Treatment with 1,25-dihydroxyvitamin D3 reduces impairment of human osteoblast functions during cellular aging in culture

Adequate responses to various hormones, such as 1,25-dihydroxyvitamin D(3) (calcitriol) are a prerequisite for optimal osteoblast functions. We have previously characterized several human diploid osteoblastic cell lines that exhibit typical in vitro aging characteristics during long-term subculturing. In order to study in vitro age-related changes in osteoblast functions, we compared constitutive mRNA levels of osteoblast-specific genes in early-passage (< 50% lifespan completed) with those of late-passage cells (> 90% lifespan completed). We found a significant reduction in mRNA levels of alkaline phosphatase (AP: 68%), osteocalcin (OC: 67%), and collagen type I (ColI: 76%) in in vitro senescent late-passage cells compared to early-passage cells, suggesting an in vitro age-related impairment of osteoblast functions. We hypothesized that decreased osteoblast functions with in vitro aging is due to impaired responsiveness to calcitriol known to be important for the regulation of biological activities of the osteoblasts. Thus, we examined changes in vitamin D receptor (VDR) system and the osteoblastic responses to calcitriol treatment during in vitro osteoblast aging. We found no change in the amount of VDR at either steady state mRNA level or protein level with increasing in vitro osteoblast age and examination of VDR localization, nuclear translocation and DNA binding activity revealed no in vitro age-related changes. Furthermore, calcitriol (10(-8)M) treatment of early-passage osteoblastic cells inhibited their proliferation by 57 +/- 1% and stimulated steady state mRNA levels of AP (1.7 +/- 0.1-fold) and OC (1.8 +/- 0.2-fold). Similarly, calcitriol treatment increased mRNA levels of AP (1.7 +/- 0.2-fold) and OC (3.0 +/- 0.3-fold) in late-passage osteoblastic cells. Thus, in vitro senescent osteoblastic cells maintain their responsiveness to calcitriol and some of the observed in vitro age-related decreases in biological markers of osteoblast functions can be reverted by calcitriol treatment.     

Lipopolysaccharide negatively modulates vitamin D action by down-regulating expression of vitamin D-induced VDR in human monocytic THP-1 cells

Vitamin D3, an important seco-steroid hormone for the regulation of body calcium homeostasis, promotes immature myeloid precursor cells to differentiate into monocytes/macrophages. Vitamin D receptor (VDR) belongs to a nuclear receptor super-family that mediates the genomic actions of vitamin D3 and regulates gene expression by binding with vitamin D response elements in the promoter region of the cognate gene. Thus by regulating gene expression, VDR plays an important role in modulating cellular events such as differentiation, apoptosis, and growth. Here we report lipopolysaccharide (LPS), a bacterial toxin; decreases VDR protein levels and thus inhibits VDR functions in the human blood monocytic cell line, THP-1. The biologically active form of vitamin D3, 1alpha,25-dihydroxy vitamin D3 [1,25(OH)2D3], induced VDR in THP-1 cells after 24 h treatment, and LPS inhibited 1,25(OH)2D3-mediated VDR induction. However, LPS and 1,25(OH)2D3 both increased VDR mRNA levels in THP-1 cells 20 h after treatment, as observed by real time RT-PCR. Moreover, LPS plus 1,25(OH)2D3 action on VDR mRNA level was additive and synergistic. A time course experiment up to 60 h showed an increase in VDR mRNA that was not preceded with an increase in VDR protein levels. Although the proteasome pathway plays an important role in VDR degradation, the proteasome inhibitor lactacystin had no effect on the LPS-mediated down-regulation of 1,25(OH)2D3 induced VDR levels. Reduced VDR levels by LPS were accompanied by decreased 1,25(OH)2D3/VDR function determined by VDR responsive 24-hydroxylase (CYP24) gene expression. The above results suggest that LPS impairs 1,25(OH)2D3/VDR functions, which may negatively affect the ability of 1,25(OH)2D3 to induce myeloid differentiation into monocytes/macrophages.     

