Luminal STAT5 mediates H2AX promoter activity in distinct population of basal mammary epithelial cells

Abstract

Deregulated STAT5 activity in the mammary gland causes parity-dependent tumorigenesis. Epithelial cell cultures transfected with constitutively active STAT5 express higher levels of the histone H2AX than their non-transfected counterparts. Higher H2AX expression may be involved in tumorigenesis. Here, we aimed to link high STAT5 activity to H2AX–GFP expression by looking for distinct types of mammary cells that express these proteins. In vitro and in transgenic mice, only 0.2 and 0.02%, respectively, of the cells expressed the H2AX–GFP hybrid gene. Its expression correlated with that of the endogenous H2AX gene, suggesting that detectable H2AX–GFP expression marks high levels of H2AX transcript. Methylation of the H2AX promoter characterized non-GFP-expressing H2AX–GFP cells and was inversely correlated with promoter activity. Administration of 5-azacytidine increased H2AX promoter activity in an activated STAT5-dependent manner. In transgenic mice, H2AX–GFP expression peaked at pregnancy. The number of H2AX–GFP-expressing cells and GFP expression decreased in a Stat5a-null background and increased in mice expressing the hyperactivated STAT5. Importantly, H2AX–GFP activity was allocated to basal mammary cells lacking stem-cell properties, whereas STAT5 hyperactivity was detected in the adjacent luminal cells. Knockdown of RANKL by siRNA suggested its involvement in signaling between the two layers. These results suggest paracrine activation of H2AX via promoter demethylation in specific populations of basal mammary cells that is induced by a signal from neighboring luminal cells with hyper STAT5 activity. This pathway provides an alternative route for the luminally confined STAT5 to affect basal mammary cell activity.

High Expression of CD200 and CD200R1 Distinguishes Stem and Progenitor Cell Populations within Mammary Repopulating Units

“…A similar transplantation procedure was performed for serial transplantations of CD200 high /CD200R1 high MRUs. Host mice were C57BL and donor mice were C57/GFP (ubiquitin-EGFP mice [ Reichenstein et al., 2016 ]). Further details can be found in Supplemental Experimental Procedures .…”

H2AX Promoter Demethylation at Specific Sites Plays a Role in STAT5-Induced Tumorigenesis

“…This was first demonstrated in vitro in mammary epithelial CID-9 cells with STAT5 hyperactivity under pregnancylike conditions [8], and later in vivo, in a population of basal cells that were juxtaposed with a rare luminal epithelial cell population with high deregulated STAT5 activity [9]. These luminal epithelial cells secreted RANKL, resulting in paracrine activation of H2AX in the adjacent basal epithelial cells via its promoter's demethylation [9]. H2AX is a member of the histone 2A (H2A) family, one of five families of histone proteins involved in the nucleosomal organization of chromatin [10].…”

“…The initial events associated with STAT5-induced tumorigenesis involve induced expression of members of the DNA-damage response (DDR) pathway, including H2AX. This was first demonstrated in vitro in mammary epithelial CID-9 cells with STAT5 hyperactivity under pregnancylike conditions [8], and later in vivo, in a population of basal cells that were juxtaposed with a rare luminal epithelial cell population with high deregulated STAT5 activity [9]. These luminal epithelial cells secreted RANKL, resulting in paracrine activation of H2AX in the adjacent basal epithelial cells via its promoter's demethylation [9].…”

Impact of silver nanoparticles on the ultraviolet radiation direct and bystander effects on TK6 cell line

“…The H2AX gene is a coding gene for DNA damage response proteins and is one of the oxidative DNA damage markers. [ 34 35 36 ] TK6 cells were exposed to UVC radiation after treatment with AgNPs (10 μg/ml) for 1 h. Then, to investigate the BSE, conditioned medium was prepared and unexposed cells were exposed to this conditioned medium, and then, the mentioned factors were measured in different groups.…”