Milk Protein Expression Research Articles

Site specific insertion of a transgene into the murine α-casein (CSN1S1) gene results in the predictable expression of a recombinant protein in milk.

Gene loci of highly expressed genes provide ideal sites for transgene expression. Casein genes are highly expressed in mammals leading to the synthesis of substantial amounts of casein proteins in milk. The α-casein (CSN1S1) gene has assessed as a site of transgene expression in transgenic mice and a mammary gland cell line. A transgene encoding an antibody light chain gene (A1L) was inserted into the α-casein gene using sequential homologous and site-specific recombination. Expression of the inserted transgene is directed by the α-casein promoter, is responsive to lactogenic hormone activation, leads to the synthesis of a chimeric α-casein/A1L transgene mRNA, and secretion of the recombinant A1L protein into milk. Transgene expression is highly consistent in all transgenic lines, but lower than that of the α-casein gene (4%). Recombinant A1L protein accounted for 0.5% and 1.6% of total milk protein in heterozygous and homozygous transgenic mice, respectively. The absence of the α-casein protein in homozygous A1L transgenic mice leads to a reduction of total milk protein and delayed growth of the pups nursed by these mice. Overall, the data demonstrate that the insertion of a transgene into a highly expressed endogenous gene is insufficient to guarantee its abundant expression.

DCas9-guided demethylation of the AKT1 promoter improves milk protein synthesis in a bovine mastitis mammary gland epithelial model induced by usingStaphylococcus aureus.

Mastitis is among the main factors affecting milk quality and yield. Although DNA methylation is associated with mastitis, its role in mastitis remains unclear. In this study, a bovine mastitis mammary epithelial cells (BMMECs) model was established via Staphylococcus aureus infection of bovine mammary gland epithelial cells (BMECs). Bisulfite sequencing PCR was used to determine the methylation status of the AKT1 promoter in BMMECs. We found that the degree of the AKT1 promoter methylation in BMMECs was significantly greater than that in BMECs, and the expression levels of genes related to milk protein synthesis were significantly decreased. We used the pdCas9-C-Tet1-SgRNA 2.0 system to regulate the methylation status of the AKT1 promoter. High-efficiency sgRNAs were screened and dCas9-guided AKT1 promoter demethylation vectors were constructed. Following transfection with the vectors, the degree of methylation of the AKT1 promoter was significantly reduced in BMMECs, while AKT1 protein levels increased. When the methylation level of the AKT1 promoter decreased, the synthesis of milk proteins and the expression levels of genes related to milk protein synthesis increased significantly. The viability of the BMMECs was enhanced. Taken together, these results indicate that demethylation guided by the pdCas9-C-Tet1-SgRNA 2.0 system on the AKT1 promoter can reactivate the expression of AKT1 and AKT1/mTOR signaling pathway-related proteins by reducing the AKT1 promoter methylation level and promoting the recovery milk protein expression in BMMECs, thereby alleviating the symptoms of mastitis.

Valine promotes milk synthesis by regulating PKM2 nuclear accumulation and histone H3 acetylation through the TAS1R1-mTOR-DDX39B signaling pathway

Valine, a branched-chain amino acid found in dairy cows, has been recognized for its critical role in milk synthesis. However, the precise effect of valine on lactation in dairy cows remains an area of investigation. In our study, bovine mammary epithelial cells (BMECs) were isolated to explore the mechanism through which valine enhances milk synthesis. The results showed that 100 μM valine significantly boosted the milk synthesis via TAS1R1-mTOR-DDX39B signaling pathway in BMECs. Subsequent investigations revealed that DDX39B governs the accumulation of PKM2 in the nuclei of BMECs. This nuclear buildup of PKM2 weakened the interaction between HDAC3 and histone H3, leading to an increase in the acetylation levels of histone H3. In an vivo context, the 0.25 % valine-enriched drinking water notably elevated in the expression of milk protein and fat in these mice. Further examination showed that 0.25 % valine drinking water considerably augmented the protein expression levels of DDX39B, PKM2, and p-mTOR in the mice mammary glands. In summary, our results suggest that valine, by modulating the TAS1R1-mTOR-DDX39B signaling pathway, directs the accumulation of PKM2 in the nucleus. This, in turn, escalates the acetylation levels of histone H3, promoting the synthesis of both milk protein and fat.

