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. 2016 Jun 20;157(9):3658–3667. doi: 10.1210/en.2015-1974

T3 Regulates a Human Macrophage-Derived TSH-β Splice Variant: Implications for Human Bone Biology

R Baliram 1,✉, R Latif 1, S A Morshed 1, M Zaidi 1, T F Davies 1
PMCID: PMC5007892  PMID: 27300765

Abstract

TSH and thyroid hormones (T3 and T4) are intimately involved in bone biology. We have previously reported the presence of a murine TSH-β splice variant (TSH-βv) expressed specifically in bone marrow-derived macrophages and that exerted an osteoprotective effect by inducing osteoblastogenesis. To extend this observation and its relevance to human bone biology, we set out to identify and characterize a TSH-β variant in human macrophages. Real-time PCR analyses using human TSH-β-specific primers identified a 364-bp product in macrophages, bone marrow, and peripheral blood mononuclear cells that was sequence verified and was homologous to a human TSH-βv previously reported. We then examined TSH-βv regulation using the THP-1 human monocyte cell line matured into macrophages. After 4 days, 46.1% of the THP-1 cells expressed the macrophage markers CD-14 and macrophage colony-stimulating factor and exhibited typical morphological characteristics of macrophages. Real-time PCR analyses of these cells treated in a dose-dependent manner with T3 showed a 14-fold induction of human TSH-βv mRNA and variant protein. Furthermore, these human TSH-βv-positive cells, induced by T3 exposure, had categorized into both M1 and M2 macrophage phenotypes as evidenced by the expression of macrophage colony-stimulating factor for M1 and CCL-22 for M2. These data indicate that in hyperthyroidism, bone marrow resident macrophages have the potential to exert enhanced osteoprotective effects by oversecreting human TSH-βv, which may exert its local osteoprotective role via osteoblast and osteoclast TSH receptors.

The human TSH gene (TSH-β) is primarily expressed in the thyrotrophs of the anterior pituitary gland and consists of three exons and two introns, with portions of exons 2 and 3 coding for the 176 TSH-β polypeptide (1, 2). TSH-β gene expression is under the control of TRH from the hypothalamus and by direct thyroid hormone regulation of pituitary TSH-β gene transcription (3–5). Extrapituitary sources of TSH have been known for more than 20 years including TSH expression in lymphocytes and enterocytes (3, 6), and widespread expression of the TSH receptor has also now been well validated (7, 8). Hence, parallel to the pituitary-thyroid circuit, it has long been postulated that additional local TSH-related loops may be functioning in extrathyroidal sites, especially within the immune system. However, functional evidence for the existence of local TSH production has been sparse. The recent descriptions of a TSH-β splice variant produced in both the mouse pituitary and peripheral blood cells have supported this concept (2, 8, 9) of an extrapituitary TSH-like molecule, and the description of an extrapituitary thyrostimulin has also added further proof to this concept (10).

Our observation of a murine TSH-β isoform (TSH-βv) expressed in bone marrow derived macrophages (8) and the osteoprotective effects that it may exert, similar to the interaction of full-length TSH with osteoblast and osteoclast TSH receptors (8), indicated a bioactive TSH-like protein is produced by bone-derived macrophages with possible skeletal effects. In addition to this extrathyroidal TSH activity, we also know that thyroid hormones (T4 and T3) themselves have a profound influence on bone biology (11), and we have shown that the TSH receptor, and indirectly a TSH receptor stimulator, is able to prevent maximum bone loss in hyperthyroid mice (12), once again suggesting the importance of a local TSH-like molecule. The identification of TSH-βv in local bone marrow macrophages therefore further supports the evidence that a TSH-like molecule may have a local osteoprotective effect. Furthermore, we have previously observed that thyroid hormones themselves may up-regulate mouse TSH-βv, once again indicating a potential local osteoprotective system.

