Adamts17 is involved in skeletogenesis through modulation of BMP-Smad1/5/8 pathway

Abstract

Fibrillin microfibrils are ubiquitous elements of extracellular matrix assemblies that play crucial roles in regulating the bioavailability of growth factors of the transforming growth factor beta superfamily. Recently, several "a disintegrin and metalloproteinase with thrombospondin motifs" (ADAMTS) proteins were shown to regulate fibrillin microfibril function. Among them, ADAMTS17 is the causative gene of Weill-Marchesani syndrome (WMS) and Weill-Marchesani-like syndrome, of which common symptoms are ectopia lentis and short stature. ADAMTS17 has also been linked to height variation in humans; however, the molecular mechanisms whereby ADAMTS17 regulates skeletal growth remain unknown. Here, we generated Adamts17-/- mice to examine the role of Adamts17 in skeletogenesis. Adamts17-/- mice recapitulated WMS, showing shorter long bones, brachydactyly, and thick skin. The hypertrophic zone of the growth plate in Adamts17-/- mice was shortened, with enhanced fibrillin-2 deposition, suggesting increased incorporation of fibrillin-2 into microfibrils. Comprehensive gene expression analysis of growth plates using laser microdissection and RNA sequencing indicated alteration of the bone morphogenetic protein (BMP) signaling pathway after Adamts17 knockout. Consistent with this, phospho-Smad1 levels were downregulated in the hypertrophic zone of the growth plate and in Adamts17-/- primary chondrocytes. Delayed terminal differentiation of Adamts17-/- chondrocytes, observed both in primary chondrocyte and primordial metatarsal cultures, and was prevented by BMP treatment. Our data indicated that Adamts17 is involved in skeletal formation by modulating BMP-Smad1/5/8 pathway, possibly through inhibiting the incorporation of fibrillin-2 into microfibrils. Our findings will contribute to further understanding of disease mechanisms and will facilitate the development of therapeutic interventions for WMS.

Keywords: Adamts17; Fibrillin; Microfibril; Skeletal formation.

Fig. 1

Adamts17-/- mice display postnatal impairment of skeletal growth. a Generation of Adamts17-flox mice. The diagrams show the Adamts17 genomic locus with exon 4 (Adamts17 locus); Adamts17 targeting vector including the neo-cassette (Targeting Vector); recombination between homologous regions of the targeting vector and the Adamts17 locus, leading to the generation of the Adamts17-targeted allele (Targeted Allele); floxed Adamts17 allele following flippase recombinase target (FRT)-mediated recombination of the Adamts17-targeted allele (Floxed allele); and Adamts17-deleted allele following Cre-mediated recombination of the floxed Adamt17 allele (Deleted allele). H, HindIII; S, SmaI; E, EcoRV; B, BamHI. b Pie chart of the genotypes detected at 7 days of age from intercrosses of Adamts17 ± mice on the C57BL/6 strain. Note the reduced viability of Adamts17-/- mice. c Alizarin Red and Alcian Blue double-staining of the whole skeleton (left), clavicle, upper extremities, and lower extremities (right) of WT and Adamts17-/- (KO) littermates at P0. Scale bars, 2 mm. d Length of clavicles and long bones of WT (n = 5) and KO (n = 6) littermates at P0. e Gross appearance of 12-week-old WT and KO mice. f Time-course of naso-anal length and body weight of male WT and KO mice (n = 6 per genotype). g Bone length of 4-week-old and 12-week-old male WT and KO mice (n = 5 per genotype). Statistical significance was calculated using a two-tailed unpaired Student’s t test. *P < 0.05 between genotypes

Fig. 2

Adamts17-/- mice recapitulate Weill-Marcehsani syndrome. a Distal forepaws and hindpaws of WT and Adamts17-/- (KO) mice at 8 months of age. Scale bars, 5 mm. b Bone length of forepaws and hindpaws of 8-month-old male WT (n = 7) and KO (n = 5) mice. *P < 0.05 versus WT. c Gross inspection of skin stiffness of 3- and 8-month-old WT and KO mice. Mice were anesthetized, shaved, and suspended by forceps. d Hematoxylin and eosin (H&E) and Masson’s trichrome staining of skin from 3- and 8-month-old WT and KO mice. Scale bars, 300 µm. Statistical significance was calculated using a two-tailed unpaired Student’s t test

Fig. 3

Histological analyses of growth plates from WT and Adamts17-/- mice. a Safranin-O staining of proximal tibiae from WT and Adamts17-/- (KO) mice at P7; right panels show higher magnifications of the boxed areas in the left panels. The white broken lines indicate the boundary between resting zone and proliferative zone (PZ), or the boundary between PZ and hypertrophic zone (HZ). Scale bars, 300 µm. b Length of hypertrophic and proliferative zones in WT and KO mice at P7 (n = 5 per genotype). *P < 0.05 versus WT. c EdU labeling (green) and phase contrast images of proximal tibiae from WT and KO mice at P7 and rates of EdU-positive cells in PZ (n = 5 per genotype). Inset boxes in the upper panels indicate the regions of enlarged images in the lower panels. Scale bars, 300 μm. d TUNEL staining of proximal tibiae from WT and KO mice at P7 and number of TUNEL-positive cells in the chondro-osseous junction (n = 4 per genotype). Scale bars, 300 µm. Statistical significance was calculated using a two-tailed unpaired Student’s t test

