However, whether hormones influence bone mass or the rate of bone remodeling via actions on osteocytes has heretofore been unknown. Sclerostin, the product of the Sost gene, is expressed exclusively by osteocytes in bone [13] — [16]. Loss of sclerostin expression in humans results in the high bone mass disorders Van Buchem's disease [17] and sclerosteosis [18] , providing compelling evidence that osteocytes can control bone mass. Moreover, targeted deletion of the Sost gene in mice results in increased bone formation and strength [19] ; and administration of an anti-sclerostin antibody increases bone formation and restores the bone lost after ovariectomy in rodents [20].
Conversely, transgenic mice overexpressing Sost exhibit low bone mass [13] , [21]. Thus, osteocytes exert negative feedback control of osteoblast number and bone formation via production of sclerostin. Abundant evidence from humans and experimental animals indicates that parathyroid hormone PTH increases the rate of bone resorption, and thereby the rate of bone remodeling.
Thus, chronic elevation of PTH levels increases bone resorption [28] , [29] whereas patients with hypoparathyroidism [30] or rodents lacking PTH [31] , [32] exhibit reduced bone resorption. Consistent with this notion, removal of the PTH responsive region of the RANKL gene is sufficient to reduce the rate of bone resorption, mimicking the effects of hypoparathyroidism [37].
Chronic PTH elevation also increases osteoblast number and bone formation. This may occur indirectly through stimulation of bone resorption which releases growth factors embedded in the bone matrix and these in turn promote osteoblast formation [38]. Intermittent administration of PTH as a therapy to induce bone anabolism also increases osteoblast number, but the mechanisms are thought to be different from those involved with chronic PTH elevation [29] , [39].
Chronic PTH elevation, as in hyperparathyroidism, causes loss of cortical bone. Cancellous bone is also lost with secondary hyperparathyroidism caused by dietary calcium deficiency, but it is preserved or even increased in primary hyperparathyroidism or with activating mutations of PTHR1 [40] , [41]. Thus, PTH always increases the rate of bone remodeling, but it can result in either loss or gain of cancellous bone mass depending on the balance between resorption and formation.
The mechanisms underlying these different outcomes of PTH action are unknown. Recent genetic studies in mice demonstrate that PTHrP also acts postnatally to control bone mass [43] , [44]. Thus, mice with PTHrP haploinsufficiency, or with deletion of the PTHrP gene specifically from osteoblasts, exhibit reduced bone formation due to increased osteoblast apoptosis. In addition, the number of osteoclasts is reduced in these animals most likely because of reduced RANKL expression.
We and others have recently determined that PTH inhibits the expression of the osteocyte specific gene Sost, raising the possibility that the hormone stimulates bone formation via direct actions on osteocytes [15] , [46]. Consistent with this idea, expression of PTHR1 has been demonstrated in osteocytes [47]. Here we demonstrate that activation of PTHR1 signaling exclusively in osteocytes in transgenic mice is sufficient to decrease sclerostin expression, increase Wnt signaling, and increase bone mass.
Unexpectedly, PTHR1 activation in osteocytes also accelerates the rate of bone remodeling. Remarkably, deletion of the Wnt co-receptor LRP5 attenuates the high bone mass phenotype induced by the transgene, but does not affect the increased remodeling.
Thus, PTH signaling in osteocytes stimulates the accrual of bone mass and increases the rate of bone remodeling by LRP5-dependent and -independent mechanisms, respectively. To determine whether hormonal signaling in osteocytes is sufficient to alter skeletal homeostasis, we generated transgenic mice expressing a constitutively active form of human PTHR1 [48] specifically in osteocytes using the dentin matrix protein-1 DMP1 promoter DMP1-caPTHR1 mice Figure 1A.
This promoter was previously shown to direct osteocyte-specific expression of transgenes in mice [5] , [49] — [52]. Arrows indicate the sites recognized by primers used for genotyping. DMP1-caPTHR1 transgenic mice were born at the expected Mendelian frequency, were fertile, exhibited normal size and weight Figure S1B , but displayed increased radiodensity in regions of the skull and long bones at 4 weeks of age Figure 2A.