Most recently it has been reported that the human pituitary expresses a TSH-β isoform (TSH-βv) that is analogous to the mouse TSH-β splice variant, consisting of a 27-nucleotide portion of intron 2 and all of exon 3, resulting in the coding for 71.2% of the native human TSH-β polypeptide (2). Because all of our earlier studies were performed with mouse models and mouse cells, we have now reassessed the relevance of our earlier observations to human physiology using pituitary, peripheral blood mononuclear cells (PBMCs), bone marrow cells (BMCs), and human macrophages. In addition, we have used a model monocyte-macrophage cell line, called the THP-1 line, derived from a patient with acute monocytic leukemia (13). These cells are highly plastic and therefore can be polarized into multiple lineages (14–16). Since their original description in 1980, THP-1 cells have been used as a model for monocyte-macrophage differentiation (15–21) because they can be easily differentiated into mature macrophages by phorbol 12-myristate 13-acetate (PMA) (13, 22) as evidenced by an increase in Toll-like receptor-4 (CD14) expression, considered a marker of mature human macrophages (22).

The studies presented in this report describe the identification and characterization of a TSH-β variant in human macrophages and its positive regulation by thyroid hormone and again provide proof of concept for a local osteoprotective circuit.

Materials and Methods

Cells, cytokines, hormones, antibodies, and human sample

The THP-1 human monocyte line was obtained from American Type Culture Collection (ATCC TIB-202), and the RAW 264.7 mouse cell line also from American Type Culture Collection (ATCC TIB-71). Anti-CD14-fluorescein isothiocyanate (11-0141-81) was obtained from e-Bioscience (antimouse-PE 480 [12-4801-80]). A high-potency rabbit antibody to TSH CKLFPKYALSQDVCTYRDF residues was prepared by Hong Kong GenicBio Co and was found to be active at a dilution of 1:10 000. Cytokines IL-10, IL-4, interferon (IFN)-γ, macrophage colony-stimulating factor (MCSF) were purchased from Peprotech and T3 hormone from Sigma-Aldrich (catalog number T 6397), respectively. T4 hormone pellets (5 mg) (T4) were purchased from Innovative Research of America. To make macrophages from peripheral blood, we used one healthy 47-year-old female East Asian with BMI 24.

Differentiation of human THP-1 monocytes

The base medium for the THP-1 cell line was RPMI 1640 containing 0.05 mM 2β-mercaptoethanol, 10% fetal bovine serum, and 2% penicillin/streptomycin. Semiconfluent cells were differentiated by treating with 100 ng/mL of PMA in complete medium for 4 days with refreshing the medium on the second day. At the end of 4 days, phase-contrast microscopy revealed macrophage-like cells in two phenotypes round and spindle shaped (Figure 1, A and B) and that stained positive for a human macrophage marker CD14 (Figure 1C). Fluorescence-activated cell sorter (FACS) analysis of these differentiated cells for CD14+ cell surface markers indicated approximately 45% positivity (Figure 1G). This differentiated macrophage population, when stimulated with PMA, expressed a variety of macrophage markers including MCSF, myristoylated alanine-rich C (MARC), chemokine ligand (CCL)-22, CCL-2, and WNT 5A (Figure 2, A–G) confirming their macrophage identity.

Figure 1.

Figure 1.

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Macrophage phenotypes. A, Human THP-1 monocytes were a suspension of cells that were then differentiated into macrophages with PMA (100 ng/mL) (×20). B, Differentiated cells were rested for 48 hours and consisted of two populations: one population was of a fried egg shape and the second population of a spindle-like shape. C, Both populations of cells stained for surface and intracellular CD14 (Toll-like receptor) expression and 4′,6′-diamidino-2-phenylindole (DAPI) stain was used to illustrate the nuclei. D, Human PBMCs were allowed to differentiate with 100 ng/mL MCSF. E, Differentiated human macrophages. F, These differentiated cells from panel E were also CD14 positive. G, FACS analysis showed that 46.1% of the differentiated THP-1 cells were CD14 positive.

Figure 2.

Figure 2.

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Macrophage gene expression. Real-time PCR analysis revealed that differentiated THP-1 cells show enhanced expression of MCSF (A), CCL-22 (B), MARC1 (C), CCL-2 (D), and WNT-5A (E) gene expression, confirming their macrophage phenotype. Conventional RT-PCR demonstrated enhanced expression of the correct-size transcripts for the same macrophage genes after differentiation from the original cell cultures (F, G). *, P < .05.

