Intermittent recombinant TSH injections prevent ovariectomy-induced bone loss

Abstract

We recently described the direct effects of thyroid-stimulating hormone (TSH) on bone and suggested that the bone loss in hyperthyroidism, hitherto attributed solely to elevated thyroid hormone levels, could at least in part arise from accompanying decrements in serum TSH. Recent studies on both mice and human subjects provide compelling evidence that thyroid hormones and TSH have the opposite effects on the skeleton. Here, we show that TSH, when injected intermittently into rodents, even at intervals of 2 weeks, displays a powerful antiresorptive action in vivo. By virtue of this action, together with the possible anabolic effects shown earlier, TSH both prevents bone loss and restores the lost bone after ovariectomy. Importantly, the osteoclast inhibitory action of TSH persists ex vivo even after therapy is stopped for 4 weeks. This profound and lasting antiresorptive action of TSH is mimicked in cells that genetically overexpress the constitutively active ligand-independent TSH receptor (TSHR). In contrast, loss of function of a mutant TSHR (Pro --> Leu at 556) in congenital hypothyroid mice activates osteoclast differentiation, confirming once again our premise that TSHRs have a critical role in regulating bone remodeling.

Fig. 1.

TSH restores ovariectomy-induced bone loss. (A and B) BMD (A) and bone strength (B) declines over 28 weeks due to ovariectomy of Sprague–Dawley rats (4–6 months old) are restored with purified rat TSH injected at the stated doses three times a week (3/wk), once a week (1/wk), and once every two weeks (1/2 wk). Bone strength parameters include maximum load and stiffness (units as stated). Data are shown as mean ± SEM (n = 12 rats per group). P values calculated by ANOVA Dunett test comparing treatment versus no treatment in ovariectomized rats (*, P < 0.05) or ovariectomy versus sham control (⋀, P < 0.05).

Fig. 2.

Loss of trabecular bone structure over 28 weeks due to ovariectomy of Sprague–Dawley rats (4–6 months), measured by μCT, is restored with purified rat TSH injected at stated doses of three times a week (3/wk), once a week (1/wk), and once every 2 weeks (1/2 wk). (A) Representative micrographs. (B) Measured parameters include BV/TV, Tb.Th, Tb.N, Tb.Sp, and cortical thickness. Data are shown as mean ± SD (n = 12 rats per group). P values calculated by ANOVA Dunett's test comparing treatment versus no treatment in ovariectomized rats (*, P < 0.05; **, P < 0.01) or ovariectomy versus sham control (⋀, P < 0.01).

Fig. 3.

TSH prevents ovariectomy-induced bone loss. (A and B) BMD declines (A) and trabecular loss (B) due to ovariectomy (OVX) of mice (12 weeks old) are restored with rhTSH, (thyrogen, doses as stated) injected three times a week, rhPTH injected three times a week, or the s.c. implantation of estrogen pellets (E₂). Treatments with rhTSH or rhPTH ended at 8 weeks, after which the mice were given a 4-week treatment-free period with the exception of E₂. Bones were harvested for μCT and bone marrow cultures at 12 weeks after BMD measurements (see Fig. 4A). (B) μCT images of trabecular bone at the distal femoral metaphysis, together with estimates of BV/TV. Data are shown as mean ± SEM (n = 8–10 mice per group). The P values are calculated by unpaired Student's t test comparing treatment versus no treatment in ovariectomized mice (*, P < 0.05; **, P < 0.01) or ovariectomy versus sham control (⋀, P < 0.01). Median value and ranges are given for BV/TV.

Fig. 4.

Gain and loss of function of TSHR results in opposite effects on osteoclast differentiation in ex vivo cultures. (A) Effect of TSH (doses as stated) injected three times a week for 8 weeks or the s.c. implantation of estrogen pellets (E₂) for 12 weeks on the expression of the osteoclast differentiation markers TRAP, cathepsin K (CathK), integrin β₃, calcitonin receptors (CTR), NFATc1, IκBα, and metalloproteinase-9 (MMP-9) after a TSH-free period of 4 weeks. (B) Endogenous murine TSHR expression (i), transgenic human TSHR expression (ii), TRAP-positive (TRAP⁺) cell formation (iii), alkaline-phosphatase-positive CFU-f (ALP⁺ CFU-f) numbers (iv), and TRAP mRNA expression (v) in bone marrow-derived hematopoetic stem cells (i, ii, iii, and v) or stromal cells (iv) obtained from mice transgenically expressing either hTSHR or caTSHR. (C) TRAP, integrin β₃, and CTR mRNA expression in hematopoetic stem cells isolated from wild-type (+/+), heterozygotic (+/mu), or homozygotic (mu/mu) hyt mice that were exposed to RANK-L and/or TSH (as shown). Data are shown as mean ± SEM. The P values were calculated by unpaired Student's t test comparing treatment versus zero-dose control or wild-type cells as appropriate (*, P < 0.05; **, P < 0.01).