CaSRs are broadly present in bone, brain, gut, skin, and endocrine glands, and changes in [Ca2+]e alter cell function in these tissues. Whether the CaSR is the mediator of all aspects of Ca2+-sensing in these tissues in vivo has not been easy to address because of the lack of suitable animal models. The floxed Casr mouse strain that we have developed is the first model that permits tissue-specific deletion of Casr.
We showed that Cre recombinase excised floxed Casr
alleles in three tissues critical to Ca2+
homeostasis and skeletal development. The PTC-specific Casr
KO mouse provided proof of concept for the gene-targeting strategy we used, because these mice showed severe HPT and hypercalcemia, despite having normal renal responsiveness to Ca2+
. Heterozygous and homozygous PTC-specific Casr
KO mice showed the expected mild and severe HPT, respectively, with a clear gene dosage effect, similar to the phenotype of generalized Casr−/−
). We noted that the degree of HPT in PTC-specific Casr
KO mice appeared to be more severe than that of the generalized KO model (10
), with serum PTH concentrations in PT-Het and PT-KO mice that were ~2- to 3- and 17-fold higher, respectively, than those of control littermates (). In contrast, the abundance of serum PTH in generalized Casr+/−
mice was only ~0.5- and 9.5-fold higher, respectively, than that of control mice (10
). This raises the possibility that the alternatively spliced Casr
mRNA, which lacks exon 5, might also be expressed in PTCs in the generalized Casr−/−
) and so mediate some degree of Ca2+
). To confirm this will require direct comparison of the responsiveness of PTCs to Ca2+
in these two mouse models, which will be challenging. Alternatively, different breeding conditions, diets, mouse strains, and PTH assays may explain the differences in the biochemical phenotypes.
Data from experiments performed in PT-KO mice confirmed that severe HPT impeded bone growth and mineralization, as was seen in generalized Casr−/−
). We further show that this is accompanied by the reduced expression of genes encoding markers of osteoblast differentiation. This recapitulates the effects of continuous PTH treatment on both the differentiation of osteoblasts and the formation of mineralized nodules in culture (26
). In contrast, the milder HPT observed in PT-Het mice produced osteopenia later in life, which is similar to the classic presentation of primary HPT in humans. The molecular basis for the effects of HPT on skeletal development remains to be elucidated. PTH mediates signaling through the receptor activator of nuclear factor κB lig-and (RANK-L)/RANK/osteoprotegerin (28
) and Wnt (30
) pathways, obvious candidate pathways to interrogate in the future.
We unexpectedly observed a marked reduction in the abundance of the CaSR in the bones of PT-KO mice, which suggests that loss of CaSR signaling in that tissue may also contribute to skeletal pathology. This idea is supported by the presence of skeletal defects in both models of Casr KO in osteoblasts (COL-KO and OSX-KO). As well as blocking differentiation, deletion of Casr in the bones of COL-KO mice reduced the expression of IGF-1 and increased the expression of IL-10 compared with that in control mice. Because IGF-1 signaling in osteoblasts is critical for cell survival and because IL-10 is proapoptotic, changes in the abundance of these factors in bone would be expected to promote cell death, particularly in cells of the osteoblast lineage. This was confirmed by the presence of increased TUNEL staining in osteoblasts and osteocytes from COL-KO mice compared with that in control mice. Taken together, these data suggest that CaSR signaling modulates the proliferation, survival, and differentiation of osteoblasts, potentially by altering growth and survival factors elaborated by bone.
The embryonic lethality of the chondrocyte-specific Casr
KO mouse was unexpected. Generalized Casr−/−
), which were developed by a different targeting strategy, live for several days postnatally and survive for a longer period if HPT is prevented (12
). This has been interpreted as evidence that the CaSR is not critical for embryonic development. In light of data from the Cart-KO mouse, we suspect that alternatively spliced Casr
mRNAs, which lack exon 5, are present in the generalized Casr−/−
mice and can compensate for as yet undefined functions of CaSRs in the embryo. The delayed cartilage maturation and mineralization observed in the two E12.5 Cart-KO embryos we studied support a role for the CaSR in the early stages of growth plate formation. It has been reported that Col(II) is transiently expressed in heart valves in mice between E10.5 and E14.5 (31
). Thus, knockdown of Casr
at that site might affect cardiac development and function and lead to embryonic death, but this remains to be determined.
Because Cart-KO embryos were nonviable, we turned to an inducible KO model. Tam-induced KO of the expression of Casr
in GPCs produced a rickets-like phenotype with expansion and reduced mineralization of the hypertrophic zone and the decreased abundance of markers of mature, terminally differentiated chondrocytes within 2 to 3 days of exposure to Tam. These observations are consistent with work by us and others that show that high [Ca2+
promotes chondrocyte differentiation and that suppressing Casr
expression with antisense or dominant-negative cDNA constructs blocks this effect (9
). Furthermore, our data indicate that reduced local IGF-1R signaling plays at least some part in delaying GPC differentiation in the KO mice. Taken together, these data are the first to definitively establish a role for the CaSR in Ca2+
sensing in GPCs.
In summary, this study provides the first evidence supporting the direct involvement of CaSR signaling in the differentiation of osteoblasts and GPCs and skeletal development in vivo and suggests that inhibition of Casr expression in bone in states of HPT maybe part of the mechanism underlying the skeletal defects in this disorder. These floxed Casr mice will enable future definitive assessment of CaSR function in other tissues.