Preparation of human macrophages and mouse bone marrow cells

In brief, 20 mL of human whole blood was collected into heparized ficoll gradient (BD 362753, BD Biosciences) tubes and was centrifuged at 1500 × g for 15 minutes. Mononuclear cells were collected, washed, and cultured with 100 ng/mL of MCSF in RPMI 1640 containing 0.05 mM 2β-mercaptoethanol, 10% fetal bovine serum, and 2% penicillin/streptomycin for 9 days during which they matured into human macrophages, which was then confirmed with CD14 staining (Figure 1, D–F). Blood samples were originally collected with the approval of the Institutional Review Board of the Icahn School of Medicine at Mount Sinai (Mt Sinai Beth Israel Medical Center, New York, New York) in accordance with Mount Sinai's Federal Wide Assurances. Mouse BMCs were flushed from murine femur and washed as previously described (8).

Standard RT-PCRs

Total RNA was isolated from differentiated, treated, and untreated macrophages from human PBMCs and THP-1 cells using TRIZOL (Invitrogen Corp; catalog number 15596026) as per the manufacturer's instructions. Human pituitary cDNA control was obtained from Biochain Inc. Chromosomal DNA was removed from the RNA using Ambion's TURBO DNA-free deoxyribonuclease I (Ambion, Inc; catalog number AM1907). The RNA concentration was determined on the basis of absorbance at 260 nm, and its purity was evaluated by the ratio of absorbance at 260:280 nm (>1.9). RNAs were kept frozen at −70 C until analyzed. Total RNA (1 μg) was reverse transcribed into cDNA with random hexamers using Advantage RT-PCR kits (CLONTECH Laboratories, Inc; catalog number 639506). All RT-PCRs were performed with TITANIUM Taq polymerase (CLONTECH Laboratories; catalog number 639506). Cycling conditions were as follows: 94ºC for 1 minute, followed by 30 cycles of amplification (94ºC denaturation for 0.5 min; annealing for 1 min, annealing temperature dependent on primers; 72ºC elongation for 2 min), with a final incubation at 72ºC for 7 minutes. The amplified PCR products were separated on a 2% agarose gels. Supplemental Table 1 details the amplimers used.

Quantitative RT-PCR

The quantitative RT-PCRs were performed using an Applied Biosystems StepOne Plus real-time PCR system (Applied Biosystems) and a series of well-characterized primers (Supplemental Table 1). The reactions were established with Power SYBR Green master mix (Applied Biosystems; catalog number 4309155), 0.4 μL (2 μM) sense/antisense gene-specific primers, 2 μL cDNA, and diethylpyrocarbonate-treated water to a final volume of 20 μL. The PCR mix was denatured at 95ºC for 60 seconds before the first PCR cycle. The thermal cycle profile was as follows: denaturing for 30 seconds at 95ºC; annealing for 30 seconds at 57ºC-60ºC (dependent on primers); and extension for 60 seconds at 72ºC. A total of 40 PCR cycles were used. PCR efficiency, uniformity, and the linear dynamic range of each quantitative RT-PCR assay were assessed by the construction of standard curves using DNA standards. An average threshold cycle from triple assays was used for further calculation. For each target gene, the relative gene expression was normalized to that of the β-actin housekeeping gene using real-time PCR software. Data presented here are the mean from three independent experiments in which each sample set was analyzed in triplicate.

Immunostaining and confocal imaging

Differentiated THP-1 cells and Chinese hamster ovary (CHO) cells were plated individually in glass bottom Delta T dishes (catalog number 12071-33) at 5 × 105, and upon 70%–80% confluence, the cells were washed with 1× PBS and then fixed in 4% paraformaldehyde for 10 minutes, prior to CD14 staining. For intracellular TSH staining, the fixed cells were permeabilized with 0.1% Triton X-100 for 30 minutes followed by blocking for 1 hour with 5% BSA and stained with unlabeled anti-TSH-β for 1 hour at room temperature followed by antirabbit Alexa 488 secondary antibody (Invitrogen; catalog number 3) at 1:100 dilution. After removal of secondary antibody by repeated washing with PBS, the stained cells were mounted using VectaShield (catalog number H-1200) and visualized under a Zeiss LSM-700 confocal microscope.

