As several studies elucidating the roles of STC1 in osteoblasts and its mechanisms in osteoblast differentiation and activity have reported controversial results, the roles of STC1 in local biomineralization by osteoblasts remain to be investigated. Yoshiko et al. reported that the overexpression and downregulation of STC1 had accelerating and inhibitory effects, respectively, on osteoblast differentiation and the mRNA expression of osteopontin and osteocalcin in mature rat osteoblast cultures but not in osteoprogenitor cell cultures 6. They also suggested that the stimulatory effect of STC1 on osteoblast differentiation was related to an increase in both sodium-dependent phosphate uptake and Pit1 gene expression in mature rat osteoblast cultures 6. In another previous study, Johnston et al. reported that calvarial cells obtained from transgenic mice constitutively expressing human STC1 exhibited reduced viability, reduced proliferation, delayed differentiation, and reduced expression of osteocalcin 27. They also reported that Pit1 gene expression did not differ between wild-type and transgenic calvarial cell cultures 27. However, they found that the expression of phosphate regulators other than Pit1, including Dmp1, Sfrp4, Mepe, and Enpp1, was decreased in calvarial cells derived from STC1 transgenic mice 27. Our findings are consistent with the latter of previous studies. We observed that STC1 negatively regulates osteoblast differentiation and the expression of late osteoblast marker genes. The overexpression of STC1 using STC1 retrovirus, recombinant STC1, and cultures of BMSCs derived from STC1 transgenic mice and the downregulation of STC1 exhibited inhibitory and accelerating effects, respectively, on osteoblast differentiation as well as Ibsp and Bglap mRNA expression. The discrepancy between results elucidating the role of STC1 in osteoblasts, including the present results, may be due to differences in the cell type used or the STC1 source, possibly due to the cell differentiation stage-dependent dual function of STC1 in osteoblasts. Nevertheless, our results indicate that STC1 inhibits osteoblast differentiation and activity in vitro, at least in osteoblast progenitor cells, although this effect may still be controversial in mature osteoblasts.
Filvaroff et al. reported that transgenic mice expressing human STC1 under the control of a muscle-specific promoter exhibited reduced cortical bone volume and total bone volume because of their smaller size than wild-type mice 17. In the STC1 transgenic mice, the rate of bone formation but not mineralization was decreased, possibly due to reduced osteoclast activity. Similarly, contrary to the expectation from the findings that STC1 could suppress osteoblast differentiation in vitro and BMP2-induced bone formation in vivo, we confirmed that the long bone of transgenic mice expressing mouse Stc1 under the control of an osteoblast-specific promoter did not exhibit a noticeably altered bone phenotype. Although Stc1-null mice display no obvious phenotype and long bones of STC1-hyperstimulated mice do not show distinct changes in bone phenotypes, severe cranial hypoplasia has been reported in pups of STC1-hyperstimulated mice 16,27. As calvarial bone hypoplasia is generally indicative of decreased osteoblast progenitor proliferation and differentiation, it seems likely that stimulated STC1 delays osteoblast proliferation and differentiation during intramembranous bone development; however, the effect is not persistent. Taken together, these results suggest that STC1 is closely involved in intramembranous bone formation during development; however, it is not necessarily required for bone formation under normal conditions.
It is known that 1,25(OH)2D3, an important hormone that regulates calcium absorption and bone mineralization, upregulates STC1 in renal proximal tubular cells 29. Hence, we examined whether 1,25(OH)2D3 could regulate the expression of STC1 in osteoblasts and whether STC1 could contribute to the function of 1,25(OH)2D3 in osteoblasts. The expression of STC1 in osteoblasts was strongly increased by 1,25(OH)2D3, as observed in renal proximal tubular cells. VDR, a functional nuclear receptor of 1,25(OH)2D3, directly bound to the VDR-binding region of the STC1 promoter, increasing the mRNA expression level of Stc1. Interestingly, STC1 increased Vdr expression by inhibiting Akt phosphorylation. This fact was confirmed by several findings, such as increased Vdr expression by STC1, decreased Akt phosphorylation by STC1, and increased Vdr expression by the inhibitor of Akt activity. The reciprocal expression regulation noted between Vdr and Stc1 suggests that 1,25(OH)2D3 and STC1 have similar functions and act cooperatively in osteoblasts. Substantively, STC1 synergistically could promote all effects of 1,25(OH)2D3 on osteoblast differentiation, RANKL secretion, and bone formation in vitro and in vivo.
