Vitamin D receptor expression in mature osteoclasts reduces bone loss due to low dietary calcium intake in male mice.

Mature osteoclasts express the vitamin D receptor (VDR) and are able to respond to active vitamin D (1α, 25-dihydroxyvitamin D3; 1,25(OH)2D3) by regulating cell maturation and activity. However, the in vivo consequences of vitamin D signalling directly within functionally mature osteoclasts is only partially understood. To investigate the in vivo role of VDR in mature osteoclasts, conditional deletion of the VDR under control of the cathepsin K promoter (CtskCre/Vdr-/-), was assessed in 6 and 12-week-old mice, either under normal dietary conditions (NormCaP) or when fed a low calcium (0.03 %), low phosphorous (0.08 %) diet (LowCaP). Splenocytes from CtskCre/Vdr-/- mice were co-cultured with MLO-Y4 osteocyte-like cells to assess the effect on osteoclastogenesis. Six-week-old CtskCre/Vdr-/- mice demonstrated a 10 % decrease in vertebral bone volume (p < 0.05), which was associated with increased osteoclast size (p < 0.05) when compared to Vdrfl/fl control mice. Control mice fed a LowCaP diet exhibited extensive trabecular bone loss associated with increased osteoclast surface, number and size (p < 0.0001). Interestingly, CtskCre/Vdr-/- mice fed a LowCaP diet showed exacerbated loss of bone volume fraction (BV/TV%) and trabecular number (Tb.N), by a further 22 % and 21 %, respectively (p < 0.05), suggesting increased osteoclastic bone resorption activity with the loss of VDR in mature osteoclasts under these conditions. Co-culture of CtskCre/Vdr-/- splenocytes with MLO-Y4 cells increased resulting osteoclast numbers 2.5-fold, which were greater in nuclei density and exhibited increased resorption of dentine compared to osteoclasts derived from Vdrfl/fl splenocyte cultures. These data suggest that in addition to RANKL-mediated osteoclastogenesis, intact VDR signalling is required for the direct regulation of the differentiation and activity of osteoclasts in both in vivo and ex vivo settings.

Evidence for altered osteoclastogenesis in splenocyte cultures from VDR knockout mice.

The indirect action of 1α,25(OH)2-vitamin-D3 (1,25D) on the osteoclast through stromal signalling is well established. The role of vitamin D in osteoclasts through direct 1,25D-VDR signalling is less well known. We showed previously that local 1,25D synthesis in osteoclasts modified osteoclastogenesis and osteoclastic resorptive activity. In this study, we hypothesised that osteoclasts lacking VDR expression would display an enhanced resorptive capacity due to the loss of 1,25D signalling. Splenocytes were cultured under osteoclast-differentiating conditions from mice with global deletion of the Vdr gene (VDRKO) and this was compared with age-matched wild-type littermate controls (WT). In VDRKO cultures, osteoclastogenesis was reduced, as indicated by fewer TRAP-positive multinucleated cells at all time points measured (p<0.05) compared to WT levels. However, VDRKO osteoclasts demonstrated greater resorption on a per cell basis than their WT counterparts. VDRKO cultures expressed greatly increased c-Fos mRNA compared to WT. In addition, the ratio of expression of the pro-apoptotic gene Bax to the pro-survival gene Bcl-2 was decreased in VDRKO cultures, implying that these osteoclasts may survive longer than WT osteoclasts. Our data indicate abnormal osteoclastogenesis due to the absence of Vdr expression, consistent with direct effects of vitamin D signalling being important for regulating the maturation and resorptive activities of osteoclasts.

Absence of vitamin D receptor in mature osteoclasts results in altered osteoclastic activity and bone loss.

Mature osteoclasts express the vitamin D receptor (VDR) and are able to synthesise and respond to 1,25(OH)2D3 via CYP27B1 enzyme activity. Whether vitamin D signalling within osteoclasts is necessary for the regulation of osteoclastic bone resorption in an in vivo setting is unclear. To determine the requirement for the VDR- and CYP27B1-mediated activity in mature osteoclasts, conditional deletion mouse models were created whereby either Vdr or Cyp27b1 gene was inactivated by breeding either Vdrfl/fl or Cyp27b1fl/fl mice with Cathepsin K-Cre transgenic mice (CstkCre) to generate CtskCre/Vdr-/- and CtskCre/Cyp27b1-/- mice respectively. To account for potential CtskCre-meaited off-target deletion of Vdr, Dmp1Cre were also used determine the effect of Vdr deletion in osteocytes. Furthermore, CtskCre/Vdr-/- mice were ovariectomised (OVX) to assess the role of VDR in osteoclasts under bone-loss conditions and bone marrow precursor cells were cultured under osteoclastogenic conditions to assess osteoclast formation. Six-week-old CtskCre/Vdr-/- female mice demonstrated a 15% decrease in femoral BV/TV (p<0.05). In contrast, BV/TV remained unchanged in CtskCre/Cyp27b1-/- mice as well as in Dmp1Cre/VDR-/- mice. When CtskCre/Vdr-/- mice were subjected to OVX, the bone loss that occurred in CtskCre/Vdr-/- was predominantly due to a diminished volume of thinner trabeculae when compared to control levels. These changes in bone volume in CtskCre/Vdr-/- mice occurred without an observable histological change in osteoclast numbers or size. However, while cultured bone marrow-derived osteoclasts from CtskCre/Vdr-/- mice were marginally increased when compared to VDRfl/fl mice, elevated expression of genes such as Cathepsin K, Nfatc1 and VATPase was observed. Collectively, these data indicate that the absence of VDR in mature osteoclasts causes exacerbated bone loss in young mice and during OVX which is associated with enhanced osteoclastic activity and without increased osteoclastogenesis.

Evidence for altered osteoclastogenesis in splenocyte cultures from Cyp27b1 knockout mice.

The association between increased serum 25-hydroxyvitamin D (25D) and reduced osteoclastic bone resorption is well known. Previously, we have demonstrated that mechanism by which this occurs, may include the conversion of 25D to 1,25-dihydroxyvitamin D (1,25D) by osteoclasts, catalysed by the CYP27B1 enzyme. Local 1,25D synthesis in osteoclasts was shown to regulate osteoclastogenesis and moderating resorptive activity. Thus, we hypothesised that osteoclasts differentiated from mice with global deletion of the Cyp27b1 gene (Cyp27b1 KO) would display enhanced resorptive capacity due to the lack of an ameliorating effect of 1,25D. Splenocytes isolated from Cyp27b1 KO mice or their wild-type (WT) littermates between 6 and 8 weeks of age were cultured under osteoclast-forming conditions for up to 14 days. Osteoclast formation was measured by staining for the osteoclast marker tartrate resistant acid phosphatase (TRAP). Bone resorption activity was measured by plating the cells on a bone-like substrate. In Cyp27b1 KO cultures, osteoclastogenesis was reduced, as indicated by fewer TRAP-positive multinucleated cells at all time points measured (p<0.05) when compared to wild-type (WT) levels. However, Cyp27b1 KO osteoclasts demonstrated greater resorption on a per cell basis than their WT counterparts (p<0.03). In addition, the ratio of expression of the pro-apoptotic gene Bax to the pro-survival gene Bcl-2 was decreased in Cyp27b1 KO cultures, implying that these smaller osteoclasts survive longer than WT osteoclasts. Our data indicate abnormal osteoclastogenesis due to the absence of CYP27B1 expression, consistent with the notion that endogenous metabolism of 25D optimises osteoclastogenesis and ameliorates the resulting activity of mature osteoclasts.