Human fibrocytes coexpress thyroglobulin and thyrotropin receptor

Abstract

Thyroglobulin (Tg) is the macromolecular precursor of thyroid hormones and is thought to be uniquely expressed by thyroid epithelial cells. Tg and the thyroid-stimulating hormone receptor (TSHR) are targets for autoantibody generation in the autoimmune disorder Graves disease (GD). Fully expressed GD is characterized by thyroid overactivity and orbital tissue inflammation and remodeling. This process is known as thyroid-associated ophthalmopathy (TAO). Early reports suggested that in TAO, both Tg and TSHR become overexpressed in orbital tissues. Previously, we found that CD34⁺ progenitor cells, known as fibrocytes, express functional TSHR, infiltrate the orbit, and comprise a large subset of orbital fibroblasts in TAO. We now report that fibrocytes also express Tg, which resolves as a 305-kDa protein on Western blots. It can be immunoprecipitated with anti-Tg Abs. Further, ¹²⁵iodine and [³⁵S]methionine are incorporated into Tg expressed by fibrocytes. De novo Tg synthesis is attenuated with a specific small interfering RNA targeting the protein. A fragment of the Tg gene promoter fused to a luciferase reporter exhibits substantial activity when transfected into fibrocytes. Unlike fibrocytes, GD orbital fibroblasts, which comprise a mixture of CD34⁺ and CD34⁻ cells, express much lower levels of Tg and TSHR. When sorted into pure CD34⁺ and CD34⁻ subsets, Tg and TSHR mRNA levels become substantially higher in CD34⁺ cells. These findings indicate that human fibrocytes express multiple “thyroid-specific” proteins, the levels of which are reduced after they infiltrate tissue. Our observations establish the basis for Tg accumulation in orbital GD.

Thyroid epithelium exhibits an array of phenotypic attributes that sets it apart from other mammalian tissues. At the heart of its highly specialized functions is the ability to produce thyroglobulin (Tg) (1). Tg is secreted into the lumen of thyroid follicles, where it is iodinated. It can then bind to endocytic receptors such as megalin (2) and undergo complex intracellular trafficking to the endoplasmic reticulum and lysosomes and undergo recycling or proteolytic cleavage to yield thyroid hormones. Several protein–protein interactions appear to determine the processing and export of Tg and thyroid hormones, such as those with GRP94 (3). In health, these functions are tightly regulated by the hypothalamic/pituitary axis. Thyroid-stimulating hormone (thyrotropin, TSH) represents a glycoprotein hormone that acts through the TSH receptor (TSHR) displayed on the surface of thyroid epithelial cells (4). The characteristics distinguishing thyroid epithelial cells from other cell types have become blurred following detection of functional TSHR in extrathyroidal tissues (5–10). This suggests that it might play physiological roles outside the thyroid such as the regulation of lipolysis (11).

Detection of “thyroid-specific” proteins in orbital tissues from individuals with thyroid-associated ophthalmopathy (TAO) has been reported previously. TAO is a localized manifestation of Graves disease (GD), an autoimmune process also affecting the thyroid gland (12). In TAO, connective tissues of the orbit become inflamed and remodeled. Observations made 40 y ago suggested that Tg accumulates in TAO and may participate in localized immune reactions (13). This concept has been revisited recently (14). Orbital TSHR expression has also been detected in TAO (7). Orbital fibroblasts (OF) display low-level surface TSHR that is increased following differentiation in vitro into adipocytes (8). A central component of GD is the generation of activating Abs directed against TSHR, and these drive the overproduction of thyroid hormones in GD. Abs directed at Tg were among the first associated with thyroid autoimmunity (15), and can be detected in many patients with GD.

