Proteolysis of fibrillin-2 microfibrils is essential for normal skeletal development

  1. Timothy J Mead
  2. Daniel R Martin
  3. Lauren W Wang
  4. Stuart A Cain
  5. Cagri Gulec
  6. Elisabeth Cahill
  7. Joseph Mauch
  8. Dieter Reinhardt
  9. Cecilia Lo
  10. Clair Baldock
  11. Suneel S Apte  Is a corresponding author
  1. Department of Biomedical Engineering and Musculoskeletal Research Center, Cleveland Clinic Lerner Research Institute, United States
  2. Division of Cell-Matrix Biology and Regenerative Medicine, Wellcome Centre for Cell-Matrix Research, School of Biological Sciences, Faculty of Biology, Medicine and Health, The University of Manchester, Manchester Academic Health Science Centre, United Kingdom
  3. Department of Developmental Biology, University of Pittsburgh School of Medicine, United States
  4. Faculty of Medicine and Health Sciences and Faculty of Dental Medicine and Oral Health Sciences, McGill University, Canada
11 figures, 4 tables and 4 additional files

Figures

Figure 1 with 10 supplements
ADAMTS6 and ADAMTS10 are subject to transcriptional adaptation and cooperate in skeletal development.

(A) qRT-PCR analysis of Adamts6 and Adamts10 mRNA levels in wild type, Adamts6-/- and Adamts10-/- limb, heart and lung show that Adamts6 mRNA is elevated in Adamts10-/- limb, heart and lung and Adamt…

Figure 1—figure supplement 1
ADAMTS6 and ADAMTS10 do not cleave each other.

(A) ADAMTS10 was co-transfected with ADAMTS6 or the catalytically inactive mutant ADAMTS6 EA. (B) Furin site-optimized ADAMTS10 (ADAMTS10-RRKR) was co-transfected with ADAMTS6 EA. No cleavage …

Figure 1—figure supplement 2
Expected Mendelian ratios are observed in mouse crosses.

Mendelian ratios obtained from Adamts6+/- intercrosses (A) and Adamts6+/-;Adamts10+/- intercrosses at E18.5 (B) are shown. Observed and expected (in parentheses) genotype percentages are shown in …

Figure 1—figure supplement 3
Reduced crown-rump length and shorter limb skeletal elements in Adamts6-/- embyros.

At E14.5 (A) and E16.5 (B), Adamts6-deficient embryos have reduced crown-rump length and shorter long bones as compared to wild type embryos. Crown-rump length, n = 4; bone length, n ≥ 6. *p ≤ 0.05; …

Figure 1—figure supplement 4
Reduced crown-rump length and shorter limb skeletal elements in combined Adamts6 and Adamts10 alleles at E18.5.

(A) Adamts6- and Adamts10-deficient embryos and embryos with various combinations of alleles have reduced crown-rump length as compared to wild type embryos. (B) Embryos with various combinations of …

Figure 1—figure supplement 5
Greater diaphyseal width in Adamts6-/- long bones.

E18.5 Adamts6-deficient embryos have wider long bones than wild type embryos. n ≥ 6. *p ≤ 0.05; **p ≤ 0.01; ***p ≤ 0.001.

Figure 1—figure supplement 6
Delayed ossification and shorter femora in Adamts6-/- embyros.

Alcian blue-stained sections show the primary ossification center in E14.5 wild type femur (lack of blue staining in the diaphysis). The primary center did not form until E16.5 in the Adamts6-/-

Figure 1—figure supplement 7
Angulation of the Adamts6-/- distal tibia.

Alcian blue-stained sections revealed marked distal tibial angulation in Adamts6-/- hindlimbs as early as E14.5, which persisted and contributed to a club-foot like appearance of the hindlimbs. n ≥ 4.

Figure 1—figure supplement 8
Severely hypoplastic fibula in Adamts6-/- embryos.

