Vitamin D exerts antiproliferative, prodifferentiation, and proapoptotic effects on nonclassic target tissues such as breast. Blood levels of 25-hydroxyvitamin D [25(OH)D], the most sensitive indicator of vitamin D status, are inversely correlated with breast cancer risk; however, a causal relationship between vitamin D deficiency and breast cancer growth in bone has not been assessed. We examined the effect of vitamin D deficiency on the intraskeletal growth of the human breast cancer cell line MDA-MB-231-TxSA in a murine model of malignant bone lesions. Subsets of mice were treated concurrently with osteoprotegerin (OPG) to abrogate bone resorption. Outcomes were assessed by repeated radiographic and end-point micro-computed tomography and histologic analyses. Mice weaned onto a vitamin D-free diet developed vitamin D deficiency within 4 weeks [mean ± SE serum 25(OH)D: 11.5 ± 0.5 nmol/L], which was sustained throughout the study and was associated with secondary hyperparathyroidism and accelerated bone turnover. Osteolytic lesions appeared earlier and were significantly larger in vitamin D-deficient than in vitamin D-sufficient mice after 2 weeks (radiographic osteolysis: +121.5%; histologic tumor area: +314%; P < 0.05). Although OPG treatment reduced the size of radiographic osteolyses and tumor area in both groups, tumors remained larger in OPG-treated vitamin D-deficient compared with OPG-treated vitamin D-sufficient mice (0.53 ± 0.05 mm 2 versus 0.19 ± 0.05 mm 2 ; P < 0.05). We conclude that vitamin D deficiency promotes the growth of human breast cancer cells in the bones of nude mice. These effects are partly mediated through secondary changes in the bone microenvironment, along with direct effects of vitamin D on tumor growth.

Elevated follicle-stimulating hormone (FSH) activity is proposed to directly cause bone loss independent of estradiol deficiency in aging women. Using transgenic female mice expressing human FSH (TgFSH), we now reveal that TgFSH dose-dependently increased bone mass, markedly elevating tibial and vertebral trabecular bone volume. Furthermore, TgFSH stimulated a striking accrual of bone mass in hypogonadal mice lacking endogenous FSH and luteinizing hormone (LH) function, showing that FSH-induced bone mass occurred independently of background LH or estradiol levels. Higher TgFSH levels increased osteoblast surfaces in trabecular bone and stimulated de novo bone formation, filling marrow spaces with woven rather than lamellar bone, reflective of a strong anabolic stimulus. Trabecular bone volume correlated positively with ovarianderived serum inhibin A or testosterone levels in TgFSH mice, and ovariectomy abolished TgFSH-induced bone formation, proving that FSH effects on bone require an ovary-dependent pathway. No detectable FSH receptor mRNA in mouse bone or cultured osteoblasts or osteoclasts indicated that FSH did not directly stimulate bone. Therefore, contrary to proposed FSH-induced bone loss, our findings demonstrate that FSH has dose-dependent anabolic effects on bone via an ovary-dependent mechanism, which is independent of LH activity, and does not involve direct FSH actions on bone cells. ) were protected from bone loss despite estradiol deficiency, whereas haploinsufficient Fshb +/− females exhibited increased bone mass (9). By inference, it was proposed that elevated circulating FSH levels caused the bone loss observed in estrogen deficient peri-and postmenopausal women (9, 10). Mechanisms proposed for this putative FSH-induced bone loss included direct FSH stimulation of osteoclastic bone resorption via the FSHR (9) and FSH-induced TNFα production by bone marrow granulocytes and macrophages by as-yet-undefined pathways (11). However, elevated androgens in Fshb −/− and Fshr −/− female mice provide an alternate explanation for the preservation of bone mass in these estradiol-deficient mouse models (12). Although direct regulation of bone remodeling through FSH would have major clinical implications, such a mechanism has yet to exclude roles of FSH-regulated ovarianderived factors, which, on a biological basis, are more likely to control bone turnover, such as sex steroids (12-15) or inhibins (16, 17).In the current study, we have directly determined the effects of elevated FSH activity on bone mass and structure by using pituitary-independent, transgenic expression of human FSH (TgFSH) in female mice. Our TgFSH mouse model provides progressively rising circulating levels of FSH with age (18), with distinct transgenic lines allowing dose-dependent analysis of FSH actions in vivo (19)(20)(21). Using this FSH model, we have determined TgFSH actions on bone (i) in isolation of LH actions, using TgFSH expressed in hypogonadal (hpg) female mice lacking gonadotropinreleasing hormone (GnRH) and therefore endog...