The mechanistic connections between the pathology occurring within the thyroid and orbit in GD have yet to be established. However, we recently reported that circulating fibrocytes are overabundant in GD, and can infiltrate the orbit and thyroid (16, 17). OF from these patients (GD-OF) comprise both CD34⁺ and CD34⁻ cell subsets, whereas OF from healthy individuals (H-OF) are uniformly CD34⁻ (16). It would therefore appear that CD34⁺ OF derive from circulating fibrocytes. In experimental animals, fibrocytes are recruited to sites of injury (18), where they participate in antigen presentation, tissue remodeling, wound healing, and fibrosis (19, 20). These bone marrow-derived progenitor cells display a characteristic array of surface markers, including CD45, CD11b, CD34, CXCR4, and collagen I (Col I) (20, 21). They exhibit substantial plasticity and can differentiate in vitro into fat cells and myofibroblasts (21). Thus, the behavior of fibrocytes resembles that of GD-OF (22–24). Fibrocytes from healthy donors and those with GD also express functional TSHR, and TSH induces the production of inflammatory cytokines in these cells (16, 17).

Using multiple approaches, we now report the unanticipated expression by human CD45⁺CD34⁺ fibrocytes of Tg as well as TSHR. Both proteins and their transcripts were detected in fibrocytes cultured from the peripheral blood mononuclear cells (PBMCs) of healthy donors and from those with GD. Moreover, Tg and TSHR mRNA expression localizes in CD34⁺ GD-OF but is absent in CD34⁻ GD-OF. Our findings suggest that Tg and TSHR detected in orbital connective tissues in TAO result from local fibrocyte biosynthetic activity. These insights may prove relevant to the pathogenesis of TAO and could identify therapeutic targets for this vexing clinical process.

Results

Tg Protein Can Be Detected in Cultured CD34⁺ Fibrocytes.

Human fibrocytes outgrowing PBMCs and cultivated for 14 d were subjected to immunohistochemical analysis. As the images in Fig. 1 demonstrate, most cultivated cells stain strongly for Tg in a diffuse cytoplasmic pattern. In contrast, staining with an isotype control Ab is nil. Moreover, fibrocytes uniformly exhibit the CD45⁺ phenotype. Cells subjected to staining with another primary anti-Tg mAb exhibited uniform immunofluorescence in all fibrocytes within the microscopic field. This was absent in GD-OF or H-OF (Fig. 2A). Staining for TSHR is also strong in fibrocytes, substantially less robust in GD-OF, and absent in H-OF. Flow cytometric analysis of fibrocytes reveals a strong shift indicating staining for Tg and TSHR in the vast majority of fibrocytes (Fig. 2B). In contrast, Tg was not detected in either GD-OF or H-OF.

Fig. 1.

Human fibrocytes express Tg protein. Fibrocytes, in this case from a healthy individual and thyroid tissue remote from a neoplasm, were stained by immunohistochemistry using an Ultraview DAB Detection Kit (Materials and Methods). Staining with either anti-Tg mAb (Upper) or an isotype control IgG (Lower). (Right Lower) Flow cytometric analysis of CD45 display on fibrocytes (Inset, CD45 immunofluorescence staining).

Fig. 2.

Analysis of TSHR and Tg expression by human fibrocytes and fibroblasts by (A) immunofluorescence staining and (B) flow cytometry. Cultivated cells were (A) fixed, blocked, and stained with either anti-TSHR, anti-Tg, or isotype control mouse IgG followed by Alexa Fluor 488-conjugated goat anti-mouse Abs. Nuclei were stained with DAPI, and thin sections were subjected to confocal fluorescence microscopy. (Insets) Isotype IgG staining. (B) Fibrocytes and fibroblasts were incubated with phycoerythrin-conjugated anti-TSHR, anti-TG, or isotype mAbs and Alexa Fluor 488 goat anti-mouse IgG. They were subjected to flow cytometry. Horizontal lines denote fluorescence intensity compared with isotype controls. Results are representative of three experiments.