Fetuin A is down-regulated in rats during heterotopic ossification after Achilles tenotomy

We investigated the role of fetuin A during heterotopic ossification (HO) in rats following Achilles tenotomy. We performed a right midpoint Achilles tenotomy on 24 rats. At 5 and 10 h after surgery, we investigated the formation of ectopic bone using X-ray and histological examination. We evaluated the mRNA level of fetuin A using real-time PCR. Presence of fetuin A in the Achilles tendon was assessed by immunohistochemical staining. We also measured the serum concentration of fetuin A using enzyme linked immunosorbent assay (ELISA). The expression of fetuin A was significantly decreased in both the liver and Achilles tendon during HO. ELISA showed a small amount of fetuin A in blood throughout the development of HO. Immunohistochemical staining showed that fetuin A was abundant in the ectopic bone. Fetuin A appears to be involved in the formation of ectopic bone induced by Achilles tenotomy, and a deficiency of fetuin A plays a role in the development of HO.     

Functionality of unliganded VDR in breast cancer cells: repressive action on CYP24 basal transcription

It is well-established that CYP24, an immediate target gene of VDR is upregulated by VDR ligands. This study is focused on the functional role of unliganded VDR by investigating the correlation between the expression of VDR protein and basal mRNA levels of CYP24 in breast cancer cell lines. Analyses of multiple breast cancer cell lines demonstrated an inverse correlation between VDR protein expression and CYP24 mRNA expression levels; while in the presence of ligand, VDR protein level was positively correlated with CYP24 expression. In MCF-7 cells, VDR was mainly distributed in the nuclei in the absence of ligand. VDR overexpression in MCF-7 cells and MDA-MB231 cells decreased CYP24 mRNA expression levels and CYP24 promoter activity. Conversely, knock-down of VDR using siRNA techniques in MCF-7 and T47D cells significantly increased CYP24 mRNA expression. We also found that overexpression of VDR with a polymorphic site (FokI-FF) at its AF-1 domain, which makes VDR shorter by three amino acids, failed to repress CYP24 promoter activity. This report provides conclusive evidence for the repressive action of unliganded VDR on the expression of its target gene CYP24 and the importance of an intact VDR AF-1 domain for its repressive action.     

Inhibition of IL-17 prevents the progression of traumatic heterotopic ossification

Traumatic heterotopic ossification (HO) is the abnormal formation of bone in soft tissues as a consequence of injury. However, the pathological mechanisms leading to traumatic HO remain unknown. Here, we report that aberrant expression of IL-17 promotes traumatic HO formation by activating β-catenin signalling in mouse model. We found that elevated IL-17 and β-catenin levels are correlated with a high degree of HO formation in specimens from patients and HO animals. We also show that IL-17 initiates and promotes HO progression in mice. Local injection of an IL-17 neutralizing antibody attenuates ectopic bone formation in a traumatic mouse model. IL-17 enhances the osteoblastic differentiation of mesenchymal stem cells (MSCs) by activating β-catenin signalling. Moreover, inhibition of IL-17R or β-catenin signalling by neutralizing antibodies or drugs prevents the osteogenic differentiation of isolated MSCs and decreases HO formation in mouse models. Together, our study identifies a novel role for active IL-17 as the inducer and promoter of ectopic bone formation and suggests that IL-17 inhibition might be a potential therapeutic target in traumatic HO.     

VDR-mediated inhibition of DKK1 and SFRP2 suppresses adipogenic differentiation of murine bone marrow stromal cells

Osteoblasts and adipocytes are thought to derive from a common bone marrow stromal cell (BMSC) precursor. Activation of the canonical Wnt signaling pathway plays a pivotal role in the differentiation of BMSCs along either of these two lineages, promoting osteogenesis and inhibiting adipogenesis. Liganded nuclear receptors, including the vitamin D receptor (VDR) and peroxisomal proliferator-activated receptor gamma (PPARgamma), can also affect BMSCs differentiation. To address whether VDR ablation modulates the differentiation of BMSCs into the osteoblast or adipogenic lineages, BMSCs were isolated from VDR null mice and from their wild-type littermates. VDR ablation did not alter osteoblastic differentiation. However, when cultured under adipogenic conditions, BMSCs from the VDR null mice expressed higher mRNA levels of PPARgamma and of markers of adipogenic differentiation. An increase in the size and number of mature adipocyte foci was also observed in cultures isolated from VDR null mice relative to those isolated from wild-type mice. To address whether the increased adipogenesis observed in the VDR null cultures was associated with inhibition of the canonical Wnt signaling pathway, mRNA levels for DKK1 and SFRP2 were examined. Cultures from the VDR null mice expressed higher levels of mRNA encoding DKK1 and SFRP2 than did the wild-type cultures. This difference is, at least in part, due to ligand-dependent actions of the VDR, since 1,25-dihydroxyvitamin D3 suppressed DKK1 and SFRP2 expression in wild-type cultures. Thus, the VDR inhibits adipogenesis of BMSCs at least in part by suppressing the expression of inhibitors of the canonical Wnt signaling pathway.     

