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Proliferation and differentiation of osteoclasts are regulated by a cytokine system that includes RANKL, which binds 2 receptors: RANK, which activates osteoclast differentiation, and osteoprotegerin (OPG), a decoy receptor that limits RANKL action. We investigated the role of the OPG/ RANKL/RANK network in the pathogenesis of skeletal metastasis in neuroblastoma. Four different neuroblastoma cell lines (NB100, CHP212, SH-SY5Y, SJ-NK-P) showed a large amount of OPG and RANKL transcripts. Soluble RANKL was detectable in all cell lines, but poor release of OPG was observed. SH-SY5Y showed the lowest OPG-to-RANKL ratio and promoted osteoclastic differentiation of FLG29.1 and peripheral mononuclear cells, inducing expression of the osteoclast markers RANK, c-src, c-fos, cathepsin-K and TRAP. SJ-N-KP, which released both OPG and RANKL, did not show the same capability. OPG, neutralizing anti-RANKL antibody and antisense oligonucleotides were evaluated for their ability to inhibit RANKL activity. The neutralizing antibody hampered osteoclastic differentiation by blocking both the juxtacrine and the paracrine activity of RANKL. Our findings confirm that neuroblastoma cells induce osteoclastogenesis via RANKL and suggest that the RANKL expression associated with lack of the decoy receptor OPG could be a peculiarity of some tumors that makes them able to induce metastatic osteolysis. Moreover, our results suggest that RANKL could be a relevant target in the adjuvant therapy of bone metastatic neuroblastoma as proper neutralization revokes completely osteoclastic differentiation. © 2004 Wiley-Liss, Inc. Key words: neuroblastoma; bone metastasis; osteoclast; RANKL; osteoprotegerinMetastasis is the most relevant prognostic factor for survival in neuroblastoma patients, and bone is one of the target organs of metastasis in advanced neuroblastoma. Patients with bone metastasis are classified as stage IV, regardless of other clinical manifestations, since the presence of bone/bone marrow metastasis is responsible for poor prognosis despite high-dose chemotherapy and autologous hematopoietic stem cell transplantation. 1 One possible reason for this ominous prognosis is the difficulty in reaching cancer cells that have colonized the bone/bone marrow microenvironment. This suggests the need for effective therapeutic modalities for patients with skeletal metastases based on a precise understanding of the pathophysiology of bone colonization in neuroblastoma. Two classical theories are generally advocated to explain the invasion of tumor cells in bone, i.e., the soil hypothesis 2 and the circulation theory. 3 The first suggests that proper factors favor the implantation of metastatic cells, including nutrients, oxygen tension, hormonal environment and hydrogen ion concentrations. The second assumes that development of cancer metastases is dependent on the blood volume inside a target organ. This hemodynamic theory alone cannot explain the high frequency of skeletal metastasis from some neoplasms because blood flow in bone is very p...

Summary Human bone marrow stromal cells (hMSCs) have the potential to differentiate into osteoblasts; there are age-related decreases in their proliferation and differentiation to osteoblasts. Parathyroid hormone (PTH), when applied intermittently in vivo, has osteoanabolic effects in a variety of systems. In this study, we compared PTH signaling and osteoanabolic effects in hMSCs from young and old subjects. There were age-related decreases in expression of PTH/PTHrP receptor type 1 (PTHR1) gene (p=0.049, n=19) and in PTH activation of CREB (p=0.029, n=7) and PTH stabilization of β-catenin (p=0.018, n=7). Three human PTH peptides, PTH1–34, PTH1-31C (Ostabolin-C, Leu27, Cyclo[Glu22-Lys26]-hPTH1–31), and PTH1–84 (10 nM) stimulated osteoblast differentiation with hMSCs. Treatment with PTH1–34 resulted in a significant 67% increase in alkaline phosphatase (ALP) activity in hMSCs obtained from younger subjects (<50-year-old, n=5), compared with an 18% increase in hMSCs from elders (>55-year-old, n=7). Both knockdown of CREB and treatment with a PKA inhibitor H-89 blocked PTH stimulation of osteoblast differentiation in hMSCs from young subjects. The PTH peptides significantly stimulated proliferation of hMSCs. Treatment with PTH1–34 resulted in an average of twice as many cells in cultures of hMSCs from young subjects (n=4), but had no effect with hMSCs from elders (n=7). Upregulation of PTHR1 by 24-hour pre-treatment with 100 nM dexamethasone rescued PTH stimulation of proliferation in hMSCS from elders. In conclusion, age-related intrinsic alterations in signaling responses to osteoanabolic agents like PTH may contribute to cellular and tissue aging of the human skeleton.