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Calcium Research Laboratory and Department of Medicine (D.M., G.N.H., D.G.), McGill University Health Centre and Royal Victoria Hospital, McGill University, Montreal, Canada H3A 1A1; Department of Medicine and Lady Davis Institute for Medical Research (B.H., X.-Y.B., X.-K.T., A.C.K.), Sir Mortimer B. Davis-Jewish General Hospital, McGill University, Montreal, Canada H3T 1E2; and Department of Oral and Developmental Biology (B.L.), Forsyth Institute and Harvard School of Dental Medicine, Boston, Massachusetts 02115
Address all correspondence and requests for reprints to: Andrew C. Karaplis, Jewish General Hospital, 3755 Cote Ste Catherine Road, Montreal, Canada H3T 1E2. E-mail: akarapli{at}ldi.jgh.mcgill.ca.
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Abstract |
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-hydroxylase expression increased despite the absence of PTH, leading to a rise in circulating 1,25(OH)2D3 levels, marked osteoclastogenesis, and profound bone resorption. These studies demonstrate the dependence of the skeletal phenotype in animals with genetically depleted PTH on the external environment as well as on internal hormonal and ionic circulatory factors. They also show that, although PTH action is the first defense against hypocalcemia, 1,25(OH)2D3 can be mobilized, even in the absence of PTH, to guard against extreme calcium deficiency. |
Introduction |
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-hydroxylation of 25-hydroxyvitamin D, and cAMP production) reside (3). PTHR1 possesses the unusual property of binding PTH as well as the paracrine factor PTHrP with nearly equal affinity. PTHrP was initially identified as the factor responsible for humoral hypercalcemia in patients with malignancy (4). The capacity of PTHR1 to bind both PTH and PTHrP is based on sequence similarity in the N-terminal portion of these two ligands. Yet, PTHrP is distinct from PTH in many structural features and certain biological effects, particularly in fetal development and physiology. Targeted disruption of either Pthrp (5) or Pthr1 (6) in mice and defective PTHrP/PTHR1 signaling in man (7, 8) leads to a form of lethal skeletal dysplasia characterized by decreased proliferation and accelerated differentiation of growth plate chondrocytes. PTH also interacts with the type 2 PTH receptor, although the natural ligand for this receptor is likely the neuropeptide tuberoinfundibular peptide of 39 residues, rather than PTH itself (9, 10). Characterization of a third PTH receptor with specificity for the carboxyl-terminal region of PTH has also been reported in osteoblasts and osteocytes that presumably exerts an antiresorptive effect on bone by impairing osteoclast differentiation (11, 12). It would seem, therefore, that several distinct properties could be attributed to PTH, likely mediated by a variety of receptors. Whether these nonclassic biological effects of PTH have potential physiological relevance remains to be determined.
To better understand the physiological actions of PTH on skeletal homeostasis, we have generated mice homozygous for a null Pth allele (13). Here, we have examined the consequences associated with PTH deficiency in the postnatal state and the influence dietary calcium has in its absence.
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Materials and Methods |
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Animal experimentation
All animal experiments were reviewed and approved by the institutional animal care committee. The mice were housed in a 12-h light/12-h dark cycle. They were maintained in cages with wooden shavings and had free access to water and either a normal-calcium diet (0.95% calcium, 0.67% phosphorus, 4.5 IU/g vitamin D3; 0.21% Mg, 23.4% protein, 4.5% fat, 5.3% fiber, 6.9% ash; PMI Feeds, Inc., St. Louis, MO) or a low-calcium diet (0.001–0.005% calcium, 0.4% phosphorus, and 2.4 IU/g vitamin D3) in pelleted form for the indicated time period. Weight was measured every month, and food consumption was assessed at 1 and 3 months of age. Fertility in mice was defined as the number of successful pregnancies after visualization of a vaginal plug.
Serum biochemistry
Serum concentrations of calcium and inorganic phosphorus were determined by routine methods using Sigma Diagnostics reagents (Sigma-Aldrich Canada Ltd., Oakville, Ontario, Canada). Serum intact PTH was measured with an ELISA (Immutopics, Inc., San Clemente, CA), whereas serum PTHrP and 1,25-dihydroxyvitamin D3 [1,25(OH)2D3]determinations were performed using commercially available RIA kits (Nichols Institute Diagnostics, San Clemente, CA and Immunodiagnostic Systems, Boldon, UK, respectively).
Skeletal radiographs
The femur was removed and dissected free of soft tissue, and radiographs were taken using a Faxitron model 805 radiographic inspection system (Faxitron X-ray Corp., Wheeling, IL) (22 kV voltage and 4 min exposure time). Kodak X-Omat TL film (Kodak, Rochester, NY) was used and processed routinely.
