The close phylogenic relationship between SIBLING family members (5
), as well as their marked structural similarities (1
), raise questions about their functional specificities, especially in the context of bone. Indeed, mouse models in which particular SIBLINGs are knocked out (20
) have displayed a variety of distinct phenotypes. For instance, both DMP1 and MEPE strongly regulate bone mineralization, but in opposite ways, with absence of the former leading to osteomalacia (23
), at least in part through up-regulation of FGF23 (24
) and MEPE (25
), whereas absence of the latter results in high bone mass (22
). Similarly, studies in vitro under carefully controlled conditions suggest that OPN is a strong inhibitor of hydroxyapatite crystal growth, whereas BSP (11
) and DMP-1 (26
) are promoters of mineralization. The recent characterization of OPN/ALP double-knockout mice, in which the osteomalacia caused by the lack of ALP activity is partly rescued by the absence of OPN, supports a role for OPN as a mineralization inhibitor (27
). Interestingly, OPN−/−
mice were also reported to have hypermineralized bone matrix (27
), in striking contrast to the hypomineralization that we document in BSP knockout mice. BSP was shown to be associated with bone acidic glycoprotein-75 and ALP in specific structures (“biomineralization foci”) that are sites of mineral nucleation in the matrix of primary membrane bone (29
). The hypomineralization of bone nodules formed in cultures of cells from BSP−/−
mice, the slight but significant mineral deficit in BSP−/−
mice at birth (~9%), and the increased MLT in adult bone are compatible with a role of BSP in early bone matrix mineralization, whereas the progressive recovery with age of matrix mineral content () would suggest that this protein has no major and/or a redundant function in mature bone. However, the reduced expression of OPN in whole BSP−/−
bones and the delayed peak of OPN in osteogenic mutant marrow cultures suggest a more complex situation in which down-regulation of OPN may be a compensatory response to loss of BSP, and more work is needed to establish the part played by each SIBLING family member in the development and regulation of matrix mineralization.
Although the affinity of SIBLINGs for hydroxyapatite and the ability to regulate crystal growth/nucleation were among the earliest characteristics identified for members of this family, it is now clear that these proteins are involved in numerous other physiological processes. For example, recent investigations have implicated SIBLINGs in the regulation of MMP activity (31
). This, together with their well-known capacity to mediate cell attachment (32
), suggests that SIBLINGs are important factors in normal and pathogenic tissue remodeling, e.g., in cancer (37
), and possibly in bone. The latter hypothesis is supported by the phenotype of BSP−/−
The shorter size of BSP−/−
mice correlates with the reduced size of long bones. The complex process of endochondral ossification involves interplay between osteoblasts and chondrocytes, two BSP-expressing cell types (the latter at the hypertrophic stage), as well as vascularization, in which BSP may play a regulatory role (41
). Detailed studies are being carried out to describe precisely at the tissue level the time course of skeletogenesis and long bone growth in BSP−/−
mice, and clarify the cell types and mechanisms affected by BSP deletion.
The reduced cortical thickness and BFR of BSP−/−
mice are consistent with both the expression of BSP in actively bone forming/remodeling cells and its known and putative functions in cell attachment and tissue processing. Together with several previous in vitro studies (42
), these results indicate that BSP is a potent regulator of osteoblast differentiation and/or activity. It is also notable that the absence of BSP does not appear to alter the total mesenchymal progenitor cell pool, osteoprogenitor recruitment, or early differentiation, as the total number of CFU-F and CFU-ALP is not detectably changed. However, the strikingly lower number of mineralized colonies indicates that BSP is required for late stages of (at least) primary osteogenesis. This is confirmed by RT-PCR results documenting an altered expression of late osteoblast markers, perhaps matrix-driven (low type I collagen), in BSP−/−
marrow cultures, which is likely to impair the deposition of mineralized matrix. Our results are in agreement with recent data from osteoblasts treated with siRNA of intact and mutant BSP (46
), which also indicate that the RGD-containing portion of the sequence is necessary for phenotypic regulation by BSP. These in vitro data are consistent with the low rate of bone formation observed in vivo, and thus the observation that BSP−/−
mice actually display a higher trabecular bone mass strongly suggests that bone resorption is concomitantly reduced.
It is notable that the loss of BSP does not affect osteoclast capacity to resorb normal (BSP-containing) dentine, suggesting that endogenous BSP production by osteoclasts is not required for resorption. On the other hand, absence of BSP does impair osteoclast differentiation in vitro, which is consistent with previous in vitro studies implicating BSP in regulation of osteoclast differentiation and activity (47
), together with RANKL (48
). That the same is true in vivo is supported by our observation that, whereas OPG and RANKL expression are normal, there is a reproducible reduction of the percentage of area covered by osteoclasts in BSP−/−
mice, and a less consistent reduction in osteoclast numbers. Osteoclasts and their precursors adhere to BSP through the αvβ3 integrin receptor, and this interaction is thought to be an important regulator of their differentiation and activity (47
). It is thus possible that the differentiation of osteoclasts and, as is the case for OPN−/−
), their resorptive activity in vivo (on matrix lacking BSP), is significantly impaired via an integrin outside-in pathway, but further studies will be necessary to clarify this point.
Overall, the phenotype of BSP−/−
mice strikingly contrasts that seen in OPN−/−
mice. The latter have a normal BFR, but a higher trabecular bone mass with increased numbers of poorly resorbing osteoclasts (50
). Also, as previously mentioned, OPN−/−
mice do not lose bone with hindlimb unloading, which is an established model of disuse osteoporosis (15
). In contrast, and despite their low turnover, mice lacking BSP respond to hindlimb unloading by a significant trabecular bone loss. As previously shown (18
), hindlimb unloading induces a transient increase of bone resorption followed by a more sustained inhibition of bone formation. After 15 d of unloading, WT mice show strongly reduced BFR with only a trend to increased osteoclast numbers and (in females) surfaces in our experiments, indicative of a late-stage bone response. In BSP−/−
mice, BFR reduction is observed only in males, whereas the increased osteoclast surface is seen only in females, likely caused by distinct kinetics between the two sexes. Collectively, our data suggest that osteoblastic formation and osteoclastic resorption are modulated by unloading in BSP−/−
mice, the latter likely accounting for most of the bone loss, given the low BFR. Although, as mentioned, our in vitro results on dentin cannot discount reduced in vivo osteoclast activity in BSP−/−
mice, older mutant animals do lose trabecular bone (not depicted), which is suggestive of efficient metaphyseal resorption in mature (>5-mo-old) animals. Collectively, these results suggests that BSP is not an absolute limiting factor for increased resorption, perhaps caused by some compensatory mechanisms, such as overexpression of related proteins (possibly another SIBLING) in challenging conditions. Whatever the compensatory mechanism, it contrasts with the cell autonomous defect in osteoclast activity of OPN−/−
), and stresses that the cellular mechanisms underlying the two phenotypes are quite different.
In conclusion, BSP−/− mice display a reduced body and long bone growth, but have a high trabecular bone mass accompanied by low bone turnover that is, nonetheless, responsive to mechanical challenges. Our data thus highlight the specificity of BSP roles in the bone context and further confirm the nonredundancy of function of SIBLING family members in skeletal biology.