Alcian blue-stained sections revealed a delay in chondrocyte hypertrophy until E16.5 in Adamts6-/- fibula whereas it occurred at E14.5 in wild type fibula. Similarily, ossification was delayed until …

Figure 1—figure supplement 9
Decreased mineralization in Adamts6-deficent hindlimb long bones.

(A,B) E14.5 von Kossa-stained sections (A) quantified in (B) revealed reduced mineral deposition (black) in Adamts6-/- hindlimb bones. Significant staining in the periosteum as well as in the center …

Figure 1—figure supplement 10
Adamts6-mutant embryos have a severely underdeveloped axial skeleton.

Alcian blue- and alizarin red -stained E18.5 axial skeleton and craniofacial preparations show that Adamts6-/- and Adamts6-/-;Adamts10-/- embryos have shortened, stout ribs (asterisks), shortened …

Adamts6 and Adamts10 mRNAs show overlapping distribution in developing mouse limbs.

(A) RNAscope in situ hybridization shows strong expression of Adamts6 and Adamts10 in E13.5 perichondrium (arrow) and weaker expression in growth plate chondrocytes (arrowheads). (B) Strong Adamts6

Figure 3 with 4 supplements
Adamts6-/- and Adamts6-/-;Adamts10-/- hindlimbs have disorganized chondrocytes and reduced cartilage proteoglycan.

(A) RGB trichrome-stained E18.5 knees show disorganized chondrocytes and reduced alcian blue staining in Adamts6-/- and Adamts6-/-;Adamts10-/- bones. (F), femur; T, tibia; f, fibula; RZ, resting …

Figure 3—figure supplement 1
Increased collagen staining in Adamts6-deficent hindlimbs.

RGB trichrome-stained E18.5 femur showed diffuse, disorganized and increased collagen staining (red) in the perichondrium and adjacent mesenchyme in Adamts6-/- and Adamts6-/-;Adamts10-/- embryos. …

Figure 3—figure supplement 2
Adamts6-deficent femora have reduced cartilage proteoglycan staining.

(A) RGB trichrome-stained E14.5 Adamts6-/- femur sections show smaller resting zone (RZ), columnar zone (CZ) and hypertrophic zone (HZ) chondrocytes with reduced alcian blue staining of the ECM. (B, …

Figure 3—figure supplement 3
Late embryonic Adamts6-mutant femora have reduced cartilage proteoglycan.

Reduced staining intensity of aggrecan (Acan), cartilage link protein (CLP) and Sox9 were evident in Adamts6-/- proximal femoral resting and columnar zone chondrocytes. No change in Col X staining …

Figure 3—figure supplement 4
No change in proliferation or cell death in Adamts6-/- cartilage.

(A–B) PCNA +nuclei (red) (A) in E18.5 distal femur were quantified in the resting zone (RZ; yellow brackets) and columnar zone (CZ; orange brackets) and quantified in (B). n = 4. (C–D) TUNEL +cells …

Figure 4 with 1 supplement
Increased MAGP1 and fibrillin-2 staining, but not fibrillin-1 staining in Adamts6-/- and Adamts6-/-;Adamts10-/- perichondrium.

(A–B) Increased staining intensity (green) of MAGP1 (A) and fibrillin-2 (B) in E18.5 Adamts6- and Adamts10-deficient knee joints. (C) No consistent change in fibrillin-1 staining (green) was seen …

Figure 4—figure supplement 1
Differential microfibril staining in Adamts6-deficient hindlimbs.

(A) Increased MAGP1 and fibrillin-2 staining intensity in Adamts6- and Adamts10-mutant hindlimbs. There was no change, however, in fibrillin-1 staining intensity. n ≥ 4. *p ≤ 0.05; **p ≤ 0.01; ***p …

Figure 5 with 1 supplement
Loss of fibrillin-2, fibrillin-1 and fibronectin microfibrils in the presence of active ADAMTS6.