To determine the molecular mass of the protein, fibrocyte and fibroblast lysates were subjected to Western blot analysis. Tg resolved as an abundant band of 305 kDa in fibrocytes, in this case from a healthy donor (Fig. 3A), consistent with the pattern established previously (25). The analysis shown in this figure demonstrates substantially less abundant Tg content in GD-OF and a virtual absence of the protein in H-OF.

Fig. 3.

Tg protein content characterized in thyroid tissue, fibrocytes, GD-OF, H-OF, and DF. (A) Cellular lysates were processed as described in Materials and Methods. Precipitated protein (200 μg) was separated by 7.5% SDS/PAGE, transferred to PVDF membrane, and probed with mouse anti-human Tg mAb (1:1,000). These were then incubated with secondary goat anti-mouse Ab. Signals were generated with ECL Plus reagent (Materials and Methods). (B) Fibrocytes, OF, and DF were metabolically labeled with 40 μCi/mL [³⁵S]methionine for 48 h. Cell extracts were immunoprecipitated with rabbit anti-Tg mAb and processed as in A. Dried gels were exposed to X-ray film. (C) Fibrocytes were labeled with 40 μCi/mL Na¹²⁵I for 48 h. Extracts were immunoprecipitated with anti-Tg Ab or isotype IgG and processed as in B. (D) Fibrocytes, GD-OF, H-OF, and DF were incubated with 40 μCi/mL Na¹²⁵I for 48 h and immunoprecipitated with anti-Tg Ab and processed as in B. (E) Fibrocyte cultures were transfected with Tg-specific or control siRNA, and then incubated with [³⁵S]methionine and processed as in B. Radioactivity was measured by liquid scintillation spectrometry. Data are expressed as mean ± SD, n = 3 (*P < 0.05 vs. control).

CD34⁺ Fibrocytes Can Iodinate Tg.

To determine whether the Tg protein expressed by fibrocytes and OF could be iodinated in situ, fibrocyte and fibroblast cultures were incubated in medium containing Na¹²⁵I (40 μCi/mL) for 24 h. Cell lysates were subjected to immunoprecipitation with anti-Tg mAb and then resolved on SDS/PAGE. As the autoradiograph shown in Fig. 3B demonstrates, the radioactivity from the iodination resolved to the same 305-kDa band, which is abundant in fibrocytes and substantially less abundant in GD-OF. These findings indicate that fibrocytes and GD-OF possess the molecular machinery to iodinate Tg.

Evidence for de Novo Synthesis of Tg in Fibrocytes.

Cells such as macrophages can accumulate Tg from their environment (26). To determine whether fibrocytes and their putative fibroblast derivatives can synthesize Tg, they were metabolically labeled with [³⁵S]methionine (40 μCi/mL) for 24 h and were found to incorporate this amino acid into Tg. As Fig. 3C demonstrates, when cell lysates were then subjected to immunoprecipitation with an anti-Tg mAb and then resolved on 8% SDS/PAGE, a discrete band of radioactivity migrated at 305 kDa in fibrocytes, whereas considerably lower levels of radioactivity were detected in GD-OF and H-OF (Fig. 3D). A very faint Tg band could also be seen in dermal fibroblasts (DF), suggesting that the protein may be expressed at extremely low levels in skin. If the radiolabeled protein bands represent de novo synthesized and bona fide Tg, knocking down the protein with a Tg-specific siRNA should attenuate [³⁵S]methionine incorporation. As the data in Fig. 3E suggest, [³⁵S]methionine incorporation in cells transfected with the Tg-specific siRNA was diminished by 48% (P < 0.05) compared with cells receiving scrambled control siRNA.

Fibrocytes Express mRNAs Encoding Tg and TSHR.

