Follicle-stimulating hormone stimulates TNF production from immune cells to enhance osteoblast and osteoclast formation

Abstract

Declining estrogen production after menopause causes osteoporosis in which the resorption of bone exceeds the increase in bone formation. We recently found that mice deficient in the beta-subunit of follicle-stimulating hormone (FSHbeta) are protected from bone loss despite severe estrogen deficiency. Here we show that FSHbeta-deficient mice have lowered TNFalpha levels. However, TNFalpha-deficient mice are resistant to hypogonadal bone loss despite having elevated FSH, suggesting that TNFalpha is critical to the effect of FSH on bone mass. We find that FSH directly stimulates TNFalpha production from bone marrow granulocytes and macrophages. We also explore how TNFalpha up-regulation induces bone loss. By modeling the known actions of TNFalpha, we attribute the high-turnover bone loss to an expanded osteoclast precursor pool, together with enhanced osteoblast formation. TNFalpha inhibits osteoblastogenesis in the presence of ascorbic acid in culture medium, but in its absence this effect becomes stimulatory; thus, ascorbic acid reverses the true action of TNFalpha. Likewise, ascorbic acid blunts the effects of TNFalpha in stimulating osteoclast formation. We propose that hypogonadal bone loss is caused, at least in part, by enhanced FSH secretion, which in turn increases TNFalpha production to expand the number of bone marrow osteoclast precursors. Ascorbic acid may prevent FSH-induced hypogonadal bone loss by modulating the catabolic actions of TNFalpha.

Fig. 1.

FSH regulates TNFα production. (A) FSHβ^−/− mice display reduced levels of serum TNFα despite estrogen deficiency (P = 0.03; n = 4; sampled twice). (B) Secretion of TNFα into cell culture supernatants was analyzed at 10 and 24 h after FSH (100 ng/ml) addition (P = 0.03 for 10 h and 0.01 for 24 h). (C–E) Effect of FSH on TNFα production in primary CD11b⁺ macrophages/granulocytes or B220⁺ cells. Bone marrow from C57BL mice was flushed and resuspended in OPTI-MEM containing 5% FBS. After 2 h of incubation, FSH (100 ng/ml) was added, and the cells were sampled at 1 h (C and E), 6 h (data not shown), and 18 h (D) after addition. The cells were fixed in PhosphoFix, permeabilized in 90% MEOH, and stained with either of two antibody combinations: CD11b-Alexa Fluor 488 and TNFα-allophycocyanin (C and D) or B220-phycoerythrin and TNFα-allophycocyanin (E). (F) A display of the time course of TNFα staining after the addition of FSH (100 ng/ml) in gated macrophages/granulocytes.

Fig. 2.

Modeling of TNFα action suggests that it induces osteoclast precursor expansion. A mathematical model of bone metabolism (14) was adapted such that osteoclast differentiation proceeded from a pool of osteoclast precursors that could be varied independent of changes in the differentiation rate of osteoclasts. (A) The pool of osteoclast precursors was increased, as indicated by the black bar, and the effects on the number of osteoclast precursors (Upper Left), the number of osteoclasts, osteoblast precursors, and osteoblasts (Upper Right), the bone turnover (Lower Left), and net bone formation/loss (Lower Left) are shown. Note that disturbing the system by increasing the number of osteoclast precursors caused a rise in the number of active osteoclasts, responding osteoblasts, and active osteoblasts, as well as an increase in bone turnover and bone loss. (B) The effects of TNFα on expanding the osteoclast precursor pool were tested experimentally by stimulating osteoclast precursors with TNFα either 24 h before the addition of the differentiation signal RANK-L (Right) or by adding TNFα at the same time RANK-L was added (Center). The number of osteoclasts formed when TNFα was allowed to expand the osteoclast precursor pool was greater than when TNFα was given at the same time as the differentiation signal RANK-L. (Left) Control, RANK-L only.

Fig. 3.