Western blots and in-cell Western assays

Proteins were resolved on 18% Tris-HCL gels (catalog number 161-1219) and electrophoretically transferred at 70 V for 90 minutes on 0.2 μm polyvinyl difluoride membranes (catalog number 162-0218). After transfer, the membranes were blocked for 120 minutes with 5% BSA and incubated with unlabeled anti-TSH-β overnight at 4°C, followed by incubation for 30 minutes with antirabbit horseradish peroxidase secondary antibody at 1:5000 dilutions. The membranes were then visualized by an Amersham Imager 600. All procedures for the in-cell Western assays were accomplished using a protocol provided by Li-COR Inc.

Generation of cAMP

THP-1 cells were plated and cultured at a density of 2 × 105 cells per 96-well plate with 100 ng/mL PMA for 48 hours to differentiate into MØ cells. After 96 hours, human embryonic kidney (HEK)-TSHR cells and nontransfected HEK cells were plated on top of the differentiated MØ cells at a density of 3 × 104 cells per 96-well plates. Cells were allowed to make contact for 48 hours and then lysed, and intracellular cAMP levels were measured by homogenous time resolved fluorescence (Cis-Bio Assays).

In vivo studies

All mice used in this study were male and from a C57/BL6 and 129SV mixed background as previously described (23). All procedures were in accordance with the Internal Animal Care and Use Committee at Icahn School of Medicine at Mount Sinai (New York). For T4 experiments there were two groups of mice. The mice were sc implanted with T4 hormone pellets containing either 0 mg or 5 mg with a 21-day time release as detailed previously (12). At day 21 after the implantation, the mice were killed, blood was collected for thyroid function testing, and the brains sectioned to contain the pituitary gland from which RNA was prepared as described above. T3 levels were measured by an immunoassay to confirm the hyperthyroid status of the treated groups. cDNA from the control and treated animals were then PCR amplified.

Statistical analyses

All data are expressed as mean ± SEM. Statistical differences were assessed by a Student t test. Results with P < .05 were considered statistically significant.

Results

Identification of a TSH-β variant mRNA in human macrophages

Using specific primers for full-length TSH-β and the TSH-β splice variant as previously published (2), we identified, as expected, a 537-bp full-length human (h) TSH-β in human pituitary cDNA (Figure 3A) but not in the human macrophage cell line THP-1 (Figure 3C). The full-length PCR product was also absent in human BMCs and human peripheral blood (not shown). In contrast, RT-PCR for the previously reported hTSH-βv (9) was positive in human pituitary (Figure 3B), human macrophage THP-1 cells (Figure 3D), BMCs (Figure 3E), peripheral blood, and macrophages (Figure 3, F and G) as evidenced by the expected 364-bp amplified fragment. The identity of this hTSH-βv amplimer from the THP-1 cells peripheral blood and BMCs was also confirmed by sequence analysis (Figure 4, A–C). Because TSH is known to exist as a heterodimer of α/β-subunits, we wanted to verify whether the α-subunit was also expressed in these cells and tissues. Although TSH-α gene expression could be detected in pituitary cDNA (Figure 3H), it was not detected in THP-1 cells (Figure 3I). Furthermore, all samples of BMCs, PBMCs, and human macrophages were negative for the TSH-α gene transcript (not shown).

Figure 3.

Figure 3.

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Identification of a novel hTSH-βv in human macrophages. RT-PCR analyses of human pituitary extract illustrated the presence of both full-length hTSH-β and the novel hTSH-βv (A and B). The THP-1 cells (C and D), human bone marrow (E), PBMCs (F), and human macrophages (G) showed only the hTSHβv. RT-PCR amplification of the TSH-α gene was positive only in the pituitary sample (H) and not in THP-1 cells (I). *, P < .05.

Figure 4.

Figure 4.

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The hTSH-β sequence identified. A schematic outlining the native and novel exon sequence of the hTSH-β vs the hTSH-βv with the corresponding amino acid sequence for exon 3 and the nine amino acids from the intronic region (italicized) as reported elsewhere (34). The sequences identified in the THP-1 macrophages (A), human PBMCs (B), and human bone marrow (C) are bold and underlined.

