Parathyroid hormone is essential for normal fetal bone formation (original) (raw)
Effects of gene deletion on PTH and PTHrP production. Using Southern blot analysis of genomic DNA, we first identified mice lacking expression of PTH and/or PTHrP amongst the newborn offspring. The expected bands of 6.2 kb and 1.0 kb were observed for wild-type PTH and wild-type PTHrP alleles, respectively, and expected bands of 4.0 kb and 1.8 kb were observed for the targeted PTH (9) and PTHrP alleles (10), respectively (Figure 1a).
Genotyping of mice and examination of the expression of PTH and PTHrP. (a) Analysis of genomic DNA isolated from pups born of PTH–/– and PTHrP+/– matings. For Southern blots, purified DNA was digested with BamHI for PTH and with PvuII for PTHrP. The mutated PTH allele (4.0 kb) and PTHrP allele (1.8 kb) are distinguished from the wild-type PTH (6.2 kb) and PTHrP (1.0 kb) alleles using the probes described in Methods. (b–d) Thyroid and parathyroid gland paraffin-embedded sections from wild-type (WT), PTH–/–; PTHrP–/–; and PTH–/–, PTHrP–/– mice were stained with H&E (b) and immunostained for PTH (c) and CaSR (d). Paraffin-embedded sections of tibial epiphyseal cartilage were immunostained for PTHrP (e). Scale bars represent 50 μm.
We next investigated the effect of PTH and PTHrP gene deletion on the size and function of the parathyroid glands (Figure 1, b–d). Parathyroid glands were greatly enlarged in the PTH–/– mice and were moderately enlarged in PTHrP–/– mice, but were most dramatically enlarged in the PTH–/–, PTHrP–/– mice compared with the wild-type mice (Figure 1b). In wild-type mice, the parathyroid glands expressed both PTH and the CaSR, which detects extracellular fluid calcium levels (17) (Figure 1, c and d). PTH expression was absent in the parathyroids of the two PTH-deficient mouse models but was detectable in the PTHrP–/– mice (Figure 1c). Enlarged parathyroid glands were therefore present in all three models of targeted gene ablation and expressed substantial CaSR, a moiety reported to be functional in fetal life (18). This supports the view that ambient hypocalcemia existed in the mutants and stimulated parathyroid growth. Indeed, in view of the fact that PTHrP is required for maintaining the transplacental calcium flux needed for fetal calcium homeostasis, elevated blood PTH levels have previously been postulated to be increased in PTHrP–/– mice (8).
We next examined PTHrP production in the mutants (Figure 1e). PTHrP expression in the chondrocytes of the growth plates was equivalent in the PTH–/– mice and wild-type littermates but was not detected in the two PTHrP–/– mouse models (Figure 1e). The absence of both PTHrP and PTH in the compound mutant led to the greatest enlargement of the parathyroid glands (Figure 1, b–d), consistent with the absence of two calcium-regulating entities (19).
Skeletal phenotypes and alterations of the cartilaginous growth plate. In PTH–/– mice, compared with wild-type mice, the skull was abnormally formed and mineralization of the bones of the skull was enhanced. The vertebral column was also abnormal, with evidence of smaller vertebral bodies, and mineralized metacarpal and metatarsal bones were shorter (Figure 2, a versus b). Although the PTH–/– mice were viable, PTHrP–/– and compound mutant mice died at birth, with skeletal malformations including short-limbed dwarfism that were most severe in the compound mutants. The whole skeleton of the compound mutant mouse was smaller than that of the PTH–/– mouse and the PTHrP–/– mouse (Figure 2, d versus b or c), and all elements of the axial and appendicular skeletons were more dramatically reduced in size. In both the PTHrP–/–, PTH–/– mice and PTHrP–/– mice, less cartilage staining relative to calcified skeletal staining was observed than was seen in the wild-type mice (Figure 2, c and d versus a).
From H&E-stained sections (Figure 2, e–h), we could identify regions of proliferating chondrocytes, hypertrophic chondrocytes, and bone tissue in the long bones of all models. The overall lengths of tibiae (Figure 2i) and of proliferating zones (Figure 2j) were normal in the PTH–/– mice, reduced in the PTHrP–/– mice compared with wild-type mice, and reduced even further in the PTH–/–, PTHrP–/– mice (Figure 2, i and j, respectively). The hypertrophic zone was enlarged in all three models compared with the wild-type mice (Figure 2j). The ratio of hypertrophic zone to proliferating zone was high in the PTHrP–/– mice and greatest in the PTH–/–, PTHrP–/– mice.
