Functional expression of the thyrotropin receptor in C cells: new insights into their involvement in the hypothalamic-pituitary-thyroid axis

Abstract

Thyroid C cells, or parafollicular cells, are mainly known for producing calcitonin, a hormone involved in calcium homeostasis with hypocalcemic and hypophosphatemic effects. Classically, the main endocrine activity of this cell population has been believed to be restricted to its roles in serum calcium and bone metabolism. Nonetheless, in the last few years evidence has been accumulating in the literature with regard to local regulatory peptides secreted by C cells, such as somatostatin, ghrelin, thyrotropin releasing hormone or the recently described cocaine- and amphetamine-related transcript, which could modify thyroid function. As thyrotropin is the main hormone controlling the hypothalamic-pituitary-thyroid axis and, accordingly, thyroid function, we have examined the functional expression of the thyrotropin receptor in C-cell lines and in thyroid tissues. We have found that rat and human C-cell lines express the thyrotropin receptor at both mRNA and protein levels. Furthermore, incubation of C cells with thyrotropin resulted in a 10-fold inhibition of thyrotropin-receptor expression, and a concomitant decrease of the steady-state mRNA levels for calcitonin and calcitonin gene-related peptide determined by quantitative real-time PCR was found. Finally, thyrotropin receptor expression by C cells was confirmed at protein level in both normal and pathological thyroid tissues by immunohistochemistry and immunofluorescence. These results confirm that C cells, under regulation by thyrotropin, are involved in the hypothalamic-pituitary-thyroid axis and suggest a putative role in local fine-tuning of follicular cell activity.

Fig. 1

RT-PCR analysis of TSH receptor mRNA in cultured rat and human cell lines. (A) Amplification of a 438-bp RT-PCR fragment of the human TSH receptor in the TT human C-cell line; line 3, positive control; lane 4, negative control. (B) Lanes 1 and 2, amplification of a 301-bp RT-PCR fragment of the rat TSH receptor in rat C-cell lines; lane 3, positive control; lanes 4 and 5, negative controls. Beta-actin gene was used as internal control for amplification.

Fig. 2

Immunofluorescent staining of TSH-R (A, B1, C1), calcitonin and TSH-R (B2, C2), and calcitonin (B3, C3) in CA77 (A1), PC-CL3 (A2), TT (A3, B, C) and FTC-133 (A4) cell lines. TSH-R immunoreactivity coincides with calcitonin in the cytoplasm of C cells but was reinforced at the membrane level. Bar = 20 µm.

Fig. 3

SYBR® Green qRT-PCR-based analysis of the effect of TSH treatment on the TSH mRNA steady state in C cells. Graphs demonstrate the functional downregulation of the TSH-R by its ligand in C cells. Mean ± SD from six different representative experiments. *P ≤ 0.001 vs. untreated cells.

Fig. 4

SYBR® Green qRT-PCR-based analysis of the effect of TSH treatment on the synthesis of calcitonin and CGRP in C cells. Graphs show the relative mRNA amount for each peptide standardized to their basal level in non-treated cells. Beta actin was used as housekeeping gene. Mean ± SD from four different representative experiments. *P ≤ 0.001 vs. control.

Fig. 6

Immunofluorescent staining of calcitonin (A1, B1, C1, D1), calcitonin and TSH-R (A2, B2, C2, D2), and TSH-R (A3, B3, C3, D3) in rat (A,B) and human (C,D) thyroid glands: normal C cells (A,C), rat C-cell carcinoma (B) and human medullary thyroid carcinoma (D). TSH-R immunoreactivity coincides with calcitonin in the cytoplasm of both normal and neoplastic C cells. Bar = 20 µm.

Fig. 5

Immunoperoxidase staining for calcitonin (A1, B1, C1, D1) and TSH-R (A2, B2, C2, D2) in rat (A, B, C) and human (D) thyroid glands: normal C cells (A), C-cell adenoma (B), rat C-cell carcinoma (C) and human medullary thyroid carcinoma (D). TSH-R immunoreactivity was found in the cytoplasm of both follicular cells and C cells, independently of being normal or neoplastic C cells. Counterstaining: Harris haematoxylin. Bar = 100 µm.