the focus of subsequent analyses. In principle, this breeding scheme allows comparison of the phenotypes from littermates. However, due to the low predicted percentages for WT and DKO mice (6.25%) per litter in these crosses, we also intercrossed Adamts10 Het or Adamts17 Het mice to generate the respective WT and individual KOs as age- and sex-matched controls for DKO mice. We first analyzed the Adamts10 and Adamts17 genotype distribution of 180 mice at postnatal day (P) 7-P10 and determined statistically significant deviations from the expected Mendelian ratios using Chi 2 calculation ( Fig. 1F ). Adamts17 KO (3.9% vs. 6.25 expected), Adamts10 KO; Adamts17 Het (6.7% vs 12.5% expected), and DKO (3.9% vs. 6.25 expected) mice were present in significantly lower numbers than expected and the percentage of Adamts10 ; Adamts17 double heterozygous mice was significantly higher (34.4% vs. 25% expected). This resulted in a Chi 2 value of 18.09 (8 degrees of freedom) and a p-value of <0.05, suggesting reduced viability or embryonic lethality due to reduced Adamts17 gene dosage or the combined absence of Adamts10 and Adamts17 . Since we observed early postnatal lethality of Adamts10 ; Adamts17 DKO mice, we quantified postnatal survival with Kaplan-Meier survival analysis, where we observed significant postnatal mortality of DKO mice with 50% survival at 23 d (+/-7.1 d) after birth (probe > Chi 2 = 0.00029, log-rank test) ( Fig. 1G ). The cause of death is unknown. In addition to reduced survival of DKO mice, we noted reduced body size, which correlated with several genotypes, most notably Adamts17 KO mice, Adamts10 KO; Adamts17 Het mice and DKO mice ( Fig. 1H ). These size differences were also apparent from body weight measurements at 4 weeks of age where the weights of Adamts10 KO; Adamts17 Het and DKO mice were significantly reduced compared to WT ( Fig. 1I ). However, after normalization of the body weight to the average femur lengths, these differences became non-significant, suggesting proportionate short stature ( Fig. 1J ). Combined Adamts10 ; Adamts17 depletion exacerbated bone shortening To determine if Adamts10 and Adamts17 gene dosage affected bone length, we quantified the lengths of forelimb and hind limb bones from X-ray images taken at 4 weeks of age ( Fig. 2A-L ). Overall, Adamts10 ; Adamts17 DKO mice had the shortest bones across all genotypes when compared to WT or individual KOs. In addition, Adamts17 KO bones were significantly shorter than WT, except for the humerus, the 1 st metacarpal, and the 1 st metatarsal. Notably, deletion of one Adamts17 allele in Adamts10 KO mice exacerbated bone shortening, but not vice versa, except in the fibula, the 1 st metacarpal, and the 1 st metatarsal. The lengths of the 1 st metacarpal and 1 st metatarsal were significantly shorter in DKO mice compared to WT, Adamts10 KO, and Adamts17 KO but the same bones were not shorter in the individual KOs compared to WT ( Fig. 2E, L ). The lengths of Adamts10;Adamts17 double heterozygous bones were not significantly shorter compared to WT bones. Since we previously observed increased bone width in another acromelic dysplasia model (geleophysic dysplasia) due to Adamtsl2 deficiency, we measured the width of humeri and femora 36 . The mid-shaft width in DKO mice was significantly greater than WT, Adamts10 KO, or Adamts17 KO (humerus), or compared to WT (femur) ( Fig. 2M, N ). In addition, the widths of the Adamts10 KO; Adamts17 Het humerus and femur were increased compared to WT, but not the individual KOs. Collectively, combined inactivation of ADAMTS10 and ADAMST17 exacerbated bone shortening compared to the individual KOs with Adamts17 having an apparently stronger gene dosage effect compared to Adamts10 . Download figure Open in new tab Figure 2. Exacerbated bone shortening in Adamts10;Adamts17 DKO mice. A) X-ray images showing WT and DKO forelimbs. B-E) Bar graphs showing lengths of humerus (B), radius (C), ulna (D), and 1 st metacarpal (E) for all genotypes. F) X-ray images showing WT and DKO femur. G) Bar graphs showing femoral length for all genotypes. H) X-ray images showing WT and DKO tibia and fibula. I, J) Bar graphs showing lengths of tibia (I) and fibula (J) for all genotypes. K) X-ray images showing WT and DKO hind paw bones. L) Bar graphs showing length of 1 st metatarsal for all genotypes. M, N) Bar graphs showing thickness of humerus (M) and femur (N) at mid-shaft. For number of mice see Fig. 1I . All mice were 4 weeks old at the time of X-ray imaging. Bones from both limbs were measured and measurement from male and female mice were combined. In B, C, D, E, G, I, J, L, M, N floating bars indicate the 25 th – 75 th percentile range, lines the mean value and whiskers the standard deviation. Statistical differences in B, C, D, E, G, I, J, L, M, N were determined using a one-way ANOVA with post-hoc Tukey test. a, p<0.05 compared to WT; b, p<0.05 compared to Adamts10 KO; c, p<0.05 compared to Adamts17 KO. Combined Adamts10 and Adamts17 inactivation compromised growth plate chondrocyte function Since bone growth is largely driven by growth plate activity, i.e. the proliferation and hypertrophic expansion of growth plate chondrocytes, we quantified growth plate dimensions and primary chondrocyte behavior in ADAMTS10 and ADAMTS17 deficient tissue and chondrocytes 37 , 38 . Compared to WT growth plates, DKO growth plates showed a narrower hypertrophic zone, while the proliferative zone was unchanged ( Fig. 3A, B ). In addition, the columnar organization of proliferating chondrocytes appeared to be irregular with more spacing between chondrocyte columns ( Fig. 3C ). By immunostaining, we localized ADAMTS17 in the pericellular matrix of hypertrophic chondrocytes in wild type growth plates, close to the cartilage-bone interface ( Fig. 3D ). These data suggested that ADAMTS17 could play a role in regulating or maintaining chondrocyte hypertrophy. Therefore, we next used high cell density pellet cultures of mouse embryo CH3/10T1/2 cells to model chondrocyte-like differentiation in vitro and to determine the temporal dynamics of Adamts10 and Adamts17 mRNA expression during chondrocyte maturation in vitro by quantitative real-time (qRT)-PCR ( Fig. 3E ). Adamts10 mRNA levels significantly decreased between 0 and 2 days after induction of differentiation and remained low thereafter ( Fig. 3F ). In contrast, Adamts17 mRNA levels started to moderately increase until 7 days after induction of differentiation followed by a strong and statistically significant increase at 10 days. This suggested downregulation of Adamts10 and induction of Adamts17 gene expression during in vitro chondrogenesis of CH3/10T1/2 pellet cultures. The strong reduction of Col2a1 mRNA levels between 0 and 2 days after induction of differentiation was consistent with CH3/10T1/2 pellet cultures undergoing differentiation. Download figure Open in new tab Figure 3. ADAMTS10 and ADAMTS17 regulate growth plate function and chondrocyte hypertrophy. A) Images of sections through growth plates of 4 week-old WT and Adamts10;Adamts17 DKO mice. Proliferative (PZ) and hypertrophic (HZ) zones are outlined with dashed lines. B) Bar graphs showing widths of PZ and HZ from WT and DKO growth plates. Data points represent the average of multiple measurements across the growth plate zone from n=3 mice. C) Higher magnification of growth plate from images in A showing disorganized proliferative zone in DKO growth plates. D) Micrograph of ADAMTS17 immunostaining of hypertrophic chondrocytes at the cartilage-bone interface. The boxed area is magnified in the right-hand panel. E) Schematic representation of experimental design for pellet culture to induce chondrocyte-like differentiation of C3H/10T1/2 cells. F) Bar graphs showing relative Adamts10, Adamts17, and Col2a1 mRNAs levels during differentiation of C3H/10T1/2 cell pellets normalized to Gapdh (n=3 replicates). G) Schematic representation of the mechanism of action of okadaic acid in de-repressing chondrocyte hypertrophy genes. H) Bar graphs showing relative changes of Adamts10 (TS10), Adamts17 (TS17), Fbn1, Fn1, and Col10a1 mRNA levels 24 h after treatment of primary chondrocytes with 50 nM okadaic acid or DMSO (n=3 replicates). I) Micrographs of immunostaining of fibrillin-1 (FBN1) and fibronectin (FN) deposition in the ECM of primary chondrocytes 3 days after treatment with okadaic acid or DMSO only. J) Quantification of mean fluorescence intensity from I (n=3 fields-of-view). K) Schematic representation of osteogenic differentiation of P5 primary rib chondrocytes isolated from Adamts10 KO or Adamts17 KO mice. The bottom panels show brightfield micrographs of freshly isolated primary chondrocytes (-3 d, left) and confluent chondrocytes (0 d, right). L) Micrographs of two individual wells/genotype of primary WT or Adamts10 KO (10KO) chondrocytes stained with alizarin red after 21 d of culture in osteogenic medium. M) Bar graph showing quantification of mean signal intensity of alizarin red deposits (isolates from n=4-5 biological replicates/genotype). N) Micrographs of two individual wells/genotype of primary WT or Adamts17 KO chondrocytes stained with alizarin red after 21 d of culture in osteogenic medium. O) Bar graph showing quantification of mean signal intensity of alizarin red deposits (isolates from n=5 biological replicates/genotype). In B, F, H, J, M, O, bars indicate mean values and whiskers the standard deviation. Statistical significance in B, H, J, M, O was calculated with a 2-sided Student t-test and in F with one-way ANOVA followed by post-hoc Tukey test. To further probe the regulation of Adamts10 and Adamts17 expression during chondrocyte hypertrophy, we treated primary WT rib chondrocytes with okadaic acid, a protein phosphatase 2A inhibitor, which results in de-repression of chondrocyte hypertrophy genes ( Fig. 3G ) 39 , 40 . qRT-PCR showed significant upregulation of Adamts10 and Adamts17 mRNA levels 24 h after treatment of primary WT chondrocytes with okadaic acid compared to DMSO-treated control chondrocytes ( Fig. 3H ). Increased Col10a1 mRNA levels, a marker for hypertrophic chondrocytes, served as a positive control for okadaic acid-mediated de-repression of chondrocyte hypertrophy genes. In addition to Adamts10 and Adamts17 , mRNAs for genes encoding the ECM proteins fibrillin-1 ( Fbn1 ) and fibronectin ( Fn1 ) were also induced. Fibrillin-1 binds to ADAMTS10 and ADAMTS17 and mutations in FBN1 cause WMS2 9 , 21 , 22 . Fibronectin forms the ECM scaffold required for fibrillin-1 deposition in the ECM of mesenchymal cells 41 . Using immunostaining, we confirmed increased fibrillin-1 and fibronectin ECM deposition in primary WT chondrocytes following okadaic acid treatment ( Fig. 3I, J ). Finally, we investigated the implications of ADAMTS10 and ADAMTS17-deficiency on primary rib chondrocyte hypertrophy and the capacity to deposit calcium as hydroxyapatite in their ECM, which is a characteristic of terminal hypertrophic chondrocytes ( Fig. 3K ). Chondrocytes isolated from Adamts10 KO ribs showed no difference in alizarin red-positive calcium mineral deposition after 21 d under differentiation and mineralization conditions ( Fig. 3L, M ). In contrast, calcium mineral deposition by ADAMTS17-deficient primary chondrocytes was significantly reduced, suggesting differential roles or differential compensation for ADAMTS10 and ADAMTS17 in chondrocyte differentiation ( Fig. 3N, O ). To gain further insights into the epigenetic and transcriptomic regulation of ADAMTS10 and ADAMTS17 during human embryonic development, we data-mined recent assays for transposase-accessible chromatin with sequencing (ATAC-seq) and RNA-transcriptomic data sets, generated from micro- dissected cartilaginous human fetal appendicular skeletal elements ( Fig. 4A ) 42 . We first identified open chromatin regions by ATAC-seq within +/- 100 kb of ADAMTS10 and ADAMTS17 ( Fig. 4B, C ); each interval containing regulatory elements, such as enhancers, promoters, and repressor sequences, likely drives expression of the nearby gene. For ADAMTS10 , out of ten total elements within 100 kbp, we identified three cartilage open chromatin regions that were shared by most or all autopod elements (phalanges, metatarsals and metacarpals), but were absent in stylopod or zeugopod elements, i.e. proximal and distal ends of all major long bones ( Fig. 4B , Table 1 ). One regulatory region was located 100 kb distal to the ADAMTS10 transcription start site (TSS), one overlapping with the final exon, and the other <1 kb proximal to the TSS. We identified two additional cartilage open chromatin regions present in all skeletal elements, located within the ADAMTS10 gene body. For ADAMTS17 , we observed a larger number of regulatory elements compared to ADAMTS10, consistent with the larger size of ADAMTS17 . At this locus, 23 cartilage open chromatin regions were identified within or in close vicinity to the ADAMTS17 gene body ( Fig. 4C , Table 2 ). While three ADAMTS17 open chromatin regions were identified as accessible in all autopod elements and eight in most, two were specific to individual skeletal elements of the hind limb autopod (metatarsal V). Moreover, in the stylopod and zeugopod elements, two open chromatin regions were shared by the proximal elements but showed mixed accessibility in the corresponding distal ones. Download figure Open in new tab Figure 4: Chromatin accessibility and putative regulation of ADAMTS10 and ADAMTS17 in human cartilage and bone development. A) Limb skeletal elements and experimental design to generate ATAC-seq and transcriptomics data from skeletal elements of human products from conception (E54&E67). B, C) Mapping of open chromatin regions identified by ATAC-seq 100 kb up- or downstream of ADAMTS10 (B) and ADAMTS17 (C). The positions of ADAMTS10 on human chromosome 19 and ADAMTS17 on human chromosome 15 are indicated. D, E) Normalized read counts indicating ADAMTS10 and ADAMTS17 mRNA abundance in skeletal elements from stylopods and zeugopods (D) and autopods (E). F, G) Location of transcription factor binding sites 5 kb upstream of the ADAMTS10 (F) or ADAMTS17 (G) transcriptional start site (TSS). View this table: View inline View popup Download powerpoint Table 1: Accessible genomic regions in ADAMTS10 in cartilage as determined by ATAC-seq. View this table: View inline View popup Download powerpoint Table 2: Accessible genomic regions in ADAMTS17 in cartilage as determined by ATAC-seq. We next analyzed ADAMTS10 and ADAMTS17 gene expression in human embryonic skeletal elements by comparing normalized read counts as a measure of ADAMTS10 and ADAMTS17 mRNA abundance ( Fig. 4D, E ). In stylopodial and zeugopodial elements, normalized read counts for ADAMTS10 were generally higher compared to ADAMTS17 , suggesting increased expression ( Fig. 4D ). We did not observe a distinct pattern of ADAMTS10 or ADAMTS17 expression based on specific skeletal elements, suggesting that both genes were expressed in autopods of the hind limb and forelimb. Lastly, the overall read counts for ADAMTS10 and ADAMTS17 in the stylopod and zeugopod were higher than in the autopods. Finally, we mapped predicted binding sites of key chondrogenic and osteogenic transcription factors, including SOX9, RUNX2, and ATF4, within 5 kb upstream of the TSS using the Search Motif Tool in the Eukaryotic Promoter Database ( Fig. 4F, G ) 43 . Overall, transcription factor binding sites upstream of ADAMTS10 mapped more frequently closer to the TSS (<2 kb) compared to ADAMTS17 , with the exception of SOX9, SOX6, and ATF4 binding sites. We also noted more SOX9 binding sites upstream of the ADAMTS17 TSS. Collectively, these data suggest specific regions of chromatin accessibility in the vicinity of ADAMTS10 and ADAMTS17 and correlating with their expression in human embryonic cartilage. In addition, the mapping of chondrogenic and osteogenic transcription factor binding sites in the ADAMTS10 and ADAMTS17 promoter region supports the regulation of Adamts10 and Adamts17 expression that was observed during chondrocyte hypertrophy. Adamts10 and Adamts17 inactivation compromised skin development and differentially regulated ECM deposition by skin fibroblasts Adamts10 ; Adamts17 DKO skin easily detached and ripped during shaving. We investigated this phenomenon systematically by measuring the thickness of the dermal sub-layers in Masson’s trichrome stained cross-sections ( Fig. 5A-E ). The overall thickness of Adamts10 KO and Adamts17 KO dorsal skin was reduced compared to WT skin and was even further reduced in DKO skin, which was significantly thinner compared to WT and Adamts10 KO or Adamts17 KO skin ( Fig. 5B ). Epidermal layer thickness was slightly but significantly reduced in DKO skin compared to WT and Adamts10 KO skin but not compared to Adamts17 KO skin ( Fig. 5C , left). The thickness of Adamts10 KO and Adamts17 KO dermis and hypodermis was significantly reduced compared to WT and further reduced in the DKO ( Fig. 5C , middle, right). The panniculus carnosus (p. carnosus) muscle, which underlies mouse skin, was similarly significantly thinner in Adamts17 KO and DKO skin sections compared to WT and Adamts10 KO ( Fig. 5D ). The proportion of the hypodermis to overall skin thickness was greatly reduced in DKO skin, whereas the proportions of the dermis and p. carnosus were both increased ( Fig. 5E ). A similar reduction in the hypodermal layer and increase in the dermal layer were evident in Adamts17 KO skin and to a lesser extent in Adamts10 KO skin. We also observed a significant reduction in the number of hair follicles in DKO skin compared to all other genotypes, but no significant changes in the Adamts10 KO or Adamts17 KO compared to WT skin ( Fig. 5F ). Download figure Open in new tab Figure 5. Adamts10;Adamts17 DKO is associated with skin alterations and aberrant ECM deposition by dermal fibroblasts. A) Micrographs of Masson’s trichrome-stained cross-sections through dorsal skin from 4-week-old WT, Adamts10 KO (10KO), Adamts17 KO (17KO), and DKO mice. ED, epidermis; D, dermis; HD, hypodermis; PC, panniculus carnosus. B-D) Bar graphs showing quantification of overall skin thickness (B) and the thicknesses of the epidermis, dermis, hypodermis (C), and panniculus carnosus (p. carnosus, D). Individual data points represent multiple measurements along the different skin layers from n=3 mice/genotype. E) Stacked bar graphs showing the relative proportions of individual skin layers. The percentage values are indicated. F) Bar graphs showing the quantification of hair follicle numbers in the skin for each genotype. G, H) Bar graphs showing normalized gene expression in fragments per kilobase of transcript per million mapped reads (FPKM) for Adamts10 and Adamts17 in individual skin cell types at E14.5 (G) and P5 (H). Data were extracted from the Hair-GEL database 44 , 45 . I-K) Micrographs showing the localization of Adamts17 mRNA (red/dark purple) in WT skin cross-sections at E13.5 (I), E16.5 (J), and P0 (K) detected by RNAscope in-situ hybridization with a probe specific for Adamts17. Sections were counterstained with hematoxylin. L) Micrograph of ADAMTS17 immunostaining (green) of cross-sections through WT skin. Nuclei were stained with DAPI (blue). M) Micrographs of primary mouse skin fibroblasts after immunostaining for fibrillin-1 (red) and fibronectin (green). Nuclei were counterstained with DAPI (blue). N) Quantification of mean fluorescence intensity from M (n=4 biological replicates). In B, C, D, F, floating bars indicate 25 th – 75 th percentile range, lines the mean value and whiskers the standard deviation. In N, the bars represent the mean value and the whiskers the standard deviation. Statistical differences in B, C, D, F, N were determined using a one-way ANOVA with post-hoc Tukey test. a, p<0.05 compared to WT; b, p<0.05 compared to Adamts10 KO; p<0.05 compared to Adamts17 KO. To identify the cellular origins of ADAMTS10 and ADAMTS17 in the skin, we data-mined the Hair-GEL database, which contains single-cell transcriptomic data from E14.5 and P5 skin with a focus on hair follicle development 44 , 45 . At E14.5, differential expression of Adamts10 and Adamts17 was noted in the dermal condensate, where Adamts10 expression was high, and in the placode and epidermis, where Adamts17 expression was high ( Fig. 5G ). In fibroblasts, Schwann cells, or melanocytes, Adamts10 and Adamts17 were expressed at similar levels. At P5, Adamts10 was strongly expressed in subtypes of dermal papilla cells and Adamts17 was strongly expressed in the bulge stem cell population ( Fig. 5H ). Expression in other cell types, including fibroblasts, was much lower and differential expression of Adamts10 and Adamts17 was less apparent. We next validated temporal Adamts17 expression dynamics in the skin by RNA in-situ hybridization and immunostaining of embryonic and postnatal mouse skin. At E13.5, Adamts17 mRNA was localized