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The butyrophilin 1a1 knock-out mouse revisited: Ablation of Btn1a1 leads to concurrent cell death and renewal in the mammary epithelium during lactation

ABSTRACT: Purpose: To assess global changes to the levels of mRNA and protein in butyrophilin-null mice (Btn1a1-/-), which were previously shown to be defective in regulated milk-lipid secretion in the lactating mammary gland. Methods: mRNA profiles of wild-type (WT) and Btn1a1-/-mice were generated by mRNA/cDNA library preparation (Illumina), qPCR quantitation, cluster generation and pair-end deep sequencing (Illumina HiSeq 4000 sequencer) using an Illumina HiSeq SBS kit. Changes in mRNA levels (null/WT ≥ ± 2-fold; adjusted p ≤ 0.05) were assesed by GSEA and IPA analysis and compared with a proteomic analysis of the same sample set.. Results: Samples had 95-167 million pass filter reads (92% above Q30). Mapping of all samples was > 97% (mm10 reference genome). Significant changes in mRNA amounts accounted for 1.4% of 11,834 transcipts (113 up and 53 down regulated). Only 10 mRNA/protein pairs were significantly upregulated and one (Btn1a1) downregulated. Pathways and networks associated with inflammation, apoptosis and the cell cycle were upregulated and lipid synthesis and metabolism were downregulated. Biochemical and histochemical analysis confirmed that lactation is maintained in null animals, in the face of significant apoptosis, by the continual regeneration of secretory epithelial cells. Cell death proceeded through several pathways including activation of caspases 8 and 3 and not by the lysosomal lysis pathway predominant in WT cells Conclusions: Ablation of Btn1a1 has multiple effects on milk-secreting mammary cells, including disruption of lipid secretion, increased cell damage and death. Lactation is maintained by continuous regeneration of the epithelium. Expression of functional Btn1a1 is essential for the maintainance of terminally differentiated mammary cells and optimal milk production throughout lactation.

ORGANISM(S): Mus musculus

PROVIDER: GSE182075 | GEO | 2021/09/20

REPOSITORIES: GEO

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Project description:Butyrophilin 1A1 (BTN1A1) was first identified as a major integral protein of both the apical surface of mammary epithelial cells during lactation and the surface membrane surrounding lipid droplets in milk. Current evidence supports a role for BTN1A1 in the secretion of milk lipids as a membrane receptor, which forms a secretion complex with the redox enzyme, xanthine oxidoreductase (XDH). Capping of cytoplasmic lipid droplets with BTN1A1 and XDH serves to envelop the droplets with cellular membranes at the cell apex as an initial step in their subsequent release from the cell. The first evidence that BTN1A1 functions in this process was the generation of Btn1a1-/- mice, in which lipid secretion was disrupted and large unstable droplets were released into alveolar spaces with fragmented surface membranes. We have revisited this mutant mouse line using RNAseq and proteomic analysis to fully assess the consequences of ablating the Btn1a1 gene on the expression of other genes and proteins. Disruption of Btn1a1 expression led to a large build-up of Xdh (6-fold over wild-type levels) in the cytoplasm, induction of acute phase response genes and Lif-activation of STAT3 phosphorylation. At peak lactation, approx. 10% of the cells were dying, as assessed by TUNEL-analysis of nuclear DNA. Cell death appeared to proceed through expression of caspase 8 and activated caspase 3 and not via lysosomal lysis, which is the principal route for elimination of cells during mammary involution in wild-type mice. Other potential death pathways include autophagy and Slc5a8-mediated inactivation of survivin (Birc5). Milk secretion was prolonged by renewal of the secretory epithelium, as evidenced by the expression of cyclins, Fos/Jun and upregulation of Ki67 in approx. 10% of secretory cell nuclei. Thus a 21-day lactation cycle can be maintained, despite significant cell death by compensatory cell division, provided a milking stimulus is maintained. These data highlight the importance of BTN1A1 expression for the maintenance of terminally differentiated mammary epithelial cells and optimal milk production throughout lactation. From a practical standpoint, the complimentary RNAseq and proteomics data should provide a useful reference database for gene and protein expression in C57/Bl6 mice at peak lactation.

