Protein Tyrosine Phosphorylation Influences Adhesive Junction Assembly and Follicular Organization of Cultured Thyroid Epithelial Cells*

Abstract

The follicular histoarchitecture of the thyroid forms the anatomical basis for thyroid physiology and is commonly disturbed in diseases of the thyroid. We have used cultured porcine thyroid cells to study thyroid epithelial morphogenesis and its regulation. When cultured in the presence of TSH, freshly isolated thyroid cells reorganize to form follicles within three-dimensional cell aggregates. However, when established follicles are washed into TSH-free medium, thyroid cells spread and migrate to convert follicles into confluent epithelioid monolayers, activating morphogenetic mechanisms, such as cell locomotility, that may be relevant to thyroid inflammation and tumor invasiveness. The phenomenon of follicle to monolayer conversion, therefore, provides an opportunity to identify morphogenetic mechanisms that 1) must be tonically inhibited to maintain follicular organization and 2) may contribute to pathogenetic disturbances of follicular architecture when functioning aberrantly. In this study we found that follicle to monolayer conversion is associated with an increase in cellular phosphotyrosine. This was particularly evident at nascent focal adhesions (cell-substrate adhesive junctions) and later at cell-cell junctions. Focal adhesion assembly was accompanied by reorganization of the actin cytoskeleton, with the appearance of prominent stress fibers. Genistein, a potent inhibitor of protein tyrosine kinases, inhibited the accumulation of phosphotyrosine, focal adhesion assembly, and follicle to monolayer conversion. We conclude that tyrosine phosphorylation exerts an important influence on thyroid epithelial organization in culture, at least partly mediated through regulation of focal adhesion assembly.

Tyrosine phosphorylated Par3 regulates epithelial tight junction assembly promoted by EGFR signaling

“…The SFK family was thought to be required for epithelial TJ dynamics (Yap et al , 1997; Tsukamoto and Nigam, 1999; Meyer et al , 2001; Chen et al , 2002). The bidirectional interaction of EGFR and SFKs generates a synergistic cellular signaling event for promoting a variety of cell actions, as well as cell transformation in pathological conditions (Ishizawar and Parsons, 2004).…”

“…The association of EGFR and SFKs results in the activation of SFKs. Meanwhile, SFK is likely to phosphorylate its downstream targets, such as Par3, possibly also occludin and ZO‐1 (Yap et al , 1997; Tsukamoto and Nigam, 1999; Chen et al , 2002), which may modify TJ protein activity thereby regulating TJ formation.…”

“…EMK1 and LKB1, serine/threonine kinases related to the C. elegans Par1 and Par4 gene products, respectively, are also emerging as key regulators of cell polarity in lower and higher eukaryotes (Spicer and Ashworth, 2004; Vinot et al , 2005). Recently, several lines of evidence indicate that the Src family kinases (SFKs) phosphorylate TJ proteins, including ZO‐1, ZO‐2, and occludin, leading to positive and negative regulation of TJ formation (Yap et al , 1997; Tsukamoto and Nigam, 1999; Chen et al , 2002). However, the regulatory mechanism of the SFKs in TJ formation is not fully understood.…”

Real-time Quantitative Analysis of E-cadherin Expression in ret/PTC-1-Activated Thyroid Neoplasms

“…Diminished E-cadherin expression has been pro- posed as a factor dictating the characteristic growth pattern of diffuse gastric carcinoma [32] on the basis of aberrant tyrosine kinase activity and resultant alteration in cell adhesion properties [33]. In the case of PTCret+ it is possible that both these factors act synergistically, though this begs the question of why PTCrettumors should be morphologically indistinguishable.…”

Assessment ofret/PTC-1 rearrangements in neoplastic thyroid tissue using TaqMan RT-PCR

“…have demonstrated alterations in the nuclear envelope and chromatin structure in primary culture of thyroid cells infected with a retroviral vector expressing activated RET/PTC‐1 and it is possible that ret /PTC‐1 activation may influence the growth pattern in PTC, firstly because failure to express the putative cytoskeletal protein H4 alters the structure of the cell, or secondly, because RET/PTC‐1 is constitutively phosphorylated. Yap et al 21. have shown that increased tyrosine phosphorylation alters thyroid epithelial organization in culture by altering the assembly of actin‐associated adhesive junctions.…”