predominantly in the developing epidermis ( Fig. 5I ). As previously described, at E16.5, high Adamts17 mRNA levels were observed in the placode and the hair peg of the developing hair follicles, while lower Adamts17 expression was observed in the epidermis and cells of the dermal layer ( Fig. 5J ) 22 . At P0, Adamts17 mRNA was concentrated in cells surrounding the base of the hair follicle and in the outer root sheath ( Fig. 5K ). ADAMTS17 immunostaining in postnatal skin confirmed ADAMTS17 protein localization in or around hair follicles and the hair shaft ( Fig. 5L ). At later postnatal time points up to P21, Adamts17 mRNA signal was generally low (data not shown). Finally, we investigated fibrillin-1 and fibronectin ECM deposition in primary skin fibroblasts isolated from WT, Adamts10 KO, Adamts17 KO, or Adamts10;Adamts17 DKO mice. Strikingly, skin fibroblasts from DKOs did not form a fibronectin network and showed abnormal intracellular accumulation of fibrillin-1 ( Fig. 5M, N ). Fibroblasts isolated from individual Adamts10 KO or Adamts17 KO mice showed intermediate phenotypes with a significant reduction of fibronectin in both KOs and a reduction of fibrillin-1 in the Adamts17 KO. While fibronectin in Adamts17 KO fibroblasts was present in globular structures or short fibers on the cell surface or in the vicinity of fibroblasts, fibronectin in Adamts10 KO fibroblasts was largely organized in an extracellular fibrillar network. Notably, in areas of Adamts10 KO fibroblasts ECM with sparse to no fibronectin network, intracellular fibrillin-1 accumulation was more prevalent. Identification of fibronectin and COL6 as ADAMTS17 binding partners It was previously reported that ADAMTS10 constitutively had poor protease activity due to a degenerated furin cleavage site (GL KR instead of a canonical RX[K/R]R↓ site), which hindered furin- mediated activation ( Fig. 6A ) 21 , 31 . In contrast, ADAMTS17 is an active protease based on extensive autoproteolysis at the cell surface 22 . To identify ADAMTS17 substrates, we used an unbiased mass spectrometry (MS)-based N-terminomics strategy, Terminal Amine Isotopic Labeling of Substrates (TAILS). We complemented this approach by yeast-2-hybrid protein-protein interaction screening. For TAILS, we co-cultured human dermal fibroblasts with HEK293 cells stably expressing ADAMTS17 or its active site mutant ADAMTS17-EA (Glu-390 to Ala), which abolishes its autocatalytic activity ( Fig. 6B , left) 22 . Following isobaric tag labelling of the samples, we identified differentially abundant N- terminally labeled peptides uniquely present or elevated in ADAMTS17 conditioned medium and prioritized the peptides with neo-N-termini from secreted and/or ECM proteins as candidate substrates ( Fig. 6C ). Amongst potential ADAMTS17 substrates, we identified peptides from the COL1, COL6A2, and COL6A3 chains, and fibronectin (FN1). Multiple ADAMTS17 peptides were identified in the wild-type ADAMTS17 samples due to its autoctalytic activity and served as positive controls. In parallel, we used the ADAMTS17 ancillary domain (17-AD) as the bait in a yeast-2-hybrid screen to identify binding partners. This approach identified the ECM proteins thrombospondin-1 (THSB1) and fibulin-3 (FBLN3), the secreted proteases ADAM12 and PAPPA, and the extracellular domain of the catalytically inactive receptor tyrosine-protein kinase ERBB3 ( Fig. 6D ). Most notably, we also identified fibronectin and COL6 as potential ADAMTS17 binding partners, which overlapped with the results from the MS screen. Therefore, we selected fibronectin and COL6 for further investigation and validation as potential ADAMTS17 substrates. Download figure Open in new tab Figure 6. Identification of fibronectin as a potential ADAMTS17 substrate. A) Domain organization of ADAMTS10 and ADAMTS17, which are identical. The degenerate (ADAMTS10) and canonical (ADAMTS17) furin processing sites and the localization of the catalytic residue Glu-390 in ADAMTS17 (17) that was mutated into Ala to generate proteolytically inactive ADAMTS17-EA (17-EA) are indicated. The domain organization of the catalytic (17-PCD) and ancillary (17-AD) domain constructs is indicated. B) Schematic representation of experimental design for co-culture of human dermal fibroblasts (HDF) with HEK293 cells stably expressing 17- or 17-EA (left) or co-transfection of 17- or 17-EA-encoding plasmids with FN1 or COL6A2-encoding plasmids in HEK293 cells (right). C) Volcano plot showing N-terminally labeled peptides identified by N-terminomics method TAILS in conditioned medium from ADAMTS17-expressing HEK293 cells co-cultured with HDFs. Peptides present only in samples from WT ADAMTS17 (red) or enriched in conditioned medium from WT ADAMTS17 co-cultures compared to the co-cultures with proteolytically inactive ADAMTS17-EA suggest ADAMTS17 substrates. D) Venn diagram showing overlap of ADAMTS17-cleaved proteins (TAILS) from co-culture systems (left) and binding partners for the ADAMTS17 ancillary domain (17-AD) identified by yeast-2-hybrid screening with a human placenta-derived cDNA library (right). Note that fibronectin (FN1) and COL6 were independently identified in both screens. E) Domain organization of fibronectin (FN1, NP_997647) showing the localization of the domains that interacted with 17-AD (grey box, bolded amino acid sequence) and the localization of the peptide identified by TAILS (red bar, red amino acid sequence). F) MS2 spectrum of the N-terminally labeled FN1 peptide (GNSVNEGLNQPTDDSCFDPYTVSHYAVGDEWER) showing b and y ions. G) Western blot detection of endogenous fibronectin in conditioned medium (Med) and cell lysates (Lys) collected after co-culture of 17 or 17-EA-expressing HEKs with HDFs. A monoclonal (green) and four different polyclonal (red) anti-fibronectin antibodies were used. F) Western blot detection of recombinant fibronectin (rFN) in conditioned medium (Med) and cell lysate (Lys) collected after co-expression of 17 or 17-EA with rFN in HEK293 cells. A polyclonal anti-fibronectin antibody (red) and a monoclonal anti V5-tag antibody (green) were used to detect rFN. Based on the yeast-2-hybrid data, the ADAMTS17 ancillary domain interacted with the C-terminal region of fibronectin comprising FNIII domain #15 and FNI domains #10-12 (amino acid residues 2130 – 2416, NP_997647) ( Fig. 6E, F ). The N-terminally labeled fibronectin peptide identified in the MS approach localized to the same region (amino acid residues 2282 – 2317) and covered the linker region between FNIII #15 and FNI #10 and the N-terminal part of FNI #10. For biochemical validation of fibronectin as an ADAMTS17 substrate, we first used western blot of conditioned medium and cell lysates collected from human dermal fibroblasts co-cultured with ADAMTS17- or ADAMTS17-EA- expressing HEK293 cells equivalent to the MS approach ( Fig. 6B , left). Using five different antibodies against fibronectin, we detected distinct fibronectin fragmentation patterns in conditioned medium in the presence of ADAMTS17-, but not ADAMTS17-EA-expressing HEK293s ( Fig. 6G ). All antibodies were raised against full length plasma fibronectin, except 15613-1-AP (ProteinTech), which was raised against a region comprising FNIII domain #15 and FNI domains #10-12. Fibronectin fragmentation in the cell lysate, which included the ECM fraction, was not observed. Together, this suggested that ADAMTS17 protease activity correlated with fibronectin fragmentation. To more directly test if ADAMTS17 can cleave fibronectin, we co-transfected HEK293 cells with ADAMTS17 or ADAMTS17-EA- encoding plasmids and a plasmid encoding V5-tagged fibronectin (rFN) ( Fig. 6B , right). When we analyzed conditioned medium and cell lysates harvested after co-expression of these plasmids, we did not detect fibronectin fragmentation in the medium or cell lysate with a polyclonal antibody against fibronectin or an antibody against the V5 tag of rFN ( Fig. 6H ). This suggested that fibronectin may not be a direct ADAMTS17 substrate, or that ADAMTS17 selectively cleaved fibronectin fibrils, which are assembled by dermal fibroblasts. For COL6A2, the yeast-2-hydrid data suggested an interaction region for 17-AD comprising the C- terminal portion of the central triple helical domain and an N-terminal portion of the von Willebrand factor A (VWA) domain #C1 (amino acid residues 480 – 652, NP_001840.3) ( Fig. 7A ). TAILS identified an N-terminally labeled peptide originating from COL6A3 (amino acid residues 1348 – 1367, NP_004360.2), which was localized in the C-terminal portion of VWA domain #N4 ( Fig. 7B, C ). For validation of COL6 as ADAMTS17 substrate, we used the cell culture setups shown in Fig. 6B . In the co- culture system of ADAMTS17-expressing HEKs with HDFs, we did not observe a different pattern of bands originating from endogenous COL6 in conditioned medium in the presence of active ADAMTS17 as detected with a COL6 antibody ( Fig. 7D ). However, we noticed the disappearance of a ∼125 kDa COL6-reactive band in the cell lysate/ECM fraction when proteolytically active ADAMTS17 was present. When visualizing COL6 ECM deposition in this co-culture system by immunostaining, we observed reduced COL6 staining in the presence of ADAMTS17 compared to ADAMTS17-EA ( Fig. 7E, F ). We observed a similar difference when fibroblasts were cultured in the presence of cell-free conditioned medium collected from ADAMTS17- or ADAMTS17-EA-expressing HEK293 cells. In the presence of ADAMTS17, COL6 in the ECM was lower than the amount of COL6 deposited in the presence of ADAMTS17-EA ( Fig. 7G, H ). These observations would be consistent with proteolytically active ADAMTS17 limiting the amount of COL6 deposited in the ECM, potentially via proteolysis, but could also be explained by inactive ADAMTS17-EA promoting COL6 deposition into the ECM. To determine, if COL6 is a direct ADAMTS17 substrate, we co-expressed FLAG-tagged recombinant (r)COL6A2 with ADAMTS17 or ADAMTS17-EA in HEK293 cells. Using western blot detection of the FLAG-tag, we did not observe rCOL6A2 fragmentation in the medium or cell lysate and only detected full length COL6A2 ( Fig. 7I ). However, we noticed an increase in the band intensity for COL6 in the ADAMTS17-EA lysate, which could be the result of decreased proteolysis or increased cellular retention or cell surface/ECM association. To determine, if ADAMTS17 co-localized with COL6, we cultured fibroblasts producing endogenous COL6, in the presence of the previously described