Expression of thyrotropin receptor, thyroglobulin, sodium-iodide symporter, and thyroperoxidase by fibrocytes depends on AIRE

Abstract

Context: CD34(+) fibrocytes, bone marrow-derived progenitor cells, infiltrate orbital connective tissue in thyroid-associated ophthalmopathy, a manifestation of Graves' disease. In the orbit, they become CD34(+) fibroblasts and coexist with native CD34(-) fibroblasts. Fibrocytes have been shown to express TSH receptor and thyroglobulin.

Objective: The objective of the study was to determine whether a broader repertoire of thyroid protein expression can be detected in fibrocytes and whether a common factor is responsible.

Design/setting/participants: Fibrocytes and fibroblasts were collected and analyzed from healthy individuals and those with Graves' disease in an academic clinical practice.

Main outcome measures: Real-time PCR, Western blot analysis, gene promoter analysis, cell transfections, and flow cytometric cell sorting were performed.

Results: We detect two additional thyroid proteins expressed by fibrocytes, namely sodium-iodide symporter and thyroperoxidase. The autoimmune regulator (AIRE) protein appears necessary for this expression. AIRE expression in fibrocytes results from an active AIRE gene promoter and stable AIRE mRNA. Knocking down AIRE with a targeting small interfering RNA reduces the expression of these thyroid proteins in fibrocytes as well as the transcription factors paired box-8 and thyroid transcription factor-1. When compared with an unaffected first-degree relative, levels of these proteins are substantially reduced in fibrocytes from an individual with an inactivating AIRE mutation. Levels of AIRE and the thyroid proteins are lower in orbital fibroblasts from patients with thyroid-associated ophthalmopathy than in fibrocytes. However, when mixed fibroblast populations are sorted into pure CD34(+) and CD34(-) subsets, the levels of these proteins are dramatically increased selectively in CD34(+) fibroblasts.

Conclusions: Fibrocytes express four proteins, the aggregate expression of which was previously thought to be restricted to thyroid epithelium. These proteins represent the necessary molecular biosynthetic machinery necessary for thyroid hormone production. Our findings implicate AIRE in the promiscuous expression of thyroid proteins in fibrocytes.

Figure 1.

Relative NIS (A) and TPO (B) mRNA levels in H-fibrocyte, GD-fibrocyte, DF, H-OF, and GD-OF. RNA was extracted from confluent culture as described in Figure 2 and subjected to real-time PCR. Values were normalized to respective GAPDH signals. Data are expressed as the mean ± SD of three independent determinations. Insets, Data are normalized to either GAPDH or 18S RNA. C, Detection of NIS and TPO proteins in the cell types indicated. For NIS detection, equivalent quantities of solubilized proteins were subjected to Western immunobloting and probed with an anti-NIS antibody. Membranes were reprobed with anti-β-actin antibody. For TPO, cells were labeled with [³⁵S]methionine (20 μCi/mL) for 48 hours and subjected to immunoprecipitation using anti-TPO. Pulled-down proteins ran on 4%–15% SDS-PAGE, and gels were dried and analyzed using autoradiography.

Figure 2.

Human fibrocytes express AIRE mRNA, the consequence of an active AIRE gene promoter and a stable transcript. A, Relative AIRE mRNA abundance in thymus, H-fibrocyte, GD-fibrocyte, DF, H-OF, and GD-OF. Confluent cultures were lysed, and RNA was extracted, reverse transcribed, and subjected to real-time PCR. Values were normalized to their respective GAPDH signals. B, Fibrocytes, GD-OF, and DF were transfected with an empty vector or one fused to a fragment of the human AIRE gene promoter. After 24-hour incubations, cells were disrupted, and luciferase activity was quantified as described in Materials and Methods. Data are expressed as mean ± SD of triplicate determinations from one experiment, representative of three performed. C, AIRE mRNA stability was assessed as described in Materials and Methods. 5,6-Dichlorobenzimidazole was added at time 0, and replicate culture wells were harvested at the times indicated along the abscissa. AIRE mRNA was reverse transcribed and cDNA was subjected to RT-PCR.

Figure 3.

Interfering with AIRE expression knocked-down levels of AIRE, Tg, TSHR, NIS, TPO, PAX8, and TTF-1 but not PGHS-2 or 18S RNA. A, Fibrocytes from four different donors were transfected, either with scrambled (control) siRNA or siRNA directed against AIRE, as described in Materials and Methods. Cultures were then incubated for 48 hours, and RNA was harvested and then subjected to real-time PCR. Data are expressed as mean ± SD of three independent determinations. B, Fibrocyte cultures were transfected with either scrambled siRNA or siRNA targeting AIRE for 96 hours as described in Materials and Methods. Tg protein was quantified by labeling with [³⁵S]methionine (40 μCi/mL) and then immunoprecipitated, as in the legend to Figure 1. TSHR and NIS were quantified by flow cytometry (14). Levels of TPO were assessed by Western blotting.

Figure 4.

Fibrocytes derived from an individual with APS-1 express lower levels of AIRE, TSHR, Tg, NIS, TPO, PAX8, and TTF-1 than do those from an unaffected first-degree relative. Cells were prepared as described in Materials and Methods from PBMCs derived from an individual with APS-1 and her unaffected mother. The proband is heterozygous for two mutations, a C>T substitution at bp 769 and a 13-base deletion from bp 967 to bp 979. Cultures were harvested after 14 days and were interrogated by real-time PCR using the specific primer sets described. Data are expressed as mean ± SD (n = 6 independent determinations).

Figure 5.

Sorting GD-OF into pure CD34⁺ subsets results in dramatic elevations in AIRE, Tg, TSHR, NIS, and TPO mRNA levels, the consequence of increased levels of AIRE. A, Parental strain of GD-OF (containing a mixture of CD34⁺ and CD34⁻ fibroblasts) was sorted into pure CD34⁺ and CD34⁻ subsets by FACS on a FACSAria III instrument (BD). These were cultured for 48 hours, RNA extracted, reverse transcribed, and subjected to real time-PCR for the amplicons indicated. B, Transfecting pure CD34⁺ GD-OF with siRNA targeting human AIRE attenuates the up-regulation of AIRE, Tg, TSHR, NIS, and TPO. Cells were sorted as in panel A and cultured with either scrambled siRNA or siRNA targeting AIRE. RNA was extracted and subjected to RT-PCR. Data are expressed as mean ± SD of three independent determinations.