Serial measurement of bone mineral density BMD by dual-energy x-ray absorptiometry DEXA revealed a remarkable and progressive increase in bone mass in the appendicular and axial skeleton in transgenic mice of both sexes compared to wild type littermates Figure 2B and S1C. Increased bone mass in the transgenic mice was maintained until at least 8 months of age Figure S1D. Micro-CT of femur and vertebra of week-old transgenic mice confirmed the high bone mass phenotype Figure 2C.
Cross-sectional area of the femur at both the distal metaphysis and midshaft was elevated by approximately 2-fold. Increased bone was also observed in the calvaria, a bone that is formed by intramembranous ossification Figure 2C. WT mice at each time point. C Representative longitudinal and cross-sectional micro-CT images of femur, 4th lumbar vertebra, and calvaria obtained from D Hematoxylin and eosin staining of tibial and E von Kossa staining of femoral bone sections from To determine the cellular basis of the increased bone mass, we performed histomorphometric measurements of cancellous bone of the distal femur in 3.
This age was selected for histomorphometric analysis because, in contrast to mice at older ages, a large amount of cancellous bone surface was available for analysis and the cellular processes leading to increased bone mass must have still been occurring.
Cancellous bone area and trabecular width were increased in femoral bone of the transgenic mice Figure 3A and B , and Table S1 ; however, trabecular spacing and number were not significantly different from wild type littermates. Both osteoblast and osteoclast perimeters were elevated by more than 2-fold in transgenic mice, whereas the number of osteocytes per cancellous bone area osteocyte density was unchanged Figure 3C. This increase in the numbers of osteoclasts and osteoblasts, and thus the rate of bone remodeling, was reflected by a decrease in quiescent surface Figure 3D.
Therefore, quantification of the rate of bone formation was not possible. The presence of diffuse labels could be due to rapid bone formation leading to woven bone, which was indeed revealed by polarized light microscopy Figure 3E. However, abnormal mineralization cannot be excluded since osteoid surface was also elevated in the transgenic mice Table S1. Nevertheless, and consistent with the elevated osteoblast number and expected high bone formation rate, circulating osteocalcin was elevated in DMP1-caPTHR1 mice Figure 3F.
Moreover, in line with the increase in osteoclast perimeter, plasma levels of carboxy-terminal crosslinked telopeptide of type I collagen CTX and urinary levels of deoxypyridinoline DPD , markers of bone resorption, were also elevated in the transgenic mice Figure 3F. No significant changes were found in circulating levels of calcium, inorganic phosphate, or PTH Figure S4 , measured as indicated in the Supplementary Methods Text S1 section.
The increase in osteoblast number induced by the transgene was associated with a decrease in the prevalence of osteoblast apoptosis Figure 3G. In contrast, osteocyte survival was not affected Figure 3G. These results indicate that PTHR1 activation in osteocytes leads to an increased rate of bone remodeling with a positive balance resulting in more bone, associated with reduced prevalence of osteoblast apoptosis.
A—D Histomorphometric measurements were determined in the distal femur, excluding the growth plate, of 3. Both sexes were included. Bone area A , trabecular architecture B , osteoblast perimeter, osteoclast perimeter, and osteocyte density C , and quiescent surface D are shown. G Osteoblast and osteocyte apoptosis measured in femoral sections from 3.
WT mice. Furthermore, expression of the osteoclast-specific genes cathepsin K, tartrate resistant acid phosphatase TRAPase , and calcitonin receptor was also elevated in the transgenic animals Figure S5C.
Each lane contains protein lysate from a single mouse. C Anti-sclerostin immunohistochemistry in ulnae sections from Micro-CT images also revealed that the transgene was still able to increase cancellous bone in animals lacking LRP5, however this effect was greatly reduced compared to animals expressing LRP5 Figure 5C.
This persistent increase in bone induced by the transgene in animals lacking LRP5 could be due to increased Wnt signaling through LRP6. This phenomenon, as well as the reduced bone material density, may be due to the formation of woven bone [61] , abnormal mineralization [51] , or both. Taken together, these results suggest that activation of the PTH receptor in osteocytes results in two separate effects: one that leads to increased bone mass and that depends in part on LRP5 signaling to favor bone formation, and another that results in increased bone remodeling independent of LRP5 signaling.