Fig. 4

Perichondrial microfibril dysregulation in the growth plate of Adamts17-/- mice.a Immunofluorescent staining of Adamts17, Fibrillin-1 (Fbn1), and Fibrillin-2 (Fbn2) in humeral distal growth plates of WT mice at E16.5. The inset box in the left panels indicates the location of the enlarged image shown on the right panels. Scale bars, 300 µm. b Semiserial histological sections of cultured pellets of primary chondrocytes were stained with safranin-O and immunostained for Fbn1 and Fbn2. The inset box in the left panels indicates the location of the enlarged image shown on the right panels. Scale bars, 300 µm. c Fbn1 and Fbn2 mRNA levels in femoral epiphyseal cartilage of WT (n = 6) and KO (n = 5) mice at P0. d Adamts17, Fbn1, and Fbn2 mRNA levels in Adamts17^fl/fl primary chondrocytes that were adenovirally transfected with GFP or Cre and cultured for an additional 3 day (n = 3 per well). mRNA levels were normalized to β-actin mRNA levels (Actb). **P < 0.01 versus GFP. Statistical significance was calculated using a two-tailed unpaired Student’s t test

Fig. 5

BMP signaling is reduced in the growth plates of Adamts17-/- mice. a Sections of proximal tibial epiphyseal cartilages before and after prehypertrophic and hypertrophic zones were microdissected by LMD. Extracted mRNA was amplified and analyzed by RNA sequencing. Scale bars, 200 µm. b Volcano plot for RNA-seq data obtained from laser microdissected samples. Red dots indicate genes that were significantly (p < 0.05) and differentially expressed in Adamts17 KO growth plates by more than twice or less than half those of WT growth plates. c Immunofluorescent staining of phosphorylated Smad1 (pSmad1) (green) and phase contrast images in proximal tibiae of WT and Adamts17-/- (KO) mice at P7 and the percentage of pSmad1-positive cells in the prehypertrophic and hypertrophic zones of WT and KO (n = 5 per genotype) mice. The inset box in the left panels indicates the location of the enlarged image shown on the right panels. Scale bars, 300 µm. d Immunoblotting of pSmad1, Smad1, and actin in protein extracts from primary chondrocytes from WT and KO mice and the ratio of pSmad1/Smad1 signal intensity (n = 3 wells per genotype). e Id1,Bmp2, Bmp4, and BMP7 mRNA levels in primary chondrocytes from WT and KO mice (n = 3 wells per genotype). mRNA levels were normalized to β-actin mRNA levels (Actb). Statistical significance was calculated using a two-tailed unpaired Student’s t test. *P < 0.05 versus WT

Fig. 6

Delayed terminal differentiation of Adamts17-/- chondrocytes is prevented by BMP treatment. a Alizarin Red staining of primary chondrocytes, from WT and Adamts17-/- (KO) mice, cultured with or without 100 ng/mL of rhBMP2 for 20 days and the ratio of Alizarin Red-positive area (n = 3 wells per genotype). b Col10a1 and Mmp13 mRNA levels in chondrocytes from WT and KO mice, cultured with or without 100 ng/mL of rhBMP2. mRNA levels were normalized to β-actin mRNA levels (Actb). c Gross appearance of metatarsal rudiments of Adamts17 ± and Adamts17-/- mice at day 0 and after 5 days of culture, with or without 500 ng/mL of rhBMP2. Scale bars, 500 µm. d The entire length of metatarsal rudiments of Adamts17 ± (n = 18) and Adamts17-/- (n = 20) mice at day 0 and day 5. e The ratio of the mineralized zone of metatarsal rudiments from Adamts17 ± (rhBMP2 [+], n = 11; rhBMP2 [−], n = 18) and Adamts17-/- (rhBMP2 [+], n = 15; rhBMP2 [−], n = 20) mice at day 5. f von Kossa staining of metatarsal rudiments of Adamts17 ± and Adamts17-/- mice at day 5. Scale bars, 300 µm. g Immunofluorescent staining of Col10 and Mmp13 in metatarsal rudiments of Adamts17 ± and Adamts17-/- mice at day 5 and the ratio of Col10a1- and Mmp13-positive areas (n = 3 per genotype). Scale bars, 300 µm. *P < 0.05, **P < 0.01 versus Adamts17 ± . h Semiserial histological sections of cultured metatarsals cultured for 5 days were immunostained for phosphorylated Smad1 (pSmad1), Fbn1 and Fbn2, and the percentage of pSmad1-positive cells in the hypertrophic zones of Adamts17 ± (n = 6) and Adamts17-/- mice (n = 7) was shown. *P < 0.05 versus Adamts17 ± . i Col10a1, Mmp13, and Id1 mRNA levels in cultured metatarsals from Adamts17 ± and Adamts17-/- mice (n = 4 per genotype). mRNA levels were normalized to β-actin mRNA levels (Actb). Statistical significance was calculated using a two-tailed unpaired Student’s t test. *P < 0.05 versus Adamts17 ±