(A) Fbn1-/- mouse embryo fibroblasts (MEFs), which can produce fibrillin-2 but not fibrillin-1, were cocultured with human embryonic kidney (HEK) cells overexpressing ADAMTS6 or ADAMTS6 EA (inactive …

Figure 5—figure supplement 1
Increased fibrillin-1, fibrillin-2 and fibronectin microfibril staining in fibroblasts lacking ADAMTS6.

(A) Adamts6-/- mouse embryo fibroblasts (MEFs) have increased fibrillin-2 and fibrillin-1 staining after 6 days in culture as compared to wild type MEFs. Fibronectin staining was similarly increased …

Figure 6 with 1 supplement
ADAMTS6 binds directly to fibrillin-2.

(A) Cartoons of the domain structures of ADAMTS6 and fibrillin-2 and the recombinant constructs used in the present work (indicated by black lines). (B–C) Biacore analysis shows dose-dependent …

Figure 6—figure supplement 1
ADAMTS6 binds directly to fibrillin-1.

(A) Domain structure of fibrillin-1 recombinant constructs. (B) Biacore analysis shows that fibrillin-1-Nt and fibrillin-1-Ct recombinant fragments each bind strongly to ADAMTS6-Ct. (C). Comparison …

Figure 7 with 2 supplements
Fibrillin-2 cleavage by ADAMTS6 and identification of the cleavage site using N-terminomics.

(A) Schematic of the experimental approach. Proteins from conditioned medium of co-cultures of HEK293F cells stably expressing FBN2-Ct and HEK293F cells expressing either ADAMTS6 WT or ADAMTS6 EA …

Figure 7—figure supplement 1
ADAMTS6 cleaves fibrillin-1.

(A) Schematic of the experimental approach. Proteins from conditioned medium of co-cultured HEK293F cells stably expressing fibrillin-1 and HEK293F cells expressing either ADAMTS6 WT or ADAMTS6 EA …

Figure 7—figure supplement 2
ADAMTS6 cleaves fibronectin at multiple sites.

(A) Schematic of the domain structure of fibronectin and locations of the identified cleavage sites. (B–C) Annotated MS2 spectra of the VREEVVTVGNSVNEG (B) and the LNQPTDDSCFDPYTVSHYAVGDEWER (C) …

Figure 8 with 5 supplements
Genetic reversal of limb anomalies in Adamts6 mutant mice by Fbn2 haploinsufficiency.

(A–B) Deletion of one Fbn2 allele reverses limb dysmorphology in E18.5 Adamts6-/- embryos, specifically, externally evident limb segment dimensions and reversal of rotational anomaly (arrow) (A), …

Figure 8—figure supplement 1
Reversal of reduced body length and long bone shortening in Adamts6-deficient embryos by Fbn2 hemizygosity.

Reduced crown-rump length (A) and reduced radius, ulna, tibia and fibula length (B) in E18.5 Adamts6-/- embryos was ameliorated by Fbn2 heterozygosity. Crown-rump length, n ≥ 3; bone length, n ≥ 6. …

Figure 8—figure supplement 2
Fbn2 haploinsufficiency reverses axial skeleton and craniofacial anomalies in Adamts6-/- mice.

The images specifically illustrate restoration of length, ossification and relative proportions of the ribs (astrisks), sternal segmental ossific centers (arrows) and xiphoid process ossification …

Figure 8—figure supplement 3
Normalized fibrillin-2, aggrecan, cartilage link protein and Sox9 staining intensity in Adamts6-/-;Fbn2+/- cartilage.

Reduction of fibrillin-2 staining intensity was noted in Adamts6-/-;Fbn2+/- hindlimbs compared to Adamts6-/- hindlimbs. Fbn2+/- hindlimbs also had reducedfibrillin-2 staining than wild type …

Figure 8—figure supplement 4
Fbn2 hemizygosity does not itself alter hindlimb long bone length or growth plate morphology and dimensions.