p53 and MDM2 are involved in the regulation of osteocalcin gene expression

Osteocalcin (OC) is a major noncollagenous bone matrix protein and an osteoblast marker whose expression is limited to mature osteoblasts during the late differentiation stage. In previous studies we have shown osteosarcomas to lose p53 function with a corresponding loss of osteocalcin gene expression. Introduction of wild type p53 resulted in re expression of the osteocalcin gene. Using gel shift and chromatin immunoprecipitation assays, we have identified a putative p53 binding site within the rat OC promoter region and observed an increase in OC promoter activity when p53 accumulates using a CAT assay. The p53 inducible gene Mdm2 is a well-known downstream regulator of p53 levels. Our results showed a synergistic increase in the OC promoter activity when both p53 and MDM2 were transiently overexpressed. We further demonstrate that p53 is not degraded during overexpression of MDM2 protein. Increased OC expression was observed with concomitantly increased p53, VDR, and MDM2 levels in ROS17/2.8 cells during treatment with differentiation promoting (DP) media, but was significantly decreased when co-treated with DP media and the small molecule inhibitor of MDM2-p53 interaction, Nutlin-3. We have also observed a dramatic increase of the OC promoter activity in the presence of p53 and Mdm2 with inclusion of Cbfa-1 and p300 factors. Our results suggest that under some physiological conditions the oncoprotein MDM2 may cooperate with p53 to regulate the osteocalcin gene during osteoblastic differentiation.     

Skeletal Unloading Induces Biphasic Changes in Insulin-Like Growth Factor-I mRNA Levels and Osteoblast Activity

To determine the local mechanisms involved in the effects of skeletal unloading on bone formation, we studied the temporal pattern of mRNA levels for insulin-like growth factor-I (IGF-I), IGF-I receptor type I (IGF-IR), and transforming growth factor beta receptor type II (TGF-betaRII) in relation to osteoblast phenotypic markers and osteoblast activity in hindlimb suspended rats. Skeletal unloading decreased bone volume and the mineralizing and osteoblastic surfaces at 4, 7, and 14 days in the tibial metaphysis, whereas the mineral appositional rate returned to normal at 14 days of suspension. RT-PCR analysis showed that skeletal unloading decreased type 1 collagen (Col 1) and osteocalcin (OC) mRNA levels in metaphyseal bone at days 4 and 7, and the levels returned to normal at 14 days of suspension. Unloading also decreased mRNA levels for IGF-I, IGF-IR, and TGF-betaRII at 4-7 days in the metaphyseal bone. However, IGF-I and IGF-IR levels rose above normal at 14 days of suspension. The biphasic changes in IGF-I mRNA levels were strongly correlated with Col 1 and OC mRNA levels. The associated biphasic pattern of IGF-I/IGF-IR expression, osteoblast markers, and osteoblast activity strongly suggests an important role for IGF-I signaling in the local effect of skeletal unloading on metaphyseal bone formation.     