Identification of a TSH-βv mRNA in human macrophages

Confirmation of TSH-βv protein expression was first obtained by immunostaining using specific polyclonal antibody to full-length TSH-β, which recognized an overlapping epitope within the TSH-βv sequence. We detected the presence of intracellular TSH protein, indicated by intense Alexa 488 staining (green), which coexpressed with CD14 as yellow staining in the merged image in both the THP-1 cells (Figure 5, A–C) and normal human macrophages (not shown). FACS analysis identified 47% of cells expressing TSH-βv (Figure 5D). Furthermore, Figure 5E shows CHO cells stained with a TSH-β antibody. Further confirmation was obtained by Western blotting of THP-1 cell lysate (Figure 5F).

Figure 5.

Figure 5.

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TSH-βv protein expression. THP-1 macrophages were differentiated and cultured for 4 days with PMA (100 ng/mL) and their nuclei labeled with 4′,6′-diamidino-2-phenylindole. Cells were counterstained for CD14 expression (A) and TSH-β (B), and the merged pictures are shown in (C) (×630). FACS analysis of these THP-1 cells (D) showed 47% of double-staining cells. E, Control CHO cells stained with 4′,6′-diamidino-2-phenylindole, anti-CD14, and anti-TSH-β within the same experiment. F, A Western blot analysis (at 40 μg protein) with anti-TSH-β, which confirmed the presence of appropriately sized TSH-βv expression in the THP-1 macrophage whole-cell lysate.

Macrophage phenotyping and regulation of human TSH-βv

THP-1 cells were exposed to both M1 and M2 activators including lipopolysaccharide (LPS) and IFNγ and IL-10 with IL-4, respectively. LPS and IFNγ induced expression of MCSF, Wnt 10a, CCL-2, and Wnt 5A, consistent with M1 polarization, and also markedly induced human TSH-βv expression (Figure 6). In contrast, the combination of IL-10 with IL-4 induced further expression of MARC1, consistent with M2 polarization, and also induced human TSH-βv expression (Figure 6). Hence, both M1 and M2 activators induced enhanced expression of human TSH-βv.

Figure 6.

Figure 6.

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Regulation of TSH-βv by LPS, LPS/IFN-γ (IFNG), and IL-4/IL-10. Differentiated THP-1 macrophage cultures were treated with LPS, LPS/IFNγ, and IL-4/IL-10 for 24 hours and gene expression examined for TSH-βv (A), MCSF (B), CCL-2 (C), Wnt5A (D), MARC1 (E), and Wnt10-A (F). These data revealed that both M1 stimulation with LPS/IFNγ and M2 stimulation with IL-4/IL-10 induced TSHβv gene expression by up to 30-fold (A). *, P < .05; **, P < .001. cont, control.

In vivo regulation of TSH-βv

Because it is known that increasing levels of thyroid hormone are a negative regulator of pituitary full-length TSH-β, we used a mouse model to examine the influence of thyroid hormone on TSH-βv (see Materials and Methods). We (12) previously reported changes in TSH-βv in mouse bone marrow cells after TSHR-knockout mice were deprived of thyroid hormone supplements for 1 and 2 weeks, rendering them severely hypothyroid. At 1 and 2 weeks after thyroid withdrawal, the levels of TSH-βv fell significantly from their expression when they were maintained in a euthyroid state by a thyroid hormone supplemented replacement diet, suggesting that thyroid hormone was a positive regulator of TSH-βv in BMCs. As a corollary to this, normal wild-type (WT) mice treated sc with a T4 pellet of 5 mg for 3 weeks showed markedly increased TSH-βv in their BMCs (Figure 7A). In contrast to the BMCs, the pituitary regulation of both full-length TSH-β and TSH-βv was as expected: with suppression by treatment of normal mice with T4 for the same 3-week period (Figure 7B). Further proof of the positive regulation of TSH-βv in the mouse macrophages was obtained in vitro using F480-positive RAW cells that expressed murine TSH-βv (Figure 8, A–C). In these cells, after 1 hour of T3 exposure, there was a dose-dependent increase in the levels of TSHβv mRNA, indicating T3 induction of TSH-βv gene expression (Figure 8D) and confirming a role for T3 in controlling the murine TSH-βv response.

