Ritsuko Masuyama

Normocalcemia is maintained in mice under conditions of calcium malabsorption by vitamin D-induced inhibition of bone mineralization

Serum calcium levels are tightly controlled by an integrated hormone-controlled system that involves active vitamin D [1,25(OH)(2)D], which can elicit calcium mobilization from bone when intestinal calcium absorption is decreased. The skeletal adaptations, however, are still poorly characterized. To gain insight into these issues, we analyzed the consequences of specific vitamin D receptor (Vdr) inactivation in the intestine and in mature osteoblasts on calcium and bone homeostasis. We report here that decreased intestinal calcium absorption in intestine-specific Vdr knockout mice resulted in severely reduced skeletal calcium levels so as to ensure normal levels of calcium in the serum. Furthermore, increased 1,25(OH)(2)D levels not only stimulated bone turnover, leading to osteopenia, but also suppressed bone matrix mineralization. This resulted in extensive hyperosteoidosis, also surrounding the osteocytes, and hypomineralization of the entire bone cortex, which may have contributed to the increase in bone fractures. Mechanistically, osteoblastic VDR signaling suppressed calcium incorporation in bone by directly stimulating the transcription of genes encoding mineralization inhibitors. Ablation of skeletal Vdr signaling precluded this calcium transfer from bone to serum, leading to better preservation of bone mass and mineralization. These findings indicate that in mice, maintaining normocalcemia has priority over skeletal integrity, and that to minimize skeletal calcium storage, 1,25(OH)(2)D not only increases calcium release from bone, but also inhibits calcium incorporation in bone.

Vitamin D receptor in chondrocytes promotes osteoclastogenesis and regulates FGF23 production in osteoblasts

Genomic actions induced by 1alpha25-dihydroxyvitamin D(3) [1,25(OH)(2)D(3)] are crucial for normal bone metabolism, mainly because they regulate active intestinal calcium transport. To evaluate whether the vitamin D receptor (VDR) has a specific role in growth-plate development and endochondral bone formation, we investigated mice with conditional inactivation of VDR in chondrocytes. Growth-plate chondrocyte development was not affected by the lack of VDR. Yet vascular invasion was impaired, and osteoclast number was reduced in juvenile mice, resulting in increased trabecular bone mass. In vitro experiments confirmed that VDR signaling in chondrocytes directly regulated osteoclastogenesis by inducing receptor activator of NF-kappaB ligand (RANKL) expression. Remarkably, mineral homeostasis was also affected in chondrocyte-specific VDR-null mice, as serum phosphate and 1,25(OH)(2)D levels were increased in young mice, in whom growth-plate activity is important. Both in vivo and in vitro analysis indicated that VDR inactivation in chondrocytes reduced the expression of FGF23 by osteoblasts and consequently led to increased renal expression of 1alpha-hydroxylase and of sodium phosphate cotransporter type IIa. Taken together, our findings provide evidence that VDR signaling in chondrocytes is required for timely osteoclast formation during bone development and for the endocrine action of bone in phosphate homeostasis.

TRPV4-Mediated Calcium Influx Regulates Terminal Differentiation of Osteoclasts

Calcium signaling controls multiple cellular functions and is regulated by the release from internal stores and entry from extracellular fluid. In bone, osteoclast differentiation is induced by RANKL (receptor activator of NF-kappaB ligand)-evoked intracellular Ca(2+) oscillations, which trigger nuclear factor-activated T cells (NFAT) c1-responsive gene transcription. However, the Ca(2+) channels involved remain largely unidentified. Here we show that genetic ablation in mice of Trpv4, a Ca(2+)-permeable channel of the transient receptor potential (TRP) family, increases bone mass by impairing bone resorption. TRPV4 mediates basolateral Ca(2+) influx specifically in large osteoclasts when Ca(2+) oscillations decline. TRPV4-mediated Ca(2+) influx hereby secures intracellular Ca(2+) concentrations, ensures NFATc1-regulated gene transcription, and regulates the terminal differentiation and activity of osteoclasts. In conclusion, our data indicate that Ca(2+) oscillations and TRPV4-mediated Ca(2+) influx are sequentially required to sustain NFATc1-dependent gene expression throughout osteoclast differentiation, and we propose TRPV4 as a therapeutic target for bone diseases.