Project description:Non-steroidal anti-inflammatory drug activated gene 1 (NAG-1) plays some role in reducing obesity in mice overexpressing human NAG-1, even on a high fat diet. Male and female hNAG-1 expressing mice have reduced body weight, increased longevity and metabolic activity. This study investigates the role of hNAG-1 in female reproduction and finds that the female mice have reduced fertility and pup survival after birth. Examination of the mammary glands in these mice suggests that hNAG-1 overexpressing mice have altered mammary epithelial development during pregnancy, including reduced occupancy of the fat pad and increased apoptosis via TUNEL positive cells at lactation day 2. Pups nursing from hNAG-1 overexpressing dams have reduced milk spots compared to pups nursing from WT dams. When CD-1 pups were cross-fostered with hNAG-1 or WT dams; reduced milk volume was observed in pups nursing from hNAG-1 dams compared to pups nursing from WT dams in a lactation challenge study. Milk was isolated from WT and hNAG-1 dams, and the milk was found to have secreted NAG-1 protein (approximately 25ng/mL) from hNAG-1 dams compared to WT dams, which had no detectable NAG-1 in the milk. A decrease in non-esterified free fatty acids in the milk of hNAG-1 dams was observed. Altered milk composition suggests that the pups were receiving inadequate nutrients during perinatal development; to examine this hypothesis serum was isolated from pups and clinical chemistry points were measured. Male and female pups nursing from hNAG-1 dams had reduced serum triglyceride concentrations. Cidea/CIDEA expression was reduced in hNAG-1 mammary glands, and microarray analysis suggests that genes involved in lipid metabolism are differentially expressed in hNAG-1 mammary glands. This study suggests that overexpression of hNAG-1 impairs lactational differentiation and pup survival due to altered milk quality and quantity 4 WT mammary glands on lactation day 2 and 5 hNAG-1 transgenic mice mammary glands on lactation day 2 were used.

Project description:The mammary gland of the lactating mouse synthesizes and secretes milk lipid equivalent to its entire body weight in a single 20 day lactation cycle, making it one of the most active lipid synthetic organs known. To test the hypothesis that multiple metabolic control points and potential regulatory mechanisms are involved in activating lipid and lactose synthesis at the onset of lactation we compared the mammary transcriptome of 130 genes involved in glucose metabolism between late pregnancy and early lactation and in response to dietary fat. We utilized data obtained from microarray analysis of mammary glands from quadruplicate FVB mice at pregnancy day 17, and lactation day 2. Diets containing 8% or 40% lipid were fed from lactation days 5 to 10 and mammary glands and livers of triplicate FVB mice prepared for microarray analysis. We also compared the metabolome obtained from magnetic resonance spectroscopy of flash frozen glands of the mammary gland at day 17 of pregnancy with that at day 2 of lactation. The results provide a global picture of the multiple metabolic strategies utilized to turn a quiescent organ into an incredibly efficient machine for massive but balanced lipid and lactose synthesis and implicate the transcription factor SREBP-1c in regulation of part of the pathway. FVB control time-series mice were on normal lab chow diets and mammary tissue was taken at Pregnant days 12 and 17, plus lactation days 1, 2, and 9 in those animals. the time-points were taken as quadruplicates for each developmental stage. for the dietary component, triplicate FVB dams were fed normal lab chow diets until lactation day 5, when they were converted to diets containing either 8% or 40% Kcal from soy oils (see supplementary table to publication for further detail) for 5 days. At lactation day 10, mammary and liver tissues were taken from these animals and preparred for microarray analysis. All tissues were stabilized for RNA content in RNALater and were then isolated and cleaned up following manufacturer's instructions (Qiagen).

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The butyrophilin 1a1 knock-out mouse revisited: Ablation of Btn1a1 leads to concurrent cell death and renewal in the mammary epithelium during lactation

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