Reactive oxygen species initiate luminal but not basal cell death in cultured human mammary alveolar structures: a potential regulator of involution

E Thomas,P Hartmann,P Rigby,N Zeps

doi:10.1038/cddis.2011.69

Abstract

Post-lactational involution of the mammary gland is initiated within days of weaning. Clearing of cells occurs by apoptosis of the milk-secreting luminal cells in the alveoli and through stromal tissue remodeling to return the gland almost completely to its pre-pregnant state. The pathways that specifically target involution of the luminal cells in the alveoli but not the basal and ductal cells are poorly understood. In this study we show in cultured human mammary alveolar structures that the involution process is initiated by fresh media withdrawal, and is characterized by cellular oxidative stress, expression of activated macrophage marker CD68 and finally complete clearing of the luminal but not basal epithelial layer. This process can be simulated by ectopic addition of reactive oxygen species (ROS) in cultures without media withdrawal. Cells isolated from post-involution alveoli were enriched for the CD49f+ mammary stem cell (MaSC) phenotype and were able to reproduce a complete alveolar structure in subcultures without any significant loss in viability. We propose that the ROS produced by accumulated milk breakdown post-weaning may be the mechanism underlying the selective involution of secretory alveolar luminal cells, and that our culture model represents an useful means to investigate this and other mechanisms further.

Highlights

The non-lactating but mature mammary epithelium in humans consists of a simple ductal network that is supported within the mammary stroma
We propose that the luminal-specific cell death we observed in response to increased reactive oxygen species (ROS)
Supports our hypothesis that ROS-induced cell death is a potential initiator of involution in the post-weaning gland

Summary

Introduction

The non-lactating but mature mammary epithelium in humans consists of a simple ductal network that is supported within the mammary stroma. At around 4 days postweaning, the second (irreversible) phase of involution begins when matrix remodeling enzymes are upregulated.[7] Expression of matrix remodeling factors is largely restricted to the stromal fibroblasts surrounding the lobulo-alveolar network, and is likely to be regulated by epithelial-mesenchymal cross-talk originating in the glandular epithelium.[2,8] By 10 days post-weaning the gland is regressed almost completely to its pre-pregnant state, with the exception of a small population of lactation-associated cells that remains at the basal surface and are believed to serve as a ‘memory’ for subsequent lactations.[9,10] there is a growing body of knowledge of the specific signals that drive luminal but not basal cell clearing during involution of animal models, such pathways are poorly understood in human systems.[11,12].

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Results

Conclusion