Cadherin mediated adhesion and barrier function in the thyroid epithelium

Abstract

The thyroid gland is mainly composed of epithelial cells (thyrocytes) organized as follicles. The cells are connected by junction complexes that link them together into a cohesive simple epithelium. The junctions are also important for the establishment of a semipermeable barrier that restricts the paracellular flow of ions and other solutes. In the first part of this thesis, the effects of thyroid stimulating hormone (TSH) and pro-inflammatory cytokines on the epithelial barrier of primary cultured human thyrocytes were investigated. Thyroid follicle segments were prepared from surgically excised paradenomatous tissue and from resected glands with Graves´ disease. The cells formed tight, but low resistant monolayers on permeable filters in bicameral chambers. Addition of TSH further increased the transepithelial resistance to approximately 1000 * x cm2 after 3-5 days. TSH also induced an ordered organization of the tight junction protein ZO-1, promoted a polarized (apical-basolateral) ultrastructure and stimulated the secretion of thyroglobulin (Tg), preferentially in the apical direction. Treatment with IL-1a for 1-2 days induced a decreased transepithelial resistance and an increased paracellular leakage of both small ([3H]inulin) and large (125I-Tg) radiotracers irrespective of TSH and without signs of cytotoxicity. Electron microscopy showed that the main target of IL-1a action was the adherens junction where large amounts of stress fibers were formed. In contrast, other pro-inflammatory cytokines, i.e. IL-6, TNF-a, IFN-g and TGF-b, had no effect on the thyroid epithelial barrier.Changes in cell-cell adhesion have previously been correlated with tumor cell invasion and metastasis. In the second part of the thesis, cadherin-mediated adhesion and morphology in thyroid carcinoma cell lines and in cultured non-neoplastic pig and human thyrocytes were examined. E-cadherin negative cell lines established from anaplastic thyroid carcinomas (ATC) showed a variable down-regulation of catenins, known to anchor cadherins to the cytoskeleton. The reduced expression was most notable for g-catenin and p120ctn. Furthermore, the electrophoretic mobility of p120ctn was altered. A demethylating agent, 5-aza-2´-deoxycytidine, increased protein expression of E-cadherin and g-catenin although some cell lines were unresponsive. N-cadherin was detected in E-cadherin negative ATC cell lines but not in freshly isolated and primary cultured human and pig follicle segments. However, N-cadherin expression was also induced by 1-2 subcultivations of non-neoplastic thyrocytes. In this process, the upregulation of N-cadherin was paralleled by downregulation of E-cadherin and morphological alterations such as multilayering and loss of barrier function. Unexpectedly, one ATC cell line, KAT-4, expressed normal levels of E-cadherin and catenins and had a maintained epithelial morphology despite loss of barrier function and lack of contact-inhibited growth. KAT-4 cells also had a unique aberration of E-cadherin.In conclusion, human thyrocytes form a highly differentiated epithelium when grown on filter in the presence of TSH. The epithelial barrier is abrogated by IL-1a. Cultured anaplastic thyroid carcioma cells display N-cadherin-mediated adhesion and reduced catenin expression. A switch from E- to N-cadherin occurs in subcultured non-neoplastic thyrocytes. The results may have importance for the understanding of thyroid autoimmunity and tumor progression