Regulation of tight junction proteins during engorgement of the mammary gland : a thesis presented in partial fulfilment of the requirements for the degree of Doctor of Philosophy in Animal Science at Massey University, Palmerston North, New Zealand

Abstract

Extended periods of milk accumulation result in loss of secretory activity, increased apoptosis and eventually, involution of mammary glands. This process is associated with increased permeability of the tight junction (TJ) complexes between adjacent mammary epithelial cells (MECs). The change in cell shape during mammary engorgement from a cuboidal to a flattened morphology may initiate changes in protein and gene expression (mechanotransduction) that trigger these processes. Therefore, this study examined the regulation of the major TJ protein components during mammary engorgement, and in particular the role of physical distension of the mammary epithelium in the regulatory process. Expression of the integral transmembrane TJ proteins, occludin and claudin-1, and the cytoplasmic TJ protein, ZO-1, were down-regulated in both bovine and rat mammary glands during the early stages of mammary apoptosis and involution following the abrupt cessation of milk removal. In the rat, these responses were locally regulated as they occurred only in teat-sealed glands in a hemi-suckled model. Furthermore, the down-regulation of TJ proteins is consistent with a loss of TJ integrity during mammary engorgement. Induced physical distension of rat mammary glands in vivo transiently up-regulated the expression levels of occludin protein and mRNA, and ZO-1 mRNA, followed by an accelerated decrease in expression compared with the effects of milk accumulation alone. This was associated with the initiation of apoptosis, the up-regulation of the pro-apoptotic factor pSTAT3, and the down-regulation of the cell-ECM survival factor βl-integrin. An in vitro model was also developed to stretch MECs, mimicking the flattening in cell shape during mammary engorgement in vivo. While stretching MECs in vitro did not conclusively alter TJ protein expression, the overall results of this project support further investigation into the role of the TJ complex in mechanotransduction pathways. In addition, the results point to crosstalk between cell-ECM survival signalling and STAT3 death signalling as a candidate for regulation by physical distension of the mammary epithelium. In conclusion, this study supports the hypothesis that physical distension during engorgement of the mammary glands with milk is a primary trigger initiating apoptosis of MECs through changes in the regulation of gene pathways controlling cell survival and death, and the disruption of TJ function.