These studies examined the role of the osteoblast CaR in the regulation of bone development and remodeling in conditional CaR knockout mice, which avoid the severe metabolic complications of prior global CaR knockout models. In our model of conditional CaR knockout, the receptor was deleted broadly across the osteoblast lineage including preosteoblasts and proliferating, differentiating, and mature osteoblasts, as well as osteocytes -- all cells that express the Col 3.6 promoter (17
). Markedly decreased CaR protein expression was evident in osteoblasts and osteocytes in CaR obKO mice. The most striking characteristic of the CaR obKO mouse phenotype was its early lethality. Considering that prevention of hyperparathyroidism in “global knockouts” of the CaR by two different strategies rescues their phenotype (9
), this was unexpected. Osteoblast-specific knockouts of the CaR (present study and reference 16
) further support the idea that the skeletal phenotype in the “global CaR knockouts” is primarily due to severe hyperparathyroidism. However, in the “global knockout” model studied previously, insight into the functionality of the osteoblast CaR is masked by the compensatory actions of the alternatively-spliced CaR, which in the skeleton, if not in the parathyroid gland, appears sufficient to mediate calcium responsiveness. As previously observed by our group using other promoter-driven Cre transgenic mice (2.3 Col 1 promoter) (16
), the expression of the full-length CaR in cells expressing type 1 collagen is essential for postnatal survival and normal skeletal development.
We studied the development of the skeletal phenotype in the first 3 weeks of life in the CaR obKO mice. The Col 3.6-Cre recombinase construct acts from embryonic day E18 (17
). We observed slightly but significantly reduced body weight and expression of osteocalcin
mRNA at 1 day of age. The phenotype progressively worsened and was hallmarked by retarded skeletal development and growth. From the second week of life, knockout mice suffered long bone fractures. Difficulties with ambulation may have contributed to their undernourishment, although the subtle changes observed in the lungs and kidneys may also have restricted development leading to their early deaths. However, renal function, as reflected by serum creatinine and electrolyte levels, was comparable to control mice and did not point to overt kidney pathology. We did note that serum calcium and or PTH values in both heterozygous and homozygous CaR obKO mice were slightly higher than control mice, as noted above. Although some of the differences were statistically significant, the pattern of changes in serum calcium and PTH did not support primary hyperparathyroidism or a gene dosage effect of CaR deletion on parathyroid funciton. Serum calcium and PTH alterations could be contributing to skeletal abnormalities in CaR obKO mice, but these alterations are unlikely to explain the bone phenotype of the CaR obKO. The heterozygous conditional parathyroid CaR KO’s previously reported (16
) have PTH levels 2-fold higher than control mice and similar to the PTH levels in heterozygous and homozygous obCaR KO. Yet bone of heterozygous conditional parathyroid CaR KO’s was only mildly osteopenic during the first 6 months of life and showed none of the dramatic alterations in mineralization that we saw in the homozygous obCaR KO’s reported herein.
These observations, together with a comparable skeletal phenotype development in mice in which the osteoblast CaR knockout is directed by the Col 2.3 promoter (16
), lead us to conclude the CaR obKO phenotype is primarily due to an osteoblast defect, although it could have been further exacerbated by a reduction of the CaR expression in lungs, kidneys, and even parathyroid glands. Further detailed examination of extraskeletal tissues, which is outside the scope of the present study, will offer moreinsight into the effects of CaR knockout in such tissues.
The most striking skeletal finding beyond growth retardation was impaired mineralization in CaR obKO mice, and it was evident early. Histological evaluation identified excess osteoid accumulation, delayed development of secondary ossification centers, and impaired mineralization of the extracellular matrix. μCT studies showed reduced mineral content of trabecular and cortical bone, decreased bone volume, and a deterioration in trabecular architecture, reflected in decreased trabecular connectivity density, leading to severe osteoporosis. Similar changes were also evident in the vertebrae. Further studies of fluorescently labeled mineralizing surfaces showed delayed and disorganized mineralization of trabecular and cortical bone, confirming problems in skeletal development in CaR obKOs. Finally, lack of periosteal labeling of cortical bone is consistent with delayed cortical bone growth (33
). These changes confirm and substantially extend the observations we reported for mice in which the CaR was knocked-out in cells expressing Col 2.3 kb promoter (16
Our findings in vitro
support the involvement of the CaR in the regulation of bone formation (3
). Immature osteoblasts are unable to form a mineralized extracellular matrix, a phenomenon we observed when we inhibited CaR activity in vitro
). These studies also showed that the functional CaR is essential for osteoblast differentiation and production of mineralized matrix in vivo
. Observations that support this include the following. (1
) Expression levels of markers of osteoblastic differentiation (collagen I, osteocalcin, sclerostin
) are significantly reduced in skeletal tissues of CaR obKO mice. (2
) Absence of the CaR resulted in changes in both the morphology of osteoblasts and a reduction in the percentage of trabecular bone surfaces lined by osteoblasts. (3
) Cultured calvarial osteoblasts recapitulated our findings in vivo.