purified recombinant catalytic ADAMTS17 domains (17-PCD) or its ancillary domains (17-AD) and co-immunostained for endogenous COL6 and recombinant ADAMTS17 (α-Myc) 22 . Endogenous COL6 in the ECM of fibroblasts costained with both ADAMTS17 protein constructs, suggesting the possibility of an interaction in the ECM that could be the basis for selective proteolysis of COL6A3 ( Fig. 7J ). Finally, we analyzed endogenous COL6 distribution and ECM deposition in dermal fibroblasts isolated from a patient with WMS due to a ADAMTS17 Thr343Ala mutation, where we previously showed intracellular retention and reduced ECM deposition of fibronectin, fibrillin-1, and COL1 10 . Compared to control adult human dermal fibroblasts, we observed a strong reduction of COL6 ECM deposition in WMS dermal fibroblasts and concurrent intracellular COL6 accumulation ( Fig. 7K, L ). This observation was confirmed by western blot analysis, where COL6 was decreased in the medium from WMS patient-derived dermal fibroblasts and increased in the cell lysate compared to adult human dermal fibroblasts ( Fig. 7M, N ). Collectively, our data suggest that fibronectin and COL6 are potential ADAMTS17 binding partners and/or substrates. Download figure Open in new tab Figure 7. Identification of collagen VI (COL6) as an ADAMTS17 interacting protein and potential substrate. A) Domain organization of COL6A2 (NP_0018403) showing the localization of the domains that interacted with 17-AD (grey box, bolded amino acid sequence. B) Domain organization of COL6A3 (NP_004360) showing the localization of the peptide identified by MS (red bar, red amino acid sequence). C) MS2 spectrum of the N-terminally labeled ADAMTS17-digested COL6A3 peptide (SDDEVDDPAVELkQFGVAPF) showing b- and y-ions. D) Western blot of endogenous (end.) COL6 (red) in conditioned medium (Med) and cell lysate (Lys) collected from co-cultures of 17 or 17-EA-expressing HEK293 cells with HDFs. E) Micrographs of endogenous COL6 deposition (red) in the ECM of HDFs co-cultured with 17- or 17-EA-expressing HEK293 cells. Nuclei were stained with DAPI (blue). F) Quantification of the mean fluorescence intensity of the COL6 signal (n=3 replicates). G) Micrographs of endogenous COL6 deposition (red) in the ECM of HDF after culture in the presence of conditioned medium from 17- or 17-EA-expressing HEK293 cells. Nuclei were stained with DAPI (blue). H) Quantification of the mean fluorescence intensity of the COL6 signal (n=3 replicates). I) Western blot of recombinant COL6 (rCOL6) in conditioned medium (Med) and cell lysate (Lys) collected after co-expression of 17 or 17-EA and rCOL6A2 in HEK293 cells using a monoclonal anti FLAG-tag antibody (green). J) Micrographs of HDFs cultured in the presence of 50 μg/ml of purified recombinant 17-PCD and 17-AD protein (see Fig. 5A for domain organization) co-stained for endogenous COL6 (red) and the Myc-tag of the recombinant ADAMTS17 protein fragments (green). Nuclei were stained with DAPI (blue). K) Micrographs of adult HDFs and WMS-patient-derived dermal fibroblasts (WMS-DF) for endogenous COL6 (red). Nuclei were counterstained with DAPI (blue). L) Quantification of the mean fluorescence intensity of the COL6 signal (n=3 replicates, 2-3 fields-of-view). M) Western blot of endogenous COL6 (red) and GAPDH (green) in conditioned medium (Med) and cell lysate (Lys) collected from HDF and WMS-DF cultures. N) Quantification of COL6 band mean fluorescence intensities normalized to GAPDH. In E, G, M, bars represent the mean value and whiskers the standard deviation. In K, the floating bars indicate the 25 th – 75 th percentile range, the lines the mean value and whiskers the standard deviation. Statistical differences in E, G, K, M were determined using a two-sided Student t-test. Discussion The fact that mutations in ADAMTS10 and ADAMTS17 can both cause short stature in WMS unequivocally implicates both genes in the regulation of bone growth via an impact on growth plate function. Since it is unclear if and how both genes interact or cooperate in this process, we analyzed the phenotypes of Adamts10 ; Adamts17 DKO mice. We showed that combined inactivation of ADAMTS10 and ADAMTS17 exacerbated bone shortening when compared to individual KOs with ADAMTS17 gene dosage having an apparently stronger effect and compromised postnatal survival. In addition, we showed that ADAMTS10 and ADAMTS17 are required for skin development, which may relate to stiff and thickened skin described in WMS patients 3 , 46 . Finally, we identified fibronectin and COL6 as potential ADAMTS17 substrates in high-throughput unbiased screens. This could point towards a potentially distinct function for ADAMTS17 as a regulator of ECM formation and homeostasis. Indeed, such a function is known for ADAMTS10, specifically in the enhancement of fibrillin-1 assembly, since endogenous ADAMTS10 is only slightly activated by furin 21 . Bone growth is largely driven by chondrocyte proliferation in the growth plate and their subsequent hypertrophic expansion. The disruption of either process can result in bone shortening and short stature 37 , 47 – 49 . The reduction in the width of the hypertrophic zone observed in the DKO growth plate could thus be attributed to decreased formation and/or accelerated turnover of hypertrophic chondrocytes, which depend on chondrocyte proliferation and matrix metalloprotease (MMP)- mediated ECM remodeling at the ossification front, respectively 50 – 52 . Since we did not observe changes in the dimensions of the proliferative zone, we suggest that hypertrophic Adamts10 ; Adamts17 DKO chondrocytes turnover faster. Hypertrophic chondrocyte turnover requires the transition of a cartilage ECM towards a bone ECM that is primed for mineralization. Key enzymes that regulate these processes include MMP13 and MMP9, which can cleave COL2 and COL10 or promote the vascularization of the growth plate 51 , 52 . When MMP13 or MMP9 were inactivated, the length of the hypertrophic zone in developing bones was increased by ∼70% and primary ossification was delayed, indicating a delay in chondrocyte turnover, in particular chondrocyte apoptosis 51 , 52 . Since we observed a shorter hypertrophic zone, we postulate that ADAMTS10 and ADAMTS17 may attenuate chondrocyte turnover potentially by regulating the activity of MMP13 or MMP9 53 . In this context, it is interesting to note that ADAMTS10 WMS knock-in and ADAMTS17 KO mice show opposite growth plate phenotypes, each resulting in reduced bone length. The knock-in of the ADAMTS10 Ser236X mutation (Arg237X in humans) resulted in an expansion of the hypertrophic zone, which correlated with a 6-10% reduction in long bone length 34 . In contrast, inactivation of Adamts17 resulted in a shorter hypertrophic zone, which also correlated with a 6-10% reduction long bone length 33 . In Adamts17 KO mice, however, chondrocyte proliferation or apoptosis was not changed 33 . These reports would suggest that ADAMTS10 and ADAMTS17 have distinct roles in regulating growth plate activity and chondrocyte hypertrophy, both resulting in shorter bones. This interpretation is further supported by our findings reported here, that maturation and mineral deposition in ADAMTS17-deficient primary chondrocytes was strongly reduced, but unaffected in ADAMTS10-deficient chondrocytes. The latter findings could also be explained by differential compensation, where ADAMTS17 can compensate for the lack of ADAMTS10 in primary chondrocytes, but not vice versa. As an alternative explanation, ADAMTS10 and ADAMTS17 could regulate signaling pathways that drive growth plate activity and bone growth. In this context, it was shown that reduced BMP signaling in Adamts17 KO mice delayed terminal differentiation of chondrocytes 33 . In the skin of Adamts10 WMS knock-in mice, BMP signaling but not TGFβ signaling was similarly reduced 34 . How ADAMTS10- or ADAMTS17-deficiency translates into reduced BMP signaling is not clear, but could be secondary to changes in fibronectin and fibrillin-1 deposition, which regulates extracellular BMP activity 54 , 55 . Taken together, our data and data from others are consistent with ADAMTS10 and ADAMTS17 regulating different aspects of growth plate activity with ADAMTS17 having a stronger effect. Skin thickening is a feature of WMS syndrome, and decreased skin elasticity in Adamts10 KO and Adamts17 KO mice as well as increased skin thickness in Adamts10 WMS knock-in mice were reported previously 31 , 33 , 34 . In contrast, we noticed fragile skin during routine handling of Adamts10 ; Adamts17 DKO mice and, accordingly, observed reduced skin thickness in Adamts10 ; Adamts17 DKO mice, which was predominantly driven by a reduction in the hypodermal layer. This suggested that ADAMTS10 and ADAMTS17 play a role in skin development or postnatal homeostasis, with likely partial compensation in the individual KOs given the much stronger phenotype observed for the Adamts10 ; Adamts17 DKO skin. One reason for these contrasting observations could be that skin thickening was previously observed in older mice, i.e. at 3 and 8 months of age in the Adamts17 KO and at 3 months of age in the Adamts10 WMS knock-in 33 , 34 . Therefore, it is possible that the skin thickness might increase in our individual KOs with age. We previously demonstrated that WMS patient-derived dermal fibroblasts harboring an ADAMTS17 mutation were deficient in secretion and deposition of ECM proteins, in particular fibronectin, fibrillin-1 and COL1 10 . When we examined ECM deposition in primary DKO mouse skin fibroblasts, we also observed compromised ECM deposition. In the absence of both ADAMTS10 and ADAMTS17, fibronectin deposition was almost completely abolished and fibrillin-1 appeared to be retained intracellularly. Individual KOs displayed intermediate phenotypes with a more profound phenotype in Adamts17 KO fibroblasts and a somewhat milder phenotype in Adamts10 KO fibroblasts. In Adamts17 KO fibroblasts, fibronectin was deposited as globular speckles, but did not form elongated fiber-like structures or ECM networks. In Adamts10 KO fibroblasts, fibronectin did form fibers and ECM networks, but they were less dense compared to WT fibroblasts and showed regions void of fibronectin networks. Interestingly, in these regions intracellular fibrillin-1 immuno-reactivity was more apparent. Collectively, these data support a model where ADAMTS17 regulates the secretion and/or assembly of fibronectin by remodeling fibronectin