(A,B) Alcian blue- and alizarin red-stained E18.5 Fbn2+/- hindlimbs have normal patterning and unchanged long bone length (n ≥ 12). No change in crown-rump length was seen in E18.5 Fbn2+/- embryos …

Figure 8—figure supplement 5
Genetic Fbn1 reduction does not affect Adamts6-/- limb and skeletal defects.

No restoration of long bone dysmorphology and shortening (A,B) or crown-rump length (B) of Adamts6-/- embryos after hemizygosity or homozygosity for a Fbn1 mutant allele, Fbn1mgR. Measurements were …

Reduced BMP signaling but unaltered canonical TGFβ signaling in Adamts6-/- distal femur.

(A) Reduced pSMAD1/5/8 staining in Adamts6-/- femur, as compared to wild type control, is restored to wild type levels in Adamts6-/-;Fbn2+/- femur as quantified in (B). n ≥ 4; *p ≤ 0.05; **p ≤ 0.01. …

Figure 10 with 1 supplement
GDF5 co-localizes with fibrillin-2 microfibrils with greater staining intensity in Adamts6-/- knee joints.

(A–B) E14.5, E16.5 and E18.5 Adamts6-/- knee joints (A) and perichondrium (B) show both increased fibrillin-2 (red) and GDF5 (green) staining in joint mesenchyme as compared to wild-type control. …

Figure 10—figure supplement 1
Increased fibrillin-2 and GDF5 staining intensity in Adamts6-/- knee joint mesenchyme.

Quantification of fluorescent staining intensity in sections of the E14.5, E16.5, and E18.5 knee joint from wild type and Adamts6-/- embyros stained with fibrillin-2 and GDF5 antibodies. n ≥ 4. *p ≤ …

Schematic illustrating the observed anomalies in the absence of ADAMTS6 and proposed roles and mechanisms of ADAMTS6 in skeletal development.

(A) Adamts6 expression (pink dots) is shown in the perichondrium, peripheral chondrocytes, and in mesenchyme surrounding the knee joint. BMP signaling (blue circles) is evident in growth plate …

Tables

Table 1
Kinetic data for ADAMTS6 construct binding to FBN2-Ct.
Ligand: FBN2-Ct
AnalyteKD (nM)R2Bmax (RU)
ADAMTS6-Ct4360.9951727
ADAMTS6-4TSR1220.8864188
ADAMTS6-S4TSR1140.9819241
Table 2
Kinetic data for binding of FBN2 constructs to ADAMTS6-Ct.
Ligand: ADAMTS6-Ct
AnalyteKD (nM)R2Bmax (RU)
FBN2-Nt430.997285
FBN2-Ct800.9933100
Table 3
Putative sites of fibronectin cleavage by ADAMTS6 determined using TAILS.

The cleavage site is indicated by the period in column 1, with flanking amino acids numbered.

Annotated sequenceNumber of PSMsWild type abundanceEA abundance
[A292].V293YQPQPHPQPPPYGHCVTDSGVVYSVGMQWLK.[T]21.38E + 060
[Y294].Q295PQPHPQPPPYGHCVTDSGVVYSVGMQWLK.[T]16.99E + 050
[P462].M463AAHEEICTTNEGVMYR.[I]21.22E + 060
[A464].A465HEEICTTNEGVMYR.[I]14.71E + 050
[G567].T568FYQIGDSWEK.[Y]11.18E + 060
[F750].R751VEYELSEEGDEPQYLDLPSTATSVNIPDLLPGRK.[Y]13.33E + 050
[K].YIVNVYQISEDGEQS800.[L801]18.57E + 050
[Y1248].T1249VKDDKESVPISDTIIPAVPPPTDLR.[F]11.21E + 060
[Y1613].A1614QNPSGESQPLVQTAVTTIPAPTDLK.[F]16.32E + 050
[P2021].F2022VTHPGYDTGNGIQLPGTSGQQPSVGQQMIFEEHGFRR.[T]21.58E + 060
[K].VREEVVTVGNSVNEG2197.[L2198]*11.56E + 060
[G2197].L2198NQPTDDSCFDPYTVSHYAVGDEWER.[M]*16.82E + 060
  1. Abbreviations used: PSM, Peptide spectrum matches.

