A mouse model of Jansen's metaphyseal chondrodysplasia for investigating disease mechanisms and candidate therapeutics

Abstract

Jansen's metaphyseal chondrodysplasia (JMC) is a rare disorder caused by activating mutations in the parathyroid hormone (PTH)/PTH-related peptide (PTHrP) receptor (PTH1R). Patients exhibit short stature, dysmorphic bones, and severe growth plate abnormalities, as well as hypercalcemia, hypercalciuria, hypophosphatemia, and reduced plasma PTH levels. Humanized PTH1R (hPTH1R) mice expressing the H223R-hPTH1R JMC mutation die early without breeding. We therefore generated and characterized a stable mouse line expressing the T410R-hPTH1R allele, which confers a milder disease phenotype in patients. Mutant mice show near-normal longevity and reproductive capacity yet exhibit a profound skeletal phenotype characteristic of the disease. The long bones of T410R mice are markedly misshapen and have expanded metaphyses with disarrayed chondrocyte zones in growth plates and reduced primary spongiosa. PET/CT scanning revealed diminished uptake of [¹⁸F]-sodium fluoride in the growth plate area, consistent with reduced mineralization and vascularization. Genetic ablation of Hdac4 rescued the growth plate abnormalities in T410R mice, thereby establishing the PTH1R-Gαs-cAMP-PKA-SIK3-HDAC4/5 pathway as the main mediator of growth plate abnormalities in JMC. Serum calcium was elevated and endogenous PTH was suppressed in T410R mice, and both parameters could be normalized by acute injection of an optimized PTH inverse agonist peptide. The T410R mouse thus represents a stable animal model of JMC that recapitulates the abnormalities in skeletal development and mineral ion homeostasis which characterize this disease. The mice should help efforts to further define the cellular and molecular mechanisms underlying the JMC phenotype and to develop a potential mode of therapy.

Keywords: Jansen’s metaphyseal chondrodysplasia; PTH; PTH receptor; PTHrP; chondrocytes.

Fig. 1.

T410R mouse generation and skeletal phenotype. (A) Portions of the WT mouse Pth1r (wt-Pth1r) gene were replaced with the cDNA encoding the human PTH receptor (hPTH1R), as described before (28). Therefore, exon 4 of the mouse Pth1r (mPth1r) (green square) was replaced by the segment of the hPTH1R cDNA encoded by exon 4 (yellow square) and the remaining cDNA (blue square), terminated by a TGA stop-codon, that was fused to the 3’ end of mouse intron 3. An influenza hemagglutinin epitope tag (HA-tag; red line) is located in the portion of the hPTH1R encoded by exon 5 (dark gray square). The position of the introduced amino acid chance T410R is indicated by the black arrow. (B) 3D reconstructions of µCT scans of WT- and T410R tibias and femurs at postnatal days (P) 12, 37, and 65 show progressive enlargement of the regions of femur segments (arrows) as well as proximal tibiae where the primary mineralization of bones takes place.

Fig. 2.

Growth plate histology. Hematoxylin/Eosin (H/E) staining and RNA in situ hybridization (ISH, RNAscope) for type II (Col2) and type X collagen (Col10) as well as Indian hedgehog (Ihh) on postnatal days (P) P3 and P65 on sections of wild-type (WT) and T410R mice. T410R mice exhibit expanded zones of Col2a1-positive proliferative chondrocytes as well as Col10a1-positive hypertrophic chondrocytes at day P3, which was not observed at day P65. Ihh expression was disrupted in T410R growth plate histology sections.

Fig. 3.

Parameters of mineral ion homeostasis. Serum measurements for total calcium (mg/dl), phosphate (mg/dl), mouse PTH(1-84) (pg/ml), N-terminal propeptide of type I procollagen (P1NP; ng/ml) and Type I Collagen Cross-Linked C-Telopeptide (CTX-1; ng/ml). Data are combined from both sexes and across different age groups (postnatal days P12, P27, P45, and P73); sex-segregated data are shown in SI Appendix, Fig. S2). Lower limit of detection (LOD) is indicated as a dashed line in the PTH panel. Data were analyzed by Student’s t test. P values are indicated in the figure. T410R mice displayed elevated total serum calcium levels, accompanied by decreased serum phosphate levels and suppressed endogenous PTH(1-84) levels, compared to WT mice.

Fig. 4.

Uptake of [¹⁸F]NaF in WT vs T410R mice. Representative coronal views of whole body [¹⁸F]NaF PET/CT images of WT (Left) and T410R (Right) mice (P28). Magnification of CT and overlay PET/CT images (SUV_{10–30 min}) of the tibia growth plates is shown. When compared to WT mice, T410R mice exhibit blunted [¹⁸F]NaF uptake in growth plate areas.

Fig. 5.

Effects of Hdac4 ablation on the growth plate. Hematoxylin/Eosin (H/E) and Safranin O/Fast Green (SafO) staining of tibia growth plate sections of 3-d-old (P3) littermate mice. The genotype for the humanized PTH receptor is indicated as WT or T410R. Hdac4 genotype is indicated as intact gene (+) or deletion of exon 6 (−), which results in loss-of-function of the protein. Hdac4 ablation partially rescued the growth plate phenotype in T410R mice in a dose-dependent manner. In addition, the T410R-hPTH1R mutation had a partial rescue effect on the phenotype seen for homozygous Hdac4 deletion.

Fig. 6.

Inverse agonist treatment affects calcium homeostasis. (A) Baseline blood ionized calcium (iCa²⁺; mmol/l) for 55-d-old WT or T410R animals. T410R mice exhibit significantly elevated blood iCa²⁺ levels as compared to WT littermate controls. (B) iCa²⁺ measurements before (time point 0) as well as 1, 2, 4, 6, and 8 h (h) after single dose injection (s.c., 500 nmol/kg body weight) of either vehicle (veh; 10 mM citric acid/150 mM NaCl/ 0.05% Tween 80, pH 5.0) or α/β-PTH-IA (peptide 3) in male littermate WT vs. T410R mice. PTH-IA injection resulted in a significant decrease in blood iCa²⁺ levels in both WT as well as T410R mice. Recovery to baseline iCa²⁺ was delayed in T410R animals as compared with WT littermate controls. (C) Serum PTH(1-84) measurements 5h after PTH-IA injection. Injection of the PTH-IA resulted in a significant increase in serum PTH levels in WT as well as T410R mice. Data in panel A were analyzed by Student’s t test, panel B was analyzed by two-way ANOVA/mixed model followed by Fisher’s Least Significant Difference (LSD) test, panel C was assessed using one-way ANOVA. Data are presented as the means ± SEM.