Cloning and sequencing of complete thyrotropin receptor transcripts in pretibial fibroblast culture cells

Abstract

Pretibial fibroblasts are considered to be targets of autoimmune attack in pretibial myxedema. A possibility of the pathogenesis of pretibial myxedema is that T cells, reacting with thyrotropin (TSH) receptor, will be targeting to the pretibial fibroblasts where, in the presence of antigen (TSH receptor), they will secrete various cytokines and stimulate fibroblasts to secrete glycosaminoglycans. We have demonstrated that TSH and TSH receptor antibody can bind to fibroblasts and the presence of RNA encoding the extracellular domain of the TSH receptor in fibroblasts derived from skin lesions of two patients with pretibial myxedema. The present study was designed to determine whether there are complete TSH receptor transcripts in pretibial fibroblasts obtained from patients with pretibial myxedema. RNA was prepared from pretibial fibroblasts obtained from 11 patients with pretibial myxedema and from four normal subjects, then reverse-transcribed by polymerase chain reaction using three sets of primers (-11/+8 and +754/+773; +353/+373 and +1265/+1285; +1000/+1017 and +2284/+2301). The overlapped 2312 bp cDNA sequence was expected to contain the genetic sequences of the signal peptide (+1/+60), extracellular domain (+61/+1254), transmembrane domain (+1255/+2046), and cytoplasmic domain (+2047/ +2292) of the TSH receptor. The sequences were determined using dideoxy sequencing method. All of the 2312 nucleotide sequences in 15 samples were consistent with the reported TSH receptor sequence of transcripts in thyroid. These data suggest that the complete TSH receptor transcripts are very possible to be present in the fibroblasts derived from pretibial skin.