Alendronate impairs the ability of parathyroid hormone to increase the bone mineral density at the lumbar spine and the femoral neck in men. This effect may be attributable to an attenuation of parathyroid hormone-induced stimulation of bone formation by alendronate.

IntroductionIn bone remodeling the activities of osteoblasts, the bone-forming cells, and osteoclasts, cells of hematopoietic origin capable of resorbing bone, must be balanced carefully in order to maintain skeletal integrity (1). The importance of understanding the factors controlling these activities is highlighted by metabolic bone disorders such as osteoporosis, in which the imbalance of bone formation and resorption leads to bone loss. Parathyroid hormone (PTH), a major regulator of calcium homeostasis, plays an important role in both bone formation and resorption. While PTH excess in hyperparathyroidism (2) and in continuous administration of PTH (3) is characterized by large numbers of osteoclasts, rapid bone turnover, and low cortical bone mass, it has long been known that intermittent dosing of PTH can lead to increased trabecular bone mass (4,5). This anabolic effect is due to increased bone formation (6, 7). Interestingly, histomorphometric studies in patients with mild hyperparathyroidism also show an increase in cancellous bone (2). Although osteoblasts likely mediate both the anabolic and catabolic actions of the peptide, the molecular mechanisms underlying this dual effect are incompletely understood.The PTH/PTH-related protein (PTH/PTHrP) receptor (PPR), a G protein-coupled receptor, is believed to mediate many of the actions of both PTH and PTHrP in bone, as shown by mutations in mice and humans. Mice in which the PPR has been ablated by homologous recombination have decreased trabecular bone and increased thickness of cortical bone during fetal development (8). These skeletal abnormalities are similar to those observed in patients with Blomstrand lethal chondrodysplasia, a rare autosomal recessive disorder caused by inactivating PPR mutations (9, 10). Consistent with this crucial role of the PPR in cells of the osteoblast lineage, expression of the mRNA encoding this receptor is detectable in relatively mature osteoblasts and in adjacent stromal cells likely to be osteoblast precursors (11).Jansen's metaphyseal chondrodysplasia (JMC) is a rare form of short-limbed dwarfism caused by activating mutations of the PPR leading to ligand-independent cAMP accumulation (12). Histomorphometric analysis of bone from a patient with this disorder shows exaggerated loss of cortical bone and preservation, or even augmentation of trabecular bone, as is seen in mild primary hyperparathyroidism (13). Parathyroid hormone (PTH), an important regulator of calcium homeostasis, targets most of its complex actions in bone to cells of the osteoblast lineage. Furthermore, PTH is known to stimulate osteoclastogenesis indirectly through activation of osteoblastic cells. To assess the role of the PTH/PTHrelated protein receptor (PPR) in mediating the diverse actions of PTH on bone in vivo, we generated mice that express, in cells of the osteoblastic lineage, one of the constitutively active receptors described in Jansen's metaphyseal chondrodysplasia. In these transgenic mice, osteoblastic function was increased in the t...

Mice in which the genes encoding the parathyroid hormone (PTH)-related peptide (PTHrP) or the PTH͞ PTHrP receptor have been ablated by homologous recombination show skeletal dysplasia due to accelerated endochondral bone formation, and die at birth or in utero, respectively. Skeletal abnormalities due to decelerated chondrocyte maturation are observed in transgenic mice where PTHrP expression is targeted to the growth plate, and in patients with Jansen metaphyseal chondrodysplasia, a rare genetic disorder caused by constitutively active PTH͞PTHrP receptors. These and other findings thus indicate that PTHrP and its receptor are essential for chondrocyte differentiation. To further explore the role of the PTH͞PTHrP receptor in this process, we generated transgenic mice in which expression of a constitutively active receptor, HKrk-H223R, was targeted to the growth plate by the rat ␣1 (II) collagen promoter. Two major goals were pursued: (i) to investigate how constitutively active PTH͞PTHrP receptors affect the program of chondrocyte maturation; and (ii) to determine whether expression of the mutant receptor would correct the severe growth plate abnormalities of PTHrP-ablated mice (PTHrP؊͞؊). The targeted expression of constitutively active PTH͞PTHrP receptors led to delayed mineralization, decelerated conversion of proliferative chondrocytes into hypertrophic cells in skeletal segments that are formed by the endochondral process, and prolonged presence of hypertrophic chondrocytes with delay of vascular invasion. Furthermore, it corrected at birth the growth plate abnormalities of PTHrP؊͞؊ mice and allowed their prolonged survival. ''Rescued'' animals lacked tooth eruption and showed premature epiphyseal closure, indicating that both processes involve PTHrP. These findings suggest that rescued PTHrP؊͞؊ mice may gain considerable importance for studying the diverse, possibly tissue-specific role(s) of PTHrP in postnatal development.