Amplifying local serotonin signaling prior to dry-off hastens mammary gland involution and redevelopment in dairy cows

The monoamine serotonin (5-hydroxytryptamine, 5-HT) has been reported to inhibit milk protein gene expression and increase mammary epithelial cell (MEC) tight junction permeability after milk stasis. We hypothesized that increasing serotonin synthesis and signaling within the mammary epithelium before milk stasis would increase systemic and local involution markers, and downregulate the expression of milk protein and tight junction during involution, leading to more efficient tissue growth during the redevelopment phase. Herein, we examined the outcomes of increasing local mammary 5-HT synthesis before milk stasis on involution biomarkers, mammary gland microstructure, and gene and protein expression during the dry period. Multiparous Holstein cows were administered intramammary infusions (via the teat canal) of sterile water (CON, 4 mL/teat, n = 7) or 5-hydroxy-l-tryptophan (5-HTP, serotonin precursor, 20 mg/teat, n = 7) once daily for 5 d before dry-off (d 0). Blood, milk, and mammary secretions were collected and analyzed for components and metabolites. Mammary secretions were collected 12 h after the last milking and on d 1 to 4 during the dry period at 1200 h. Mammary gland biopsies were performed on d 4 (i.e., involution phase) and d 36 (i.e., redevelopment phase) of the dry period for histological and molecular evaluation. Milk protein and tight junction gene expression was quantified via real-time PCR. Hematoxylin and eosin staining, immunohistochemistry (Ki67), and immunofluorescence (serotonin, cleaved caspase 3) were performed to visualize tissue microstructure and to quantify serotonin intensity and cell turnover. Data were analyzed in SAS (SAS Institute Inc.) using 2-way ANOVA. After d 0, mammary secretions of 5-HTP cows had increased concentrations of 5-HT, lactoferrin, and bovine serum albumin. On d 1, 5-HTP cows had greater α-lactalbumin concentrations in plasma relative to CON. Serotonin intensity was increased in the mammary tissue of 5-HTP cows on d 4, relative to CON. On d 4, milk protein and tight junction gene expression was downregulated, MEC number was reduced, and cleaved caspase 3 protein was greater in mammary tissue of 5-HTP cows, relative to CON. On d 36, milk protein genes were upregulated, and the lumen:outer alveolar area and Ki67-positive cells were increased in the mammary tissue of 5-HTP cows, relative to CON. Amplifying serotonin signaling in the mammary epithelium before milk stasis at dry-off achieves greater apoptosis, leading to a reduction in MEC, allowing for greater cell proliferation, which results in more MEC during the redevelopment phase preceding the onset of lactation.

SIRT4 Expression Ameliorates the Detrimental Effect of Heat Stress via AMPK/mTOR Signaling Pathway in BMECs.

Sirtuin 4 (SIRT4), a member of the SIRT family, has been reported to be a key factor involved in antioxidant defense in mitochondria. This study aimed to explore the potential molecular mechanism via which SIRT4 regulates heat stress-induced oxidative stress and lactoprotein synthesis in bovine mammary epithelial cells (BMECs). Our results showed that SIRT4 was significantly decreased in heat stressed mammary tissue. Depletion of SIRT4 in BMECs induced the generation of ROS, which, as exhibited by the decreased activity of antioxidant enzymes, changed mitochondrial morphology through mediating protein and mRNA levels related to mitochondrial fission and fusion. Moreover, we found that depletion of SIRT4 or stress conditions inhibited the expression of milk proteins, as well as lipid and glucose synthesis-related genes, and activated the AMPK/mTOR signaling pathway. Increased SIRT4 expression was found to have the opposite effect. However, blocking the AMPK/mTOR signaling pathway could inhibit the regulatory function of SIRT4 in milk synthesis-related gene expression. In summary, our results suggest that SIRT4 may play critical roles in maintaining mammary gland function by regulating the AMPK/mTOR signaling pathway in dairy cows, indicating that SIRT4 may be a potential molecular target for curing heat stress-induced BMEC injury and low milk production in dairy cows.

Effect of Delta-9-tetrahydrocannabinol and cannabidiol on milk proteins and lipid levels in HC11 cells.

