Methods for investigating STAT3 regulation of lysosomal function in mammary epithelial cells

Abstract

The transcription factor STAT3 is activated by multiple cytokines and other extrinsic factors. It plays a key role in immune and inflammatory responses and, when dysregulated, in tumourigenesis. STAT3 is also an indispensable mediator of the cell death process that occurs during post-lactational regression of the mammary gland, one of the most dramatic examples of physiological cell death in adult mammals. During this involution of the gland, STAT3 powerfully enhances the lysosomal system to efficiently remove superfluous milk-producing mammary epithelial cells via a lysosomal-mediated programmed cell death pathway. The lysosome is a membrane-enclosed cytoplasmic organelle that digests and recycles cellular waste, with an important role as a signalling centre that monitors cellular metabolism. Here, we describe key strategies for investigating the role of STAT3 in regulating lysosomal function using a mammary epithelial cell culture model system. These include protocols for lysosome enrichment and enzyme activity assays, in addition to microscopic analyses of the vesicular compartment in cell lines. Collectively, these approaches provide the tools to investigate multiple aspects of lysosome biogenesis and function, and to define both direct and indirect roles for STAT3.

Keywords: Cell death; Lysoptosis; Lysosome; Lysosome-mediated programmed cell death; Mammary gland; STAT3 signalling.

Fig. 1

The distinctive gene expression profile of STAT3 target genes in mouse mammary tissues during pregnancy/lactation/involution. Profiles of selected genes from a microarray analysis of 12 time points during a murine mammary gland developmental cycle [12] showing the characteristic spike in expression at day 0 lactation followed by a dramatic increase during involution. The biological function of this lactation increase is not known but may be related to the sudden drop in progesterone at birth, and the switch to milk production. Some of these genes have been confirmed to be STAT targets either in vivo (Cd14, Pik3r1, Slpi, Lrg1) and/or in cell culture (Cd63) [–17]

Fig. 2

Isolation of EpH4 cell lysosomes using magnetic iron nanoparticles. Schematic representation of the magnetic iron nanoparticle lysosomal purification and fractionation protocol. Adapted from Lloyd-Lewis et al. (2018) [15]. Conditions have been optimised for isolating lysosomes from EpH4 cells to investigate the impact of STAT3 activation (via OSM stimulation) on the lysosomal compartment, which may be optimised or modified for different cell lines. For example, the efficiency of iron nanoparticle uptake can be cell-type dependent, and some cells may require a longer incubation time (provided cells remain healthy with longer iron exposures) or require more starting material to increase the yield of lysosomes isolated for downstream analysis. Isolated lysosomes can be analysed using a variety of downstream assays, including western blotting (illustrated), lysosomal enzyme activity assays or in lysosome leakiness assays. Alternatively, isolated lysosomes can be analysed by mass spectrometry or TEM

Fig. 3

Schematic overview of the lysosomal leakiness assay. The permeability of isolated lysosomes can be assessed by western blotting for cathepsin B and L antibodies to visualise the degree of leakiness of cathepsin hydrolases into the supernatant fraction under different treatment conditions (i.e. vehicle and OSM stimulated conditions in EpH4 cells). Immunoblotting for LAMP2, a lysosomal membrane protein, can be used to standardise the amount of lysosomal material in a pellet during the assay. Alternatively, cathepsin activity levels can be assessed in supernatant and pellet fractions using enzyme activity assays

Fig. 4

STAT3-mediated upregulation of the vesicular compartment in EpH4 cells. A Fluorescence microscopy of LAMP1 and LAMP2 immunostaining in EpH4 cells treated with OSM for 72 h show upregulation of the lysosomal compartment with STAT3 activation. Scale bars: 20 μm. B Fluorescence microscopy of LysoTracker® staining in EpH4 cells treated with OSM for 72 h show upregulation of the acidic compartment with STAT3 activation. Scale bars: 20 μm. C Transmission electron microscopy images of EpH4 cells showing an increased number of degradative vesicles after 72 h of OSM stimulation. Scale bars: 500 nm