Tight Junction Protein Abundance and Apoptosis During Involution of Rat Mammary Glands

Abstract

To examine tight junction protein abundance and apoptosis of epithelial cells at the onset of involution in rodent mammary glands, milk accumulation and mammary engorgement were induced by teat-sealing with an adhesive for 0, 6, 12, 18, 24, and 36 h (n = 6 per group) at peak lactation. In non-sealed control glands, histological analysis confirmed a lactating phenotype, indicating suckling by pups throughout the experiment. In contrast, alveoli of teat-sealed glands were distended within 6 h, with maximal luminal size observed by 12 h of non-suckling. By 18 h following teat-sealing, an involuting phenotype was observed, indicated by alveolar lumina engorged with milk vesicles and increased leukocytes. Relative to non-sealed glands, mammary apoptosis was increased in engorged glands 18 h following teat-sealing. The abundance of ZO-1 and occludin proteins was decreased in engorged glands by 12 and 18 h, respectively, following teat-sealing. In contrast, the claudin-1 22 kDa band was increased by 6 h and peaked at 12-18 h, whereas the 28 kDa band declined by 36 h, relative to controls. There were no temporal changes in ZO-1, occludin, and claudin-1 22 kDa proteins within control glands, although there were minor differences in claudin-1 28 kDa. These data indicate that intramammary milk accumulation due to cessation of milk removal is associated with mammary apoptosis. The apoptotic event is preceded by a rapid loss of abundance of ZO-1, occludin and an initial increase in claudin-1. The loss of cell-cell communication may initiate involution and apoptosis of mammary epithelial cells and is a localized intramammary event, occurring only in non-suckled glands. J. Cell. Physiol. 232: 2075-2082, 2017. © 2016 Wiley Periodicals, Inc.

Induced Physical Distension of Rat Mammary Glands Accelerates the Onset of Apoptosis and Involution

“…Instead, there were decreases in occludin, claudin-1 ~28 kDa band and ZO-1 by 6 h in engorged teat-sealed glands compared with suckled controls, which is similar, albeit earlier, to the recently reported gradual decrease in these proteins in response to mammary engorgement [25]. However, only changes in the protein abundance of occludin, but not claudin-1 and ZO-1, appear to be accelerated by physical distension compared with milk accumulation alone.…”

“…The infusion volume of 0.8 ml isosmotic sucrose solution was calculated as follows using raw mammary weight data kindly provided by Dr C. McMahon from previous mammary engorgement studies [22,25]. Milk secretion rate was measured from the increase in mammary mass of three abdominal inguinal glands after 6 h of teatsealing (mean 8.17 ± 0.52 g) compared with three suckled contralateral glands (mean 5.65 ± 0.23 g) from rats (n=6) at peak lactation (d 16).…”

“…Haematoxylin and eosin stained sections of alveolar mammary tissue from each time point (n=6 per group) were prepared from fixed tissue, examined under a microscope, and representative serial sections were chosen for ISEL studies described previously [23,25]. The ISEL was performed on suckled (control), teat-sealed (engorged) and acutely distended (infused) mammary sections, as described previously [23].…”

“…Protein was isolated from 100 mg aliquots of ground mammary tissue and the concentration determined as described previously [22,25]. Western analyses were performed according to Laemmli [35] using 8%, 15% and 7% SDS-PAGE separating gels for occludin, claudin-1 or ZO-1 detection, respectively.…”

“…Integrins have been shown to be mechanosensors in other cell types [20], and occludin, an integral transmembrane TJ protein, was downregulated during stretch in lung alveolar epithelial cells [21]. Thus, the down-regulation of β1-integrin [22,23] and TJ proteins [24,25] during mammary engorgement could be implicated in this mechanotransduction cascade. Furthermore, the stretching of MEC in vitro, results in the induction of apoptotic pathways [26], and morphological changes in the mechanosensors primary cilia occur during involution of the mammary gland [27,28].…”