The physiological role of the TNF receptor (TNFR) family member, RANK, was investigated by generating RANK-deficient mice. RANK −/− mice were characterized by profound osteopetrosis resulting from an apparent block in osteoclast differentiation. RANK expression was not required for the commitment, differentiation, and functional maturation of macrophages and dendritic cells from their myeloid precursors but provided a necessary and specific signal for the differentiation of myeloid-derived osteoclasts. RANK −/− mice also exhibited a marked deficiency of B cells in the spleen. RANK −/− mice retained mucosal-associated lymphoid tissues including Peyer's patches but completely lacked all other peripheral lymph nodes, highlighting an additional major role for RANK in lymph node formation. These experiments reveal that RANK provides critical signals necessary for lymph node organogenesis and osteoclast differentiation.

BACKGROUND. Metastases to bone are a frequent complication of human prostate cancer and result in the development of osteoblastic lesions that include an underlying osteoclastic component. Previous studies in rodent models of breast and prostate cancer have established that receptor activator of NF-kB ligand (RANKL) inhibition decreases bone lesion development and tumor growth in bone. RANK is essential for osteoclast differentiation, activation, and survival via its expression on osteoclasts and their precursors. RANK expression has also been observed in some tumor cell types such as breast and colon, suggesting that RANKL may play a direct role on tumor cells. METHODS. Male CB17 severe combined immunodeficient (SCID) mice were injected with PC3 cells intratibially and treated with either PBS or human osteprotegerin (OPG)-Fc, a RANKL antagonist. The formation of osteolytic lesions was analyzed by X-ray, and local and systemic levels of RANKL and OPG were analyzed. RANK mRNA and protein expression were assessed on multiple prostate cancer cell lines, and events downstream of RANK activation were studied in PC3 cells in vitro. RESULTS. OPG-Fc treatment of PC3 tumor-bearing mice decreased lesion formation and tumor burden. Systemic and local levels of RANKL expression were increased in PC3 tumor bearing mice. PC3 cells responded to RANKL by activating multiple signaling pathways which resulted in significant changes in expression of genes involved in osteolysis and migration. RANK activation via RANKL resulted in increased invasion of PC3 cells through a collagen matrix.CONCLUSION. These data demonstrate that host stromal RANKL is induced systemically and locally as a result of PC3 prostate tumor growth within the skeleton. RANK is expressed on prostate cancer cells and promotes invasion in a RANKL-dependent manner.

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Signaling through receptor activator of nuclear factor-B (RANK) is essential for the differentiation and activation of osteoclasts, the cell principally responsible for bone resorption. Animals genetically deficient in RANK or the cognate RANK ligand are profoundly osteopetrotic because of the lack of bone resorption and remodeling. RANK provokes biochemical signaling via the recruitment of intracellular tumor necrosis factor receptor-associated factors (TRAFs) after ligand binding and receptor oligomerization. To understand the RANK-mediated signal transduction mechanism in osteoclastogenesis, we have designed a system to recapitulate osteoclast differentiation and activation in vitro by transfer of the RANK cDNA into hematopoietic precursors genetically deficient in RANK. Gene transfer of RANK constructs that are selectively incapable of binding different TRAF proteins revealed that TRAF pathways downstream of RANK that affect osteoclast differentiation are functionally redundant. In contrast, the interaction of RANK with TRAF6 is absolutely required for the proper formation of cytoskeletal structures and functional resorptive activity of osteoclasts. Moreover, signaling via the interleukin-1 receptor, which also utilizes TRAF6, rescues the osteoclast activation defects observed in the absence of RANK/TRAF6 interactions. These studies are the first to define the functional domains of the RANK cytoplasmic tail that control specific differentiation and activation pathways in osteoclasts.The structural and metabolic integrity of bone is maintained through the dynamic process of bone remodeling that results from the coordinate action of bone resorption by osteoclasts and the formation of new bone by osteoblasts. Osteoclasts are large, multinuclear cells that develop from a hematopoietic progenitor and are highly specialized for the resorptive process (1). Regulation of bone remodeling occurs through multiple mechanisms that ultimately converge on the interaction of osteoclasts or their precursors with osteoblasts and bone marrow stromal cells. Two key factors supplied by the stromal environment are CSF-1 1 and the TNF family member, RANKL (also called TRANCE, ODF, OPGL) (2), as confirmed by the osteopetrotic phenotypes of the op/op mice that are mutated in the CSF-1 gene (3) and the RANKL knockout mice (4).It is now widely accepted that most osteotropic agents including IL-1, IL-6, IL-11, IL-17, TNF-␣, prostaglandin E 2 , parathyroid hormone, and 1,25-dihydroxyvitamin D 3 (5) affect bone resorption primarily by enhancing stromal cell production of RANKL. RANKL affects bone resorption and bone density by influencing the osteoclast population at multiple stages. Not only does RANKL drive the differentiation of osteoclasts from multipotential progenitors, thereby expanding the pool of osteoclasts available for bone resorption (6), RANKL also activates resorption and enhances survival of existing mature osteoclasts in vitro (7,8) and in vivo (9). An essential role for the RANKL receptor, RANK, in osteoclast differenti...

