Real-time PCR provides evidence for thyrotropin receptor mRNA expression in orbital as well as in extraorbital tissues

Abstract

Objective: The TSH receptor (TSHr) expressed on thyroid follicular cells is the autoantigen involved in the pathogenesis of Graves' hyperthyroidism. Whether this receptor is expressed in extrathyroidal tissues, and whether it participates directly in the pathogenesis of thyroid-associated ophthalmopathy (TAO) is unclear. Design: The aim of the present study was to measure TSHr mRNA in retro-orbital tissues, retro-orbital adipose tissue, extraocular muscle, and skin from patients with TAO and in several tissues from patients not affected by thyroid diseases using RT-PCR and real-time PCR. Methods: Total RNA was isolated from tissue specimens, reverse transcribed, and amplified using specific primers for the extracellular portion and a part of a 1.3 kbp variant form of the TSHr gene. Determination of TSHr mRNA levels using real-time PCR was also performed by the TaqMan system; to normalize for differences in the amount of total RNA added to the reaction, amplification of b-actin gene was performed as an endogenous control. Results: A single-round RT-PCR amplification using specific primers for the extracellular portion of the TSHr gene demonstrated an amplification product of 1.2 kbp in the thyroid, but not in all other tissues. A second-round RT-PCR amplification using the same primers and starting from the previous amplification product demonstrated a band of the size expected for the TSHr gene in all tissue specimens analyzed irrespective of their origin. Similar results were obtained using primers specific for a part of the variant form of 1.3 kbp of the TSHr gene. The amount of TSHr mRNA measured by real-time PCR with the TaqMan probe and expressed as TSHr/b-actin ratio was similar in the tissues from TAO patients with respect to the tissues from subjects not affected by thyroid diseases. Conclusions: We measured TSHr mRNA in tissues from patients with TAO using the very sensitive and quantitative method of real-time PCR. The level of transcription was similar to that measured in extraorbital tissues from patients who were not affected by thyroid diseases. These data suggest an illegitimate TSHr mRNA transcription in all the tissues examined apart from thyroid.

Evidence for protein and mRNA TSHr expression in fibroblasts from patients with thyroid-associated ophthalmopathy (TAO) after adipocytic differentiation

“…A TSHr variant transcript comprising essentially exons 1 -8 was also described in many locations, including ocular muscle, fat, lymphocytes and fibroblasts (11). Recently, we have detected by real-time PCR similar levels of TSHr mRNA in TAO orbital tissues and extraorbital tissues from patients not affected by thyroid diseases (12). The expression and function of the TSHr in cultured rat preadipocytes are closely related to cellular differentiation (13), so fibroblasts of the human connective orbital tissue may be important in the development of TAO not only because they are the major source of glycosaminoglycans, but also because the are able to differentiate into mature adipocytes and increase the expression of a functional TSHr, the hypothetical common antigen in thyroid gland and retro-ocular tissue (14,15).…”

“…The nucleotide sequence of the primers and probes (Applied Biosystems) are shown in Table 1. Quantitative PCR reactions were carried out on 96-well optical reaction plates using a cDNA equivalent of 50 ng total RNA for each sample in a volume of 50 ml using the TaqMan Universal PCR Master Mix (Applied Biosystems) as described previously (12). To minimize errors arising from variations in the amount of starting RNA among samples, amplification of b-actin mRNA was performed as an internal reference against which other RNA values can be normalized.…”

“…To validate the real-time PCR method, a standard curve for the TSHr gene was constructed from cDNA obtained from normal thyroid tissue as described previously (12). A standard curve using the same cDNA was also constructed for the b-actin gene.…”

Predictive Value of the TSH/FT4 Ratio in Women with Suspected PE or GH

“…Non as in endothelial cells obtained from the human aorta [14][15][16][17]. Studies performed in a rat model of thyroid goitrogenesis found that TSH treatment increases thyroid capillary density [18].…”

TSH-induced gene expression involves regulation of self-renewal and differentiation-related genes in human bone marrow-derived mesenchymal stem cells

“…Stimulation of the TSHR via TSH on thyroid cells leads to production of the thyroid hormones (THs), tri-iodothyronine (T 3 ) and thyroxine (T 4 ) (Toccafondi et al 1982). The TSHR expression by other tissue cells has also been reported (Haraguchi et al 1996, Hoermann 1996, Dutton et al 1997, Bell et al 2000, Agretti et al 2002, Tsai et al 2004, Ellerhorst et al 2006. However, exact function of its expression in extrathyroidal tissues has not been clarified.…”