To study in vivo the role of the PPR in cells of the osteoblast lineage, we generated mice that express the human mutant PPR, HKrk-H223R, under the control of the 2.3-kb fragment of the mouse α1(I) collagen promoter. This fragment has been shown previously to drive expression specifically in cells of the osteoblast lineage and in odontoblasts (14
). The HKrk-H223R mutant was chosen rather than the other two mutant receptors described in JMC because of its higher levels of ligand-independent cAMP accumulation (12
Specific expression of the transgene could be detected already at birth in transgenic mice. However, a change in bone structure was noted only starting from 1 week of postnatal age. Previous animal models have shown the important role of the PPR in fetal bone development (8
). The lack of a clear phenotype at birth may be due to insufficient levels of prenatal expression of the transgene or to insufficient time after transgene activation to detect accumulation of abnormal cells and matrix.
Expression of transgene mRNA was detected at similar levels in trabecular bone and at both the periosteal and endosteal surfaces of cortical bone. This pattern of localization overlapped with that of the native PPR mRNA in bone (11
), even if, as is different from the latter, it was more diffuse. These data are consistent with expression of the transgene being driven by a fragment of the collagen gene promoter. In addition, low but detectable expression of the transgene mRNA was present in the growth plate, and this might have contributed to the shortening of the limbs seen in the CL2 mice.
The actions of PTH in bone, while extensively studied (6
), are still controversial. PTH has been shown to either stimulate or inhibit the growth of osteoblastic cells and exert differentiating and dedifferentiating effects, depending on the model system studied (21
). This study shows, we believe for the first time, that activation of the PPR in cells of the osteoblast lineage stimulates a vivid anabolic response in trabecular bone. This finding is consistent with the decreased trabecular bone seen prenatally in mice lacking the PPR (8
). The increase in trabecular bone volume was related to an increase in both osteoblast number and function. In addition, an abundant population of stroma-like cells was noted among the trabeculae of the transgenic bones. In situ hybridization suggested that these cells are likely to represent cells committed to the osteoblast lineage, since they express markers of osteoblast differentiation, such as alkaline phosphatase, osteopontin (24
), and collagenase 3 (18
). Interestingly, a population of cells with fibrotic morphology was noted in trabecular bone from mice given a high dose of PTH(1–34) continuously (7
) and is a pathognomonic finding in patients with severe hyperparathyroidism (26
). Our study supports the hypothesis that the “fibrosis” described in these cases may be due to an expansion of cells of the osteoblast lineage.
Consistent with activation of the adenylate cyclase pathway by HKrk-H223R, the stroma-like cells in our transgenic mice were morphologically similar to the pathologic findings typical of polyostotic fibrous dysplasia of bone (27
). These bone lesions are associated with activating mutation of the GNAS gene, leading to constitutive activity of Gs
). As in our mice, the cells isolated from fibrous dysplasia lesions were also identified as a heterogeneous population of preosteoblasts (28
). However, in contrast with what we observe in our transgenic model, accumulation of cAMP in fibrous dysplasia severely inhibited the differentiation of preosteoblasts into fully mature osteoblasts (28
). The difference between these two models could depend on many variables, including onset of transgene expression and relative levels of intracellular cAMP, as well as potential activation of additional signaling pathways. Furthermore, a partial maturational arrest in a subset of the heterogeneous preosteoblast population present in our transgenic model cannot be excluded.
The increased proliferation rate detected in the trabecular osteoblastic cells of our transgenic mice suggests that it is the activation of the PPR that mediates the proliferative effect of PTH reported previously (29
). Interestingly, an increase in proliferation rate is also noted in cells from fibrous dysplasia lesions (27
). In CL2 transgenic mice, the proportion of osteoblasts and stroma-like cells undergoing apoptosis was decreased compared with wild-type littermates, suggesting that the PPR mediates the recently described (20
) antiapoptotic effect of PTH on cells of the osteoblast lineage. Therefore, in our transgenic model the increased osteoblast number was, at least in part, the result of both augmented proliferation and decreased apoptosis in cells of the osteoblast lineage. The rate of commitment of mesenchymal stem cells also contributes to the pool of osteoblastic cells, and some evidence (29
) has suggested that PTH may increase this rate. Further studies are needed to investigate whether the commitment rate of mesenchymal cells is modified in our transgenic model.
In CL2 mice, endosteal and trabecular bone formation rates were increased, suggesting that there was an increase in osteoblast function at these sites. An increased bone formation rate was also observed at the endosteal surface of the skull, which is formed differently from the long bones through a process of intramembranous ossification. These data show that the PPR is able to mediate an anabolic action in osteoblasts present in skeletal compartments of different developmental origin.
In sharp contrast to the increased bone-forming activity in both trabecular bone and the endosteal surface of the skull, an inhibition of both proliferation and function was noted in periosteal osteoblasts of the CL2 mice. Similarly, bone formation rate was decreased in periosteal osteoblasts from the skulls of transgenic mice. CL2 transgenic animals have therefore a dramatically different phenotype in the periosteal compartment when compared with the trabecular and endosteal compartments. Interestingly, similar levels of transgene mRNA expression were seen by in situ hybridization in these compartments. These findings could be helpful in explaining the differential effects of PTH in trabecular versus cortical bone in primary hyperparathyroidism (2
). Changes in periosteal activity may play a role in the decreased cortical thickness in the CL2 transgenic animals, as well as in the increased cortical thickness seen in mice lacking the PPR (8
). Additional experimental models are needed in order to investigate whether the differences among cells of the osteoblast lineage in these separate districts are due to intrinsic characteristics of the cells or to different bone microenvironments.
One of the most prominent actions of PTH is to increase, albeit indirectly, number and activity of osteoclasts (2
). By histologic and histomorphometric analysis, mature osteoclasts were increased dramatically in the trabecular and cortical compartments of our transgenic animals. Consistent with the increased number of osteoclasts, there was increased porosity of the cortical bone from transgenic mice. In addition, transgenic mice were normocalcemic, but had partially suppressed levels of circulating PTH, implying increased calcium release from bone. Therefore, expression of the constitutively active PPR solely in cells of the osteoblast lineage was sufficient to increase dramatically the number of mature osteoclasts in the transgenic mice. This fact points out that expression of this receptor in osteoblasts/stromal cells is critical for PTH-induced osteoclastogenesis, as has been suggested by in vitro evidence (30
In summary, targeted expression of the constitutively active PPR led to increased osteoblast function in trabecular bone and at the endosteal surface of cortical bone, while the osteoblastic activity in the periosteum was inhibited. Mature osteoblasts, as well as a heterogeneous population of preosteoblasts, were increased in the trabecular compartment by a mechanism of increased proliferation and decreased apoptosis. Osteoblastic expression of the constitutively active PPR was sufficient to lead to a dramatic increase in osteoclast number. The net effect of these actions was a substantial increase in trabecular bone volume and a decrease in cortical thickness of the long bones.
Our data are dramatically different from the decreased net bone mass observed after continuous administration of PTH. It is plausible that dose of the peptide, as well as involvement of other signaling pathways and/or other receptors, play an important role in this catabolic effect of PTH. However, our findings, for the first time to our knowledge, identify the PPR as a crucial mediator of both bone-forming and bone-resorbing actions of PTH, as well as pointing out the complexity and heterogeneity of the osteoblast population and/or their regulatory microenvironment.