Histology and histochemistry
Thyroparathyroidal tissue, femurs, tibiae, and vertebrae were removed from 2-month-old mice (Pth+/+ and Pth-/- taken from the same litter) and fixed in PLP fixative (2% paraformaldehyde containing 0.075 M lysine and 0.01 M sodium periodate solution) overnight at 5 C before processing. Occasionally, bones were decalcified in EDTA glycerol solution for 5–7 d at 5 C. Tissue samples were dehydrated and embedded in paraffin, after which 5-µm sections were cut on a rotary microtome. The sections were stained with hematoxylin and eosin, for tartrate-resistant acid phosphatase (TRAP), or immunostained, as described below. Undecalcified bones were embedded in LR White acrylic resin (London Resin Co. Ltd., Theale, UK). Sections of 1 µm were cut on an ultramicrotome and stained for mineral with the von Kossa staining procedure using toluidine blue as counterstain.
Double immunohistochemical staining for type I and II collagen (COL1A1 and COL2A1, respectively) was performed on paraffin-embedded sections of decalcified skeleton using the avidin-biotin-peroxidase complex (ABC) technique. Briefly, sections were first treated with 1% bovine testicular hyaluronidase (Sigma Chemical Co., St. Louis, MO) for 30 min at 37 C to increase antibody penetration and access to epitopes. An affinity-purified goat antihuman type I collagen antibody (Southern Biotechnology Associates, Inc., Birmingham, AL) was applied to sections overnight at room temperature. After washing, the sections were incubated with biotinylated rabbit antigoat IgG (Sigma), washed, and processed using the Vectastain ABC-AP kit (Vector Laboratories, Inc., Burlingame, CA). Red pigmentation to demarcate regions of immunostaining was produced by a 10- to 15-min treatment with Fast Red TR/Naphthol AS-MX phosphate (Sigma; containing 1 mM levamisole as endogenous alkaline phosphatase inhibitor). The sections were then treated with 3% H2O2 as endogenous peroxidase inhibitor for 5 min and incubated with an affinity-purified goat antihuman type II collagen antibody (Southern Biotechnology Associates) overnight at room temperature. After washing, the sections were incubated with biotinylated rabbit antigoat IgG (Sigma), washed, and processed using the Vectastain Elite ABC kit (Vector Laboratories). Gray pigmentation was likewise produced using a Vector SG kit (Vector Laboratories). After washing with distilled water, the sections were counterstained with methyl green and mounted with Kaiser’s glycerol jelly.
Calcein labeling was performed by ip injection with 10 µg calcein/g bodyweight (C-0875; Sigma Chemical Co.) at 10 and 3 d before sacrifice. Bones were harvested and embedded in LR White acrylic resin. Serial sections were cut, and the freshly cut surface of each section was imaged using fluorescence microscopy. The double calcein interlabel width in cortical and trabecular bone was measured using Northern Eclipse v6.0 (Empix Imaging Inc., Mississauga, Ontario, Canada) image software, and the mineral apposition rate (MAR; MAR = interlabel width/labeling period) was calculated.
For immunohistochemistry, paraffin sections of thyroparathyroidal tissue were stained for PTH and calcium-sensing receptor (CaSR) immunoreactivity by the ABC technique using goat serum against PTH (1–34) and mouse anti-CaSR monoclonal antibody, as described (13). Kidney sections were immunostained for 25-hydroxyvitamin D3 1-hydroxylase (Cyp27b1) using purified rabbit antiserum.
Computer-assisted image analysis
Computer-assisted image analysis was performed, as previously described (14). For determining the area of the mineralized and unmineralized matrix and the number and size of osteoclasts in stained bone sections, images of primary spongiosa and cortical bone were digitally recorded using a rectangular template and three different fields. In the primary spongiosa, each image was photographed from the edge of the metaphyseal border of the growth plate (i.e. at the level of the zone of vascular invasion). In cortical bone, images were taken from the diaphyseal bone close to the metaphysis. All digital images were captured with a Sony digital camera. The positive and negative areas staining in trabecular and cortical bone were measured by digital image analysis using Northern Eclipse v6.0 image software.
