Cristina Vincent

Pro-Inflammatory Cytokines TNF-Related Weak Inducer of Apoptosis (TWEAK) and TNFα Induce the Mitogen-Activated Protein Kinase (MAPK)-Dependent Expression of Sclerostin in Human Osteoblasts †‡

We have recently shown that TNF‐related weak inducer of apoptosis (TWEAK) is a mediator of inflammatory bone remodeling. The aim of this study was to investigate the role of TWEAK in modulating human osteoblast activity, and how TWEAK and TNFα might interact in this context. Recombinant TWEAK and TNF were both mitogenic for human primary osteoblasts (NHBC). TWEAK dose‐ and time‐dependently regulated the expression of the osteoblast transcription factors RUNX2 and osterix. TWEAK inhibited in vitro mineralization and downregulated the expression of osteogenesis‐associated genes. Significantly, TWEAK and TWEAK/TNF induced the expression of the osteoblast differentiation inhibitor and SOST gene product, sclerostin. Sclerostin induction was mitogen‐activated protein kinase (MAPK) dependent. The SOST mRNA levels induced by TWEAK were equivalent to or exceeded those seen in steady‐state human bone, and the TWEAK/TNF induction of SOST mRNA was recapitulated in fresh cancellous bone explants. TWEAK‐induced sclerostin expression was observed in immature osteoblastic cells, both in cycling (Ki67+) primary NHBC and in the cell lines MC3T3‐E1 and MG‐63, as well as in human osteocyte‐like cells and in the osteocyte cell line, MLO‐Y4. Treatment of NHBC with recombinant human sclerostin mimicked the effects of TWEAK to suppress RUNX2 and osteocalcin (OCN). TWEAK, TNF, and sclerostin treatment of NHBC similarly altered levels of phosphorylated and total GSK3β and active and total levels of β‐catenin, implying that the Wnt signaling pathway was affected by all three stimuli. Sclerostin also rapidly activated ERK‐1/2 MAPK signaling, indicating the involvement of additional signaling pathways. Together, our findings suggest that TWEAK, alone and with TNF, can regulate osteoblast function, at least in part by inducing sclerostin expression. Our results also suggest new roles and modes of action for sclerostin.

RANK Expression as a cell surface marker of human osteoclast precursors in peripheral blood, bone marrow, and giant cell tumors of bone.

RANK expression in vivo on hematopoietic subsets including pre‐osteoclasts, identified by monoclonal antibodies, has not been described. We describe the lineages that express RANK in bone marrow, peripheral blood, and GCTs. We show that CD14+RANKhigh cells constitute a circulating pre‐osteoclast pool.

Osteoclastic Metabolism of 25(OH)-Vitamin D3: A Potential Mechanism for Optimization of Bone Resorption

The extrarenal synthesis of 1α,25 dihydroxyvitamin D3 (1,25D) has been demonstrated in a number of cell types including osteoblasts and cells of the monocyte/macrophage lineage. The skeleton appears responsive to serum levels of the 1,25D precursor, 25 hydroxyvitamin D3 (25D), in terms of bone mineralization parameters. The effect of metabolism of 25D into active 1,25D by osteoclast lineage cells is unknown. We found that CYP27B1 mRNA expression increased with exposure of human peripheral blood mononuclear cells (PBMCs) to macrophage colony-stimulating factor in the presence or absence of receptor activator of nuclear factor-κB ligand. Consistent with this, human osteoclast cultures incubated with 25D produced measurable quantities of 1,25D. Osteoclast formation from either mouse RAW264.7 cells or human PBMCs in the presence of physiological concentrations of 25D resulted in significant up-regulation of the key osteoclast transcription factor, nuclear factor of activated T cells-c1 in PBMCs and a number of key osteoclast marker genes in both models. The expression of the osteoblast coupling factor, ephrin-b2, was also increased in the presence of 25D. Levels of CYP27B1 and nuclear factor of activated T cells-1 mRNA correlated during osteoclastogenesis and also in a cohort of human bone samples. CYP27B1 short-hairpin RNA knockdown in RAW264.7 cells decreased their osteoclastogenic potential. 25D dose dependently reduced the resorptive capacity of PBMC-derived osteoclasts without compromising cell viability. 25D also reduced resorption by RAW264.7- and giant cell tumor-derived osteoclasts. Conversely, osteoclasts formed from vitamin D receptor-null mouse splenocytes had increased resorptive activity compared with wild-type cells. We conclude that 25D metabolism is an important intrinsic mechanism for optimizing osteoclast differentiation, ameliorating osteoclast activity, and potentially promoting the coupling of bone resorption to formation.

