Thyroid and bone: macrophage-derived TSH-β splice variant increases murine osteoblastogenesis

Abstract

It is now firmly established that TSH may influence the physiology and patho-physiology of bone by activating osteoblasts and inhibiting osteoclast activity resulting in relative osteoprotection. Whether this influence is directly exerted by pituitary-derived TSH in vivo is less certain, because we have previously reported that the suppression of pituitary TSH does not remove such protection. Here, we have characterized the functional relevance of a novel form of the TSH-β subunit, designated TSH-βv, known to be produced by murine bone marrow cells. We found that fresh bone marrow-derived macrophages (MØs) preferentially produced TSH-βv and, when cocultured with CHO cells engineered to overexpress the full-length TSH receptor, were able to generate the production of intracellular cAMP; a phenomenon not seen in control CHO cells, such results confirmed the bioactivity of the TSH variant. Furthermore, cocultures of MØs and osteoblasts were shown to enhance osteoblastogenesis, and this phenomenon was markedly reduced by antibody to TSH-β, suggesting direct interaction between MØs and osteoblasts as observed under the electron microscope. These data suggest a new paradigm of local modulation of bone biology by a MØ-derived TSH-like molecule and raise the question of the relative contribution of local vs pituitary-derived TSH in osteoprotection.

Figure 1.

BM cells produce TSH-βv. (A) A schematic comparison of native TSH-β and novel TSH-βv. Of note is a missing exon IV in the splice variant resulting in a smaller peptide of 8 vs 17 kDa for the full-length peptide. The sequence of TSH-βv is also shown with the intronic region in bold. (B) The result of native TSH-β and novel TSH-βv PCR amplimer generation in C57/BL murine pituitary and BM samples. The PCR product size of 470 bp was exclusively found in the pituitary sample and corresponds to that of native TSH-β and contrasts with the PCR product size for the TSH-βv of 300 bp, which was found in both the pituitary and BM. (C) PCR amplification of the TSH-α gene was only positive in the pituitary sample. (D) BM cells were flushed from the femur and the red blood cells lysed and stained for TSH followed by FACS analysis. These figures depict TSH protein expression in fixed BM cells detected with anti-TSH-β (bottom) or control IgG (top) with a green fluorescent protein-labeled second antibody. GAPDH, glyceraldehydes-3-phosphate dehydrogenase. Ctrl, control; FL-1, fluorescence intensity; Pit, pituitary.

Figure 2.

TSH-βv is produced by MØs. (A) Upper panel, fresh BM cells from C57/BL6 mice were subfractioned into the myeloid and the lymphoid lineage by FACS sorting and then PCR amplified for the novel TSH-βv. As depicted, the CD-11b⁺ cells of the myeloid origin predominantly transcribed the novel TSH-βv by conventional PCR analysis of cDNA. (A) Lower panel, real-time PCR analysis confirmed that the CD-11b⁺ cells were the primary source of the variant. (B) Further subfractionation of CD-11b⁺ cells into MØs, lymphocytes, neutrophils, and MØs for the purpose of identifying the cell responsible for producing the novel TSH-βv revealed that, by real-time PCR, the MØ population was the primary source. (C) Fresh BM cells were isolated, FACS sorted into the same 4 fractions, and then immunostained for TSH-β with anti-TSH (Santa Cruz Biotechnology, Inc), showing that 11% of the MØs expressed the variant protein.

Figure 3.

Intracellular staining for TSH-βv. BMDMØs were cultured from fresh BM for 7 days with MCSF (10 ng/mL) and their nuclei labeled with 4′,6′-diamidino-2-phenylindole (DAPI) (A) and stained for F480⁺ surface antigen (B) and intracellular TSH-βv with anti-TSH (C). The overlay (D) illustrates that MØs contain intracellular TSH-βv (×630).

Figure 4.