Figure 7.

Figure 7.

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Thyroid hormone regulation of mouse TSH-βv in vivo. A, Bone marrow cells from normal WT mice treated with a T4 hormone pellet for 21 days showed greatly increased TSH-βv gene expression. B, Pituitary tissue from WT mice treated with T4 pellets for 21 days showed suppression of both full-length TSH-β and TSH-βv in contrast to the bone marrow cell data. *, P < .05; **, P < .001.

Figure 8.

Figure 8.

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T3 hormone regulation of mouse TSH-βv in vitro. Raw cells (264.7 mouse macrophage cell) were stained for F480, a macrophage marker (A), TSH-β (B), and the results overlaid (C) to see the coexpression of both markers. Control staining of CHO cells is shown in the insert to panel C. Raw cells were then treated with T3 hormone for 1 hour and PCR analyzed. The T3 hormone increased TSH-βv gene expression in a dose-dependent manner (D). **, P < .001, also enhanced inducible nitric oxide synthase- and arginase-2 (E, F).

In vitro regulation of human TSH-βv and bioactivity assessment

In agreement with the mouse data, exposing the human THP-1 cells to increasing concentrations of T3 showed a dose-dependent increase in TSH-βv mRNA and protein expression (Figure 9, A and B). Furthermore, T3 induced a variety of macrophage activities including marked induction of CCL-22 and MCSF (Figure 9A), also showing that T3 induced both M1 and M2 polarization in these THP-1 cells as seen previously. The bioactivity of the human TSH-βv was also illustrated by the enhanced generation of cAMP when THP-1 macrophages were cocultured with TSHR-expressing cells, a phenomenon not seen in the absence of the TSHR (Figure 9C).

Figure 9.

Figure 9.

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T3 hormone regulation of human TSH-βv in vitro. A, THP-1 cells were treated with T3 hormone for 1 hour in serum-free macrophage media (Gibco) and PCR analyzed for hTSH-βv. T3 hormone increased TSH-βv gene expression in a dose-dependent manner as well as CCL-22 and MCSF gene expression, suggesting that T3 hormone increased both M1- and M2-polarized states (see also Figure 6). *, P < .05. B, THP-1 macrophages also showed enhanced TSH-βv protein expression when treated with increasing concentrations of T3 hormone as shown by in-cell Western blot assay. C, When THP-1 macrophages (MØs) were cocultured with HEK293 cells transfected with the TSHR (HEK293 +TSHR), a cAMP response was elicited that was approximately 5-fold greater than when THP-1 macrophages were cocultured with HEK293 cells without the TSHR. The TSHR-related cAMP generation in response to MØs in coculture was greater than that generated by 1 mU/mL of TSH. Note that MØs with HEK293 cells alone induced a small cAMP response likely secondary to local chemokine release. Data were analyzed by a one-way ANOVA shown as mean ± SEM of two experiments carried out in duplicates. *, P < .001.

Discussion

The potential of pituitary TSH to modulate bone remodeling, independent of thyroid hormone, was supported by the fact that functional TSH receptors are expressed on osteoblasts and osteoclasts as established by several studies (7, 24). Our original suggestion that pituitary TSH may have a direct role in local control of bone cell interactions, as evidenced by using TSHR null mice, provided strong evidence that TSH has an osteoprotective action (7, 24), and transgenic and knockout mouse models (12, 25) and several human studies (26, 27) have cemented the role of the pituitary-bone axis as a major endocrine skeletal regulator, particularly in the context of osteoporotic bone loss. The troubling question was always why an anterior pituitary hormone would regulate bone biology from such a distance. The discovery of a TSH-β isoform in immune cells in 2009 (9) led to our observation that such a TSH-β splice variant was specifically made by murine bone marrow macrophages (8) and led to the speculation of it being an active local thyroid stimulator. Since the variant retained thyroid stimulating activity, this molecule fulfilled the criteria for providing local bone protection, at least in the mouse (8). The fact that mice are not human led us in the present study to try and identify and characterize a human homolog of the TSH-β splice variant in human macrophages that would support the paradigm of local TSH involvement in bone biology. The identification of a 364-bp human TSH-β splice variant in various human tissues, especially macrophages derived from human PBMCs, and the total absence of the full-length TSH-β or TSH-α established the TSH-βv as involved in a locally produced bone-immune endocrine network.