Proliferation of chondrocytes was not altered in the PTH–/– mice, but was dramatically decreased in the two PTHrP-deficient mouse models compared with wild-type mice (Figure 3, c and d versus a, and Figure 3m). The number of apoptotic chondrocytes was increased fivefold in the two PTHrP-deficient mice compared with wild-type mice (Figure 3, g and h versus e, and Figure 3m), consistent with previous reports (20, 21), but was not changed in the PTH–/– mice (Figure 3, f versus e, and Figure 3m).
Assessment of indices of chondrocyte proliferation, apoptosis, and differentiation. (a–d) Paraffin-embedded sections of tibiae from wild-type; PTH–/–; PTHrP–/–; and PTH–/–, PTHrP–/– mice were immunostained for PCNA as described in Methods. (e–h) Sections were stained for apoptosis using TUNEL. (i–l) Sections were immunostained for type X collagen (Col X). Scale bars in d, h, and l represent 50 μm. (m) The numbers of PCNA-positive chondrocytes, of TUNEL-positive chondrocytes, and of total chondrocytes per field were determined by image analysis; the PCNA-positive and TUNEL-positive percentages of total chondrocytes counted are presented as the mean ± SEM of triplicate determinations. (n) Immunostaining for type X collagen was performed as described in Methods, and the immunopositive area as a percentage of the growth plate field was determined. The percent-positive area is presented as mean ± SEM of triplicate determinations. *P < 0.05 in the mutant mice relative to the wild-type mice.
The deposition of type X collagen in the matrix of the hypertrophic zone was slightly enhanced in the PTH–/– mice (Figure 3, j versus i, and Figure 3n) but was substantially increased in both the PTHrP–/– mice and the PTH–/–, PTHrP–/– mice, in keeping with accelerated rates of differentiation and enlargement of the hypertrophic zones (Figure 3, k and l versus i, and Figure 3n).
Alterations of the chondro-osseous junction. Mineralization of cartilage matrix was enhanced in the PTHrP–/– mice (Figure 4, c versus a, and Figure 4m), but was reduced in the PTH–/– mice (Figure 4, b versus a, and Figure 4m) and in the PTH–/–, PTHrP–/– mice (Figure 4, d versus a, and Figure 4m).
Examination of cartilage matrix mineralization and angiogenesis regulators. (a–d) Undecalcified sections of femurs from wild-type; PTH–/–; PTHrP–/–; and PTH–/–, PTHrP–/– mice were stained with von Kossa stain as described in Methods. (e–h) Paraffin-embedded sections were immunostained for VEGF as described in Methods. (i–l) Sections were immunostained for Ang-1. Scale bars in d, h, and l represent 50 μm. (m) The mineralized area as a percent of the cartilage matrix per field was determined by image analysis as described in Methods and is presented as the mean ± SEM of triplicate determinations. (n) The VEGF-immunopositive area as a percentage of the growth plate field was determined by image analysis and is presented as mean ± SEM of triplicate determinations. (o) The immunopositive area of Ang-1 as a percentage of the growth plate field was determined by image analysis and is presented as the mean ± SEM of triplicate determinations. *P < 0.05, mutant mice relative to wild-type mice. #P < 0.05, compound mutant mice relative to PTH–/– mice or PTHrP–/– mice.
In view of the fact that mRNA encoding VEGF is expressed by hypertrophic chondrocytes in the epiphyseal growth plate and VEGF-dependent blood vessels are essential for coupling cartilage resorption with bone formation (22), we assessed the presence of VEGF by immunostaining. The results confirmed that VEGF was indeed expressed in chondrocytes of the hypertrophic zone in the wild-type mice (Figure 4e) and was also seen in this zone in each of the mutant models (Figure 4, f–h). The VEGF-positive area was significantly increased in all three animal models compared with the wild-type mice (Figure 4n).
Despite abundant VEGF expression, however, vascular invasion in the growth plate was reduced in both the PTH–/– mice and the compound mutants (Figure 2, f and h versus e) but not in the PTHrP–/– animals (Figure 2, g versus e). Ang-1 is an angiogenic regulator that recruits and interacts with periendothelial support cells and is required for blood vessel integrity (23). Ang-1 was detected in chondrocytes in the growth plate of wild-type mice (Figure 4i) and was increased in maturing and hypertrophic chondrocytes of PTHrP–/– mice (Figure 4, k and o), but was diminished in both PTH–/– models (Figure 4, j, l, and o). Consequently, reduced Ang-1 levels correlated with the decreased vascular invasion.