Cells isolated from the knockouts had an immature differentiation profile, evident by both reduced expression of osteoblast and osteocyte markers and an impaired ability to form mineralized nodules. Proliferation in these cultures was unaffected. Taken together, we propose that the inability of osteoblasts to reach full maturity results in their defective mineralizing capacity and that CaR is required for this differentiation to occur.
Although expression of osteocyte molecules sclerostin and DMP-1 is decreased in CaR obKOs, it is unclear whether this is secondary to the retarded osteoblast differentiation resulting in reduced numbers of osteocytes, or a direct consequence of absent CaR signaling in osteocytes. Osteocyte-specific CaR knockouts will directly address this question in the future studies.
We investigated the molecular mechanism responsible for the mineralization defect in CaR obKO mice. In the bones of CaR obKO mice, we detected elevated expression of NPP1
, membrane proteins that regulate mineralization in a complex but highly integrated fashion (29
). On the other hand, expression of alkaline phosphatase
, an enzyme thought to stimulate mineralization, was not significantly affected. Such directional alterations in these key mediators are predicted to result in an overall inhibition of mineralization, which was observed in the CaR obKO mice. Alkaline phosphatase, NPP1 and ANK coordinately regulate levels of OPN, the levels of which are decreased in alkaline phosphatase
knockouts and increased in mice with inactivated NPP1
). Among its many functions, OPN is thought to inhibit mineral growth directly (34
). We confirmed elevated OPN
expression in the CaR obKO model, both in vivo
and in cultures of calvarial osteoblasts in vitro
, and propose that the reduced mineralization, at least in part, results from its inhibitory effects on crystal growth. Further work, using double CaR/OPN, CaR/ANK and CaR/NPP1 knockouts, will be required to dissect out the contribution of each of these molecules to the mineralization defects in these mice. As well, direct measurements of alkaline phosphatase enzymatic activity in tissue and cultured osteoblasts will be informative as to the meaning of the PCR results.
Another mechanism that may fuel the development of osteopenia in CaR obKOs is altered control of bone resorption. We observed increased osteoclast numbers and activity, reflected in the % erosion surface in CaR obKO mice. Resorption is regulated by the interplay of a large number of local autocrine and paracrine factors. The most critical local factors are RANK-L, a stimulator of osteoclast formation and activity, and OPG, its antagonist. Both are produced by osteoblasts (31
). In femoral cortices from these mice, RANK-L
mRNA expression was increased, indicating that lack of CaR signaling in osteoblasts affects this important local stimulator of remodeling. Over time, the increase in RANK-L/OPG ratio would promote bone loss and osteopenia (36
). Furthermore, such changes could be exacerbated by the increased levels of OPN
that we observed in bone tissue and in cultured calvarial osteoblasts isolated from the knockout animals, as OPN has been shown to stimulate osteoclast maturation and bone resorption [reviewed in (37
)]. The increase in cortical porosity shown in CaR obKO mice may be caused or exacerbated by the elevated osteoclast activity, in concert with the mineralization defect.
To confirm that absence of the osteoblast CaR is responsible for changes in osteoclast numbers and activity that we observed in vivo
, we explored the osteogenic potential of the CaR obKO osteoblasts in vitro
. Cultured osteoblasts isolated from the knockouts exhibited an elevated osteoclastogenic potential, stimulating the development of up to 5-fold more TRAP-positive osteoclasts in the coculture experiments, compared to cells from control mice. High [Ca2+
has been shown to directly inhibit osteoclast function, via the osteoclast CaR, in vitro
). This is the first study which provides evidence for an inhibitory role of the osteoblast CaR in regulation of osteoclastogenesis and bone resorption in vivo
Why is bone resorption in the CaR obKO mice increased? The matrix formed by the osteoblasts in these mice is highly undermineralized, evident by osteoid accumulation. We hypothesize that such abnormalities may trigger matrix remodeling. Alternatively, these findings may support the hypothesis that, during resorption, Ca2+
is released from the mineral and acts as a coupling factor, to inhibit osteoclast function and stimulate bone formation, via the CaR. When the CaR is deleted in the CaR obKO mice, resorption increases and formation decreases. Our prior studies with a transgenic mouse model, in which constitutively active CaRs were targeted only to mature osteoblasts and osteocytes under the control of the osteocalcin promoter (Act-CaR mice), showed increased bone resorption and elevated RANK-L levels (24
), findings that are seemingly contradictory to the present study. However, combining the findings from these animal models, we propose that CaR signaling in a broad population of young to mature osteoblasts promotes mineralized bone formation and inhibits resorption, by regulating RANK-L expression, thereby aiding accretion of newly formed bone. On the other hand, activation of the CaR exclusively in mature osteoblasts and osteocytes (which express constitutively active CaRs under control of the osteocalcin promoter in Act-CaR mice) contributes to remodeling of old bone, by stimulating osteoclast differentiation and function (24
We present evidence for the essential role of the osteoblast CaR in postnatal survival, as well as osteoblast differentiation and function, including regulation of bone formation and resorption. Elucidation of the roles of the osteoclast and osteocyte CaRs awaits generation and study of osteoclast- and osteocyte-specific CaR knockout models, a task in which Flox-CaR mice will prove a valuable tool.