Effect of Copper, Zinc, and Selenium on the Migration of Bovine Neutrophils

Neutrophils represent the first line of mammary gland defense against invading pathogens by transmigration across the mammary epithelial cell barrier. The effect of trace elements on the migration of bovine neutrophils is not clear. In this study, we investigated the effect of copper (Cu; 0.5, 1.0 and 1.5 mg/L), zinc (Zn; 1.0, 5.0 and 10 mg/L) and selenium (Se; 0.1, 1.0 and 2.0 mg/L) on the migration of bovine neutrophils by using a Transwell assay. The results showed that Cu, Zn and Se promoted the number of neutrophils in the trans-mammary epithelium. With the increased concentration of Cu at 1.5 mg/L, the number of neutrophils in the trans-mammary epithelium was increased significantly (p < 0.05). Zn (5.0 mg/L) and Se (0.1 mg/L) increased the migrated number of neutrophils (p < 0.01) to an extremely significant degree. These findings provided a theoretical and experimental basis for mammary gland immunity in dairy cows. Thus, we suggest that adding moderate amounts of different trace elements can improve the immune function of dairy cows.

Hyperactive WNT/CTNNB1 signaling induces a competing cell proliferation and epidermal differentiation response in the mouse mammary epithelium

In the past forty years, the WNT/CTNNB1 signaling pathway has emerged as a key player in mammary gland development and homeostasis. While also evidently involved in breast cancer, much unclarity continues to surround its precise role in mammary tumor formation and progression. This is largely due to the fact that the specific and direct effects of hyperactive WNT/CTNNB1 signaling on the mammary epithelium remain unknown. Here we use a primary mouse mammary organoid culture system to close this fundamental knowledge gap. We show that hyperactive WNT/CTNNB1 signaling induces competing cell proliferation and differentiation responses. While proliferation is dominant at lower levels of WNT/CTNNB1 signaling activity, higher levels cause reprogramming towards an epidermal cell fate. We show that this involves de novo activation of the epidermal differentiation cluster (EDC) locus and we identify master regulatory transcription factors that likely control the process. This is the first time that the molecular and cellular dose-response effects of WNT/CTNNB1 signaling in the mammary epithelium have been dissected in such detail. Our analyses reveal that the mammary epithelium is exquisitely sensitive to small changes in WNT/CTNNB1 signaling and offer a mechanistic explanation for the squamous differentiation that is observed in some WNT/CTNNB1 driven tumors.

RAL GTPases mediate EGFR-driven intestinal stem cell proliferation and tumourigenesis

RAS-like (RAL) GTPases function in Wnt signalling-dependent intestinal stem cell proliferation and regeneration. Whether RAL proteins work as canonical RAS effectors in the intestine, and the mechanisms of how they contribute to tumourigenesis remain unclear. Here, we show that RAL GTPases are necessary and sufficient to activate EGFR/MAPK signalling in the intestine, via induction of EGFR internalisation. Knocking down Drosophila RalA from intestinal stem and progenitor cells leads to increased levels of plasma membrane-associated EGFR and decreased MAPK pathway activation. Importantly, in addition to impacting stem cell proliferation during damage-induced intestinal regeneration, this role of RAL GTPases impacts on EGFR-dependent tumorigenic growth in the intestine and in human mammary epithelium. However, the effect of oncogenic RAS in the intestine is independent from RAL function. Altogether, our results reveal previously unrecognised cellular and molecular contexts where RAL GTPases become essential mediators of adult tissue homeostasis and malignant transformation.

Mammary cell gene expression atlas links epithelial cell remodeling events to breast carcinogenesis

The female mammary epithelium undergoes reorganization during development, pregnancy, and menopause, linking higher risk with breast cancer development. To characterize these periods of complex remodeling, here we report integrated 50 K mouse and 24 K human mammary epithelial cell atlases obtained by single-cell RNA sequencing, which covers most lifetime stages. Our results indicate a putative trajectory that originates from embryonic mammary stem cells which differentiates into three epithelial lineages (basal, luminal hormone-sensing, and luminal alveolar), presumably arising from unipotent progenitors in postnatal glands. The lineage-specific genes infer cells of origin of breast cancer using The Cancer Genome Atlas data and single-cell RNA sequencing of human breast cancer, as well as the association of gland reorganization to different breast cancer subtypes. This comprehensive mammary cell gene expression atlas (https://mouse-mammary-epithelium-integrated.cells.ucsc.edu) presents insights into the impact of the internal and external stimuli on the mammary epithelium at an advanced resolution.