Ascorbic acid reverses the actions of TNFα on bone metabolism. A and B examine the role of ascorbic acid in modulating TNFα action on osteoblast formation. (A) TNFα dose-dependently increases CFU-F colony formation from total bone marrow in the absence of the ascorbic acid derivative ascorbate-2-phosphate. (B) In the presence of ascorbate-2-phosphate, TNFα dose-dependently decreases CFU-F colony formation from total bone marrow. C and D examine the role of ascorbic acid in modulating TNFα action on osteoclast precursor expansion and osteoclast formation. (C) Treatment of total bone marrow with TNFα leads to an expansion in the number of CD11b⁺ cells, as assessed by flow cytometry; this effect is blunted in media containing ascorbate-2-phosphate (A-2-P). The y axis denotes the percentage of change in the percentage of all bone marrow cells that were CD11b⁺. (D) Blunting of the effect of TNFα on the number of cells displaying CD38, a marker of TNFα action on murine macrophages. Total bone marrow from C57BL mice was plated in media either with ascorbate-2-phosphate (A-2-P) or without it in the presence of various doses of TNFα (0–50 ng/ml). After washing to remove nonadherent cells, EDTA was used to lift the adherent cells; these cells were stained with antibodies to CD11b and CD38 after which 30,000 cells were analyzed by flow cytometry. The frequency of CD38⁺ cells was calculated and plotted as a percentage change from the non-TNFα-treated group. (E) The effects of TNFα on increasing osteoclast formation (Upper) are reversed in the presence of ascorbate-2-phosphate, such that TNFα decreases RANK-L-induced osteoclast formation (Lower). P = 0.02 for 10 ng/ml TNFα and 0.03 for 30 ng/ml in Upper; P = 0.008 for 10 ng/ml and 0.007 for 30 ng/ml in Lower. (F) The appearance of osteoclasts formed in ascorbate-2-phosphate- (A-2-P)-containing medium. Murine bone marrow was flushed and plated with M-CSF (5 ng/ml) for 24 h. Nonadherent cells were used for purification on a Ficoll column. The interface layer was then plated at 3 × 10⁴ cells per well in media containing A-2-P or in media without A-2-P (data not shown). M-CSF (30 ng/ml) was added with RANK-L at 80 ng/ml (Upper Right) or 40 ng/ml (Upper Left and Lower). To some wells, murine TNFα was added at 10 ng/ml (Lower Left) or 30 ng/ml (Lower Right). After 5 days of culture, the cells were fixed and stained for tartrate-resistant acid phosphatase (TRAP).

Fig. 4.

Integrated hypothesis for hypogonadal bone loss. Ovarian dysfunction and the loss of estrogen lead to decreased inhibin levels and dramatic increases in FSH levels. FSH, in turn, directly stimulates osteoclast differentiation and TNFα production from bone marrow macrophages/granulocytes. TNFα (shown as T) acts to increase M-CSF levels and/or M-CSF receptor expression, resulting in an expansion of the number of osteoclast precursors. Additionally, TNFα may prime macrophages to induce the proliferation of activated T lymphocytes, which highly express RANK-L and further contribute to TNFα production. The overabundance of osteoclast precursors, coupled with the osteoclastic differentiation agents FSH and RANK-L, which is expressed on T lymphocytes and stromal cells, likely compose the proosteoclastic component of high-turnover bone loss. TNFα-induced increases in the number of osteoblasts, as well as resorption-induced osteoblast formation, likely compose the proosteoblastic component of high-turnover bone loss. TNFα action can be blocked by treatment with etanercept or analogs, or, as we have found in this report, through supplementation with ascorbic acid (vitamin C). The proosteoclastogenic actions of RANK-L can be blocked by RANK-Fc or OPG. The resorptive function of osteoclasts can be blocked by bisphosphonates, which are taken up by resorbing osteoclasts and modulate their sensitivity to apoptotic stimuli. Estrogen replacement therapy or selective estrogen receptor modulator (SERM) therapy, per our hypothesis, may decrease FSH levels to reduce TNFα expression.