Northern blot analysis
A cDNA fragment corresponding to nucleotides 421-1474 of mouse Cyp27b1 (GenBank accession no. AB006034) was prepared by RT-PCR of mouse kidney RNA, subcloned, and sequenced. DNA probes for Cyp27b1 and glyceraldehyde-3-phosphate dehydrogenase were prepared by Random Primed DNA Labeling Kit (Roche Molecular Biochemicals, Basel, Switzerland) and [-32P] deoxycytidine triphosphate (800 Ci/mmol; NEN Life Science Products, Boston, MA). Total RNA was isolated from kidney with Tripure Isolation Reagent (Roche), and 20-µg aliquots were fractionated by electrophoresis on a 1% formaldehyde agarose gel, transferred to nitrocellulose membranes, and hybridized to the radiolabeled cDNA fragments (48% formamide, 10% dextran sulfate, 5x sodium chloride/sodium citrate, 1x Denhardt’s, and 100 µg/ml salmon sperm DNA) at 42 C overnight. The membranes were washed, and autoradiograms were prepared using Kodak BioMax film (Kodak) at -80 C with intensifying screens. Quantification of signal intensity on autoradiograms was performed by Molecular Dynamics Personal Densitometer (Amersham Biosciences, Piscataway, NJ) using ImageQuant software (Amersham Biosciences).
Statistical analysis
Data from biochemical and image analyses are presented as means ± SEM. Statistical comparisons were made using the Student’s t test, with P < 0.05 being considered significant.
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Results |
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). Despite abnormalities in mineral homeostasis, mice grew normally (similar food consumption and weight gain) and were fertile, although females were much less so than wild-type littermates (
50–60% success rate).
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). PTH immunoreactivity was absent in the homozygous Pth-null glands, thereby confirming the successful targeted disruption of the Pth gene. In contrast, expression of the CaSR protein was not markedly altered.
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Because PTH has major effects on bone remodeling, we studied the skeletons of Pth-/- animals fed a normal-calcium diet at 2, 4, 6, and 9 months of age. All data reported here are from 4-month-old animals, which are representative. Radiographically, bones from mutant mice were of normal shape and size, although there was clear evidence for increased trabecular bone content (Fig. 3A). Histologically, cartilage development at the growth plate was typical (Fig. 3B), with proper zone organization and adequate mineralization, suggesting that, in the postnatal state, PTH does not play a major role in chondrocyte biology (13). However, major bony alterations were observed in the mutant animals. Trabecular and cortical bone volume was increased (1.8-fold) in these mice compared with sex-matched littermates, as assessed by static histomorphometry (Fig. 3, C and D).
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, A and B). Endosteal and trabecular MAR, a parameter of bone formation, was profoundly decreased in the mutant mice compared with normal littermates, and osteoblasts lining bone surfaces were also diminished (Fig. 4C). Additionally, the number of osteoclasts was reduced (50%) in the Pth-null animals (Fig. 4D), indicating that loss of PTH is associated with a generalized state of low bone turnover. This was further substantiated by the persistent presence of cartilage remnants in the trabecular bone of the mutant specimen (Fig. 4E).
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). Circulating levels of PTHrP also remained unchanged. In contrast, serum 1,25(OH)2D3 levels rose markedly in both groups after institution of a low-calcium diet (4.9-fold in wild-type vs. 6.0-fold in mutant mice), indicating that mechanisms had been set in motion in the Pth-null animals to overcome the absence of the stimulatory effect of PTH on vitamin D synthesis. This increase was reflected, in part, by a rise in renal proximal tubule Cyp27b1 transcript levels (Fig. 5B) and protein immunoreactivity (Fig. 5C), the former being more pronounced in wild-type animals likely due to the prevailing secondary hyperparathyroidism. Interestingly, under these dietary conditions, female Pth-/- mice completely lost their capacity to conceive, whereas the reproductive capability of male animals remained unaffected.
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). Trabecular bone volume in the wild-type lumbar vertebrae was reduced to 54% of the pretreatment content, whereas in the mutant specimens, the corresponding reduction was 29% of the basal level. Cortical bone thickness was unaffected in the wild-type mice but significantly decreased in the Pth-null animals (97% vs. 56% of pretreatment thickness, respectively; Fig. 6B). Because of the changes in bone volume that arose as a consequence of low dietary calcium, we then examined TRAP staining (Fig. 6C) and quantified osteoclast number and size in these animals (Fig. 6D). In Pth-/- mice fed the normal-calcium diet, these parameters were decreased compared with the wild-type littermates. However, when animals were moved to a low-calcium diet for 3 d, TRAP staining and the number and size of osteoclasts increased, considerably more so in the Pth-/- than in wild-type mice. These findings imply that a low-calcium diet increases 1,25(OH)2D3 levels in vivo, even in the absence of PTH, and enhances bone resorption for maintenance of calcium homeostasis, but in the process, it can impact negatively on bone mass.
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Discussion |
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Although it is unclear at present whether the observed decreased fertility in the Pth-null female mice is a consequence of the lack of PTH per se, the prevailing hypocalcemia, or the decreased circulating 1,25(OH)2D3 levels, the available experimental evidence supports hypocalcemia. For example, the reproductive dysfunction of the vitamin D receptor-null mice was corrected by a high-calcium diet (16). Interestingly, the impaired reproductive capacity observed in the Pth-/- mice has also been described in women with untreated hypoparathyroidism (17). Although successful pregnancies are reported in patients receiving vitamin D and calcium replacement in this setting, lack of therapy (17) or inadequate therapy (calcium concentration 
7 mg/dl) (18) places the pregnancy at considerable risk.