  2. *

    Peptides with termini identifying the same cleavage site (G2197-L2198).

  3. Peptide unique to the FN1-8 isoform.

Key resources table
Reagent type (species) or resourceDesignationSource or referenceIdentifiersAdditional information
Gene (Mus musculus)Adamts6 Genbank NCBI Gene: 108,154 ADAM metallopeptidase with thrombospondin type 1 motif 6
Gene (Mus musculus)Adamts10 Genbank NCBI Gene: 224,697 ADAM metallopeptidase with thrombospondin type 1 motif 10
Gene (Mus musculus)Adamts17 Genbank NCBI Gene: 233,332 ADAM metallopeptidase with thrombospondin type 1 motif 17
Gene (Mus musculus)Adamts19 Genbank NCBI Gene: 240,322 ADAM metallopeptidase with thrombospondin type 1 motif 19
Gene (Mus musculus)Fbn1 Genbank NCBI Gene: 14,118 Fibrillin-1
Gene (Mus musculus)Fbn2 Genbank NCBI Gene: 14,119 Fibrillin-2
Gene (Mus musculus)Fn1 Genbank NCBI Gene: 14,268 Fibronectin
Strain, strain background (Mus musculus)Adamts6b2b2029Clo (C57BL/6 J) Mouse Genome Informatics RRID: MGI:5487397Adamts6 null allele
Strain, strain background (Mus musculus)Adamts10tm1Dgen (C57BL/6 J) Mouse Genome Informatics RRID: MGI:6355992Adamts10 null allele
Strain, strain background (Mus musculus)Fbn1tm2Rmz (C57BL/6 J) Mouse Genome Informatics RRID: MGI:1934906Fbn1mgR allele
Strain, strain background (Mus musculus)Fbn2tm1Rmz (C57BL/6 J) Mouse Genome Informatics RRID: MGI:3652417Fbn2 null allele
Strain, strain background (Mus musculus)Fbn1tm3Rmz (C57BL/6 J) PMCID:PMC3875392 RRID: MGI:3641232Fbn1 null allele MEFs used for cell culture experiments
Strain, strain background (Mus musculus)Fbn2tm1Rmz (C57BL/6 J) This paper RRID: MGI:3652417Fbn2 null MEFs used for in vitro microfibril staining
Strain, strain background (Mus musculus) C57BL/6 J strain This paper RRID: IMSR_JAX:000664 Wild type MEFs used for in vitro microfibril staining
Strain, strain background (Mus musculus)Adamts6b2b2029Clo (C57BL/6 J) This paper RRID: MGI:5487397Adamts6 mutant MEFs for in vitro microfibril staining
Transfected construct (Mus musculus) ADAMTS6 WT PMCID:PMC6048820 ADAMTS6 plasmid
Transfected construct (Mus musculus) ADAMTS6 EA This paper This paper Catalytically- inactive ADAMTS6 plasmid
Cell line (human) FBN1-expressing cells Dieter Reinhardt, Ph.D. PMID:12399449 Stable HEK293 cell line expressing full length FBN1
Cell line (human) FBN2-Nt-expressing cells Dieter Reinhardt, Ph.D. PMID:12399449 Stable HEK293 cell line expressing the N-terminal half of FBN2
Cell line (human) FBN2-Ct-expressing cells Dieter Reinhardt, Ph.D. PMID:12399449 Stable HEK293 cell line expressing the C-terminal half of FBN2
Peptide, recombinant proteinFBN2-Nt-expressing cellsDieter Reinhardt, Ph.D. rFBN2-N; PMID:12399449Used for Biacore analysis
