Mechanism of shortened bones in mucopolysaccharidosis VII

Abstract

Mucopolysaccharidosis VII (MPS VII) is a lysosomal storage disease in which deficiency in beta-glucuronidase results in glycosaminoglycan (GAG) accumulation in and around cells, causing shortened long bones through mechanisms that remain largely unclear. We demonstrate here that MPS VII mice accumulate massive amounts of the GAG chondroitin-4-sulfate (C4S) in their growth plates, the cartilaginous region near the ends of long bones responsible for growth. MPS VII mice also have only 60% of the normal number of chondrocytes in the growth plate and 55% of normal chondrocyte proliferation at 3weeks of age. We hypothesized that this reduction in proliferation was due to C4S-mediated overactivation of fibroblast growth factor receptor 3 (FGFR3). However, MPS VII mice that were FGFR3-deficient still had shortened bones, suggesting that FGFR3 is not required for the bone defect. Further study revealed that MPS VII growth plates had reduced tyrosine phosphorylation of STAT3, a pro-proliferative transcription factor. This was accompanied by a decrease in expression of leukemia inhibitory factor (LIF) and other interleukin 6 family cytokines, and a reduction in phosphorylated tyrosine kinase 2 (TYK2), Janus kinase 1 (JAK1), and JAK2, known activators of STAT3 phosphorylation. Intriguingly, loss of function mutations in LIF and its receptor leads to shortened bones. This suggests that accumulation of C4S in the growth plate leads to reduced expression of LIF and reduced STAT3 tyrosine phosphorylation, which results in reduced chondrocyte proliferation and ultimately shortened bones.

Fig. 1

Mouse bone lengths. A and B. Representative radiographs of femurs and caudal vertebrae (6 through 9) from 6 week-old mice. C. Bone length measurements at 6 and 3 weeks. Measurements from 6 week normal (N=11), MPS I (N=9), and MPS VII (N=6) and 3 week normal (N=6) and MPS VII (N=7) bones are shown. For 6 week old mice, data for male and female mice were pooled and were normalized to the average length of the same bone in 6 week old normal mice, using one way ANOVA with Tukey post-hoc analysis for statistical comparisons. For 3 week old mice, all data were from males and the Student’s t test was used for statistical comparisons. P values compared to normal are labeled as ** for p<0.01, and * for p=0.01-0.05. Error bars indicate SEM, and the dashed line indicates normal. The 2^nd lumbar vertebrae and the 8^th caudal vertebrae were measured.

Fig. 2

Histopathology and labeling index of proximal tibia growth plates. A. H & E stains of tibia growth plates at 3 weeks of age. Black boxes indicate the region enlarged at the right. Brackets indicate hypertrophic zone (HZ), proliferating zone (PZ), and resting zone (RZ). Black brackets in column 2 show a representative chondrocyte column that was evaluated for the number of cells in the proliferating zone. B. BRDU staining of tibia growth plates at 3 weeks of age. Brown cells are BRDU-positive, as exemplified by black arrows. C. Quantification of the number of chondrocytes per column in normal (N=9), MPS I (N=5), and MPS VII (N=6) growth plates. Error bars represent SEM, and **: p<0.01 for data sets connected by brackets using one way ANOVA with Tukey all-pairwise post-hoc analysis. D. Quantification of the percentage of BRDU positive cells in the proliferating zone of normal (N=5), MPS I (N=4), and MPS VII (N=4) growth plates.

Fig. 3

GAG immunohistochemistry on tibias of 3 week old mice. Chondroitin-4-sulfate was detected in growth plates by digestion with chondroitinase AC-I (digests C4S and C6S) followed by incubation with Δdi-4S antibody. Growth plate zones are designated as in Fig. 2. Boxes in A-C indicate the regions of magnification shown in D-F. White arrows in panel F show examples of cells with excess C4S. C4S is also present in the extracellular space. Photographs are representative of results from 3 mice of each genotype.

Fig. 4

FGFR3 does not mediate bone shortening in MPS VII. A. Radiographs of normal (MPS VII +/-, FGFR3 +/-), MPS VII (MPS VII -/-, FGFR3 +/-), FGFR3-deficient (MPS VII +/-, FGFR3 -/-), and MPS VII and FGFR3-deficient (MPS VII -/-, FGFR3 -/-) mouse tibias at 3 weeks of age. B. Length measurements of tibias from 3 week old male mice. Statistics were performed as in Fig. 2.

Fig. 5

Immunohistochemistry of mouse tibia growth plates. Immunohistochemistry for the indicated antibodies was performed on 5 normal and 5 MPS VII growth plates at 3 weeks, and representative photographs of the growth plate and the proliferative zone are provided. Brown color signifies positive cells, which are indicated by black arrows. Black boxes in columns 1 and 3 indicate the region magnified in the image to its right. Growth plate zones are designated as in Fig. 2.

Fig. 6

Evaluation of growth plate RNA from MPS VII and normal mice at 3 weeks of age. RNA was extracted from the head of the tibia, real-time reverse-transcription PCR was performed, and transcript expression was normalized to β actin. Results are given here as a percentage of normal expression, while relative abundance of each gene is listed in the supplemental methods. **: p<0.01, *: p=0.01-0.05 using Student’s t test. Error bars indicate SEM. N=7 or more animals for each group. Genes tested are as follows: collagen X (Col X), collagen II (Col II), indian hedgehog (IHH), parathyroid hormone related peptide receptor (PTHrPR), insulin-like growth factor 1 (IGF1), receptor activator for nuclear factor κB ligand (RANKL), osteoprotegerin (OPG), cathepsin S (CathS), matrix metallopeptidase 3 (MMP3), matrix metallopeptidase 13 (MMP13), leukemia inhibitory factor (LIF), interleukin 6 (IL6), oncostatin M (OSM), interleukin 11 (IL11), ciliary neurotrophic factor (CNTF), cardiotrophin 1 (CTF1), cardiotrophin-like cytokine factor 1 (CLCF1), interferon γ (IFNγ), interleukin 1β (IL1β), tumor necrosis factor α (TNFα), tumor necrosis factor ligand superfamily member 10 (TNFSF10), tumor necrosis factor receptor-associated factor 6 (TRAF6), SH-domain containing phosphatase 1 (SHP1), SH-domain containing phosphatase 2 (SHP2), protein tyrosine phosphatase - receptor type C (PTPRC), suppressor of cytokine signaling 1 (SOCS1), suppressor of cytokine signaling 2 (SOCS2), suppressor of cytokine signaling 3 (SOCS3), protein inhibitor of activated STAT1 (PIAS1), and protein inhibitor of activated STAT3 (PIAS3).

Fig. 7

In-situ hybridization for Col X (A, D), Col II (B, E), and IHH (C, F). Representative photographs chosen from 3 normal and 3 MPS VII mice are shown, using identical exposure times for each gene in all samples. Red signal represents areas positive for the RNA of interest. The color was obtained by converting the signal seen in dark field to false color red, followed by overlaying the signal on an identical image in bright field. Growth plate zones are designated as in Fig. 2.