Pregnant and lactating women have been discouraged from using cannabis by Health Canada. However, the increasing rate of cannabis use among pregnant women has presented an urgent need to investigate its physiological effects during the perinatal period. During pregnancy, the mammary gland (MG) undergoes remodeling, which involves alveolar differentiation of mammary epithelial cells (MECs), which is essential for breast milk production and secretion. Limited evidence has been reported on the impact of cannabis or its components, delta-9-tetrahydrocannabinol (THC) and cannabidiol (CBD), on MG development or MEC differentiation. In this study, we investigated the effects of THC and CBD on the differentiation of MECs by assessing changes in cellular viability, lipid accumulation, and gene and protein expression of major milk protein and lipid synthesizing markers. using the HC11 cells as a model. We hypothesized that THC and CBD will negatively impact the synthesis of milk proteins and lipids, as well as lipid markers in HC11 cells. Our results demonstrated that THC and CBD reduced cellular viability at concentrations above 30μM and 20μM, respectively. Relative to control, 10μM THC and 10μM CBD reduced mRNA levels of milk proteins (CSN2 and WAP), lipid synthesizing and glucose transport markers (GLUT 1, HK2, FASN, FABP4, PLIN2 and LPL), as well as whey acidic protein and lipid levels. In addition, co-treatment of a CB2 antagonist with THC, and a CB2 agonist with CBD, reversed the impact of THC and CBD on the mRNA levels of key markers, respectively. In conclusion, 10μM THC and CBD altered the differentiation of HC11 cells, in part via the CB2 receptor.

Intramammary rapamycin administration to calves induces epithelial stem cell self-renewal and latent cell proliferation and milk protein expression

Mammary epithelial stem cells differentiate to create the basal and luminal layers of the gland. Inducing the number of differentiating bovine mammary stem cells may provide compensating populations for the milk-producing cells that die during lactation. Inhibition of mTOR activity by rapamycin signals self-renewal of intestinal stem cells, with similar consequences in the mouse mammary gland and in bovine mammary implants maintained in mice. The implementation of these results in farm animals for better mammary development and production was studied in 3-month-old calves. mTOR activity decreased by ~50% in mammary epithelial cells subjected to 3-week rapamycin administration, with no negative consequences on mammary morphology or β-casein expression. Subsequently, stem cell self-renewal was induced, reflected by a higher propagation rate of cultures from rapamycin-treated glands compared to respective controls and higher expression of selected markers. Followed by 4-day estrogen and progesterone administration, rapamycin significantly induced proliferation rate. Higher numbers of basal and luminal PCNA+ cells were detected in small ducts near the elongating sites as compared to large ducts, in which only luminal cells were affected. Rapamycin administration resulted in induction of individual milk protein genes' expression, which was negatively correlated to their endogenous levels. The inductive effect of rapamycin on luminal cell number was confirmed in organoid cultures, but milk protein expression decreased, probably due to lack of oscillation in rapamycin levels. In conclusion, intramammary rapamycin administration is an effective methodology to reduce mTOR activity in bovine mammary epithelial cells and consequently, induce stem cell self-renewal. The latent positive effect of rapamycin on epithelial cell proliferation and its potential to improve milk protein expression in calves may have beneficial implications for mature cows.

Prosit-TMT: Deep Learning Boosts Identification of TMT-Labeled Peptides.

The prediction of fragment ion intensities and retention time of peptides has gained significant attention over the past few years. However, the progress shown in the accurate prediction of such properties focused primarily on unlabeled peptides. Tandem mass tags (TMT) are chemical peptide labels that are coupled to free amine groups usually after protein digestion to enable the multiplexed analysis of multiple samples in bottom-up mass spectrometry. It is a standard workflow in proteomics ranging from single-cell to high-throughput proteomics. Particularly for TMT, increasing the number of confidently identified spectra is highly desirable as it provides identification and quantification information with every spectrum. Here, we report on the generation of an extensive resource of synthetic TMT-labeled peptides as part of the ProteomeTools project and present the extension of the deep learning model Prosit to accurately predict the retention time and fragment ion intensities of TMT-labeled peptides with high accuracy. Prosit-TMT supports CID and HCD fragmentation and ion trap and Orbitrap mass analyzers in a single model. Reanalysis of published TMT data sets show that this single model extracts substantial additional information. Applying Prosit-TMT, we discovered that the expression of many proteins in human breast milk follows a distinct daily cycle which may prime the newborn for nutritional or environmental cues.