Mutations in UVSSA cause UV-sensitive syndrome and impair RNA polymerase IIo processing in transcription-coupled nucleotide-excision repair

UV-sensitive syndrome (UVSS) is a genodermatosis characterized by cutaneous photosensitivity without skin carcinoma. Despite mild clinical features, cells from individuals with UVSS, like Cockayne syndrome cells, are very UV sensitive and are deficient in transcription-coupled nucleotide-excision repair (TC-NER), which removes DNA damage in actively transcribed genes. Three of the seven known UVSS cases carry mutations in the Cockayne syndrome genes ERCC8 or ERCC6 (also known as CSA and CSB, respectively). The remaining four individuals with UVSS, one of whom is described for the first time here, formed a separate UVSS-A complementation group; however, the responsible gene was unknown. Using exome sequencing, we determine that mutations in the UVSSA gene (formerly known as KIAA1530) cause UVSS-A. The UVSSA protein interacts with TC-NER machinery and stabilizes the ERCC6 complex; it also facilitates ubiquitination of RNA polymerase IIo stalled at DNA damage sites. Our findings provide mechanistic insights into the processing of stalled RNA polymerase and explain the different clinical features across these TC-NER–deficient disorders.

Dietary calcium and phosphorus ratio regulates bone mineralization and turnover in vitamin D receptor knockout mice by affecting intestinal calcium and phosphorus absorption

The effects of the dietary Ca and P ratio, independent of any vitamin D effects, on bone mineralization and turnover was examined in 60 VDRKO mice fed different Ca/P ratio diets. High dietary Ca/P ratio promoted bone mineralization and turnover with adequate intestinal Ca and P transports in VDRKO mice.

Calcemic actions of vitamin D: Effects on the intestine, kidney and bone

The analysis of mice that lack systemically the actions of the active form of vitamin D, 1,25(OH)₂D, has shown that 1,25(OH)₂D is an essential regulator of calcium homeostasis and that its actions are aimed at maintaining serum calcium levels within narrow limits. Especially the stimulation of intestinal calcium transport by 1,25(OH)₂D is important for calcium and bone homeostasis. The involved transporters are however still elusive. The targeted deletion of 1,25(OH)₂D action in chondrocytes has provided compelling evidence for a paracrine control of bone development and endocrine regulation of phosphate homeostasis by 1,25(OH)₂D. Targeting vitamin D receptor (VDR) function in other tissues will further enhance our understanding of the cell-type specific action of 1,25(OH)₂D. In this review, we will discuss the current understanding and remaining questions concerning the calcemic actions of 1,25(OH)₂D in the intestine, kidney and bone, with special focus on the evidence obtained by the use of transgenic mouse models.

IFN-γ directly inhibits TNF-α-induced osteoclastogenesis in vitro and in vivo and induces apoptosis mediated by Fas/Fas ligand interactions

Cytokines secreted by T cells play a pivotal role in inflammatory bone destruction. Tumor necrosis factor-α (TNF-α) is a major proinflammatory cytokine produced by macrophages following T cell activation, and directly promotes osteoclast differentiation resulting in accelerated bone resorption. Interferon-γ (IFN-γ) attenuates RANKL-initiated cellular signals through osteoclast formation and counterbalances aberrant bone resorption. With respect to this crosstalk during osteoclastogenesis, the direct interruption of IFN-γ in TNF-α-induced osteoclast formation still requires elucidation. We have demonstrated that IFN-γ directly inhibits osteoclastogenesis induced by TNF-α stimulation and accelerates apoptosis mediated by Fas/Fas ligand signals. There were a decreased number of osteoclasts and reduced mRNA levels encoding Nfatc1 in cultured bone marrow macrophages. Apoptotic responses of cultured cells were observed, with accelerated nuclear fragmentation in osteoclast precursor cells and increased FasL mRNA levels in bone marrow cells stimulated with TNF-α evident. IFN-γ reduced the level of osteoclastogenesis in response to TNF-α treatment in vivo. IFN-γ inhibited TNF-α-induced osteoclastogenesis in mice with T cells that had been exposed to anti-CD4 and -CD8 antibodies. These results provide evidence that IFN-γ directly inhibits osteoclastogenesis and induces cells apoptosis by Fas/FasL signals, leading to the indirect regulation of bone resorption, which is required for protective roles in bone destruction at an inflammation site.