CD34⁺ fibrocytes cultivated from patients with GD and those derived from healthy donors express detectable Tg and TSHR mRNAs (Fig. 4A). Levels of both transcripts are dramatically lower (three to four orders of magnitude) than those found in thyroid tissue. However, steady-state levels of both mRNAs were significantly higher in many fibrocyte strains than those observed in OF and DF. Thirteen fibrocyte strains (six healthy and seven GD) were examined, each from a different donor. No significant differences exist between mRNA levels in fibrocytes from patients with GD compared with those from control donors, but levels vary greatly among individuals [TSHR mRNA; H fibrocytes, 7.35 ± 1.8-fold vs. GD fibrocytes, 5.24 ± 3.1-fold (mean ± SD, P = 0.17); Tg mRNA; H fibrocytes, 45.76 ± 33.0-fold vs. GD fibrocytes, 30.23 ± 24.7-fold (P = 0.37)].

Fig. 4.

Comparatively low-level TSHR and Tg mRNA is expressed by fibrocytes. (A and B) TSHR and Tg mRNA in thyroid tissue, fibrocytes, GD-OF, H-OF, and DF was quantified by real time-PCR. Values were normalized to their respective GAPDH signals. Data from individual strains are shown as mean ± SD. (C) Fibrocytes were treated as indicated with bTSH (5 mIU/mL), and RNA was extracted and processed as in A and B. Data are expressed as mean ± SD of triplicate independent determinations. *P < 0.05, **P < 0.01 vs. baseline. (D) Fibrocytes and OF were transfected with empty reporter vector or one fused to a fragment of the Tg promoter. Data are expressed as mean ± SD of three independent determinations. **P < 0.01 compared with controls.

TSH regulates the expression of Tg in thyroid epithelium (1). We next assessed its impact on Tg and IL-6 mRNA levels in fibrocytes by treating confluent cultures with TSH (5 IU/mL) for graded intervals. Tg transcript is detectable at time 0 and insignificantly varies over the next 96 h (Fig. 4C). In contrast, the IL-6 transcript is undetectable at baseline but is dramatically induced at least 100-fold at 16 h (P < 0.005) by TSH and remains elevated for 72 h before returning to baseline by 96 h.

Tg Gene Promoter Activity in Fibrocytes.

To determine whether the Tg gene promoter was more active in fibrocytes than in OF, a luciferase reporter gene construct containing a fragment of human Tg gene promoter, extending from −181 nt to +16 nt, was transfected into fibrocytes and OF. Its activity is sixfold above that of the empty reporter (Fig. 4D). In contrast, no reporter activity could be detected in OF. It would therefore appear that higher levels of Tg gene expression underlie the greater Tg production in fibrocytes than in OF.

Tg and TSHR mRNAs Segregate in GD-OF to CD34⁺ Cells.

Unsorted (parental) GD-OF cultures comprise a mixed population of CD34⁺ and CD34⁻ cells (16). We postulate that CD34⁺ fibroblasts derive from circulating CD34⁺ fibrocytes that express Tg and TSHR mRNAs (Fig. 4). Very low levels of both transcripts were detected in parental strains of GD-OF. When sorted into pure CD34⁺ and CD34⁻ subsets and cultured for 48 h, levels of Tg and TSHR mRNAs increase dramatically in pure CD34⁺ GD-OF compared with parental strain cells (P < 0.05) or isolated CD34⁻ GD-OF (P < 0.05) (Fig. 5). This result strongly suggests that CD34⁺ GD-OF fibroblasts revert to a phenotype resembling that of circulating fibrocytes when cultured in the absence of CD34⁻ GD-OF. Thus, CD34⁻ GD-OF appear to down-regulate Tg and TSHR expression in their CD34⁺ counterparts.

Fig. 5.

Discordant TSHR and Tg mRNA levels in CD34⁺ and CD34⁻ orbital fibroblast subsets. An unsorted (parental) strain of GD-OF was sorted into pure CD34⁺ and CD34⁻ subsets by FACS. Cells were cultured for 48 h, and RNA was extracted and subjected to real-time PCR for TSHR (A) and Tg (B) mRNA. Values were normalized to their respective GAPDH signals and expressed as mean ± SD of three independent determinations. *P < 0.05 vs. either parental or pure CD34⁻ strains.

