The vitamin D receptor is involved in the regulation of human breast cancer cell growth via a ligand-independent function in cytoplasm

Vitamin D has pleiotropic effects on multiple tissues, including malignant tumors. Vitamin D inhibits breast cancer growth through activation of the vitamin D receptor (VDR) and via classical nuclear signaling pathways. Here, we demonstrate that the VDR can also function in the absence of its ligand to control behaviour of human breast cancer cells both outside and within the bone microenvironment. Stable shRNA expression was used to knock down VDR expression in MCF-7 cells, generating two VDR knockdown clonal lines. In ligand-free culture, knockdown of VDR in MCF-7 cells significantly reduced proliferation and increased apoptosis, suggesting that the VDR plays a ligand-independent role in cancer cell growth. Implantation of these VDR knockdown cells into the mammary fat pad of nude mice resulted in reduced tumor growth in vivo compared with controls. In the intra-tibial xenograft model, VDR knockdown greatly reduced the ability of the cells to form tumors in the bone microenvironment. The in vitro growth of VDR knockdown cells was rescued by the expression of a mutant form of VDR which is unable to translocate to the nucleus and hence accumulates in the cytoplasm. Thus, our data indicate that in the absence of ligand, the VDR promotes breast cancer growth both in vitro and in vivo and that cytoplasmic accumulation of VDR is sufficient to produce this effect in vitro. This new mechanism of VDR action in breast cancer cells contrasts the known anti-proliferative nuclear actions of the VDR-vitamin D ligand complex

Single-Limb Irradiation Induces Local and Systemic Bone Loss in a Murine Model

Increased fracture risk is commonly reported in cancer patients receiving radiotherapy, particularly at sites within the field of treatment. The direct and systemic effects of ionizing radiation on bone at a therapeutic dose are not well-characterized in clinically relevant animal models. Using 20-week-old male C57Bl/6 mice, effects of irradiation (right hindlimb; 2 Gy) on bone volume and microarchitecture were evaluated prospectively by microcomputed tomography and histomorphometry and compared to contralateral-shielded bone (left hindlimb) and non-irradiated control bone. One week postirradiation, trabecular bone volume declined in irradiated tibias (-22%; p < 0.0001) and femurs (-14%; p = 0.0586) and microarchitectural parameters were compromised. Trabecular bone volume declined in contralateral tibias (-17%; p = 0.003), and no loss was detected at the femur. Osteoclast number, apoptotic osteocyte number, and marrow adiposity were increased in irradiated bone relative to contralateral and non-irradiated bone, whereas osteoblast number was unchanged. Despite no change in osteoblast number 1 week postirradiation, dynamic bone formation indices revealed a reduction in mineralized bone surface and a concomitant increase in unmineralized osteoid surface area in irradiated bone relative to contralateral and non-irradiated control bone. Further, dose-dependent and time-dependent calvarial culture and in vitro assays confirmed that calvarial osteoblasts and osteoblast-like MC3T3 cells were relatively radioresistant, whereas calvarial osteocyte and osteocyte-like MLO-Y4 cell apoptosis was induced as early as 48 hours postirradiation (4 Gy). In osteoclastogenesis assays, radiation exposure (8 Gy) stimulated murine macrophage RAW264.7 cell differentiation, and coculture of irradiated RAW264.7 cells with MLO-Y4 or murine bone marrow cells enhanced this effect. These studies highlight the multifaceted nature of radiation-induced bone loss by demonstrating direct and systemic effects on bone and its many cell types using clinically relevant doses; they have important implications for bone health in patients treated with radiation therapy

Hyperactive transforming growth factor-β1 signaling potentiates skeletal defects in a neurofibromatosis type 1 mouse model

Dysregulated transforming growth factor beta (TGF-β) signaling is associated with a spectrum of osseous defects as seen in Loeys-Dietz syndrome, Marfan syndrome, and Camurati-Engelmann disease. Intriguingly, neurofibromatosis type 1 (NF1) patients exhibit many of these characteristic skeletal features, including kyphoscoliosis, osteoporosis, tibial dysplasia, and pseudarthrosis; however, the molecular mechanisms mediating these phenotypes remain unclear. Here, we provide genetic and pharmacologic evidence that hyperactive TGF-β1 signaling pivotally underpins osseous defects in Nf1(flox/-) ;Col2.3Cre mice, a model which closely recapitulates the skeletal abnormalities found in the human disease. Compared to controls, we show that serum TGF-β1 levels are fivefold to sixfold increased both in Nf1(flox/-) ;Col2.3Cre mice and in a cohort of NF1 patients. Nf1-deficient osteoblasts, the principal source of TGF-β1 in bone, overexpress TGF-β1 in a gene dosage-dependent fashion. Moreover, Nf1-deficient osteoblasts and osteoclasts are hyperresponsive to TGF-β1 stimulation, potentiating osteoclast bone resorptive activity while inhibiting osteoblast differentiation. These cellular phenotypes are further accompanied by p21-Ras-dependent hyperactivation of the canonical TGF-β1-Smad pathway. Reexpression of the human, full-length neurofibromin guanosine triphosphatase (GTPase)-activating protein (GAP)-related domain (NF1 GRD) in primary Nf1-deficient osteoblast progenitors, attenuated TGF-β1 expression levels and reduced Smad phosphorylation in response to TGF-β1 stimulation. As an in vivo proof of principle, we demonstrate that administration of the TGF-β receptor 1 (TβRI) kinase inhibitor, SD-208, can rescue bone mass deficits and prevent tibial fracture nonunion in Nf1(flox/-) ;Col2.3Cre mice. In sum, these data demonstrate a pivotal role for hyperactive TGF-β1 signaling in the pathogenesis of NF1-associated osteoporosis and pseudarthrosis, thus implicating the TGF-β signaling pathway as a potential therapeutic target in the treatment of NF1 osseous defects that are refractory to current therapie

Recql4 haploinsufficiency in mice leads to defects in osteoblast progenitors: Implications for low bone mass phenotype

The cellular and molecular mechanisms that underlie skeletal abnormalities in defective Recql4-related syndromes are poorly understood. Our objective in this study was to explore the function of Recql4 in osteoblast biology both in vitro and in vivo. Immunohistochemistry on adult mouse bone showed Recql4 protein localization in active osteoblasts around growth plate, but not in fully differentiated osteocytes. Consistent with this finding, Recql4 gene expression was high in proliferating mouse osteoblastic MC3T3.E1 cells and decreased as cells progressively lost their proliferation activity during differentiation. Recql4 overexpression in osteoblastic cells exhibited higher proliferation activity, while its depletion impeded cell growth. In addition, bone marrow stromal cells from male Recql4+/- mice had fewer progenitor cells, including osteoprogenitors, indicated by reduced total fibroblast colony forming units (CFU-f) and alkaline phosphatase-positive CFU-f colonies concomitant with reduced bone mass. These findings provide evidence that Recql4 functions as a regulatory protein during osteoprogenitor proliferation, a critical cellular event during skeleton development