The skeletal findings in the Pth-null mice are of interest. First, they indicate that, in the postnatal state, PTH does not play a major role in cartilage development or in the development of the primary spongiosa, roles that may be subserved by locally produced PTHrP (5, 13). Second, in the presence of a normal-calcium diet, the absence of PTH in the postnatal state was notable for decreased bone turnover, with resorption being particularly compromised. This leads to the noted marked increases in bone volume. Observations in patients with hypoparathyroidism tend to support these findings. Increased bone mineral density is reported in patients with chronic idiopathic hypoparathyroidism or after surgery for either thyroid cancer or hyperparathyroidism (19, 20). In addition, this condition provides protection against age-related bone loss in postmenopausal women, perhaps due to attenuation of the high-turnover bone loss associated with menopause (21). Although supplementation with vitamin D and calcium may contribute to the increased bone mass in these patients, it would appear that high bone mineral density is a feature of hypoparathyroidism per se because it is also observed in untreated individuals with the disorder (22, 23). Consequently, in the postnatal state, where the maintenance of normal circulating calcium concentration in the organism is to a great extent dependent on more direct access to calcium in the external environment, the function of PTH appears to have evolved to primarily defend against decreases in the ambient calcium. This involves stimulation of Cyp27b1 expression to raise 1,25(OH)2D3 levels and induction of a catabolic action on bone to maintain normocalcemia.
Our findings in the postnatal Pth-/- mice and clinical observations in hypoparathyroid patients raise the possibility that regulation of PTH secretion can provide a novel therapeutic avenue for the treatment of metabolic bone disease. Preliminary studies in animals tend to add credence to this hypothesis because daily transient decreases in PTH levels after administration of the calcimimetic NPS R-568, a CaSR agonist, had an anabolic effect on uremic bones (24, 25) and slowed the rate of bone loss after ovariectomy (26).
Finally, our studies of the Pth-null mice exposed to limiting amounts of calcium in the external environment point to additional mechanisms mobilized to retain circulating calcium levels. On a low-calcium diet, even in the absence of PTH, Cyp27b1 expression in the kidney was increased, circulating 1,25(OH)2D3 concentrations were augmented, bone resorption was enhanced, and the increased bone volume noted in the hypoparathyroid mice on a normal-calcium diet was converted to an osteopenic state. Most likely, limiting amounts of dietary calcium resulted in transient further reduction of the hypocalcemia observed in the hypoparathyroid animals on normal calcium intake. This initiated Cyp27b1 stimulation, increased 1,25(OH)2D3 synthesis, augmented osteoclastogenesis in bone (27), and mobilized calcium stores from bone. Hence, a new steady state was reached in which severe hypocalcemia was re-set to the moderate levels but at the expense of extreme osteopenia. This is consistent with previous reports suggesting that extracellular calcium concentrations can, independently of PTH, regulate Cyp27b1 activity in vivo (28) and in vitro (29). However, our studies suggest that, in the presence of a normal calcium intake, the ensuing moderate hypocalcemia is less effective in enhancing Cyp27b1 expression than in the presence of a reduced calcium intake where more extreme hypocalcemia may transiently exist. Consequently, the first line of defense in stimulating Cyp27b1 transcription and maintaining a normal circulating calcium concentration is augmentation of PTH levels, whereas 1,25(OH)2D3 is directly mobilized, even in the absence of PTH, as hypocalcemia becomes more extreme.
An additional possibility is that intestinal epithelial cells directly play a role in defending against a further fall in calcium when dietary calcium is reduced. It is possible that enterocytes have the capacity to sense the decreasing levels of dietary calcium intake and, in turn, release a signal, perhaps a circulating agent that acts at the kidney to increase Cyp27b1 expression. A concomitant effect of such a factor on the skeleton to directly promote bone resorption cannot be excluded.
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Acknowledgments |
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Footnotes |
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D.M. and B.H. contributed equally to this work.
Abbreviations: ABC, Avidin-biotin-peroxidase complex; CaSR, calcium-sensing receptor; Cyp27b1, 25-hydroxyvitamin D 1 -hydroxylase; 1,25(OH)2D3, 1,25-dihydroxyvitamin D3; MAR, mineral apposition rate; PTHR1, type 1 PTH receptor; TRAP, tartrate-resistant acid phosphatase.
Received August 22, 2003.
Accepted for publication December 12, 2003.
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References |
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