fibrils, which are required for the formation of stable fibrillin-1 networks, whereas ADAMTS10 plays a more “downstream” role where it could enhance fibrillin-1 assembly as suggested previously 21 , 41 , 56 . Since ADAMTS10 and ADAMTS17 are members of the ADAMTS protease family they are presumed to fulfil their biological function through their respective protease activities. However, ADAMTS10 is the only ADAMTS protease with a degenerated furin cleavage site (GLKR↓, lacking a canonical Arg residue at the P4 position) and is incompletely activated by furin-mediated removal of the inhibitory prodomain 21 . It was shown that ADAMTS10 could cleave fibrillin-1 and fibrillin-2 efficiently after restoring a canonical furin recognition site (RRKR↓) by mutagenesis, suggesting that ADAMTS10 has intrinsic protease activity when activated 21 , 31 . In Adamts10 KO and Adamts10 WMS knock-in mice, fibrillin-2 accumulation in the ciliary zonule of the eye was observed and could be explained by the absence of “fibrillin-ase” activity in ADAMTS10-deficient tissue. Indeed, furin activated ADAMTS10 cleaves fibrillin-2 31 . However, fibrillin-2 accumulation could also be explained by ADAMTS10 promoting the assembly of fibrillin-1, which would be reduced in the Adamts10 KO and result in increased fibrillin-2 exposure to antibodies in the fibrillin microfibril bundles of the ciliary zonule 21 , 31 , 34 . Alternatively, ADAMTS10 could be involved in promoting the switch from developmental fibrillin-2-rich microfibrils to postnatal fibrillin-1-rich microfibrils, which similarly would be lacking or be reduced in ADAMTS10-deficient tissues. Therefore, it is plausible that ADAMTS10 may primarily regulate ECM formation both via protease-dependent and protease-independent activities on fibrillin-2 and fibrilin-1 respectively, modulating the formation and isoform composition of fibrillin microfibrils. For ADAMTS17, we showed that it cleaves itself at multiple sites, including in the active site, and is thus proteolytically active 22 . Therefore, we used a complementary MS and yeast-2-hybrid approach in the quest to uncover ADAMTS17 substrates. In both screens, fibronectin and COL6 were identified as potential substrates and binding partners for ADAMTS17. In summary, this study provides evidence that ADAMTS10 and ADAMTS17 may operate in distinct ways in the pathways that regulate bone growth and skin development through regulation of ECM deposition and turnover and provide additional understanding of the mechanisms underlying WMS. Materials and Methods Mouse models Adamts10 KO mice generated by Deltagen Inc. and their genotyping were described previously 31 . In brief, 41 bp of Adamts10 exon 5 were replaced with an IRES-lacZ-neo cassette resulting in a frameshift and a premature stop codon triggering nonsense-mediated mRNA decay of the Adamts10 mRNA. The mice were licensed from Deltagen Inc. (agreement #AGR-17486) and maintained in the C57BL/6 strain. Adamts17 KO mice were generated in C57BL/6 ES cells by CRISPR/Cas9-induced non-homologous end-joining mutagenesis by contract to Applied StemCell, Inc. A guide RNA targeting Adamts17 exon 3 (5’-GTCCCTCCACCTCCGTAGCA-3’) was co-injected with Cas9 protein into blastocysts to generate F0 mice, which were screened for frameshift-causing indels. F0 mice harboring an AT insertion in Adamts17 exon 3 were identified by Sanger sequencing and used to generate F1 mice with the germline-transmitted Adamts17 AT insertion. F1 mice harboring the AT insertion were identified by Sanger sequencing of a PCR product generated with primers adjacent to exon 3 (F primer: 5’-GAGAGCATCTGATCAGACGCAAATGG-3’, R primer: 5’-CATGTGACCCACAGAGTGTCAGC-3’). Adamts17 KO mice were rederived at the Icahn School of Medicine. Adamts17 Het and KO mice were genotyped using DNA from clipped toes as template for a PCR reaction with the following primers: TS17-F 5’-CAGCAGACAGAAGCACAAGCC-3’ and TS17-R 5’-TAGAATCATGGCCCTGACACC-3’. The resulting PCR product was isolated and submitted for Sanger sequencing using the TS17-F primer (Psomagen). The Adamts17 mutant was maintained in the C57BL/6 strain. Mice heterozygous for the Adamts10 KO and Adamts17 KO alleles were crossbred to generate Adamts10 Het; Adamts17 Het mice, which were then crossbred to generate Adamts10 ; Adamts17 DKO mice. Adamts10 Het or Adamts17 Het mice were crossbred to generate age- and sex-matched WT, Adamts10 KO, or Adamts17 KO mice. Mice were used between 4-8 weeks of age and data from both sexes were combined. All mouse experiments were approved by the Institutional Animal Care and Use Committee (IACUC) of the Icahn School of Medicine at Mount Sinai (protocol numbers IACUC-2018-0009, PROTO202000259, and TR202300000105). X-ray imaging After sacrifice, intact mouse limbs were imaged using a high-resolution radiographic system (UltraFocus digital X-ray cabinet, Faxitron Bioptics). A 10 mm metal rod was used as a scale to enable quantification of bone length. Histology Limbs from 4-week-old mice were disarticulated and the tibia and femur cut mid-shaft to dissect the knee joint. Muscle and other soft tissue were removed and the knee was fixed in Z-fix (Electron Microscopy Science) for 48 h. After decalcification in 14% EDTA solution, the knee joints were dehydrated, embedded in paraffin and sectioned through the middle of the knee. Sections were then stained with hematoxylin & eosin (H&E) for histomorphometry. Proximal tibial growth plates were imaged and the dimensions of the growth plate regions measured at multiple points across the growth plate using ImageJ Fiji (NIH) 57 , 58 . Dorsal skin was first shaved to remove hair, and a full thickness rectangular strip was dissected and flattened on filter paper. The filter paper with the skin was then immersed in Z-Fix Zinc Formalin Fixative for 24h, processed and paraffin embedded. Cross sections were stained with Masson’s trichrome stain and imaged. Skin thickness and the thickness of individual skin layers was measured from micrographs using ImageJ Fiji. Cell culture assays Human embryonic kidney (HEK) 293 cells (CRL-1573) and adult human dermal fibroblasts (HDF, PCS- 201-012) were purchased from ATCC. Isolation and characterization of the WMS patient-derived dermal fibroblasts (WMS-DF) harboring the ADAMTS17 c.1027A>G (p.Thr343Ala) mutation was described previously 10 . Cells were cultured in Dulbecco’s Modified Eagle Medium (DMEM) supplemented with 10% fetal bovine serum (FBS), 1% L-glutamine, 100 units/ml penicillin and 100 mg/ml streptomycin (PSG) (complete DMEM) in a 5% CO 2 atmosphere in a humidified incubator at 37°C. Upon reaching confluence, cells were split in a 1:10 (HEK293) or 1:3 (fibroblasts) ratio. Primary fibroblasts were used up to passage 5-7. HEK293 cells stably expressing ADAMTS17 or ADAMTS17-EA were described previously and maintained in complete DMEM supplemented with geneticin (G418) 22 . For co-culture assays, HDFs and 2 × 10 6 ADAMTS17 or ADAMTS17-EA (4 × 10 6 cells total) were combined and seeded on a 10 cm cell culture dish. After reaching confluency the cell layer was rinsed with PBS and cultured in serum free DMEM. After 48 h, conditioned medium was collected and proteins were precipitated with 10% trichloroacetic acid. The cell layer was lysed in RIPA buffer (0.1% NP40, 0.05% DOC, 0.01% SDS in PBS). Equal volumes were mixed with 5x reducing SDS loading buffer, boiled at 100 °C and separated via SDS-PAGE for western blotting. For direct ADAMTS17 proteolysis assays, HEK293 cells were co-transfected with ADAMTS17 or ADAMTS17-EA and plasmids encoding FN1 or COL6A2 using Lipofectamine 3000. The plasmid encoding V5-tagged fibronectin was described recently and kindly provided by Dr. Dieter Reinhardt (McGill University, Montreal, Canada) 59 . The COL6A2-encoding plasmid was purchased from Genscript (OHu18654D). After 24 h, cell layers were rinsed with PBS and cultured in serum-free DMEM for an additional 48 h. Conditioned medium was collected and cleared from cell debris by centrifugation. The cell layer was lysed in RIPA buffer, transferred into an Eppendorf tube and cleared of debris by centrifugation. Equal volumes were combined with 5x reducing SDS loading buffer, boiled at 100 °C and separated via SDS-PAGE for western blotting. Western blotting The proteins in equal volumes of media or cell lysates were separated on polyacrylamide gels using SDS-PAGE and blotted onto polyvinylidene difluoride (PVDF) membranes (Immobilon-FL, Merck Millipore Ltd.) using the semi-dry Bio-Rad trans-Blot® Turbo transfer system for 33 min at 25 V (Bio- Rad) or a wet transfer system for 1.5 h at 70 V at 4 °C both operated with 25 mM Tris, 192 mM glycine, 20% methanol as transfer buffer. Membranes were blocked with 5% (w/v) milk in TBS (10 mM Tris-HCl, pH 7.2, 150mM NaCl) for 1 h at RT and incubated with the following primary antibodies diluted in 5% (w/v) milk in TBS-T (TBS + 0.1% Tween 20) at 4 °C overnight: polyclonal antibodies against fibronectin (F3648, 1:1,000, Sigma; ab2033, 1:200, Millipore; 15613-1-AP, 1:1000, ProteinTech; ab61214, 1:200, Abcam), monoclonal antibody against fibronectin (cl 15, 1:400, Sigma), polyclonal antibody against COL6 (ab6588, 1:500, Abcam), monoclonal antibodies against the V5-tag (mAB, 1:500, Invitrogen) and the FLAG-Tag (M2, 1:500, Sigma), or a monoclonal antibody against GAPDH (1:1,000, Millipore). After incubation with the primary antibody, membranes were rinsed with TBS-T 3 × 5 min at RT and incubated with IRDye-goat-anti-mouse or goat-anti-rabbit secondary antibodies (1:10,000 in 5% (w/v, Jackson ImmunoResearch Laboratories) in TBS-T for 2 h at RT. Membranes were then rinsed 3 × 5 min with TBS-T, once in TBS and imaged using an Azure c600 Western blot imaging system (Azure Biosystems). Band intensities were quantified using the AzureSpot analysis software and normalized to GAPDH. Immunostaining of tissue sections and cell layers Growth plate and skin sections were de-paraffinized and rehydrated followed by protease-mediated antigen retrieval using HistoZyme (Diagnostic BioSystems). After blocking with 5% BSA in PBS, sections were incubated with a mouse monoclonal antibody against ADAMTS17, which was raised against human ADAMTS17 (3B7, 1:500, Novus Biologicals) in blocking buffer in a humidified chamber overnight at 4 °C. Sections were rinsed in PBS 3 × 10 min at RT and incubated in AlexaFluor 488-labeled secondary goat-anti-mouse antibody in blocking buffer at for 1 h at RT. Slides were cover-slipped with