(A and B) TSH-βv binding to the TSHR ECD. Computer modeling of the TSH-βv binding with the TSHR ECD showed docking of the peptide (green) at the concave surface of the leucine-rich repeat region (gray/black). Two rotated views are illustrated. The binding affinity of the variant was comparable with that of the native TSH-β subunit (638 vs 649 μm, respectively). (C–F) MØ phenotyping. (C) Validation of MØ content in BMDMØ cultures was obtained by staining for F480⁺ surface antigen (red) in a similar way to RAW cells, a MØ cell line, as a positive control. (D) By conventional PCR, the EMR1 gene, a specific MØ marker, was expressed in both BMDMØ and RAW cell cultures, and both were transcribed TSH-βv. (E and F) The BMDMØs were then subjected to FACS analysis to determine their M1 (C) vs M2 (D) phenotype. Analyses showed that 14.6% of the cells were of the M1 phenotype (CD-11c⁺), and 35.6% of the cells were of the M2 phenotype (CD14⁺). GAPDH, glyceraldehydes-3-phosphate dehydrogenase; DAPI, 4′,6′-diamidino-2-phenylindole. GFP, green fluorescence protein.

Figure 5.

M2 phenotype is the major source of TSH-βv transcription. BMDMØs were cultured from fresh BM for 7 days under the influence of MCSF 10 ng/mL and further inducted into M1- and M2-like cells by 24 hours of treatment with an IL-4/IL-10 (20 ng/mL each) combination and lipopolysaccharide (100 ng/mL), respectively. Cells were then extracted with TRIzol and real-time PCR amplified for M2 markers (arginase 1 and Mus musculus secretory protein precursor, YM-1) (A and B) and for M1 (i-NOS) (C). Testing for TSH-βv itself (D) revealed transcription of both phenotypes. LPS, lipopolysaccharide; Ctrl, control.

Figure 6.

Bioactivity of TSH-βv. (A) A schematic representation of 5 experimental conditions used in studying the ability of BMDMØs to release TSH-βv and signal at the TSHR as evidenced by cAMP generation. The following controls were used: 1) MØs alone, 2) CHO cells alone, 3) CHO-TSHR cells alone, 4) CHO cells cocultured with MØs, and 5) the experiment itself consisted of CHO-TSHR cells with MØs. (B) When BMDMØs were cocultured with CHO cells transfected with the TSHR (CHO+TSHR), a cAMP response was elicited that was 5-fold greater than when MØs were cocultured with CHO cells without the TSHR. The cAMP generation in response to MØs in coculture was comparable with that of 1 mU/mL of TSH. Data are shown ±SEM of 2 replicate experiments. *P < .01.

Figure 7.

Bone EM shows intracellular TSH-containing vesicles. EM studies revealed the interaction of MØs, containing TSH secretary vesicles, localized adjacent to osteoblasts in bone slices, suggesting paracrine regulation of bone cells by MØ-derived TSH-βv. The MØ and osteoblast in the figure are as marked, and the inset shows a magnified view of the now gold-labeled intracellular TSH-containing vesicles detected using our specific TSH-β peptide antibody (no. 1).

Figure 8.

(A) To determine the influence of TSH-βv on osteoblast formation, calvarial cells were stimulated for 22 days under 4 experimental conditions as indicated. The inhibitory effect of anti-TSH on osteoblast formation was shown in comparison with control IgG by measuring collagen gene expression. Osteoblast gene expression of collagen was enhanced by osteogenic stimulation but down-regulated by anti-TSH-β antibody. At 22 days, the cultures contained 16.6% of MØs by FACS analysis with anti-F480. *P < .02; **P < .001. (B) Femoral preosteoblast-like cells were sequentially digested from bone, then cultured with osteogenic stimulation for 22 days, by which time they contained approximately 25% MØs by FACS analysis with anti-F480. Cultured cells were then treated with anti-TSH to illustrate the effect of blocking TSH-βv secreted by MØ. Significant inhibition of osteoblast formation was again observed. *P < .02; **P < .001. (C) Calvarial preosteoblast-like cells were sequentially digested from bone then cultured with osteogenic stimulation for 11 days. Cultured cells were then treated with conditioned medium from BMDMØ with osteogenic stimulatory factors and ±anti-TSH (no. 1) to illustrate the effect of blocking TSH-βv secreted by MØ. Significant stimulation of ALP activity occurred with conditioned medium and control IgG, and inhibition of this effect was seen with rabbit anti-TSH indicative of inhibition of TSH-induced osteoblastogenesis. *P < .02; **P < .04. OS, Osteogenic stimulation.