Estrogen prevents bone loss through transforming growth factor beta signaling in T cells

Abstract

Estrogen (E) deficiency leads to an expansion of the pool of tumor necrosis factor (TNF)-producing T cells through an IFN-gamma-dependent pathway that results in increased levels of the osteoclastogenic cytokine TNF in the bone marrow. Disregulated IFN-gamma production is instrumental for the bone loss induced by ovariectomy (ovx), but the responsible mechanism is unknown. We now show that mice with T cell-specific blockade of type beta transforming growth factor (TGFbeta) signaling are completely insensitive to the bone-sparing effect of E. This phenotype results from a failure of E to repress IFN-gamma production, which, in turn, leads to increased T cell activation and T cell TNF production. Furthermore, ovx blunts TGFbeta levels in the bone marrow, and overexpression of TGFbeta in vivo prevents ovx-induced bone loss. These findings demonstrate that E prevents bone loss through a TGFbeta-dependent mechanism, and that TGFbeta signaling in T cells preserves bone homeostasis by blunting T cell activation. Thus, stimulation of TGFbeta production in the bone marrow is a critical "upstream" mechanism by which E prevents bone loss, and enhancement of TGFbeta levels in vivo may constitute a previously undescribed therapeutic approach for preventing bone loss.

Fig. 1.

ovx decreases BMM TGFβ1 mRNA and plasma TGFβ1 levels (mean ± SE). (a) BMMs from sham-operated and ovariectomized mice were treated with vehicle, 17β estradiol (10^–8 M), or 17β estradiol plus ICI 182720 (10^–8 M) for 3 days. *, P < 0.05, as compared with vehicle-treated sham-operated BMMs. (b) BM plasma TGFβ1 levels. *, P < 0.05 as compared with sham-operated mice (n = 6).

Fig. 2.

Silencing of TGFβ signaling in T cells decreases BMD in growing mice. (a) BMD (mean ± SE) was measured in intact WT and CD4dnTGFβIIR mice (n = 6) between the ages of 4 and 16 weeks. *, P < 0.05 as compared with age-matched WT mice. (b) Silencing of TGFβ signaling in T cells increases bone resorption in adult mice. Serum C-terminal telopeptide (CTX) (mean ± SE), a marker of bone resorption, was measured in WT and CD4dnTGFβIIR mice (n = 6) at 16 weeks of age. *, P < 0.05 as compared with age-matched WT mice. (c) Silencing of TGFβ signaling in T cells induces bone loss in E-replete mice (n = 6). BMD was measured in vivo at baseline and 4 weeks after surgery. Data (mean ± SE) are expressed as percentage change from baseline. *, P < 0.05 as compared with sham-operated WT controls. (d and e) Transfer of T cells insensitive to TGFβ into E-replete nude mice leads to bone loss (d) and increases bone resorption (e). T cells from WT or CD4dnTGFβIIR mice were transferred into nude recipients (n = 6), which were then subjected to ovx or sham operation. BMD was measured in vivo at baseline and at 4 weeks. Data (mean ± SE) are expressed as percentage change from baseline. Serum C-terminal telopeptide levels (mean ± SE) were measured at 4 weeks after surgery. *, P < 0.05 as compared with sham-operated WT mice.

Fig. 3.

Silencing of TGFβ signaling in T cells increases secretion of IFN-γ by phorbol 12-myristate 13-acetate and ionomycin-stimulated T cells (a), CIITA expression in BMMs (b), and BMM Ag presentation (c). Data are expressed as mean ± SE. *, P < 0.05 as compared with WT mice. (d and e) TGFβ1 neutralization blocks the repressive effect of E on BMM CIITA mRNA expression (d) and Ag presentation activity (e). BMMs from WT-operated sham and ov mice were treated for 3 days in vitro with either 17β estradiol or control vehicle and anti-TGFβ1 antibody or irrelevant antibody. *, P < 0.05 as compared with sham-operated controls. DPM, disintegrations per minute.

Fig. 4.

Silencing of TGFβ signaling in T cells increases T cell production of osteoclastogenic cytokines. Levels (mean ± SE) of TNF (a) and RANKL (b) were measured in the culture media of phorbol 12-myristate 13-acetate and ionomycin-stimulated T cells harvested from WT and CD4dnTGFβIIR mice. *, P < 0.05 compared with WT controls.

Fig. 5.

(a) Treatment with pCMV-TGFβ1 prevents ovx-induced bone loss. BMD was measured at baseline and 2 and 4 weeks after surgery in mice treated with pCMV-TGFβ1 plasmid or pCMV-Null (n = 10). Data (mean ± SE) are expressed as percent change from baseline. *, P < 0.05 compared with sham-operated mice treated with pCMV-Null. (b and c) Histomorphometric analysis (mean ± SE) of proximal tibia. Osteoclast surface/bone surface (b) and mineral apposition rate (MAR) (c) from sham-operated and ovariectomized mice were measured at 4 weeks. *, P < 0.05 as compared with sham-operated mice treated with pCMV-Null.