Calcitonin Receptor Plays a Physiological Role to Protect Against Hypercalcemia in Mice

It is well established that calcitonin is a potent inhibitor of bone resorption; however, a physiological role for calcitonin acting through its cognate receptor, the calcitonin receptor (CTR), has not been identified. Data from previous genetically modified animal models have recognized a possible role for calcitonin and the CTR in controlling bone formation; however, interpretation of these data are complicated, in part because of their mixed genetic background. Therefore, to elucidate the physiological role of the CTR in calcium and bone metabolism, we generated a viable global CTR knockout (KO) mouse model using the Cre/loxP system, in which the CTR is globally deleted by >94% but <100%. Global CTRKOs displayed normal serum ultrafiltrable calcium levels and a mild increase in bone formation in males, showing that the CTR plays a modest physiological role in the regulation of bone and calcium homeostasis in the basal state in mice. Furthermore, the peak in serum total calcium after calcitriol [1,25(OH)2D3]‐induced hypercalcemia was substantially greater in global CTRKOs compared with controls. These data provide strong evidence for a biological role of the CTR in regulating calcium homeostasis in states of calcium stress.

The generation of osteoclasts from RAW 264.7 precursors in defined, serum-free conditions

Osteoclasts are the unique cell type capable of resorbing bone. The discovery of the TNF-ligand family member, RANKL, has allowed more reliable study of these important cells. The mouse monocytic cell line, RAW 264.7, has been shown to readily differentiate into osteoclasts upon exposure to recombinant RANKL. Unlike primary osteoclast precursors, there is no requirement for the addition of macrophage colony stimulating factor (M-CSF). However, to date, their differentiation has always been studied in the context of added foetal calf serum (FCS). FCS is a complex and largely undefined mixture of growth factors and matrix proteins, and varies between batches. For this reason, osteoclastogenesis would ideally be studied in the context of a defined, serum-free medium. RAW 264.7 cells were cultured in serum-replete α-MEM or serum-deprived medium (SDM) shown previously to support the growth of human osteoclasts in a co-culture with normal osteoblasts. In SDM, in the presence of recombinant RANKL, RAW 264.7 cells readily differentiated into tartrate resistant acid phosphatase (TRAP) positive multinucleated osteoclast-like cells, a process that was enhanced with the addition of 1α,25-dihydroxyvitamin D3 (1,25D). While the osteoclasts grown in SDM were smaller in size compared with those derived in serum-replete media, their resorptive capacity was significantly increased as indicated by a twofold increase in average resorption pit size. In conclusion, we describe a defined model for studying osteoclast differentiation and activity in the absence of serum, which will be ideal for studying the role of agonistic and antagonistic molecules in this process.

RNAi-mediated silencing of CYP27B1 abolishes 1,25(OH)2D3 synthesis and reduces osteocalcin and CYP24 mRNA expression in human osteosarcoma (HOS) cells.

Although local synthesis of 1,25D has been postulated to regulate parameters of cell growth and differentiation in non-renal cells, the physiological role of 1,25D production in bone cells remains unclear. We used the technique of RNA interference to inhibit the mRNA encoding the enzyme responsible for 1,25D synthesis, 25-hydroxyvitamin D 1alpha-hydroxylase (CYP27B1). Human osteosarcoma (HOS) cells were transfected with siRNA for CYP27B1 or non-silencing RNA before being treated with 25D for 48h under normal growth conditions. De novo synthesis of 1,25D was measured in the media as well as mRNA levels for CYP27B1, osteocalcin (OCN) and 25-hydroxyvitamin D 24-hydroxylase (CYP24). We demonstrated that HOS cells express CYP27B1 mRNA, metabolize 25D and secrete detectable levels of de novo synthesized 1,25D. CYP27B1 mRNA silencing by RNAi, resulted in the suppression of 1,25D production and subsequent reduction of OCN and CYP24 mRNA expression. Our findings suggest that local 1,25D synthesis has paracrine effects in the bone microenvironment implying that vitamin D metabolism in human osteoblasts represents a physiologically important pathway, possibly regulating the maturation of osteoblasts.

TWEAK and Fn14 expression in the pathogenesis of joint inflammation and bone erosion in rheumatoid arthritis

IntroductionTNF-like weak inducer of apoptosis (TWEAK) has been proposed as a mediator of inflammation and bone erosion in rheumatoid arthritis (RA). This study aimed to investigate TWEAK and TWEAK receptor (Fn14) expression in synovial tissue from patients with active and inactive rheumatoid arthritis (RA), osteoarthritis (OA) and normal controls and assess soluble (s)TWEAK levels in the synovial fluids from patients with active RA and OA. Effects of sTWEAK on osteoclasts and osteoblasts were investigated in vitro.MethodsTWEAK and Fn14 expression were detected in synovial tissues by immunohistochemistry (IHC). Selected tissues were dual labelled with antibodies specific for TWEAK and lineage-selective cell surface markers CD68, Tryptase G, CD22 and CD38. TWEAK mRNA expression was examined in human peripheral blood mononuclear cells (PBMC) sorted on the basis of their expression of CD22. sTWEAK was detected in synovial fluid from OA and RA patients by ELISA. The effect of sTWEAK on PBMC and RAW 264.7 osteoclastogenesis was examined. The effect of sTWEAK on cell surface receptor activator of NF Kappa B Ligand (RANKL) expression by human osteoblasts was determined by flow cytometry.ResultsTWEAK and Fn14 expression were significantly higher in synovial tissue from all patient groups compared to the synovial tissue from control subjects (P < 0.05). TWEAK was significantly higher in active compared with inactive RA tissues (P < 0.05). TWEAK expression co-localised with a subset of CD38+ plasma cells and with CD22+ B-lymphocytes in RA tissues. Abundant TWEAK mRNA expression was detected in normal human CD22+ B cells. Higher levels of sTWEAK were observed in synovial fluids isolated from active RA compared with OA patients. sTWEAK did not stimulate osteoclast formation directly from PBMC, however, sTWEAK induced the surface expression of RANKL by human immature, STRO-1+ osteoblasts.ConclusionsThe expression of TWEAK by CD22+ B cells and CD38+ plasma cells in RA synovium represents a novel potential pathogenic pathway. High levels of sTWEAK in active RA synovial fluid and of TWEAK and Fn14 in active RA tissue, together with the effect of TWEAK to induce osteoblastic RANKL expression, is consistent with TWEAK/Fn14 signalling being important in the pathogenesis of inflammation and bone erosion in RA.