Upregulation of heme oxygenase‐1 inhibits the maturation and mineralization of osteoblasts

Heme‐oxygenase‐1 (HO‐1), an important enzyme involved in vascular disease, transplantation, and inflammation, catalyzes the degradation of heme into carbon monoxide and biliverdin. It has been reported that overexpression of HO‐1 inhibits osteoclastogenesis. However, the effect of HO‐1 on osteoblast differentiation is still not clear. We here used adenoviral vector expressing recombinant human HO‐1 and HO‐1 inducer hemin to study the effects of HO‐1 in primary cultured osteoblasts. The results showed that induction of HO‐1 inhibited the maturation of osteoblasts including mineralized bone nodule formation, alkaline phosphatase activity and decreased mRNA expression of several differentiation markers such as alkaline phosphatase, osteocalcin, and RUNX2. Furthermore, downstream products of HO‐1, bilirubin, carbon monoxide, and iron, are involved in the inhibitory action of HO‐1. HO‐1 can be induced by H₂O₂, lipopolysaccharide and inflammatory cytokines such as TNF‐α and IL‐1β in osteoblasts and also in STZ‐induced diabetic mice. In addition, endogenous PPARγ ligand, 15‐deoxy‐Δ^12,14‐prostaglandin‐J2 (15d‐PGJ2) markedly increased both mRNA and protein levels of HO‐1 in osteoblasts via PI3K‐Akt and MAPK pathways. Blockade of HO activity by ZnPP IX antagonized the inhibitory action on osteocalcin expression by hemin and 15d‐PGJ2. Our results indicate that upregulation of HO‐1 inhibits the maturation of osteoblasts and HO‐1 may be involved in oxidative‐ or inflammation‐induced bone loss. J. Cell. Physiol. 222: 757–768, 2010. © 2009 Wiley‐Liss, Inc.     

Promoter and 3'-untranslated-region haplotypes in the vitamin d receptor gene predispose to osteoporotic fracture: the rotterdam study

Polymorphisms of the vitamin D receptor gene (VDR) have been shown to be associated with several complex diseases, including osteoporosis, but the mechanisms are unknown and study results have been inconsistent. We therefore determined sequence variation across the major relevant parts of VDR, including construction of linkage disequilibrium blocks and identification of haplotype alleles. We analyzed 15 haplotype-tagging SNPs in relation to 937 clinical fractures recorded in 6,148 elderly whites over a follow-up period of 7.4 years. Haplotype alleles of the 5' 1a/1e, 1b promoter region and of the 3' untranslated region (UTR) were strongly associated with increased fracture risk. For the 16% of subjects who had risk genotypes at both regions, their risk increased 48% for clinical fractures (P = .0002), independent of age, sex, height, weight, and bone mineral density. The population-attributable risk varied between 1% and 12% for each block and was 4% for the combined VDR risk genotypes. Functional analysis of the variants demonstrated 53% lower expression of a reporter construct with the 1e/1a promoter risk haplotype (P = 5 x 10(-7)) in two cell lines and 15% lower mRNA level of VDR expression constructs carrying 3'-UTR risk haplotype 1 in five cell lines (P = 2 x 10(-6)). In a further analysis, we showed 30% increased mRNA decay in an osteoblast cell line for the construct carrying the 3'-UTR risk haplotype (P = .02). This comprehensive candidate-gene analysis demonstrates that the risk allele of multiple VDR polymorphisms results in lower VDR mRNA levels. This could impact the vitamin D signaling efficiency and might contribute to the increased fracture risk we observed for these risk haplotype alleles.     

Loss of RUNX3 expression may contribute to poor prognosis in patients with chondrosarcoma

Chondrosarcoma is the second most common type of bone cancer. Loss of RUNX3 expression has been demonstrated in many other cancers. However, no studies have shown the relationship between RUNX3 expression and chondrosarcoma. In this study, we detected RUNX3 expression in the progression of chondrosarcoma. In patient samples, the levels of RUNX3 mRNA and protein were lower in cancer tissues than in normal tissues. Down-regulation of RUNX3 mRNA in tumor tissues was associated with an increase in RUNX3 promoter methylation. Loss of RUNX3 expression was significantly associated with more aggressive chondrosarcoma types and decreased survival time of patients. To examine the effects of exogenous expression of RUNX3 in vitro, chondrosarcoma cells were transfected with the pcDNA3.1-RUNX3 expression vector. Relative to control cells, RUNX3-expressing cells exhibited lower proliferation and higher apoptosis rates as assessed by colony formation and Annexin V-FITC/PI double staining, respectively. Taken together, these results suggest that RUNX3 acts a tumor suppressor in chondrosarcoma and that RUNX3 promoter methylation may be the molecular mechanism for its decreased expression.