Vitamin D supplements are widely recommended for bone health in the general population 30. It is well established that vitamin D supplementation can improve bone health because vitamin D deficiency can cause rickets in children and osteomalacia in adults. Moreover, vitamin D indirectly stimulates bone formation by increasing calcium absorption from the intestines 30. Several studies have reported that vitamin D contributes to bone health by directly acting on osteoblasts; however, this remains controversial. Studies using human osteoblasts have shown that 1,25(OH)2D3 positively regulates bone formation and mineralization 31,32. Moreover, 1,25(OH)2D3 induces the production of ALP-positive matrix vesicles during osteoblast differentiation to enhance mineralization 32,33. In contrast, accumulating evidence indicates that 1,25(OH)2D3 negatively regulates bone formation and mineralization in murine osteoblasts 31,32. Murine osteoblasts lacking VDR exhibit increased osteogenic potential. The hormone 1,25(OH)2D3 increases pryosphate (PPi) levels by inducing the expression of progressive ankylosis (ANK) and ectonucleotide pyrophosphatase phosphodiesterase (ENPP1) proteins, in addition to increasing osteopontin levels in murine osteoblasts 32. These effects in turn decrease mineralization. The discrepancy between results regarding the direct effects of 1,25(OH)2D3 on osteoblasts cannot be solely explained by species differences. This is because different effects of 1,25(OH)2D3 have been reported in different murine models. Specific transgenic mice with osteoblast-specific VDR overexpression have been found to exhibit increased bone formation and mineralization, while global VDR-knockout mice have been found to exhibit similar bone phenotypes 31,34–36. Instead, the discrepancy in the direct effects of 1,25(OH)2D3 on osteoblasts may be partly explained by the complex functions of 1,25(OH)2D3. In particular, 1,25(OH)2D3 stimulates carboxylated osteocalcin and activin A, established inhibitors of mineralization, and inhibits bone sialoprotein (IBSP), an established stimulator of mineralization, in human osteoblasts to prevent pathological overmineralization 32. In this study, we demonstrated the negative roles of 1,25(OH)2D3 in osteoblast differentiation and bone formation in vitro. We found that 1,25(OH)2D3 inhibited nodule formation and the expression of osteoblast-related genes in calvarial osteoblasts. The direct roles of 1,25(OH)2D3 and STC1 in osteoblasts are very similar. Under our culture conditions, 1,25(OH)2D3 and STC1 negatively regulated osteoblast differentiation. Interestingly, both 1,25(OH)2D3 and STC1 could positively regulate osteoblast differentiation, possibly due to their complex dual functions that differ depending on the differentiation stage of osteoblasts. Therefore, in addition to reciprocal expression regulation between Vdr and Stc1, the similar functions of 1,25(OH)2D3 and STC1 in osteoblasts suggest that 1,25(OH)2D3 and STC1 complementarily or synergistically regulate osteoblast differentiation.
In a recent study, compared with the placebo, vitamin D supplements did not result in a significantly decreased risk of fractures among generally healthy midlife and older adults who were not selected vitamin D deficiency, low bone mass, or osteoporosis 37. In addition, among older community-dwelling women, the annual oral administration of high-dose vitamin D resulted in an increased risk of fractures 38. In this study, 1,25(OH)2D3 significantly attenuated BMP2-induced bone formation in an ectopic bone formation experiment, which was performed to exclude the possibility that 1,25(OH)2D3 plays a role in calcium absorption from the intestines. Moreover, the administration of a high dose of 1,25(OH)2D3 dramatically reduced long bone mass. Collectively, these results suggest that 1,25(OH)2D3 has a detrimental effect on bone health by exhibiting a direct effect on osteoblasts under certain conditions. Another interesting result of our study is that STC1 inhibits BMP2-induced ectopic bone formation and that this effect is amplified in the presence of 1,25(OH)2D3. Furthermore, we found that the normal long bone mass of STC1 transgenic mice was significantly lower than that of wild-type mice following 1,25(OH)2D3 administration. Thus, the simultaneous activation of the STC1 and 1,25(OH)2D3 signaling pathways in osteoblasts can lead to decreased bone formation.
Possible differences between species pose an obstacle to the application of our experimental results obtained from murine models to humans. Further studies will be needed to confirm the effect of the interaction between STC1 and 1,25(OH)2D3 on the differentiation and activity of human osteoblasts. These future studies will help consider STC1 levels as a remarkable indicator for determining the appropriate dose and duration of vitamin D when taking vitamin D supplements for bone health.