Thymic stromal lymphopoietin (TSLP) is a novel cytokine that was found to promote the development of murine B cells in vitro.Here we describe the cloning and characterization of the human homologue of murine TSLP. This protein, which is expressed in a number of tissues including heart, liver and prostate, prevented apoptosis and stimulated growth of the human acute myeloid leukemia (

The Tax protein, encoded by the human

RANK and RANKL, the key regulators of osteoclast differentiation and activation, also play an important role in the control of proliferation and differentiation of mammary epithelial cells during pregnancy. Here, we show that RANK protein expression is strictly regulated in a spatial and temporal manner during mammary gland development. RANK overexpression under the control of the mouse mammary tumor virus (MMTV) promoter in a transgenic mouse model results in increased mammary epithelial cell proliferation during pregnancy, impaired differentiation of lobulo-alveolar structures, decreased expression of the milk proteins ␤-casein and whey acidic protein, and deficient lactation. We also show that treatment of three-dimensional in vitro cultures of primary mammary cells from MMTV-RANK mice with RANKL results in increased proliferation and decreased apoptosis in the luminal area, resulting in bigger acini with filled lumens. Taken together, these results suggest that signaling through RANK not only promotes proliferation but also inhibits the terminal differentiation of mammary epithelial cells. Moreover, the increased proliferation and survival observed in a three-dimensional culture system suggests a role for aberrant RANK signaling during breast tumorigenesis.The tumor necrosis factor (TNF) family member RANKL and its receptor RANK are key regulators of osteoclast differentiation and activation (16,30). The balance between RANKL, RANK, and osteoprotegerin (OPG), a soluble decoy receptor that competes with RANK for binding to RANKL (32), regulates osteoclast differentiation and activation and therefore bone remodeling and mobilization of calcium from the skeleton (27). In mice, RANK and RANKL are also essential for the development of the mammary gland during pregnancy (18), in particular, for the formation of lobuloalveolar structures after pregnancy day 14 (P14) which are required for a functional lactating mammary gland.Deregulation in the RANKL/OPG system has been reported in malignant bone disease including bone metastasis and humoral hypercalcemia of malignancy (13,21,23). Numerous lines of evidence in animal models indicate that blocking RANK/RANKL interaction effectively prevents tumor-induced hypercalcemia or reduces tumor-induced bone lesions, due to its critical role in osteoclast differentiation and activation (reviewed in references 38 and 41). While RANK expression has been detected in breast cancer lines and in primary tumors including breast tumors (45) and may play a functional role in tumor cell migration and metastasis to the bone (24), it is not clear whether activation of the RANK/RANKL pathway may also contribute to primary tumor growth.The mammary gland defects observed in RANK-and RANKL-deficient mice support the notion that the RANK/ RANKL pathway plays an active role in mammary cell proliferation and survival; however, the direct effect of increased RANK signaling in mammary cells is unknown. To determine if activation of the RANK/RANKL pathway can directly increase proliferation or surv...

Signaling through the receptor activator of nuclear factor B (RANK) is required for both osteoclast differentiation and mammary gland development, yet the extent to which RANK utilizes similar signaling pathways in these tissues remains unclear. Mice expressing a kinase-inactive form of the inhibitor of B kinase ␣ (IKK␣) have mammary gland defects similar to those of RANKnull mice yet have apparently normal osteoclast function. Because mice that completely lack IKK␣ have severe skin and skeletal defects that are not associated with IKK␣-kinase activity, we wished to directly examine osteoclastogenesis in IKK␣ ؊/؊ mice. We found that unlike RANK-null mice, which completely lack osteoclasts, IKK␣ ؊/؊ mice did possess normal numbers of TRAP ؉ osteoclasts. However, only 32% of these cells were multinucleated compared with 57% in wild-type littermates. A more profound defect in osteoclastogenesis was observed in vitro using IKK␣ ؊/؊ hematopoietic cells treated with colony-stimulating factor 1 and RANK ligand (RANKL), as the cells failed to form large, multinucleated osteoclasts. Additionally, overall RANKL-induced global gene expression was significantly blunted in IKK␣ ؊/؊ cells, including osteoclastspecific genes such as TRAP, MMP-9, and c-Src. IKK␣ was not required for RANKL-mediated I B␣ degradation or phosphorylation of mitogen-activated protein kinases but was required for RANKL-induced p100 processing. Treatment of IKK␣ ؊/؊ cells with tumor necrosis factor ␣ (TNF␣) in combination with RANKL led to partial rescue of osteoclastogenesis despite a lack of p100 processing. However, the ability of TNF␣ alone or in combination with transforming growth factor ␤ to induce osteoclast differentiation was dependent on IKK␣, suggesting that synergy between RANKL and TNF␣ can overcome p100 processing defects in IKK␣ ؊/؊ cells.Skeletal mass homeostasis is maintained by the coupled activities of bone-building osteoblasts and bone-resorbing osteoclasts. Dysregulation of osteoclast differentiation or function underlies many diseases affecting the skeletal system, including osteoporosis, joint inflammation, tumor metastasis to bone, and Paget disease (1). Osteoclasts are highly specialized multinucleated cells that differentiate from monocyte/macrophage lineage hematopoietic precursors. The key factors regulating osteoclastogenesis are the TNF 1 receptor family member RANK, its ligand (RANKL), and the RANKL inhibitor, osteoprotegerin. Ablation of RANK or RANKL in mice results in a complete absence of osteoclasts and severe osteopetrosis, whereas lack of osteoprotegerin leads to excessive osteoclast activity and osteoporosis (2-5).Genetic studies in mice, as well as in vitro culture of osteoclasts using CSF-1 and RANKL, have elucidated many key components of RANK signaling during osteoclastogenesis. Binding of RANKL to RANK stimulates recruitment of TRAF2, -5, and -6 (6) followed by activation of mitogenactivated protein kinases and I B kinases, which ultimately lead to activation of the transcription factors AP-1 and NF-B. Osteo...