Calcium/calmodulin‐signaling supports TRPV4 activation in osteoclasts and regulates bone mass

Osteoclast differentiation is critically dependent on calcium (Ca2+) signaling. Transient receptor potential vanilloid 4 (TRPV4), mediates Ca2+ influx in the late stage of osteoclast differentiation and thereby regulates Ca2+ signaling. However, the system‐modifying effect of TRPV4 activity remains to be determined. To elucidate the mechanisms underlying TRPV4 activation based on osteoclast differentiation, TRPV4 gain‐of‐function mutants were generated by the amino acid substitutions R616Q and V620I in TRPV4 and were introduced into osteoclast lineage in Trpv4 null mice to generate Trpv4R616Q/V620I transgenic mice. As expected, TRPV4 activation in osteoclasts increased the number of osteoclasts and their resorption activity, thereby resulting in bone loss. During in vitro analysis, Trpv4R616Q/V620I osteoclasts showed activated Ca2+/calmodulin signaling compared with osteoclasts lacking Trpv4. In addition, studies of Trpv4R616Q/V620I mice that lacked the calmodulin‐binding domain indicated that bone loss due to TRPV4 activation was abrogated by loss of interactions between Ca2+/calmodulin signaling and TRPV4. Finally, modulators of TRPV4 interactions with the calmodulin‐binding domain were investigated by proteomic analysis. Interestingly, nonmuscle myosin IIa was identified by liquid chromatography–tandem mass spectroscopy (LC‐MS/MS) analysis, which was confirmed by immunoblotting following coimmunoprecipitation with TRPV4. Furthermore, myosin IIa gene silencing significantly reduced TRPV4 activation concomitant with impaired osteoclast maturation. These results indicate that TRPV4 activation reciprocally regulates Ca2+/calmodulin signaling, which involves an association of TRPV4 with myosin IIa, and promotes sufficient osteoclast function.

Overexpression of Bcl2 in Osteoblasts Inhibits Osteoblast Differentiation and Induces Osteocyte Apoptosis

Bcl2 subfamily proteins, including Bcl2 and Bcl-XL, inhibit apoptosis. As osteoblast apoptosis is in part responsible for osteoporosis in sex steroid deficiency, glucocorticoid excess, and aging, bone loss might be inhibited by the upregulation of Bcl2; however, the effects of Bcl2 overexpression on osteoblast differentiation and bone development and maintenance have not been fully investigated. To investigate these issues, we established two lines of osteoblast-specific BCL2 transgenic mice. In BCL2 transgenic mice, bone volume was increased at 6 weeks of age but not at 10 weeks of age compared with wild-type mice. The numbers of osteoblasts and osteocytes increased, but osteoid thickness and the bone formation rate were reduced in BCL2 transgenic mice with high expression at 10 weeks of age. The number of BrdU-positive cells was increased but that of TUNEL-positive cells was unaltered at 2 and 6 weeks of age. Osteoblast differentiation was inhibited, as shown by reduced Col1a1 and osteocalcin expression. Osteoblast differentiation of calvarial cells from BCL2 transgenic mice also fell in vitro. Overexpression of BCL2 in primary osteoblasts had no effect on osteoclastogenesis in co-culture with bone marrow cells. Unexpectedly, overexpression of BCL2 in osteoblasts eventually caused osteocyte apoptosis. Osteocytes, which had a reduced number of processes, gradually died with apoptotic structural alterations and the expression of apoptosis-related molecules, and dead osteocytes accumulated in cortical bone. These findings indicate that overexpression of BCL2 in osteoblasts inhibits osteoblast differentiation, reduces osteocyte processes, and causes osteocyte apoptosis.

Vitamin D endocrine system and the intestine

Calcium and phosphate regulate numerous biological processes and they are essential for bone mass and bone quality. The calcium and phosphate balance largely depends on intestinal absorption, and the dietary content of these ions determines the type of transport. High dietary intake of calcium and phosphate enables absorption by passive transport, but often the dietary content of these ions is in the low-normal range, especially for calcium. In this condition, the contribution of active intestinal calcium transport will increase to maintain normal serum levels. This adaptation is mainly regulated by the active form of vitamin D, 1,25 dihydroxyvitamin D, and requires normal concentrations of the precursor 25-hydroxyvitamin D. When intestinal calcium absorption is insufficient, hormonal adaptations will release calcium from bones to secure normocalcemia, not only by increasing bone loss but also by decreasing bone mineralization. These data underline the fact that adequate calcium intake is critical to secure skeletal integrity. Despite the insights that sufficient dietary calcium intake and normal 25-hydroxyvitamin D levels are critical for calcium and bone homeostasis, surprisingly little is known on the proteins that mediate intestinal calcium transport. Also, the interaction between the intestine and the kidney to control serum phosphate levels is still incompletely understood.