ProLong Antifade Gold mounting medium and imaged using a Zeiss Axio Observer Z1 Fluorescence Motorized Microscope w/Definite Focus. HEK293 cells and HDFs or co-cultures thereof, or primary chondrocytes were seeded in 8-well chamber slides (50,000 cells/well) (Celltreat Scientific Products) and cultured in complete DMEM for 3-4 days. For the analysis of COL6, complete DMEM was supplemented with 100 μM ascorbic acid (ThermoFisher). After medium removal, cell layers were rinsed with PBS and fixed for 5 min with 150 μl ice-cold 70% methanol/30% acetone (Thermo-Fisher), which permeabilized the cells, or with 4% PFA for 20 min to only stain cell surface and ECM proteins, followed by permeabilization with 0.1% Triton X- 100 prior to incubation with the secondary antibody. After fixation, cells were rinsed with PBS and blocked with 10% normal goat serum (Jackson ImmunoResearch Laboratories) in PBS for 1 h at RT. For Okadaic acid (Sigma Aldrich #459620) chondrocytes were treated with 50nM Okadaic acid and the controls were treated with DMSO for 24 hours and allowed to differentiate. For immunostaining cells were fixed with 4% PFA without permeabilization. Cells were incubated with primary antibodies against fibrillin-1, fibronectin, COL6, anti-Myc-tag, or ADAMTS17 diluted in blocking buffer overnight at 4 °C. Cells were rinsed 3 × 5 min with PBS and incubated with AlexaFluor labeled secondary goat-anti- mouse or goat-anti-rabbit antibodies (1:350 in blocking buffer) (Jackson ImmunoResearch Laboratories) for 2 h at RT followed by 3 × 5 min rinses with PBS and mounting with ProLong Gold Antifade Reagent with DAPI (Thermo-Fisher). Slides were imaged using a Zeiss Axio Observer Z1 Fluorescence Motorized Microscope w/Definite Focus and Zeiss Zen Microscope Software and ImageJ Fiji were used to quantify fluorescence pixel intensities. 10T1/2 pellet culture for chondrocyte-like differentiation C3H/10T1/2 cells (CCL-226) were purchased from ATCC and cultured according to ATCC’s instructions in Eagle’s Basal medium 60 , 61 . For maintenance, cells were sub-cultured prior to confluence. For pellet cultures, C3H/10T1/2 cells were detached with trypsin-EDTA and 5 × 10 5 cells/ml were centrifuged at 500 g for 5 min in 15-ml tubes and cultured in serum-free DMEM supplemented with 0.1 μM dexamethasone, 0.2 mM L-ascorbic acid-2 phosphate, insulin-transferrin-selenium supplement and 10 ng/ml TGF-β1. The medium was changed every 48 h. mRNA isolation and quantitative real-time PCR Chondrocyte pellets were removed at the experimental time points, immersed in TRIzol reagent and RNA was extracted according to the manufacturer’s instructions. Pellets were lysed by pipetting the TRIzol up and own several times to ensure complete homogenization. The lysate was collected into sterile tubes and incubated at room temperature for 5 min. Following lysis, 0.2 ml chloroform was added per 1 ml of TRIzol, the Eppendorf tubes were inverted several times and incubated at RT for 2-3 minutes. To separate the organic and inorganic phase, samples were centrifuged at 12,000 g for 15 min at 4 °C. The aqueous phase containing RNA was carefully transferred to a new Eppendorf tube and the RNA was precipitated by adding 0.5 ml of isopropanol per 1 ml of TRIzol reagent and samples were incubated for 10 min at RT. The RNA was pelleted by centrifugation at 12,000 g for 10 min at 4 °C. The supernatant was discarded and the RNA pellet was washed with 1 ml 75% ethanol per 1 ml of TRIzol, followed by centrifugation at 7,500 g for 5 min at 4 °C. The RNA pellet was air-dried for 5-10 min at RT and dissolved in 30 – 50 μl of RNase-free water depending on pellet size. RNA concentration and purity were measured using a Nanodrop OneC spectrophotometer (ThermoFisher). RNA preparations were further purified using DNAse I to remove traces of DNA. Reverse transcriptase was used to convert 1 μg of RNA into cDNA. Quantitative real-time (qRT)-PCR was performed in triplicates in a 384-well plate format. For each reaction, 2 μl cDNA, 0.5 μl of forward and reverse primers (100 μM stock) and SYBR Green PCR Master Mix (Applied Biosystems) were combined in a total reaction volume of 10 μl. The amplification and detection were performed on an ABI PRISM 7900HT Sequence Detection System (Applied Biosystems). All reactions were run under standard cycling conditions. The following PCR primer pairs were used: Gapdh (F: 5’-AGGTCGGTGTGAACGGATTTG-3’, R: 5’GGGGTCGTTGATGGCAACA-3’ or F: 5’- CTTTGTCAAGCTCATTTCCTGG-3’, R: 5’-TCTTGCTCAGTGTCCTTGC-3’), Adamts10 (F: 5’-CCCGCCTATTCTACAAGGTGG-3’, R: 5’- GCCTTCCCGTGTCCAGTATTC-3’ or F: 5’-GTAGTGGAGTGCCGAAATCAG-3’, R: 5’-CAGCGTGACCAGTTTCCTAC-3’), Adamts17 (F: 5’-CTGCTGTATTTGTGACCAGGAC-3’, R: 5’- AGCACACATTTCCTCTTAGCAC-3’ or F: 5’-CCTTTACCATCGCACATGAAC-3’, R: 5’-ATTCCGTCCTTTTACCCACTC-3’), Fbn1 (F: 5’-GGACAGCTCAGCGGGATTG-3’, R: 5’- AGGACACATCTCACAGGGGT-3’), Fn1 (F: 5’- GCTCAGCAAATCGTGCAGC -3’, R: 5’- CTAGGTAGGTCCGTTCCCACT -3’), Col2a1 (F: 5’- GGGAATGTCCTCTGCGATGAC-3’, R: 5’-GAAGGGGATCTCGGGGTT-3’), Col10a1 (F: 5’- TTCTGCTGCTAATGTTCTTGACC-3’, R: 5’- GGGATGAAGTATTGTGTCTTGGG-3’). Isolation and differentiation of primary chondrocytes Primary costal chondrocytes were isolated as described previously 62 . The ribcage of P5 mouse pups was dissected, flattened and soft tissue was removed. The cartilaginous portions of the ribs were then transferred into PBS containing 10× penicillin and streptomycin (Gibco). The ribs were digested with 15 ml pronase (2mg/ml, Sigma-Aldrich) in a 50 ml Falcon tube for 1 h at 37 °C in a tissue culture incubator under a 5% CO 2 atmosphere. The pronase solution was replaced with 15 ml of collagenase D (3 mg/ml, Sigma-Aldrich) followed by incubation for 1 h under the same conditions with gentle agitation every 10 min. After 45 min of incubation, the collagenase D solution was vigorously pipetted up and down over the thoracic cages. The solution was finally transferred into a 50 ml falcon tube. The soft tissue debris was removed as it sediments slower and the process was repeated with sterile PBS buffer. The cleaned cartilage was then digested again with 15 ml collagenase D solution for 4-5 hours at 37 °C in the cell culture incubator. Finally, primary chondrocytes were released by pipetting the solution containing the cartilage fragments up and down ∼10 times. This digest was passed through 40 μm cell strainer and pelleted by centrifugation at 300 g for 5 min. The chondrocyte pellet was rinsed twice with PBS and chondrocytes were resuspended in 10 ml complete DMEM medium, plated at a density of 10 5 /cm 2 on 6-well plates and cultured in complete DMEM. Upon reaching confluency, the medium was replaced with chondrocyte maturation medium (complete DMEM supplemented with 50 μg/ml ascorbic acid and 10 mM β-glycerophosphate). The medium was changed every 2 days and mineral deposition was visualized after 21 d by alizarin red staining. For this, the differentiated and matured chondrocytes were rinsed with PBS and fixed with 10% formaldehyde (MP Biomedicals) for 15 minutes at RT. The cell layer was rinsed twice with distilled water and incubated in 1 mL of 40 mM alizarin red staining solution (pH 4.1) per well for 20 min at RT under gentle agitation on a shaker. Plates were incubated at room temperature for 20 minutes with gentle shaking. The alizarin red staining solution was removed and the wells were rinsed several times with 5 mL of distilled water. Prior to brightfield imaging, excess water was removed and the cell layer was dried at RT. The cell layers were imaged and the stained mineral deposition was quantified using the ImageJ Fiji. ATAC-seq and transcriptomics analyses of ADAMTS10 and ADAMTS17 expression Sample generation, ATAC-seq, RNA sequencing and data analyses for the data set have been described recently 42 . No additional human products of conceptions that were not previously described, were used for this manuscript. In brief, epiphyses (long bones) or whole elements (pooled phalanges or metapodials) were micro-dissected and RNA was extracted after tissue homogenization followed by RNA sequencing. For ATAC-seq, tissues were digested with collagenase to generate single cell suspensions, which were then subjected to the ATAC-seq protocol. The data sets for the stylopodial and zeugopodial elements are deposited in the Gene Expression Omnibus repository under the accession numbers GSE252289 (human long bone skeleton ATAC-seq) and GSE252288 (human long bone skeleton RNA-seq). The data sets for the autopodial elements will be published elsewhere (Okamoto & Capellini, manuscript in preparation). RNAScope in-situ hybridization WT mouse embryos at E13.5, E16.5, and P0 were fixed in 4% paraformaldehyde in PBS overnight at 4°C, dehydrated, and embedded in paraffin. Fresh 6 μm sections were used for in-situ hybridization using RNAscope (Advanced Cell Diagnostics) following the manufacturer’s protocol. Tissue localization of Adamts17 mRNA was achieved with a probe recognizing the mRNA from mouse Adamts17 (#316441) in combination with the RNAscope 2.0 HD detection kit “RED”. Tissue sections were counterstained with hematoxylin and cover-slipped with Cytoseal 60 (Electron Microscopy Science). After curation of the mounting medium, sections were observed using an Olympus BX51 upright microscope equipped with a CCD camera (Leica Microsystems) for imaging. Isolation of primary mouse skin fibroblasts Mice were euthanized using CO2 inhalation followed by cervical dislocation and skin fibroblasts were isolated using enzymatic digestion. Following euthanasia, the dorsal skin was shaved and 1-2cm2 section was excised using sterile scissors and forceps. The excised skin was washed in phosphate- buffered saline (PBS). The skin was minced into 1-2mm2 fragments and transferred into a 50ml conical tube. The tissue was digested using 2mg/ml Collagenase type II (Worthington, LS004202) in DMEM. The tube was incubated at 37oC for 1 hour with gentle agitation to release the fibroblasts. After digestion, the cell suspension was triturated with a 10ml pipette to further dissociate the tissue. The suspension was then filtered through a 70um cell strainer to remove undigested fragments. The filtered cell suspension was centrifuged at 300g for 5 minutes at room temperature. The resulting cell pellet was resuspended in DMEM. The cell suspension was plated onto sterile 10cm culture dishes and incubated at 37oC, 5% CO2. 