14-3-3β is essential for milk composition stimulated by Leu/IGF-1 via IGF1R signaling pathway in BMECs

The cell proliferation of bovine mammary epithelial cells (BMECs) and consequent milk synthesis are regulated by multiple factors. The purpose of this study was to examine the effect of 14-3-3β on cellular proliferation and milk fat/β-casein synthesis in BMECs and reveal its underlying mechanisms. In this study, we employed gene function analysis to explore the regulatory effect and molecular mechanisms of 14-3-3β on milk synthesis and proliferation in BMECs. We found that leucine and IGF-1 enhance cell proliferation and milk synthesis in a 14-3-3β-dependent manner and only exhibiting such effect in the presence of 14-3-3β. We further determined that 14-3-3β interacts with the IGF1R self-phosphorylation site and it additionally mediated leucine and IGF-1 to stimulate the synthesis of milk through the IGF1R-AKT-mTORC1 signaling pathway. In summary, our data indicated that 14-3-3β mediates the expression of milk fat and protein stimulated by leucine and IGF-1, leading to lactogenesis through IGF1R signaling pathway in BMECs.

Dynamic miRNA Landscape Links Mammary Gland Development to the Regulation of Milk Protein Expression in Mice.

Simple SummaryMilk synthesis is vital for maintaining the normal growth of newborn animals. Abnormal mammary gland development leads to a decrease in female productivity and the overall productivity of animal husbandry. This study characterized the dynamic miRNA expression profile during the process of mammary gland development, and identified a novel miRNA regulating expression of β-casein—an important milk protein. The results are valuable for studying mammary gland development, increasing milk production, improving the survival rate of pups, and promoting the development of animal husbandry.Mammary gland morphology varies considerably between pregnancy and lactation status, e.g., virgin to pregnant and lactation to weaning. Throughout these critical developmental phases, the mammary glands undergo remodeling to accommodate changes in milk production capacity, which is positively correlated with milk protein expression. The purpose of this study was to investigate the microRNA (miRNA) expression profiles in female ICR mice’s mammary glands at the virgin stage (V), day 16 of pregnancy (P16d), day 12 of lactation (L12d), day 1 of forced weaning (FW1d), and day 3 of forced weaning (FW3d), and to identify the miRNAs regulating milk protein gene expression. During the five stages of testing, 852 known miRNAs and 179 novel miRNAs were identified in the mammary glands. Based on their expression patterns, the identified miRNAs were grouped into 12 clusters. The expression pattern of cluster 1 miRNAs was opposite to that of milk protein genes in mammary glands in all five different stages. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analyses revealed that the predicted target genes of cluster 1 miRNAs were related to murine mammary gland development and lactation. Furthermore, fluorescence in situ hybridization (FISH) analysis revealed that the novel-mmu-miR424-5p, which belongs to the cluster 1 miRNAs, was expressed in murine mammary epithelial cells. The dual-luciferase reporter assay revealed that an important milk protein gene—β-casein (CSN2)—was regarded as one of the likely targets for the novel-mmu-miR424-5p. This study analyzed the expression patterns of miRNAs in murine mammary glands throughout five critical developmental stages, and discovered a novel miRNA involved in regulating the expression of CSN2. These findings contribute to an enhanced understanding of the developmental biology of mammary glands, providing guidelines for increasing lactation efficiency and milk quality.

UFL1 regulates milk protein and fat synthesis–related gene expression of bovine mammary epithelial cells probably via the mTOR signaling pathway

UFL1 is an ufmylation (a novel post-translational modification) E3 ligase, mainly located in the endoplasmic reticulum (ER), that has emerged as a significant regulator of several physiological and pathological processes. Yet its physiological function in milk synthesis in bovine mammary epithelial cells (BMECs) remains unknown. In this study, we investigated the effects of UFL1 in milk protein and fat synthesis-related gene expression, with a particular emphasis on the role of UFL1 in LPS-treated BMECs. Results showed that UFL1 depletion significantly reduced the expression of milk protein and fat synthesis-related gene and mTOR phosphorylation in both normal and LPS-treated BMECs. Overexpression of UFL1 enhanced the activation of the mTOR and milk protein and fat synthesis-related gene expression. Collectively, these above results strongly demonstrate that UFL1 could regulate milk protein and fat synthesis-related gene expression of BMECs probably via the mTOR signaling pathway.

Protein Profile and Simulated Digestive Behavior of Breast Milk from Overweight and Normal Weight Mothers.