Peptide, recombinant proteinFBN2-CtDieter Reinhardt, Ph.D. rFBN2-C; PMID:12399449Used for Biacore analysis
Peptide, recombinant proteinFBN1-NtDieter Reinhardt, Ph.D. rFBN1-N; PMID:12399449Used for Biacore analysis
Peptide, recombinant proteinFBN1-CtDieter Reinhardt, Ph.D. rFBN1-C; PMID:12399449Used for Biacore analysis
Peptide, recombinant proteinFN1Deane Mosher, M.D. PMD:6133865Used for Biacore analysis
Peptide, recombinant proteinADAMTS6-CtStuart Cain, Ph.D. PMCID:PMC5078793Used for Biacore analysis
Peptide, recombinant proteinADAMTS6-4TSRStuart Cain, Ph.D. This paperUsed for Biacore analysis
Peptide, recombinant proteinADAMTS6-S4TSRStuart Cain, Ph.D. This paperUsed for Biacore analysis
Peptide, recombinant proteinADAMTS6-TCSStuart Cain, Ph.D. This paperUsed for Biacore analysis
AntibodyAnti-Fibrillin-2-Gly(rabbit polyclonal)Robert Mecham, Ph.D. PMID:10825173IF (1:300)
Antibody Anti-mFbn1-C (rabbit polyclonal)Dieter Reinhardt, Ph.D. PMID:33039488IF (1:500)
Antibody Anti-rFBN2-C (rabbit polyclonal)Dieter Reinhardt, Ph.D. PMID:12399449WB (1:500)
Antibody Anti-MAGP1 (rabbit polyclonal)Robert Mecham, Ph.D. PMCID:PMC14862IF (1:200)
Antibody Anti-Sox9 (rabbit polyclonal) Millipore AB5535 RRID: AB_2239761 IF (1:300)
Antibody Anti-Acan (rabbit polyclonal) Millipore AB1031 RRID: AB_90460 IF (1:400)
Antibody Anti-CLP (mouse monoclonal) DSHB 9/30/8 A-4 RRID: AB_2248142 IF (1:100)
Antibody Anti-Col X (rabbit polyclonal) Abcam ab58632 RRID: AB_879742 IF (1:1000)
Antibody Anti-PCNA (mouse monoclonal) Cell Signaling 2,586 RRID: AB_2160343 IF (1:200)
Antibody Anti-His (mouse monoclonal) R&D MAB050 RRID: AB_357353 IF (1:400) WB (1:1000)
Antibody Anti-pSmad5 (rabbit polyclonal) Abcam ab92698 RRID: AB_10561456 WB (1:1000)
Antibody Anti-pSmad1/5 (rabbit polyclonal) Cell Signaling 9,516 RRID: AB_491015IF (1:200)
AntibodyAnti-pSmad2(rabbit polyclonal) Cell Signaling 3,108 RRID: AB_490941IF (1:200)WB (1:1000)
Antibody Anti-GDF5 (goat polyclonal) R&D AF853 RRID: AB_355662 IF (1:100)
Antibody Anti-GAPDH (mouse monoclonal)Millipore MAB374 RRID: AB_2107445 WB (1:5000)
Commercial assay or kitAdamts6 RNAscope probe ACD Bio 428,301Mouse ISH probe
Commercial assay or kitAdamts10 RNAscope probe ACD Bio 585,161Mouse ISH probe
Commercial assay or kit RNAscope 2.5 HD Red in situ detection kit ACD Bio 322,350Used to detect Adamts6 and Adamts10 probes
Software, algorithm GraphPad Prism GraphPad RRID: SCR_002798 Utilized for statistical computing of data
Software, algorithm Fiji NIH RRID: SCR_002285Used to quantify IF tissue sections
  1. IF, Immunofluorescence.; WB, western blot.; ISH, in situ hybridization.

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