Discussion

Human fibrocytes express both Tg and TSHR, two proteins linked to normal thyroid function and autoimmunity (1, 4, 15). Using several complementary approaches, we report that Tg is expressed by fibrocytes. These studies used multiple mAbs directed against Tg, and thus have strengthened the evidence that bona fide Tg is detected in these cells. Considerably lower levels of Tg could be detected in GD-OF and H-OF using a sensitive radiometric method (Fig. 3D). This figure demonstrates an extremely faint band among proteins from normal DF. These findings are congruent with the recent report of Cianfarani et al. (10), who found transcripts encoding Tg, TSHR, sodium-iodine symporter (NIS), and thyroid peroxidase (TPO) in healthy skin. Primary cultured keratinocytes also express TSHR, TPO, and NIS mRNAs. Klein and coworkers have detected expression of a TSH variant and functional TSHR in distinct populations of mouse bone marrow cells (27, 28). During the course of these studies, Meischl et al. (29) reported that cultured H9c2 cardiomyoblasts and rat heart tissue express Tg mRNA and protein. These authors detected the apparent synthesis of thyroxine and triiodothyronine by cardiomyoblasts in vitro. Thus, the aggregate evidence suggests that these proteins are expressed widely.

An unresolved question pertains to the identity of the shared self-antigen(s) underlying infiltration of the orbit and thyroid by immune cells. Although recent attention has focused primarily on TSHR, other candidate orbital antigens have emerged. In 1970, Kriss proposed that thyroid-derived antigens might be involved in the pathogenesis of TAO (13). His concept revolved around the accumulation of these proteins in orbital tissues as a consequence of hyperthyroidism and/or its treatment. Among the candidates he considered was Tg. Using radioisotopic thyroidolymphography, lymph drainage from hyperplastic thyroids was found to be abnormal. [^99mTc]Sulfur colloid injected directly into the thyroid was rapidly visualized in adjacent lymph nodes (13). This same group generated anti-Tg mAbs by immunizing mice with extraocular muscle membranes (30). More recently, Marinò et al. (14) detected intact Tg by Western blot analysis and ELISA in orbital tissues from three patients with TAO. The protein was absent in healthy orbital tissues. Because thyroid hormones were detected within the Tg molecule, the authors concluded that it derived from the thyroid. The current findings do not preclude an influx of Tg to the orbit from the thyroid, but they suggest strongly the potential for its local generation in the orbit by fibrocytes. In so doing, they implicate this protein in TAO by identifying a putative mechanism for its accumulation in affected tissue.

Fibrocytes exhibit phenotypic plasticity, can efficiently present antigens, produce several cytokines, and generate collagen and vitronectin (19, 31). In experimental models of lung fibrosis, they traffic specifically to affected tissues through the CXCL12/CXCR4 chemokine pathway (32). Evidence for their potential involvement in autoimmunity is relatively recent. For instance, Galligan et al. found excessive phosphorylation of signaling molecules in fibrocytes isolated from patients with rheumatoid arthritis (33). In asthma, circulating fibrocytes become more frequent (34) and might serve as myofibroblast precursors (35). More recently, Bucala has suggested participation of fibrocytes in the pathogenesis of nephrogenic systemic fibrosis (36). Our current findings raise the important possibility that Tg as well as TSHR might play unanticipated roles in fibrocyte function and tissue remodeling such as occurs in GD and TAO. Because fibrocytes present antigen (37), it is possible that they participate in the generation of Abs associated with those disease processes. Further scrutiny into their seemingly complex interactions with other components of the immune system is warranted, including whether autoimmune reactivity against Tg and TSHR expressed by fibrocytes might occur. Another aspect of the current findings relates to whether CD34⁺ GD-OF derive from circulating fibrocytes. Further characterization of the phenotypic transition that appears to occur once fibrocytes infiltrate the orbit and how CD34⁻ fibroblasts might impose their negative influence on the expression of Tg and TSHR in vivo awaits the development of an animal model of TAO.