24h post-plating, the medium was replaced to remove non-adherent cells. N-terminomics via TAILS For iTRAQ labelling, we co-cultured HDFs with HEK293 cells expressing ADAMTS17 or ADAMTS17-EA (1 × 10 6 cells each, two 10 cm dishes). After reaching confluence, the cell layer was rinsed with PBS and phenol-free serum-free DMEM was added. After 2 d and 4 d, the medium was collected and EDTA (10 mM final concentration) and one tablet of Complete EDTA-free protease inhibitors (Roche), dissolved in 250 μl water, were added. The medium was cleared of cellular debris by centrifugation at 500 rpm for 5 min, sterile filtrated through a 0.22 μm filter, and stored at -80 °C. Media (∼40 ml) were thawed and concentrated to 2.5 mL with centrifugal filter devices (Amicon Ultra-4, 3kDa cut-off; #UFC800324). Proteins were then precipitated by adding 20 mL of ice-cold acetone and 2.5 mL ice cold methanol followed by vortexing and incubation at -80 °C for 3 h. Protein precipitates were collected by centrifugation at 14,200 g in a Beckman JS-13.1 outswing rotor at 4 °C for 20 min. The supernatant was decanted and the protein pellets were air dried. Air-dried pellets were dissolved in 360 μL 8 M guanidine-HCl, 504 μL double-distilled water, and 288 μL 1M HEPES buffer resulting in terminal amine isotopic labeling of substrates (TAILS) buffer (final concentration: 2.5 M GuHCl, 250 mM HEPES, pH7.8). 300 µg of protein from the TS17X1-WT and TS17X1-mut samples were prepared for iTRAQ-TAILS as previously described 63 . In brief, protein was reduced and alkylated with DTT and IAA respectively. Proteins samples were mixed at a 1:1 ratio with iTRAQ labels 113 (TS17X1-WT) or 115 (TS17X1-mut) in DMSO at 37° C overnight and quenched with 1 M tris pH 8. Samples were then combined prior to overnight digestion with trypsin. N-terminally labeled peptides were enriched as previously described using the hyper-dendritic polyglycerol aldehyde 64 . Prior to MS analysis, the samples were desalted using a C18 Ziptip and reconstituted in 50 μL 1% acetic acid. Peptides were identified with a Dionex Ultimate 3000 UHPLC interfaced with a Thermo LTQObitrap Elite hybrid mass spectrometer system. The HPLC column was a Dionex 15 cm x 75 μm id Acclaim Pepmap C18, 2μm, 100 Å reversed- phase capillary chromatography column. 5 μL volumes of the extract were injected and the peptides eluted from the column by an acetonitrile/0.1% formic acid gradient at a flow rate of 0.3 μL/min were introduced into the source of the mass spectrometer on-line operated at 1.9 kV. The digest was analyzed using a data dependent MS method acquiring full scan mass spectra to determine peptide molecular weights and product ion spectra to determine amino acid sequence in successive instrument scans. Both collision-induced dissociation (CID) and higher-energy collisional dissociation (HCD) fragmentation methods were performed on the top-8 most abundant precursor ions in each scan cycle. HCD fragmentation is required to quantify the reporter ions of the iTRAQ labels on peptides. The samples were analyzed using a data dependent acquisition (DDA) using both HCD and CID fragmentations. The resulting data were searched using Sequest program which integrated in Proteomics Discoverer 2.5 software package against Uni-prot human protein sequence database (March, 2024). Trypsin (semi) was set as protease, carbomidomethylation of Cys was set as a static modification and iTRAQ 8-plex of peptide N-terminal, Lys and Tyr, oxidation of Met, and N-term Gln cyclization were set as dynamic modifications. Yeast-2-hybrid screening An ULTImate Y2H SCREEN was performed by Hybrigenics Services using ADAMTS17-AD (aa 546-1122) as bait together with the Prey Library Human Placenta_RP6. The bait was cloned into the pB27 (N- LexA-bait-C fusion) vector. 60 clones were processed and 170 million interactions analyzed. Statistical analyses Statistical analyses were performed using the OriginPro 2018 software. N=2 samples were compared with a two-sided Student’s t-test and n≥3 samples with a one-sided ANOVA. P-values <0.05 were considered statistically different. Author contributions NT, SZK, ZB, LWW, BBW and DRM, performed experiments and analyzed data. SSA and DH analyzed data and contributed to study design. DR, ASO, TDC analyzed ATAC-seq and transcriptomics data. DH conceptualized the study, performed experiments, analyzed data, drafted the manuscript and prepared the figures. All authors edited the manuscript and figures and approved the final version. Data Availability The data sets for the stylopodial and zeugopodial elements are deposited in the Gene Expression Omnibus repository under the accession numbers GSE252289 (human long bone skeleton ATAC-seq) and GSE252288 (human long bone skeleton RNA-seq). The data sets for the autopodial elements will be published elsewhere (Okamoto & Capellini, manuscript in preparation) and made publicly available. Conflict of Interest The authors declare no financial or non-financial conflict of interest. Acknowledgements This research was in part supported by a grant from the National Institutes of Health (R01AR070748 to D.H.). We thank Dr. Dieter Reinhardt (McGill University, Montreal, Canada) for kindly providing the fibrillin-1 antibody and the V5-tagged rFN1 expression plasmid. We thank Damien Laudier (Orthopedic Research Laboratories, Icahn School of Medicine at Mount Sinai, New York, NY) for growth plate and skin histology. References 1. ↵ Stanley , S. , Balic , Z. & Hubmacher , D . Acromelic dysplasias: how rare musculoskeletal disorders reveal biological functions of extracellular matrix proteins . Ann. N. Y. Acad. Sci . , doi: 10.1111/nyas.14465 ( 2020 ). OpenUrl CrossRef PubMed 2. ↵ Le Goff , C. & Cormier-Daire , V . Genetic and molecular aspects of acromelic dysplasia . Pediatr Endocrinol Rev 6 , 418 – 423 ( 2009 ). OpenUrl PubMed 3. ↵ Marzin , P. et al. Weill-Marchesani syndrome: natural history and genotype-phenotype correlations from 18 news cases and review of literature . J. Med. Genet . , doi: 10.1136/jmg-2023-109288 ( 2023 ). OpenUrl Abstract / FREE Full Text 4. ↵ Morales , J. et al. Homozygous mutations in ADAMTS10 and ADAMTS17 cause lenticular myopia, ectopia lentis, glaucoma, spherophakia, and short stature . Am. J. Hum. Genet . 85 , 558 – 568 , doi: 10.1016/j.ajhg.2009.09.011 ( 2009 ). OpenUrl CrossRef PubMed Web of Science 5. Kutz , W. E. et al. Functional analysis of an ADAMTS10 signal peptide mutation in Weill-Marchesani syndrome demonstrates a long-range effect on secretion of the full-length enzyme . Hum. Mutat . 29 , 1425 – 1434 , doi: 10.1002/humu.20797 ( 2008 ). OpenUrl CrossRef PubMed Web of Science 6. ↵ Dagoneau , N. et al. ADAMTS10 mutations in autosomal recessive Weill-Marchesani syndrome . Am. J. Hum. Genet . 75 , 801 – 806 , doi: 10.1086/425231 ( 2004 ). OpenUrl CrossRef PubMed Web of Science 7. ↵ Haji-Seyed-Javadi , R. et al. LTBP2 mutations cause Weill-Marchesani and Weill-Marchesani-like syndrome and affect disruptions in the extracellular matrix . Hum. Mutat . 33 , 1182 – 1187 , doi: 10.1002/humu.22105 ( 2012 ). OpenUrl CrossRef PubMed 8. ↵ Cecchi , A. et al. Missense mutations in FBN1 exons 41 and 42 cause Weill-Marchesani syndrome with thoracic aortic disease and Marfan syndrome . American journal of medical genetics. Part A 161A , 2305 – 2310 , doi: 10.1002/ajmg.a.36044 ( 2013 ). OpenUrl CrossRef 9. ↵ Faivre , L. et al. In frame fibrillin-1 gene deletion in autosomal dominant Weill-Marchesani syndrome . J. Med. Genet . 40 , 34 – 36 , doi: 10.1136/jmg.40.1.34 ( 2003 ). OpenUrl Abstract / FREE Full Text 10. ↵ Karoulias , S. Z. et al. A novel ADAMTS17 variant that causes Weill-Marchesani syndrome 4 alters fibrillin-1 and collagen type I deposition in the extracellular matrix . Matrix Biol . 88 , 1 – 18 , doi: 10.1016/j.matbio.2019.11.001 ( 2020 ). OpenUrl CrossRef PubMed 11. ↵ Evans , D. R. et al. A novel pathogenic missense ADAMTS17 variant that impairs secretion causes Weill- Marchesani Syndrome with variably dysmorphic hand features . Sci Rep 10 , 10827 , doi: 10.1038/s41598-020-66978-8 ( 2020 ). OpenUrl CrossRef 12. ↵ Faivre , L. et al. Clinical homogeneity and genetic heterogeneity in Weill-Marchesani syndrome . American journal of medical genetics. Part A 123A , 204 – 207 , doi: 10.1002/ajmg.a.20289 ( 2003 ). OpenUrl CrossRef PubMed 13. ↵ Kuchtey , J. et al. Screening ADAMTS10 in Dog Populations Supports Gly661Arg as the Glaucoma-Causing Variant in Beagles . Invest Ophth Vis Sci 54 , 1881 – 1886 , doi: 10.1167/iovs.12-10796 ( 2013 ). OpenUrl Abstract / FREE Full Text 14. Kuchtey , J. et al. Mapping of the disease locus and identification of ADAMTS10 as a candidate gene in a canine model of primary open angle glaucoma . PLoS genetics 7 , e1001306 , doi: 10.1371/journal.pgen.1001306 ( 2011 ). OpenUrl CrossRef PubMed 15. Oliver , J. A. , Forman , O. P. , Pettitt , L. & Mellersh , C. S . Two Independent Mutations in ADAMTS17 Are Associated with Primary Open Angle Glaucoma in the Basset Hound and Basset Fauve de Bretagne Breeds of Dog . PloS one 10 , e0140436 , doi: 10.1371/journal.pone.0140436 ( 2015 ). OpenUrl CrossRef PubMed 16. Gould , D. et al. ADAMTS17 mutation associated with primary lens luxation is widespread among breeds . Vet Ophthalmol 14 , 378 – 384 , doi: 10.1111/j.1463-5224.2011.00892.x ( 2011 ). OpenUrl CrossRef PubMed 17. ↵ Farias , F. H. et al. An ADAMTS17 splice donor site mutation in dogs with primary lens luxation . Invest. Ophthalmol. Vis. Sci . 51 , 4716 – 4721 , doi: 10.1167/iovs.09-5142 ( 2010 ). OpenUrl Abstract / FREE Full Text 18. ↵ Jeanes , E. C. , Oliver , J. A. C. , Ricketts , S. L. , Gould , D. J. & Mellersh , C. S . Glaucoma-causing ADAMTS17 mutations are also reproducibly associated with height in two domestic dog breeds: selection for short stature may have contributed to increased prevalence of glaucoma . Canine Genet Epidemiol 6 , 5 , doi: 10.1186/s40575-019-0071-6 ( 2019 ). OpenUrl CrossRef 19. ↵ Karoulias , S. Z. , Taye , N. , Stanley , S. & Hubmacher , D . The ADAMTS/Fibrillin Connection: Insights into the Biological Functions of ADAMTS10 and ADAMTS17 and Their Respective Sister Proteases . Biomolecules 10 , doi: 10.3390/biom10040596 ( 2020 ). OpenUrl CrossRef PubMed 20. ↵ Hubmacher , D. & Apte , S. S . Genetic and functional linkage between ADAMTS superfamily proteins and fibrillin-1: a novel mechanism influencing microfibril assembly and function . Cell. Mol. Life Sci . 68 , 3137 – 3148 , doi: 10.1007/s00018-011-0780-9 ( 2011 ). OpenUrl CrossRef PubMed 21. ↵ Kutz , W. E. et al. ADAMTS10 protein interacts with fibrillin-1 and promotes its deposition in extracellular matrix of cultured fibroblasts . J. Biol. Chem . 