To establish a well-controlled model, we also extended our studies on the regulation of this TSH-βv using the well-characterized THP-1 monocytic cell line, which could be induced to show the presence of the variant by RT-PCR confirmed by sequencing, Western blot with TSH control, immunohistochemistry, and bioactivity assessment with TSHR-expressing cells. The phenotypic appearance of the THP-1 cells after differentiation was consistent with macrophages (28, 29), and our flow cytometric analysis of these cells showed that nearly 50% transformed into macrophages by our established protocol and were capable of expressing the TSH-βv on differentiation.

Macrophages in different tissue sites can exist in more than one phenotype (30, 31). M1 and M2 are the two major phenotypic classes with opposing roles. The TSH-βv was markedly up-regulated by enhancement of both the M1 phenotype (using LPS and/or IFNγ) and the M2 phenotype (IL-10 and/or IL-4), indicating that both forms of activated macrophages produce the variant and suggesting that TSH-βv has a role in both osteoinflammatory and osteoprotective responses. As shown in the mouse, the proximity of the TSH-βv-producing cells to osteoblasts, and likely also osteoclasts, may be a critical factor in a local bone-immune axis (8) and was also suggested in the present studies by cocultures of human macrophages and TSHR-expressing cells.

Thyroid hormones (THs) are known to act via the nuclear TH-β receptor in the hypothalamus and pituitary to inhibit TRH and TSH production and secretion (4–5) and thus completing a negative feedback loop that maintains systemic thyroid status. However, it is also known that THs exert their action on bone via the TH-α receptor (32), unlike in the pituitary (33), although it is not known whether this is the case for bone-derived macrophages. Enhanced expression of the TSH-βv in TH-induced hyperthyroidism in normal mice suggested that THs were important regulators of TSH-βv activity, and our observation that the macrophage TSH variant was also markedly up-regulated by TH in vitro confirmed that the production of TSH-βv can be regulated by THs. It is well established that TSH-α and -β subunit genes are negatively regulated by TH at the transcriptional level by the interaction of the TH receptors with the respective promoter immediately downstream of the TATA box to inhibit transcription. However, in macrophages the positive transcriptional effect of T3 indicates a different molecular mechanism when compared with the pituitary. Elucidation of the exact mechanism of this regulation requires promoter activity assays using deletion mutants and promoter binding assays.

In summary, the macrophage, which has been shown to be intimately involved with bone homeostasis and which is known to secrete a wide variety of active factors and cytokines, also includes in its armamentarium a unique TSH-β variant, which has the potential to influence bone biology and serve as a local osteoprotective resource for bone remodeling in disease states and in fracture repair. In particular, the fact that this variant is up-regulated by TH suggests that it may also have an important role in helping to resist the well-known counter effects of TH excess in skeletal biology.

Acknowledgments

This work was supported in part by Grants DK069713 and DK052464 from the National Institutes of Health (to T.F.D.) and the Veterans Affairs Merit Review Program (to T.F.D.).

Disclosure Summary: T.F.D. is a member of the Board of Kronus Inc (Star, Idaho). The other authors have nothing to disclose.

For News & Views see page 3402

Abbreviations:
BMC
bone marrow cell
CCL
chemokine ligand
CHO
Chinese hamster ovary
DAPI
4′,6′-diamidino-2-phenylindole
FACS
fluorescence-activated cell sorter
h
human
HEK
human embryonic kidney
IFN
interferon
LPS
lipopolysaccharide
MARC
myristoylated alanine-rich C
MCSF
macrophage colony-stimulating factor
PBMC
peripheral blood mononuclear cell
PMA
phorbol 12-myristate 13-acetate
TH
thyroid hormone
TSHR
TSH receptor
TSH-βv
TSH-β isoform
WT
wild type.

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