Our observations carry substantial biological implications beyond those necessarily related to GD. They indicate that cultured fibrocytes express functional Tg. This raises the possibility that thyroid hormones might be generated, not only in the thyroid gland, but elsewhere in the body. Taurog and Evans (38) had suggested that possibility many years ago. Congruent with their earlier assertions and our current findings, it is possible that fibrocytes produce iodothyronines, including thyroid hormones. Thus, where they infiltrate, fibrocytes could provide locally generated hormones during tissue remodeling. Moreover, they might play a role in the abnormal metabolism associated with chronic inflammation.

Materials and Methods

Materials.

Plasmid containing a fragment of the human Tg gene promoter extending from −181 nt to +16 nt fused to the luciferase gene was kindly provided by S. Refetoff (University of Chicago, Chicago, IL). FacLyse buffer, Cytofix, anti-CXCR4, anti-CD34, anti-CD45, anti-CD31, Col I, TSHR, isotype mouse IgG 1 FITC, phycoerythrin (PE), allophycocyanin (APC), anti-Col I, anti-TSHR, and CyChrome were purchased from BD Biosciences. Mouse and rabbit anti-Tg mAbs were from Abcam (ab80783 and ab92467), as was anti-TSHR mAb (ab49702). FBS was supplied by Life Technologies. bTSH was obtained from Sigma-Aldrich.

Fibrocyte and Fibroblast Cultivation.

Procurement of human tissues and blood was conducted after obtaining informed consent as approved by the Institutional Review Board of the University of Michigan Health System. Fibrocytes were generated from blood provided by the American Red Cross (n = 41), from patients with GD (n = 7), or healthy donors (n = 6). They were isolated and cultivated as described by Bucala et al. (19) in 6-well plates inoculated with 1 × 10⁷ cells in DMEM with 5% FBS (vol/vol). After 12–14 d in culture, adherent cells (<5% of starting PBMC population) were washed and removed by scrapping. Culture purity and viability were verified to be >90% fibrocytes by FACS analysis and >90% by trypan blue exclusion, respectively.

OF were cultivated from surgical waste from individuals with TAO (n = 2) or healthy donors (n = 2). GD-OF typically contain 30–60% CD34⁺ cells. DF were derived from healthy donors (n = 3). They were used between the 2nd and 12th passages, during which they maintained a stable phenotype (24).

Immunohistochemical and Immunofluorescence Staining.

Tg staining was performed on an immunostainer (Ventana) with an Ultraview DAB Detection Kit (760-500). Anti-Tg Ab was purchased from Dako (1:2,000, A0251). Antigen retrieval was performed at high pH for 32 min. For immunofluorescence studies, cells were processed as previously reported (16, 17). They were incubated with mouse anti-TSHR mAb (1:300, ab49702; Abcam), anti-Tg (1:300, ab80783; Abcam), or isotype control IgG at 4 °C overnight, washed, and incubated for 1 h with Alexa Fluor 488-conjugated goat Ab to mouse IgG (1:500; Invitrogen). Cells were stained with DAPI and examined by confocal microscopy (Leica).

Flow Cytometry and Cell Sorting.

Details were published previously (16, 17). Dispersed fibroblasts and fibrocytes were incubated with heat-inactivated mouse serum for 5 min and then with PerCP-conjugated anti-human CD34, CXCR4, TSHR, Col I, and Tg mAbs. They were washed, resuspended in staining buffer at 4 °C, and subjected to FACS analysis in a Calibur flow cytometer (BD). For intracellular Tg staining, cells were permeabilized as reported (39). Mean fluorescent intensity is the ratio of mean fluorescence sample:isotype control fluorescence. Viable cells were gated on the basis of forward light scatter. Data were analyzed with FCS Express software (De Novo Software). Studies were performed at least three times. For generating pure CD34⁺ and CD34⁻ GD-OF subsets, mixed unsorted (parental) cultures were stained with FITC-conjugated anti-human CD34 for 30 min at 4 °C. Washed cells were sorted under sterile conditions with a FACSAria III (BD).