The concept that PTH can act directly on osteocytes was proposed in the 's based on evidence that injected radiolabeled-PTH localizes in osteocytes and that the hormone changes osteocyte morphology [62] , [63]. Recently we and others have shown that PTH suppresses the expression of the osteocyte-specific product sclerostin, an antagonist of Wnt signaling [15] , [46]. This evidence, together with the well documented role of Wnt signaling in osteoblastogenesis, suggested a mechanism by which PTH may control the production of osteoblasts through actions on osteocytes.
In the present report, we have addressed the functional consequences of PTH actions in osteocytes by taking advantage of a fragment of the DMP1 promoter that confers osteocyte-specific expression of transgenes [5] , [49] , [52] , [64].
The evidence presented herein demonstrates that PTH receptor signaling exclusively in osteocytes is sufficient to increase osteoblast number and dramatically increase bone mass via down-regulation of sclerostin and thereby elevation of LRP5 signaling.
Remarkably, activation of PTHR1 in osteocytes also caused an increase in bone resorption, and thereby the rate of bone remodeling. Thus, our findings reveal for the first time that osteocytes could be the targets and mediators of the two most important effects of PTH on bone Figure 7. Whether this is the case outside the context of the genetic manipulation used here will await osteocyte-specific deletion of the PTH receptor. In the proposed model, PTHR1 signaling in osteocytes activates at least two distinct pathways: one leading to increased bone mass and the other leading to increased bone remodeling.
PTH can clearly increase bone mass when administered intermittently, and this effect has been attributed by us to a direct and potent anti-apoptotic effect of the hormone on osteoblasts [29] , [39] , [65]. In the present report, we determined that the increased osteoblast number and bone formation caused by PTH signaling exclusively in osteocytes is also associated with decreased osteoblast apoptosis. Attenuation of osteoblast apoptosis in our model can be readily explained by unleashing Wnt signaling secondary to suppression of Sost expression.
Indeed, several studies demonstrate that Wnt signaling increases osteoblast number and bone formation at least in part by inhibiting osteoblast apoptosis. Specifically, the increased bone mass observed in mice deficient in the Wnt antagonist secreted frizzled related protein-1 SFRP-1 , or mice expressing a LRP5 mutant GV unable to bind sclerostin, is associated with decreased osteoblast and osteocyte apoptosis [66] — [68].
We developed a mathematical model that accounts for net bone loss with continuous PTH administration and net bone formation with intermittent PTH administration, based on the differential effects of PTH on the osteoblastic and osteoclastic populations of cells. Bone, being a major reservoir of body calcium, is under the hormonal control of PTH. The overall effect of PTH is to raise plasma levels of calcium, partly through bone resorption.
Osteoclasts resorb bone and liberate calcium, but they lack receptors for PTH. The preosteoblastic precursors and preosteoblasts possess receptors for PTH, upon which the hormone induces differentiation from the precursor to preosteoblast and from the preosteoblast to the osteoblast. The osteoblasts generate IL-6; IL-6 stimulates preosteoclasts to differentiate into osteoclasts. We developed a mathematical model for the differentiation of osteoblastic and osteoclastic populations in bone, using a delay time of 1 hour for differentiation of preosteoblastic precursors into preosteoblasts and 2 hours for the differentiation of preosteoblasts into osteoblasts.
The ratio of the number of osteoblasts to osteoclasts indicates the net effect of PTH on bone resorption and deposition; the timing of events producing the maximum ratio would induce net bone deposition. Secondary hyperparathyroidism occurs in response to low blood calcium levels and is caused by other mechanisms, for example, kidney disease and vitamin D deficiency.
Mild primary hyperparathyroidism often causes few if any symptoms and is frequently diagnosed by finding a high calcium concentration on a routine blood test. Treatment may be by surgical removal of the affected gland s parathyroidectomy. Further information on the symptoms for each condition can be found in the individual articles.
Too little parathyroid hormone or hypoparathyroidism , is a rare medical condition. It can result in low levels of calcium in the blood hypocalcaemia. It is usually treated medically with oral calcium and vitamin D analogues but the availability of parathyroid hormone replacement therapy may change the approach to treatment for some patients. About Contact Events News.
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