286 , 17156 – 17167 , doi: 10.1074/jbc.M111.231571 ( 2011 ). OpenUrl Abstract / FREE Full Text 22. ↵ Hubmacher , D. et al. Unusual life cycle and impact on microfibril assembly of ADAMTS17, a secreted metalloprotease mutated in genetic eye disease . Sci. Rep . 7 , 41871 , doi: 10.1038/srep41871 ( 2017 ). OpenUrl CrossRef PubMed 23. ↵ Satz-Jacobowitz , B. & Hubmacher , D . The quest for substrates and binding partners: A critical barrier for understanding the role of ADAMTS proteases in musculoskeletal development and disease . Dev. Dyn . 250 , 8 – 26 , doi: 10.1002/dvdy.248 ( 2021 ). OpenUrl CrossRef PubMed 24. ↵ Apte , S. S. ADAMTS Proteins: Concepts, Challenges, and Prospects . Methods Mol. Biol . 2043, 1-12 , doi: 10.1007/978-1-4939-9698-8_1 ( 2020 ). OpenUrl CrossRef PubMed 25. ↵ Mead , T. J. & Apte , S. S . ADAMTS proteins in human disorders . Matrix Biol . 71 - 72 , 225-239 , doi: 10.1016/j.matbio.2018.06.002 ( 2018 ). OpenUrl CrossRef PubMed 26. ↵ Santamaria , S . ADAMTS-5: A difficult teenager turning 20 . Int. J. Exp. Pathol . 101 , 4 – 20 , doi: 10.1111/iep.12344 ( 2020 ). OpenUrl CrossRef 27. ↵ Longpre , J. M. et al. Characterization of proADAMTS5 processing by proprotein convertases . Int. J. Biochem. Cell Biol . 41 , 1116 – 1126 , doi: 10.1016/j.biocel.2008.10.008 ( 2009 ). OpenUrl CrossRef PubMed Web of Science 28. Koo , B. H. et al. Regulation of ADAMTS9 secretion and enzymatic activity by its propeptide . J. Biol. Chem . 282 , 16146 – 16154 , doi: 10.1074/jbc.M610161200 ( 2007 ). OpenUrl Abstract / FREE Full Text 29. Koo , B. H. et al. Cell-surface processing of pro-ADAMTS9 by furin . J. Biol. Chem . 281 , 12485 – 12494 , doi: 10.1074/jbc.M511083200 ( 2006 ). OpenUrl Abstract / FREE Full Text 30. ↵ Wang , P. et al. Proprotein convertase furin interacts with and cleaves pro-ADAMTS4 (Aggrecanase-1) in the trans-Golgi network . J. Biol. Chem . 279 , 15434 – 15440 , doi: 10.1074/jbc.M312797200 ( 2004 ). OpenUrl Abstract / FREE Full Text 31. ↵ Wang , L. W. et al. Adamts10 inactivation in mice leads to persistence of ocular microfibrils subsequent to reduced fibrillin-2 cleavage . Matrix Biol . 77 , 117 – 128 , doi: 10.1016/j.matbio.2018.09.004 ( 2019 ). OpenUrl CrossRef PubMed 32. ↵ Balic , Z. et al. Alternative splicing of the metalloprotease ADAMTS17 spacer regulates secretion and modulates autoproteolytic activity . FASEB J . 35 , e21310 , doi: 10.1096/fj.202001120RR ( 2021 ). OpenUrl CrossRef PubMed 33. ↵ Oichi , T. et al. Adamts17 is involved in skeletogenesis through modulation of BMP-Smad1/5/8 pathway . Cell. Mol. Life Sci . , doi: 10.1007/s00018-019-03188-0 ( 2019 ). OpenUrl CrossRef 34. ↵ Mularczyk , E. J. et al. ADAMTS10-mediated tissue disruption in Weill-Marchesani syndrome . Hum. Mol. Genet . 27 , 3675 – 3687 , doi: 10.1093/hmg/ddy276 ( 2018 ). OpenUrl CrossRef PubMed 35. ↵ Wu , H. J. , Mortlock , D. P. , Kuchtey , R. W. & Kuchtey , J . Altered Ocular Fibrillin Microfibril Composition in Mice With a Glaucoma-Causing Mutation of Adamts10 . Invest. Ophthalmol. Vis. Sci . 62 , 26 , doi: 10.1167/iovs.62.10.26 ( 2021 ). OpenUrl CrossRef 36. ↵ Hubmacher , D. et al. Limb- and tendon-specific Adamtsl2 deletion identifies a role for ADAMTSL2 in tendon growth in a mouse model for geleophysic dysplasia . Matrix Biol . 82 , 38 – 53 , doi: 10.1016/j.matbio.2019.02.001 ( 2019 ). OpenUrl CrossRef 37. ↵ Breur , G. J. , VanEnkevort , B. A. , Farnum , C. E. & Wilsman , N. J . Linear relationship between the volume of hypertrophic chondrocytes and the rate of longitudinal bone growth in growth plates . J. Orthop. Res . 9 , 348 – 359 , doi: 10.1002/jor.1100090306 ( 1991 ). OpenUrl CrossRef PubMed Web of Science 38. ↵ Kember , N. F . Comparative patterns of cell division in epiphyseal cartilage plates in the rat . J. Anat . 111 , 137 – 142 ( 1972 ). OpenUrl PubMed 39. ↵ Dy , P. et al. Sox9 directs hypertrophic maturation and blocks osteoblast differentiation of growth plate chondrocytes . Dev. Cell 22 , 597 – 609 , doi: 10.1016/j.devcel.2011.12.024 ( 2012 ). OpenUrl CrossRef PubMed Web of Science 40. ↵ Kozhemyakina , E. , Cohen , T. , Yao , T. P. & Lassar , A. B . Parathyroid hormone-related peptide represses chondrocyte hypertrophy through a protein phosphatase 2A/histone deacetylase 4/MEF2 pathway . Mol. Cell. Biol . 29 , 5751 – 5762 , doi: 10.1128/MCB.00415-09 ( 2009 ). OpenUrl CrossRef PubMed 41. ↵ Sabatier , L. et al. Fibrillin assembly requires fibronectin . Mol. Biol. Cell 20 , 846 – 858 , doi: 10.1091/mbc.E08-08-0830 ( 2009 ). OpenUrl Abstract / FREE Full Text 42. ↵ Richard , D. et al. Functional genomics of human skeletal development and the patterning of height heritability . Cell , doi: 10.1016/j.cell.2024.10.040 ( 2024 ). OpenUrl CrossRef 43. ↵ Dreos , R. , Ambrosini , G. , Groux , R. , Cavin Perier , R. & Bucher , P . The eukaryotic promoter database in its 30th year: focus on non-vertebrate organisms . Nucleic Acids Res . 45 , D51 – D55 , doi: 10.1093/nar/gkw1069 ( 2017 ). OpenUrl CrossRef PubMed 44. ↵ Rezza , A. et al. Signaling Networks among Stem Cell Precursors, Transit-Amplifying Progenitors, and their Niche in Developing Hair Follicles . Cell reports 14 , 3001 – 3018 , doi: 10.1016/j.celrep.2016.02.078 ( 2016 ). OpenUrl CrossRef PubMed 45. ↵ Sennett , R. et al. An Integrated Transcriptome Atlas of Embryonic Hair Follicle Progenitors, Their Niche, and the Developing Skin . Dev. Cell 34 , 577 – 591 , doi: 10.1016/j.devcel.2015.06.023 ( 2015 ). OpenUrl CrossRef PubMed 46. ↵ Marzin , P. , Cormier-Daire , V. & Tsilou , E. in GeneReviews((R)) (eds M. P. Adam et al. ) ( 1993 ). 47. ↵ Cooper , K. L. et al. Multiple phases of chondrocyte enlargement underlie differences in skeletal proportions . Nature 495 , 375 – 378 , doi: 10.1038/nature11940 ( 2013 ). OpenUrl CrossRef PubMed 48. Wilsman , N. J. , Farnum , C. E. , Leiferman , E. M. , Fry , M. & Barreto , C . Differential growth by growth plates as a function of multiple parameters of chondrocytic kinetics . J. Orthop. Res . 14 , 927 – 936 , doi: 10.1002/jor.1100140613 ( 1996 ). OpenUrl CrossRef PubMed Web of Science 49. ↵ Wilsman , N. J. , Farnum , C. E. , Green , E. M. , Lieferman , E. M. & Clayton , M. K . Cell cycle analysis of proliferative zone chondrocytes in growth plates elongating at different rates . J. Orthop. Res . 14 , 562 – 572 , doi: 10.1002/jor.1100140410 ( 1996 ). OpenUrl CrossRef PubMed Web of Science 50. ↵ Kennedy , A. M. et al. MMP13 mutation causes spondyloepimetaphyseal dysplasia, Missouri type (SEMD(MO) . J. Clin. Invest . 115 , 2832 – 2842 , doi: 10.1172/JCI22900 ( 2005 ). OpenUrl CrossRef PubMed Web of Science 51. ↵ Inada , M. et al. Critical roles for collagenase-3 (Mmp13) in development of growth plate cartilage and in endochondral ossification . Proc Natl Acad Sci U S A 101 , 17192 – 17197 , doi: 10.1073/pnas.0407788101 ( 2004 ). OpenUrl Abstract / FREE Full Text 52. ↵ Vu , T. H. et al. MMP-9/gelatinase B is a key regulator of growth plate angiogenesis and apoptosis of hypertrophic chondrocytes . Cell 93 , 411 – 422 , doi: 10.1016/s0092-8674(00)81169-1 ( 1998 ). OpenUrl CrossRef PubMed Web of Science 53. ↵ Knauper , V. et al. Cellular activation of proMMP-13 by MT1-MMP depends on the C-terminal domain of MMP-13 . FEBS Lett . 532 , 127 – 130 , doi: 10.1016/s0014-5793(02)03654-2 ( 2002 ). OpenUrl CrossRef PubMed Web of Science 54. ↵ Wohl , A. P. , Troilo , H. , Collins , R. F. , Baldock , C. & Sengle , G . Extracellular Regulation of Bone Morphogenetic Protein Activity by the Microfibril Component Fibrillin-1 . J. Biol. Chem . 291 , 12732 – 12746 , doi: 10.1074/jbc.M115.704734 ( 2016 ). OpenUrl Abstract / FREE Full Text 55. ↵ Sengle , G. et al. Targeting of bone morphogenetic protein growth factor complexes to fibrillin . J. Biol. Chem . 283 , 13874 – 13888 , doi: 10.1074/jbc.M707820200 ( 2008 ). OpenUrl Abstract / FREE Full Text 56. ↵ Sabatier , L. et al. Complex contributions of fibronectin to initiation and maturation of microfibrils . Biochem. J . 456 , 283 – 295 , doi: 10.1042/BJ20130699 ( 2013 ). OpenUrl Abstract / FREE Full Text 57. ↵ Schindelin , J. , Rueden , C. T. , Hiner , M. C. & Eliceiri , K. W . The ImageJ ecosystem: An open platform for biomedical image analysis . Mol. Reprod. Dev . 82 , 518 – 529 , doi: 10.1002/mrd.22489 ( 2015 ). OpenUrl CrossRef PubMed 58. ↵ Schindelin , J. et al. Fiji: an open-source platform for biological-image analysis . Nat. Methods 9 , 676 – 682 , doi: 10.1038/nmeth.2019 ( 2012 ). OpenUrl CrossRef PubMed Web of Science 59. ↵ Lee , C. S. et al. Mutations in Fibronectin Cause a Subtype of Spondylometaphyseal Dysplasia with "Corner Fractures" . Am. J. Hum. Genet . 101 , 815 – 823 , doi: 10.1016/j.ajhg.2017.09.019 ( 2017 ). OpenUrl CrossRef PubMed 60. ↵ Reznikoff , C. A. , Bertram , J. S. , Brankow , D. W. & Heidelberger , C . Quantitative and qualitative studies of chemical transformation of cloned C3H mouse embryo cells sensitive to postconfluence inhibition of cell division . Cancer Res . 33 , 3239 – 3249 ( 1973 ). OpenUrl Abstract / FREE Full Text 61. ↵ Reznikoff , C. A. , Brankow , D. W. & Heidelberger , C . Establishment and characterization of a cloned line of C3H mouse embryo cells sensitive to postconfluence inhibition of division . Cancer Res . 33 , 3231 – 3238 ( 1973 ). OpenUrl Abstract / FREE Full Text 62. ↵ Mirando , A. J. , Dong , Y. , Kim , J. & Hilton , M. J . Isolation and culture of murine primary chondrocytes . Methods Mol. Biol . 1130, 267 – 277 , doi: 10.1007/978-1-62703-989-5_20 ( 2014 ). OpenUrl CrossRef 63. ↵ Martin , D. R. et al. Proteomics identifies a convergent innate response to infective endocarditis and extensive proteolysis in vegetation components . JCI Insight 5 , doi: 10.1172/jci.insight.135317 ( 2020 ). OpenUrl CrossRef PubMed 64. ↵ Nandadasa , S. et al. Degradomic Identification of Membrane Type 1-Matrix Metalloproteinase as an ADAMTS9 and ADAMTS20 Substrate . Mol Cell Proteomics 22 , 100566 , doi: 10.1016/j.mcpro.2023.100566 ( 2023 ). OpenUrl CrossRef View the discussion thread. Back to top Previous Next Posted January 24, 2025. Download PDF Email Thank you for your interest in spreading the word about bioRxiv. NOTE: Your email address is requested solely to identify you as the sender of this article. Your Email * Your Name * Send To * Enter multiple addresses on separate lines or separate them with commas. 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