Isolation and Western Blot Analysis of Tg.

Tg was partially purified from pooled fibrocytes derived from 16–20 Red Cross leukocyte filters, each from a different donor. Cellular material was suspended in PBS (pH 7.4) containing 1 mM PMSF and protease inhibitor mixture (Sigma). Samples were sonicated on ice and centrifuged for 10 min at 10,000 × g and supernatants were collected. Homogenized human thyroid (2.3 g) served as a positive control. Protein concentrations were determined using the Dc Protein Assay Kit (Bio-Rad). Ammonium sulfate (AS) (pH 6.8) was added to the supernatant to a concentration of 1.6 M, incubated at 4 °C for 30 min, and centrifuged at 10,000 × g, and the AS concentration was brought to 1.8 M and recentrifuged after 6 h. The dissolved precipitate was dialyzed overnight at 4 °C against PBS.

For Western blots, samples were solubilized in lysis buffer (Invitrogen) containing PMSF (1 mM), boiled in 4× NuPAGE sample buffer with 8 M urea, layered on 7.5% Tris⋅HCl gels, subjected to SDS/PAGE, and transferred to PVDF membrane (Millipore). Primary antibody against Tg (1:1,000, ab80783; Abcam) was incubated overnight at 4 °C. Washed membranes were incubated with horseradish peroxidase-conjugated secondary Ab (P0447; DAKO). ECL Plus reagent (Thermo Scientific) was used for signal generation.

Metabolic labeling was conducted by incubating confluent cultures for 48 h in DMEM with 10% FBS and 40 μCi/mL Na¹²⁵I (PerkinElmer) and 1 μM unlabeled NaI (Sigma). Alternatively, [³⁵S]methionine (40 μCi/mL; PerkinElmer) in methionine- and cysteine-free DMEM (Invitrogen) covered confluent monolayers for 48 h. Cell pellets were collected by centrifugation at 4,000 × g for 10 min and stored at −70 °C. They were resuspended in 100 μL ice-cold RIPA buffer (Thermo Scientific) with protease inhibitor mixture and incubated at 4 °C for 10 min. Extracts were incubated with 50 μL of protein A agarose beads (SC-2001; Santa Cruz Biotechnology) coupled with anti-rabbit Tg Ab (ab92467; Abcam) at 4 °C. Immunoprecipitates were rinsed, resuspended in Laemmli buffer (Bio-Rad), and loaded on 7.5% Tris⋅HCl SDS/PAGE gels. Dried gels were subjected to autoradiography. Radioactivity was quantified by liquid scintillation spectrometry (Winspectral; PerkinElmer).

RNA Preparation and Real-Time PCR.

Total cellular RNA was extracted with ULTRASPEC RNA reagent (Biotecx Laboratories) or RNeasy (Qiagen). RNA (2 μg) was reverse-transcribed, and real-time PCR was performed with an Applied Biosystems instrument using a QuantiTect SYBR Green PCR Kit (Qiagen). The following primer sequences, synthesized by Life Technologies, were used: Tg, forward 5′-GAGCCCTACCTCTTCTGGCA-3′ and reverse 5′-GAGGTCCTCATTCCTCAGCC-3′; TSHR, forward 5′-AGCCACTGCTGTGCTTTTAAG-3′ and reverse 5′-CCAAAACCAATGATCTCATCC-3′. Sample values were generated against a standard curve and normalized to GAPDH signals.

Fibrocyte transfections were conducted as described by Hong et al. (21), whereas fibroblasts were treated as reported by Tsui et al. (40).

Statistics.

Statistical significance was determined with the two-tailed Student’s t test.