The role of Gs
α in governing osteoblast differentiation is of particular relevance for several human skeletal disorders (46
). Heterozygous loss-of-function mutations in Gs
α result in Albright hereditary osteodystrophy (AHO), with short stature, brachydactyly, and obesity. A subset of patients with AHO, frequently those with paternal inheritance of the mutated allele, experience ectopic ossifications that can involve subcutaneous or deep soft tissues as well as skeletal muscle (progressive osseous heteroplasia). Conversely, activating somatic mutations of Gs
α are found in fibrous dysplasia, skeletal lesions that can occur alone or in association with other endocrinopathies (McCune-Albright syndrome). Fibrous dysplasia lesions are characterized by impaired osteoblast differentiation and woven bone. Therefore Gs
α plays a crucial role in osteoblast development and function, and understanding the molecular mechanisms will be of great clinical significance.
To that end we have generated mice with conditional deletion of Gsα early in the osteoblast lineage. GsαOsxKO mice have severe osteoporosis characterized by impaired endochondral and intramembranous ossification. The dramatic reduction in bone mass is accompanied by a failure of normal bone formation, with production of predominantly woven bone of inadequate mass and strength, and a marked decrease in osteoblast number. Gsα is downstream of multiple GPCRs and therefore may participate in various signaling pathways. We have identified at least two distinct mechanisms that may underlie the diminished osteoblast number in GsαOsxKO mice: (a) accelerated differentiation of osteoblasts into osteocytes and (b) decreased commitment of mesenchymal progenitors to the osteoblast lineage, in association with attenuated Wnt signaling that results at least in part from increased expression of the Wnt inhibitors sclerostin and Dkk1.
We have found that the population of osteoblast precursors (CFU-ALP) was significantly decreased among both BMSCs and calvarial cells of Gs
mice, suggesting reduced commitment of mesenchymal progenitors to the osteoblast lineage. Wnt signaling is required for osteoblast commitment and early osteoblast differentiation (18
), and PKA signaling downstream of the PPR can suppress expression of the canonical Wnt inhibitors sclerostin and Dkk1. Sclerostin was overexpressed in calvariae of Gs
mice, and both Sost
mRNA levels were increased in calvarial osteoblasts from Gs
mice, with a concomitant decrease in expression of Wnt target genes in these same cells. Therefore, the inhibition of Wnt signaling may contribute to the dramatic decrease in the pool of osteoprogenitors in Gs
mice. In addition to sclerostin and Dkk1, several laboratories have demonstrated other points of interaction between the PTH/PPR/PKA and Wnt signaling pathways (21
). PTH is able to activate β-catenin targets in osteoblasts even in the absence of Dkk1 suppression (25
), implicating other actions downstream of the PPR in regulating Wnt signaling. Indeed, several potential mechanisms have been identified, including direct interactions between PPR and the Wnt coreceptor LRP6 (48
), as well as phosphorylation of β-catenin by PKA (49
). Therefore, the effects of PKA on Wnt signaling may be both indirect, mediated by soluble inhibitors such as sclerostin and Dkk1, as well as cell autonomous, by direct phosphorylation of components of the Wnt signaling pathway. More detailed investigations into the effects of Gs
α ablation on Wnt signaling in Gs
mice are underway.
Once mesenchymal progenitors are committed to the osteoblast lineage, however, the loss of Gsα somewhat paradoxically results in accelerated osteogenic maturation. Although the near absence of osteocalcin mRNA initially suggested a failure of osteoblast differentiation, instead the reduced expression of osteocalcin mRNA reflected attenuated PKA signaling in osteoblasts lacking Gsα. In support of this, expression of Mmp13 mRNA, another PKA target gene, was also reduced in GsαOsxKO bones, while other markers of osteogenic differentiation were still expressed.