Human trabecular bone-derived osteoblasts support human osteoclast formation in vitro in a defined, serum-free medium

While it has been assumed that osteoblasts in the human support osteoclast formation, in vitro evidence of this is currently lacking. We tested the ability of normal human trabecular bone‐derived osteoblasts (NHBCs) to support osteoclast formation from human peripheral blood mononuclear cells (PBMC) in response to treatment with either 1α,25‐dihydroxyvitamin D3 (1,25D) or parathyroid hormone (PTH), using a serum‐replete medium previously used to support human osteoclast formation on a stroma of murine ST‐2 cells. Under these conditions, NHBC did not support osteoclast formation, as assessed by morphological, histochemical, and functional criteria, despite our previous results demonstrating a link between induction of RANKL mRNA expression and NHBC phenotype in these media. We next tested a defined, serum‐free medium (SDM) on NHBC phenotype, their expression of RANKL and OPG, and their ability to support osteoclast formation. SDM, containing dexamethasone (DEX) and 1,25D, induced phenotypic maturation of NHBC, based on the expression of STRO‐1 and the bone/liver/kidney isoform of alkaline phosphatase (AP). PTH as a single factor did not induce phenotypic change. 1,25D and DEX induced the greatest ratio of RANKL:OPG mRNA, predictive of supporting osteoclast formation. Consistent with this, co‐culture of NHBC with CD14+ PBMC, or bone marrow mononuclear cell (BMMC), or CD34+ BMMC precursors in SDM + 1,25D + DEX, resulted in functional osteoclast formation. Osteoclast formation also occurred in PTH + DEX stimulated co‐cultures. Interestingly, SDM supplemented with recombinant RANKL (25–100 ng/ml) and M‐CSF (25 ng/ml), did not induce osteoclast formation from any of the osteoclast precursor populations in stromal‐free cultures, unlike serum‐replete medium. This study demonstrates that under the appropriate conditions, adult human primary osteoblasts can support de novo osteoclast formation, and this model will enable the detailed study of the role of both cell types in this process.

Apo2L/TRAIL inhibits tumor growth and bone destruction in a murine model of multiple myeloma.

Purpose: Multiple myeloma is an incurable disease, for which the development of new therapeutic approaches is required. Here, we report on the efficacy of recombinant soluble Apo2L/tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) to inhibit tumor progression and bone destruction in a xenogeneic model of human multiple myeloma. Experimental Design: We established a mouse model of myeloma, in which Apo2L/TRAIL-sensitive RPMI-8226 or KMS-11 cells, tagged with a triple reporter gene construct (NES-HSV-TK/GFP/Luc), were transplanted directly into the tibial marrow cavity of nude mice. Tumor burden was monitored progressively by bioluminescence imaging and the development of myeloma-induced osteolysis was measured using high resolution in vivo micro-computed tomography. Results: Tumor burden increased progressively in the tibial marrow cavity of mice transplanted with Apo2L/TRAIL-sensitive RPMI-8226 or KMS-11 cells associated with extensive osteolysis directly in the area of cancer cell transplantation. Treatment of mice with recombinant soluble Apo2L/TRAIL reduced myeloma burden in the bone marrow cavity and significantly protected against myeloma-induced osteolysis. The protective effects of Apo2L/TRAIL treatment on bone were mediated by the direct apoptotic actions of Apo2L/TRAIL on myeloma cells within the bone microenvironment. Conclusions: This is the first in vivo study that investigates the efficacy of recombinant Apo2L/TRAIL on myeloma burden within the bone microenvironment and associated myeloma-induced bone destruction. Our findings that recombinant soluble Apo2L/TRAIL reduces myeloma burden within the bone microenvironment and protects the bone from myeloma-induced bone destruction argue against an inhibitory role of osteoprotegerin in Apo2L/TRAIL-induced apoptosis in vivo and highlight the need to clinically evaluate Apo2L/TRAIL in patients with multiple myeloma.

Does Apo2L/TRAIL play any physiologic role in osteoclastogenesis?

To the editor: Apo2L/TRAIL is a promising new cancer therapeutic with demonstrated efficacy against a variety of malignancies.[1][1] A number of recent studies have demonstrated the ability of different versions of recombinant Apo2L/TRAIL to inhibit both human and mouse osteoclastogenesis, and