TNFalpha mediates the skeletal effects of thyroid-stimulating hormone

Abstract

We have shown recently that by acting on the thyroid-stimulating hormone (TSH) receptor (TSHR), TSH negatively regulates osteoclast differentiation. Both heterozygotic and homozygotic TSHR null mice are osteopenic with evidence of enhanced osteoclast differentiation. Here, we report that the accompanying elevation of TNFalpha, an osteoclastogenic cytokine, causes the increased osteoclast differentiation. This enhancement in TSHR-/- and TSHR+/- mice is abrogated in compound TSHR-/-/TNFalpha-/- and TSHR+/-/TNFalpha+/- mice, respectively. In parallel studies, we find that TSH directly inhibits TNFalpha production, reduces the number of TNFalpha-producing osteoclast precursors, and attenuates the induction of TNFalpha expression by IL-1, TNFalpha, and receptor activator of NF-kappaB ligand. TSH also suppresses osteoclast formation in murine macrophages and RAW-C3 cells. The suppression is more profound in cells that overexpress the TSHR than those transfected with empty vector. The overexpression of ligand-independent, constitutively active TSHR abrogates osteoclast formation even under basal conditions and in the absence of TSH. Finally, IL-1/TNFalpha and receptor activator of NF-kappaB ligand fail to stimulate AP-1 and NF-kappaB binding to DNA in cells transfected with TSHR or constitutively active TSHR. The results suggest that TNFalpha is the critical cytokine mediating the downstream antiresorptive effects of TSH on the skeleton.

Fig. 1.

Osteoclast formation and TRAP expression in the mice with TSHR and TNFα ablation, and cytokine expression in TSHR null mice. (A and B) Rescue experiments showing osteoclast formation (A) and TRAP mRNA expression (B) in bone marrow cell cultures isolated from various genotypes, namely TSHR^+/+/TNF^+/+ (w/w), TSHR^−/−/TNF^+/+ (k/w), TSHR^−/−/TNF^+/−(k/h), and TSHR^−/−/TNF^−/− (k/k) mice. (C) Real-time PCR measurements showing the expression of interleukin-1α (IL-1α), IL-1β, IL-6, and TNFα in bone marrow cell cultures from TSHR^+/− (+/−) and TSHR^−/− (−/−) mice, relative to TSHR^+/+ (+/+) cultures. In both experimental protocols, bone marrow cell cultures were incubated with RANKL (60 ng/ml) and M-CSF (30 ng/ml) for up to 6 days. ∗, P < 0.05, comparisons with wild-type (w/w or +/+) mice in all cases; +, P < 0.05, compared with k/w mice.

Fig. 2.

RAW-C3 (A and C) and CD11b⁺ macrophages (B and D) were cultured with IL-1α (10 ng/ml) and TNFα (50 ng/ml) in the presence or absence of TSH (10 milliunits per ml). TNFα expression (A and B) or murine TNFα levels in the supernatants (C and D) were measured, respectively, by real-time PCR (after 2 h) and ELISA (after 1 day). Note that human recombinant TNFα was used for the ELISA experiments. ∗, P < 0.05, comparisons with respective controls; +, P < 0.05, compares TSH effects with IL-1/TNFα alone.

Fig. 3.

Regulation of TNFα mRNA expression and TNFα promoter activity. (A) TNFα mRNA levels quantitated by real-time PCR in RAW-C3 cells overexpressing the TSHR or the constitutively active TSHR (caTSHR) after treatment with IL-1α (10 ng/ml) and TNFα (50 ng/ml) (IL-1/TNFα) or RANKL (100 ng/ml) for 2 h. ∗, P < 0.05, comparisons with respective controls; +, P < 0.05, compares the IL-1/TNFα and RANKL effects of TSHR-C3 and caTSHR-C3 cells with those elicited in empty-C3 cells. (B) Empty-C3, TSHR-C3, or caTSHR-C3 transformants were transfected with the TNFα promoter (−197 to +115 bp) luciferase fragment. Luciferase activity was measured in duplicate 24 h after treatment with PMA (3 × 10⁻⁸ M) or PMA plus TSH (10 milliunits per ml).