Although the number of mature osteoblasts was markedly reduced in Gs
mice, this was not due to complete failure of osteoblast differentiation, as these mice have abundant osteocytes, which are derived from terminally differentiated osteoblasts (40
). Rather, we have shown that in the absence of Gs
α, cells of the osteoblast lineage differentiate rapidly into osteocytes. Whereas PTH has been demonstrated to decrease apoptosis in osteoblasts in adult mice (50
), we could find no discernible alteration in osteoblast apoptosis in early postnatal Gs
mice in vivo. The effects of PTH on osteoblast apoptosis vary greatly by anatomic location (51
) as well as means of administration (10
). During early postnatal growth, a time of rapid bone formation, perhaps changes in apoptosis may have lesser effects on cell numbers. In addition, there was no evidence of increased proliferation of Gs
α-deficient osteoblasts in vivo. Thus, accelerated differentiation results in depletion of osteoblasts and accumulation of osteocytes in Gs
mice. It therefore appears that in cells of the osteoblast lineage, Gs
α restrains cellular differentiation. Of note, the bone that is present in Gs
mice is mainly woven bone. By regulating the pace of osteoblast differentiation, Gs
α may function to promote the formation of a larger number of osteoblasts and production of more orderly lamellar bone. In the absence of Gs
α, the resulting bone is largely woven, typically associated with embryonic development and states of pathologically rapid bone formation such as fracture repair.
Our data are in keeping with the findings that continuous exposure of osteoblasts to PTH inhibits differentiation (11
). Moreover, in chondrocytes, ablation of either PPR or Gs
α leads to accelerated chondrocyte differentiation and hypertrophy, with adverse consequences for skeletal development (13
). Whether similar mechanisms underlie the effects of Gs
α and PKA in inhibiting differentiation of both osteoblasts and chondrocytes is under investigation.
The mechanisms mediating the effects of Gs
α on osteoblast lineage commitment and differentiation are distinct and involve both Wnt-dependent and Wnt-independent effects. In particular, like Wnt signaling and perhaps through its effects on Wnt signaling, Gs
α favors osteogenic commitment of mesenchymal progenitors. In contrast, whereas Wnt signaling positively regulates osteoblast differentiation, osteoblast maturation is accelerated in Gs
mice even in the face of reduced Wnt signaling, suggesting that absence of Gs
α can override the inhibition of Wnt signaling in driving osteoblast differentiation forward. The accelerated differentiation of osteoblasts into osteocytes in the absence of Gs
α is unlikely to be related to alterations in Wnt signaling. In differentiated osteoblasts, manipulation of Wnt signaling does not affect bone formation, but only affects bone resorption via disruption of osteoprotegerin expression (52
). Furthermore, reduced Wnt signaling in osteocytes due to either ablation of β-catenin or overexpression of Dkk1 in osteoblasts does not lead to an increase in osteocyte density or woven bone (25
). Together these studies suggest that Wnt signaling probably does not regulate differentiation of osteoblasts into osteocytes.
The actions of Gsα on both commitment to and differentiation of the osteoblast lineage are at work in both intramembranous and endochondral bone. When plated at low cell densities, osteoprogenitor numbers, as determined by CFU-ALP, were reduced in both BMSCs and calvarial cells of GsαOsxKO mice. In cells committed to the osteoblast lineage, however, the absence of Gsα resulted in accelerated osteoblast differentiation. In keeping with a similar role for Gsα in both types of cells, when equal numbers of osteoprogenitors were plated in vitro, followed by ablation of Gsα, mineralization was dramatically increased in both BMSCs and calvarial cells. Therefore, we conclude that Gsα acts to enhance osteoblast lineage commitment but restrain osteoblast differentiation in both intramembranous and endochondral bone.
The relevant receptors upstream of Gs
α are of great interest. The PPR is a prime candidate, since PTH has important effects on osteoblasts, and many of these are mediated by Gs
). In vitro, deletion of the PPR in calvarial osteoblasts also resulted in accelerated osteogenic differentiation; however, ablation of the PPR in osteoprogenitors did not lead to the presence of fractures at birth (data not shown). Therefore, other GPCRs may well be involved. Other Gs
α-coupled GPCRs reported in osteoblasts include TSHR and β2AR. However, ablation of TSHR leads to a high-turnover osteoporosis (31
), while deletion of β2AR results in high bone mass (32
). Thus, the actions of these receptors are unlikely, at least during embryonic development, to be the predominant GPCRs signaling to Gs
α in early cells of the osteoblast lineage. PGE2
also has anabolic effects on bone (55
), and in osteoblasts the PGE2
receptors EP2 and EP4 are coupled to Gs
α and may serve crucial functions in regulating bone formation.
In summary, our studies demonstrate that Gsα signaling early in the osteoblast lineage is crucial for the formation of normal bone. In the absence of Gsα, decreased commitment of mesenchymal progenitors to the osteoblast lineage and pathologically accelerated osteogenic differentiation yield bone of striking fragility. That Gsα mediates multiple functions in osteoblasts suggests that more specific targeted interventions may be possible to increase bone mass, quality, and strength.