Regulation of E-cadherin mediated adhesion in thyroid epithelial cells

Abstract

Cell adhesion is one of multicellular organisms most fundamental features. Cells connect to each other by transmembrane proteins arranged in junction complexes. E-cadherin is a Ca2+ dependent transmembrane protein of the adherens junction between epithelial cells, and a key component in the regulation of cell-cell adhesion that may fail in diseases like cancer. Thyrocytes are epithelial cells arranged as follicles within the thyroid gland. Their primary function is to produce the thyroid hormones, T3 and T4, in a process that require a tight epithelial barrier to be able to accumulate iodide and thyroglobulin within the follicle lumen. Thyrotropin (TSH) stimulates many thyroid functions involved in hormone biosynthesis as well as thyroid growth. The aim of this thesis was to investigate whether TSH also has a regulatory role in E-cadherin mediated adhesion in the thyroid. Studies were performed on primary cultures of porcine thyrocytes cultured on permeable filters in a two chamber system. Measurements of transepithelial resistance and potential difference were used to monitor epithelial barrier function. Disruption of cadherin-mediated adhesion was studied using a calcium switch protocol. E-cadherin and cadherin-associated proteins were studied with immunofluorescence, biotinylation, immunoprecipitation and Western blotting techniques.E-cadherin requires Ca2+ for binding. Reduction of the extracellular Ca2+ level caused rapid cleavage of E-cadherin at the cell surface, internalization of remaining mature E-cadherin and accumulation of the E-cadherin precursor in the cytoplasm. TSH prevented all low Ca2+ induced changes and stabilized full-length E-cadherin at the cell surface through a cyclic AMP mediated pathway (Paper I). TSH signalling can operate both through cyclic AMP dependent protein kinase (PKA) and by activation of the exchange protein directly activated by cyclic AMP (Epac). Using specific agonists both PKA and Epac were found to stabilize E-cadherin in low Ca2+ conditions. The results further suggest that inhibition of Rho kinase (ROCK) activity acting on the junction-associated actin-myosin-II ring might be involved in the protection of E-cadherin by TSH (Paper II). IQGAP1 is a scaffolding protein that regulates cadherin-mediated adhesion. In vivo thyroid cells were found to express high levels of IQGAP1 mainly located in the cytoplasm. In cultured cells IQGAP1 transiently associated with maturing adherens junctions and when preformed junctions were disrupted by switch to low Ca2+. In this process IQGAP1 bound to the cadherin-catenin complex. TSH via cyclic AMP displaced IQGAP1 from E-cadherin accompanying recovery of adhesion (Paper III).In conclusion, TSH strengthens thyroid epithelial adhesion by stabilizing E-cadherin at the cell surface. Several signalling pathways converging on mechanisms that regulate E-cadherin turnover probably mediate this protective effect. Actin-regulating molecules, i.e. Rho GTPases and IQGAP1, are possible effectors. All together, the thesis work provides a novel mechanism by which TSH supports the thyroid epithelial phenotype.