Human milk proteins have shown to vary in concentration and distribution through lactation. However, while some regulatory components, such as hormones, have shown associations with regard to the mothers’ body mass index, there is limited information on the possible influence of this condition on the whole protein distribution. The objective of this study was to evaluate the protein profile of human milk from normal weight and overweight or obese mothers to identify differences in protein expression in colostrum, transitional and mature milk. The mass spectrometry analysis showed the ability to class with a high degree of confidence the lactation state and the milk profile according to the mother’s condition. Individual milk samples were subjected to a digestion in vitro model that takes into account the specificities of the gastrointestinal conditions of full-term newborn infants. The digestion products were compared with available data from the digestive contents in newborns. The behavior of the most abundant proteins and the overall peptide generation and survival, showed good correspondence with in vivo data.

Bisphenol A and benzophenone-3 exposure alters milk protein expression and its transcriptional regulation during functional differentiation of the mammary gland in vitro

The plastic monomer and plasticizer bisphenol A (BPA), and the UV-filter benzophenone-3 (BP3) have been shown to have estrogenic activities that could alter mammary gland development. Our aim was to analyze whether BPA or BP3 direct exposure affects the functional differentiation of the mammary gland using an in vitro model. Mammary organoids were obtained and isolated from 8 week-old virgin female C57BL/6 mice and were differentiated on Matrigel with medium containing lactogenic hormones and exposed to: a) vehicle (0.01% ethanol); b) 1 × 10−9 M or 1 × 10−6 M BPA; or c) 1 × 10−12 M, 1 × 10−9 M or 1 × 10−6 M BP3 for 72 h. The mRNA and protein expression of estrogen receptor alpha (ESR1) and progesterone receptor (PR) were assessed. In addition, mRNA levels of PR-B isoform, glucocorticoid receptor (GR), prolactin receptor (PRLR) and Stat5a, and protein expression of pStat5a/b were evaluated at 72 h. The mRNA and protein expression of milk proteins and their DNA methylation status were also analyzed. Although mRNA level of PRLR and GR was similar between treatments, mRNA expression of ESR1, total PR, PR-B and Stat5a was increased in organoids exposed to 1 × 10−9 M BPA and 1 × 10−12 M BP3. Total PR expression was also increased with 1 × 10−6 M BPA. Nuclear ESR1 and PR expression was observed in all treated organoids; whereas nuclear pStat5a/b alveolar cells was observed only in organoids exposed to 1 × 10−9 M BPA and 1 × 10−12 M BP3. The beta-casein mRNA level was increased in both BPA concentrations and 1 × 10−12 M BP3, which was associated with hypomethylation of its promoter. The beta-casein protein expression was only increased with 1 × 10−9 M BPA or 1 × 10−12 M BP3. In contrast, BPA exposure decreased alpha-lactalbumin mRNA expression and increased DNA methylation level in different methylation-sensitive sites of the gene. Also, 1 × 10−9 M BPA decreased alpha-lactalbumin protein expression. Our results demonstrate that BPA or BP3 exposure alters milk protein synthesis and its transcriptional regulation during mammary gland differentiation in vitro.

Hoxd10 Is Required Systemically for Secretory Activation in Lactation and Interacts Genetically with Hoxd9

Targeted disruption of the murine Hoxd10 gene (ΔHoxd10) leads to a high frequency of localized (gland-to-gland or regionally within a gland) lactation impairment in homozygous mutant mice as a single gene mutation. The effect of Hoxd10 disruption was enhanced by simultaneous disruption of Hoxd9 (ΔHoxd9/d10), a mutation shown previously to have no effect on mammary function as a single gene alteration. Mammary glands of homozygous ΔHoxd10 and ΔHoxd9/d10 females were indistinguishable from those of wild type littermate and age-matched control mice in late pregnancy. However, in lactation, 47% of homozygous ΔHoxd10 females, and 100% of homozygous ΔHoxd9/d10 females, showed localized or complete failure of two or more glands to undergo lactation-associated morphological changes and to secrete milk. Affected regions of ΔHoxd10 and ΔHoxd9/d10 mutants showed reduced prolactin receptor expression, reduced signal transducer and activator transcription protein 5 (STAT5) phosphorylation, reduced expression of downstream milk proteins, mislocalized glucose transporter 1 (GLUT1), increased STAT3 expression and phosphorylation, recruitment of leukocytes, altered cell cycle status, and increased apoptosis relative to unaffected regions and wild type control glands. Despite these local effects on alveolar function, transplantation results and hormone analysis indicate that Hoxd10 primarily has systemic functions that confer attenuated STAT5 phosphorylation on both wild type and ΔHoxd10 transplants when placed in ΔHoxd10 hosts, thereby exacerbating an underlying propensity for lactation failure in C57Bl/6 mice.