To determine the role of PTHrP in fetal calcium metabolism, blood calcium was measured in mice homozygous (HOM) 45 Ca accumulation in HOM P THrP-ablated fetuses, but P THrP-(1-34), PTH-(1-84), and the diluent had no effect. Finally, similar studies were performed on fetal mice that lacked the PTH͞PTHrP receptor gene. Ionized calcium was significantly reduced in HOM PTH͞PTHrP receptor-ablated fetuses. However, 5 min after maternal injection of 45 Ca and 51 Cr, relative accumulation of 45 Ca was significantly increased in these fetuses. It was concluded that PTHrP is an important regulator of fetal blood calcium and placental calcium transport. In addition, the bioactivity of PTHrP for placental calcium transport is specified by a mid-molecular region that does not use the PTH͞PTHrP receptor.

The calcium-sensing receptor (CaSR) regulates PTH secretion to control the extracellular calcium concentration in adults, but its role in fetal life is unknown. We used CaSR gene knockout mice to investigate the role of the CaSR in regulating fetal calcium metabolism. The normal calcium concentration in fetal blood is raised above the maternal level, an increase that depends upon PTH-related peptide (PTHrP). Heterozygous (+/-) and homozygous (-/-) disruption of the CaSR caused a further increase in the fetal calcium level. This increase was modestly blunted by concomitant disruption of the PTHrP gene and completely reversed by disruption of the PTH/ PTHrP receptor gene. Serum levels of PTH and 1, 25-dihydroxyvitamin D were substantially increased above the normal low fetal levels by disruption of the CaSR. The free deoxypyridinoline level was increased in the amniotic fluid (urine) of CaSR-/- fetuses; this result suggests that fetal bone resorption is increased. Placental calcium transfer was reduced, and renal calcium excretion was increased, by disruption of the CaSR. These studies indicate that the CaSR normally suppresses PTH secretion in the presence of the normal raised (and PTHrP-dependent) fetal calcium level. Disruption of the CaSR causes fetal hyperparathyroidism and hypercalcemia, with additional effects on placental calcium transfer.

Indian hedgehog (Ihh) and Parathyroid Hormone-related Protein (PTHrP) play a critical role in the morphogenesis of the vertebrate skeleton. Targeted deletion of Ihh results in short-limbed dwarfism, with decreased chondrocyte proliferation and extensive hypertrophy, features shared by mutants in PTHrP and its receptor. Activation of Ihh signaling upregulates PTHrP at the articular surface and prevents chondrocyte hypertrophy in wild-type but not PTHrP null explants, suggesting that Ihh acts through PTHrP. To investigate the relationship between these factors during development of the appendicular skeleton, mice were produced with various combinations of an Ihh null mutation (Ihh(−/−)), a PTHrP null mutation (PTHrP(−/−)), and a constitutively active PTHrP/Parathyroid hormone Receptor expressed under the control of the Collagen II promoter (PTHrPR*). PTHrPR* rescues PTHrP(−/−) embryos, demonstrating this construct can completely compensate for PTHrP signalling. At 18.5 dpc, limb skeletons of Ihh, PTHrP compound mutants were identical to Ihh single mutants suggesting Ihh is necessary for PTHrP function. Expression of PTHrPR* in chondrocytes of Ihh(−/−) mice prevented premature chondrocyte hypertrophy but did not rescue either the short-limbed dwarfism or decreased chondrocyte proliferation. These experiments demonstrate that the molecular mechanism that prevents chondrocyte hypertrophy is distinct from that which drives proliferation. Ihh positively regulates PTHrP, which is sufficient to prevent chondrocyte hypertrophy and maintain a normal domain of cells competent to undergo proliferation. In contrast, Ihh is necessary for normal chondrocyte proliferation in a pathway that can not be rescued by PTHrP signaling. This identifies Ihh as a coordinator of skeletal growth and morphogenesis, and refines the role of PTHrP in mediating a subset of Ihh's actions.

Two heterozygous PTH/PTH-related peptide (PTHrP) receptor missense mutations were previously identified in patients with Jansen's metaphyseal chondrodysplasia (JMC), a rare form of short limb dwarfism associated with hypercalcemia and normal or undetectable levels of PTH and PTHrP. Both mutations, H223R and T410P, resulted in constitutive activation of the cAMP signaling pathway and provided a plausible explanation for the abnormalities in skeletal development and mineral ion homeostasis. In the present study we analyzed genomic DNA from four additional sporadic cases with JMC to search for novel activating mutations in the PTH/PTHrP receptor, to determine the frequency of the two previously identified missense mutations, H223R and T410P, and to determine whether different mutations present with different severity of the disease. The H223R mutation was identified in three novel JMC patients and is, therefore, to date the most frequent cause of JMC. In the fourth patient, a novel heterozygous missense mutation was found that changes isoleucine 458 in the receptor's seventh membrane-spanning region to arginine (I458R). In COS-7 cells expressing the human PTH/PTHrP receptor with the I458R mutation, basal cAMP accumulation was approximately 8 times higher than that in cells expressing the wild-type receptor despite impaired surface expression of the mutant receptor. Furthermore, the I458R mutant showed higher responsiveness to PTH than the wild-type receptor in its ability to activate both downstream effectors, adenylyl cyclase and phospholipase C. Like the H223R and the T410P mutants, the I458R mutant had no detectable effect on basal inositol phosphate accumulation. Overall, the patient with the I458R mutation exhibited clinical and biochemical abnormalities similar to those in patients with the previously identified H223R and T410P mutations.