Defective postnatal endochondral bone development by chondrocyte-specific targeted expression of parathyroid hormone type 2 receptor

Abstract

The human parathyroid hormone type 2 receptor (PTH2R) is activated by PTH and by tuberoinfundibular peptide of 39 residues (TIP39), the latter likely acting as its natural ligand. Although the receptor is expressed at highest levels in the nervous system, we have observed that both PTH2R and TIP39 are expressed in the newborn mouse growth plate, with the receptor localizing in the resting zone and the ligand TIP39 localizing exclusively in prehypertrophic and hypertrophic chondrocytes. To address the role of PTH2R in postnatal skeletal growth and development, Col2a1-hPTH2R (PTH2R-Tg) transgenic mice were generated. The mice were viable and of nearly normal size at birth. Expression of the transgene in the growth plate was limited to chondrocytes. We found that chondrocyte proliferation was decreased, as determined by in vivo BrdU labeling of proliferating chondrocytes and CDK4 and p21 expression in the growth plate of Col2a1-hPTH2R transgenic mice. Similarly, the differentiation and maturation of chondrocytes was delayed, as characterized by decreased Sox9 expression and weaker immunostaining for the chondrocyte differentiation markers collagen type II and type X and proteoglycans. As well, there was altered expression of Gdf5, Wdr5, and β-catenin, factors implicated in chondrocyte maturation, proliferation, and differentiation.These effects impacted on the process of endochondral ossification, resulting in delayed formation of the secondary ossification center, and diminished trabecular bone volume. The findings substantiate a role for PTH2R signaling in postnatal growth plate development and subsequent bone mass acquisition.

Fig. 1.

Generation of the collagen type II (Col2a1)-human parathyroid hormone type 2 receptor (hPTH2R) mice. A: schematic representation of the Col2a1-hPTH2R transgene; 2.3-kb promoter B: restriction enzyme digestion of the transgene plasmid showing the release of the 4.8-kb Col2a1-hPTH2R transgene. C: identification of the 3 founder transgenic mice (indicated by the nos. on top) by Southern blot analysis of tail genomic DNA restricted with either BamHI or EcoRI using radiolabeled hPTH2R cDNA as probe. D: Western blot analysis of PTH2R protein expression in growth plates of long bones from newborn transgenic mice and control littermates. The antibody recognizes both murine and human receptor proteins. Mouse brain expression was utilized as positive control. β-Actin levels were used as loading control. E: Northern blot analysis showing the expression of the transgene mRNA in the epiphyses of long bones from transgenic mice but not in the diaphysis. Similar samples from wild-type mice failed to show transcript expression in any of the indicated tissues (data not shown). Kidney expression of PTH2R was examined as a negative control. Lane loading was assessed using Gapdh. F: immunohistochemistry using anti-PTH2R antibody illustrates the more pronounced expression and extensive distribution of the receptor protein in the transgenic growth plate compared with wild type, including cells in the resting (R) and proliferative (P) zones.

Fig. 2.

Radiographic and histological analyses of long bones from transgenic (Tg) mice. A: radiographs of tibiae from 6-wk-old wild-type and Tg litter mates showing increased radiolucency of the Tg bones. B: comparison of microcomputed tomography-scanned sections through the tibiae from wild-type and Col2a1-hPTH2R Tg littermates illustrating the decrease in trabecular bone volume in the Tg specimens. C: quantitative histomorphometric analysis showing the decrease in total bone volume over total tissue volume (BV/TV), trabecular thickness, and trabecular number and the increase in trabecular separation in the Tg bones. Cortical thickness and porosity were not different in Tg samples. Each value is the mean ± SE of determinations in 6 animals from each group. *P < 0.05; **P < 0.01 relative to wild-type mice. D: von Kossa-stained calcified sections illustrating the decrease in mineralized bone content in the Tg sections.

Fig. 3.

Impaired chondrocyte proliferation in Col2a1-hPTH2R Tg growth plates. A: bromodeoxyuridine (BrdU) staining of growth plate chondrocytes (A) and quantitative assessment of BrdU staining index (B) showing a decrease in the number of BrdU-positive cells in the Tg sections. Data shown represent means ± SE of 4 animals/group. *P < 0.05 relative to wild-type (WT) littermate sections C: immunohistochemical staining of decalcified paraffin-embedded sections of growth plates for cyclin-dependent kinase 4 (CDK4; arrows). D: Western blot analysis and relative quantitation (Syngene Bioimaging system) of CDK4 protein in wild-type and Tg growth plates. β-Actin levels were used as lane loading control. E: immunohistochemical staining of decalcified paraffin-embedded sections of growth plates for p21 (arrows).

Fig. 4.

Analyses of collagen markers in Tg long bones. A: detection of Sox9 by in situ hybridization. B: immunostaining of WT and Col2a1-hPTH2R long bone sections for type II collagen (Col II), type X collagen (Col X), proteoglycans (PG), and aggrecan (ACAN), the major proteoglycan.

Fig. 5.

Delayed formation of secondary ossification center in Col2a1-hPTH2R mice. A: sequential temporal analysis (P6 to P10) of paraffin-embedded sections of bones from WT (top) and Tg littermates (bottom) for the development of the secondary SOC. *Presence of hypertrophic chondrocytes and the beginning of the secondary ossification center formation. B: comparative secondary ossification center development in WT and Tg growth plates at P9 and P10.

Fig. 6.

Altered expression of chondrocyte differentiation markers in Tg growth plates. Immunohistochemical staining for Gdf5 (A) and Wdr5 (B) in long bones from P10 mice (arrows). SA, subarticular region; SOC, secondary ossification center; R, resting zone. C: Western blot analyses substantiating the decrease in Wdr5 protein expression in Tg growth plates. D: PTH2R-transfected CFK2 chondrocytic cells recapitulate the decrease in Wdr5 expression following treatment with tuberoinfundibular peptide of 39 residues (TIP39; 10⁻⁷ M). E: Western blot analyses illustrating the increased Mmp13 protein expression in Tg growth plates. F: β-catenin expression in the SOC in growth plates of long bones from Col2a1-hPTH2R (P9) and WT (P8) littermates.

Fig. 7.

Decreased osteoblast number and function in Tg bones. A: decalcified paraffin bone sections stained histochemically for tartrate-resistant acid phosphatase (TRAP) activity in osteoclasts. B: micrographs from decalcified paraffin sections stained with hematoxylin and eosin (H & E) to assess osteoblast number. C: quantitative assessment by histomorphometric analysis of osteoblast number per millimeter of bone perimeter (N.Ob/B.Pm). D: double-calcein labeling of WT and Tg trabecular bone. E: quantitative assessment of the mineral apposition rate (MAR; μm/day), a parameter of bone formation. Data shown represent means ± SE of 5–6 animals/group at 4 wk of age; *P < 0.05; **P < 0.01 relative to WT littermate sections.