Overall, PTH deficiency in the two PTH–/– models was associated with reductions in both cartilage matrix mineralization and vascular invasion. The opposite was observed in the PTHrP–/– mice, possibly reflecting elevated circulating PTH levels in that model.
Effects on cortical and trabecular bone. The length of bone tissue was reduced in the long bones of PTH–/– mice and PTHrP–/– mice, and was reduced even more dramatically in the PTH–/–, PTHrP–/– mice compared with the wild-type mice (Figure 2, f versus e; g versus e; and h versus e, respectively; and Figure 2k). The cortical thickness of long bones was increased in all three mutant models compared with that in wild-type mice (Figure 5, a–d and m). In contrast, trabecular bone volume, although somewhat increased in the PTHrP–/– mice, was diminished in the PTH–/– mice, and was even more dramatically diminished in the PTH–/–, PTHrP–/– mice compared with the wild-type mice (Figure 5, a–d and n).
Quantitation of bone parameters. (a–d) Undecalcified sections of femurs stained with von Kossa stain as described in Methods and photographed at a magnification of 25. T, trabecular bone (red square). C, cortical bone (arrows). (e–h) Primary spongiosa from wild-type; PTH–/–; PTHrP–/–; and PTH–/–, PTHrP–/– mice stained with H&E. (i–l) Diaphyseal region from wild-type and mutant mice stained with H&E. (m and n) The cortical thickness and trabecular bone volume were measured from three each of the wild-type; PTH–/–; PTHrP–/–; and PTH–/–, PTHrP–/– mice and presented as mean ± SEM. (o) The number of osteoblasts per field were counted in triplicate in the primary spongiosa of H&E-stained femurs of the mice and presented as mean ± SEM. *P < 0.05, mutant mice relative to wild-type mice. #P < 0.05, compound mutant mice relative to PTH–/– mice or PTHrP–/– mice.
Decreased osteoblast numbers were found in the primary spongiosa of the PTH–/– mice and the PTH–/–, PTHrP–/– mice, whereas osteoblast numbers were increased in the primary spongiosa of the PTHrP–/– mice, possibly reflecting secondary hyperparathyroidism (Figure 5, e–h and Figure 5o). In the endosteum, changes in osteoblast numbers were similar to those in the primary spongiosa (Figure 5, i–l). In contrast, periosteal cells were not altered significantly in any of the three models compared with wild-type mice (Figure 5, i–l). Consequently, reduced osteoblast numbers in the primary spongiosa appeared to account for the reduced trabecular bone volume in the two PTH–/– models, and PTH appears to be essential for optimal osteoblast production in this region of fetal trabecular bone.
Nitric oxide has been implicated in the local regulation of skeletal metabolism, and mice with ablation of the gene encoding eNOS develop osteoblast defects and reduced bone formation (24). We therefore examined the expression of eNOS in chondrocytes and osteoblasts from the gene knockout animals. In wild-type mice, eNOS was expressed at high levels in osteoblasts in the metaphysis and was also detected in some hypertrophic chondrocytes. The expression of this enzyme in both chondrocytes and metaphyseal osteoblasts was enhanced in the PTHrP–/– mice, but was reduced in both the PTH–/– mice and the PTH–/–, PTHrP–/– mice (Figure 6, a–d and m). Whether this reduction is a consequence of PTH deficiency that contributes to diminished osteoblast production or is simply a reflection of decreased osteoblast numbers remains to be determined.
We found increased levels of apoptosis in osteoblasts and osteocytes in the endosteum of all three models compared with those in wild-type mice (Figure 6, e–h and n). Consequently, both PTH and PTHrP appear to protect osteoblasts from apoptosis.
Using histochemical staining for tartrate-resistant acid phosphatase (TRAP) (Figure 6, i–l) and subsequent image analysis, we found that the number (Figure 6o) and size (Figure 6p) of osteoclasts were decreased in all three mutant models compared with those in wild-type mice, but were most dramatically reduced in the compound homozygous mice. Consequently, diminished bone resorption in the absence of either PTH or PTHrP appears to explain the increased cortical thickness observed in all three models.