Induction of Serum Amyloid A3 in Mouse Mammary Epithelial Cells Stimulated with Lipopolysaccharide and Lipoteichoic Acid

In this study, to establish whether serum amyloid A (SAA) 3 plays a role in the defense against bacterial infection in mouse mammary epithelium, normal murine mammary gland (NMuMG) epithelial cells were stimulated with lipopolysaccharide (LPS) and lipoteichoic acid (LTA). LPS and LTA significantly enhanced mRNA expression level of the Saa3 gene, whereas no significant change was observed in the Saa1 mRNA level. Furthermore, LPS induced SAA3 protein expression more strongly than LTA, whereas neither LPS nor LTA significantly affected SAA1 protein expression. These data indicate that the expression of SAA3 in mouse mammary epithelial cells was increased by the stimulation with bacterial antigens. SAA3 has been reported to stimulate neutrophils in the intestinal epithelium and increase interleukin-22 expression, which induces activation of the innate immune system and production of antibacterial proteins, such as antimicrobial peptides. Therefore, collectively, these data suggest that SAA3 is involved in the defense against bacterial infection in mouse mammary epithelium.

The miR-424/503 cluster modulates Wnt/β-catenin signaling in the mammary epithelium by regulating the expression of the LRP6 co-receptor

During the female lifetime, the enlargement of the epithelial compartment dictated by the ovarian cycles is supported by a transient increase in the MaSC population. Notably, activation of Wnt/β-catenin signaling is an important trigger for MaSC expansion. Here, we report that the miR-424/503 cluster is a novel modulator of canonical Wnt-signaling in the mammary epithelium that exerts its function by targeting the LRP6 co-receptor. Additionally, we show that the loss of this microRNA cluster is associated with breast cancers possessing high levels of Wnt/β-catenin signaling.

ScRNA-seq Reveals a Role of Mammary Luminal Epithelium in Adipocyte Adaptations

Almost four decades of research suggest a dynamic role of ductal epithelial cells in adipocyte adaptation in mammary gland white adipose tissue (mgWAT), but factors that mediate such communication are not known. Here, we identify a complex intercellular crosstalk in mgWAT revealed by single-cell RNA-seq (scRNA-seq) and comprehensive data analysis suggest that epithelial luminal cells during cold exposure undergo major transcriptomic changes that lead to the expression of an array of genes that encode for secreted factors involved in adipose metabolism such as Adropin (Enho), neuregulin 4 (Nrg4), angiopoietin-like 4 (Angptl4), lipocalin 2 (Lcn2), milk fat globule-EGF factor 8 (Mfge8), Insulin-like growth factor-binding protein 1 (Igfbp1), and haptoglobin (Hp). To define the mammary epithelial secretome, we coin the phrase “mammokines”. We validated our cluster annotations and cluster-specific transcriptomics using eight different adipose scRNA-seq datasets including Tabula Muris and Tabula Muris Senis. In situ mRNA hybridization and ex vivo isolated mgWAT luminal cells show highly localized expression of mammokines in mammary ducts. Trajectory inference demonstrates that cold-exposed luminal cells have similar transcriptional profiles to lactation post-involution (PI), a phase defined by reappearance and maintenance of adipocytes in the mammary gland. Concomitantly, we found that under cold exposure female mgWAT maintains more adipogenic and less thermogenic potential than male scWAT and ex vivo removal of luminal epithelial cells from mgWAT markedly potentiates beige adipocyte differentiation. Conditioned media from isolated mammary epithelial cells treated with isoproterenol suppressed thermogenesis in differentiated beige/brown adipocytes and treatment of beige/brown differentiated adipocyte with mammokine LCN2 suppresses thermogenesis and increases adipogenesis. Finally, we find that mice lacking LCN2 show markedly higher cold-dependent thermogenesis in mgWAT than controls, and reconstitution of LCN2 in the mgWAT of LCN2 knockout mice promotes inhibition of thermogenesis. These results show a previously unknown role of mammary epithelium in adipocyte metabolism and suggest a potentially redundant evolutionary role of mammokines in maintaining mgWAT adiposity during cold exposure. Our data highlight mammary gland epithelium as a highly active metabolic cell type and mammokines could have broader implications in mammary gland physiology and lipid metabolism.