Fig. 4.

Osteoclast formation in TSHR or caTSHR overexpressing macrophages. (A) RAW-C3 cells stably transfected with empty vector, TSHR, or caTSHR were cultured with 100 ng/ml RANKL with or without human TSH (0.1–10 milliunits per ml) for 6 days. TRAP-positive osteoclasts were counted. ∗, P < 0.05 compares TSHR and caTSHR transformants with Empty-C3 cells at each concentration of TSH; +, P < 0.05, compares the effect of each concentration of TSH against zero-TSH in empty-C3 cells. (B) Bone marrow macrophages were transiently infected with either an empty retrovirus (empty) or a retrovirus containing either the TSHR or caTSHR construct. The infected cells were treated with RANKL (60 ng/ml) and M-CSF (30 ng/ml) for 6 days and examined for TRAP-positive osteoclast formation. ∗, P < 0.05, compares the effect of RANKL or RANKL plus TSH in TSHR-C3 and caTSHR-C3 cells versus empty-C3 cells; +, P < 0.05, compares the effect of RANKL plus TSH with that of RANKL alone (control) for each transformant.

Fig. 5.

Increased macrophage population in bone marrow of TSHR null mice and inhibition of macrophage proliferation by TSH. (A) Cell types present in fresh bone marrow from TSHR^+/+ (+/+), TSHR^+/− (+/−), and TSHR^−/− (−/−) mice analyzed by specific antibodies using flow cytometry and expressed as percentages of positive cells. (B) Fresh bone marrow cells from TSHR^+/+ (+/+), TSHR^+/− (+/−), and TSHR^−/− (−/−) mice were cultured with M-CSF (30 ng/ml) in Metcalf for 7 days, and colony numbers were determined. (C) Fresh bone marrow cells from TSHR^+/+ mice were similarly cultured with M-CSF and TSH (0.1–10 milliunits per ml), and colony numbers were determined. (D) Isolated and precultured CD11b⁺ cells were treated with M-CSF alone or M-CSF plus TSH (10 milliunits per ml) for 3 days. Proliferating cells were measured by BrdU incorporation (expressed as absorbance at 450 nm). ∗, P < 0.05, comparison with TSHR^+/+ mice (B) or with zero-TSH (C and D).

Fig. 6.

TSHR expression in primary macrophages and its increase by cytokines and RANKL. (A) CD11b⁺, B220⁺, and CD106⁺ cells were isolated from fresh bone marrow, CD3⁺ cells were obtained from spleen, and primary osteoblasts (POB) were obtained from neonatal calvaria. The cells were incubated with a TSHR antibody, RSR-1 (mouse IgG, negative control), followed by a second incubation with FITC-labeled anti-mouse IgG antibody. Both nonspecific and specific binding are shown as percentages. (B and C) Bone marrow macrophages were treated with IL-1 (10 ng/ml) and TNFα (50 ng/ml) (IL-1/TNFα) or with RANKL (60 ng/ml) in the presence of M-CSF (30 ng/ml) for 24 h. TSHR expression was assessed by real-time PCR (B) or flow cytometry (using MS-1 antibody) (C). Please refer to Materials and Methods for details on the antibodies. ∗, P < 0.05, compares IL-1/TNFα and RANKL against untreated (control).

Fig. 7.

Electrophoretic mobility shift assay (EMSA) for AP-1 and NF-κB binding of TSHR or caTSHR overexpressing RAW-C3 cells. RAW-C3 cells transfected with empty plasmid, TSHR, or caTSHR were treated with IL-1α (10 ng/ml) and TNFα (50 ng/ml) (IL-1/TNFα) or with RANKL (100 ng/ml) for 20, 40, or 60 min. Nuclear fractions prepared from each group were incubated with biotin-labeled AP-1 or NF-κB for DNA binding studies.