Bacterial Endotoxin Induces Oxidative Stress and Reduces Milk Protein Expression and Hypoxia in the Mouse Mammary Gland.

The aim of this study was to investigate the mechanisms underlying the reduced milk production during mastitis. We hypothesized that bacterial endotoxin induces hypoxia, oxidative stress, and cell apoptosis while inhibiting milk gene expression in the mammary gland. To test this hypothesis, the left and right sides of the 4th pair of mouse mammary glands were alternatively injected with either lipopolysaccharide (LPS, E. coli 055: B5, 100 μL of 0.2 mg/mL) or sterile PBS through the teat meatus 3 days postpartum. At 10.5 and 22.5 h postinjection, pimonidazole HCl, a hypoxyprobe, was injected intraperitoneally. At 12 or 24 h after the LPS injection, the 4th glands were individually collected (n = 8) and analyzed. LPS treatment induced mammary inflammation at both 12 and 24 h but promoted cell apoptosis only at 12 h. Consistently, H2O2 content was increased at 12 h (P < 0.01), but dropped dramatically at 24 h (P < 0.01) in the LPS-treated gland. Nevertheless, the total antioxidative capacity in tissue tended to be decreased by LPS at both 12 and 24 h (P = 0.07 and 0.06, respectively). In agreement with these findings, LPS increased or tended to increase the mRNA expression of antioxidative genes Nqo1 at 12 h (P = 0.05) and SLC7A11 at 24 h (P = 0.08). In addition, LPS inhibited mammary expression of Csn2 and Lalba across time and protein expression of Csn1s1 at 24 h (P < 0.05). Furthermore, hypoxyprobe staining intensity was greater in the alveoli of the PBS-treated gland than the LPS-treated gland at both 12 and 24 h, demonstrating a rise in oxygen tension by LPS treatment. In summary, our observations indicated that while intramammary LPS challenge incurs inflammation, it induces oxidative stress, increases cell apoptosis and oxygen tension, and differentially inhibits the milk protein expression in the mammary gland.

Controlled synchronization of prolactin/STAT5 and AKT1/mTOR in bovine mammary epithelial cells.

The prolactin/STAT5 and AKT1/mTOR pathways play a key role in milk protein transcription and translation, respectively. However, the correlation between them in bovine mammary epithelial cells remains unclear. Here, mRNA and protein expression levels of AKT1, STAT5, and mTOR and the phosphorylation of these proteins were determined. Cell proliferation and viability were examined using the CASY-TT assay. Purified bovine mammary epithelial cells were cultured in differentiation media for different periods. The basic differentiation medium contained a lactogenic hormone cocktail of insulin (5μg/mL), hydrocortisone (1μg/mL), and prolactin (5μg/mL). The cells cultured in this medium grew slowly and expressed higher levels of p-STAT5, p-AKT1, and p-mTOR (activated form) than those of control cells. Although the phosphorylation ratio was not increased, transcription and translation of these proteins were upregulated by the addition of insulin-like growth factor-1 or growth hormone, which further increased β-casein mRNA expression. Furthermore, the three proteins were upregulated or downregulated synchronously, even after RNA interference silencing of either Stat5 or Akt1. These findings indicate that a few hormones and other factors play lactogenic and galactogenic roles by promoting two key lactogenic signaling associated with milk protein expression. We provide evidence of prolactin/STAT5 and AKT1/mTOR synchronization, establishing a direct correlation between transcription regulation and translation regulation of milk protein in bovine mammary epithelial cells.

Remodeling of Zn2+ homeostasis upon differentiation of mammary epithelial cells

Zinc is the second most abundant transition metal in humans and an essential nutrient required for growth and development of newborns. During lactation, mammary epithelial cells differentiate into a secretory phenotype, uptake zinc from blood circulation, and export it into mother's milk. At the cellular level, many zinc-dependent cellular processes, such as transcription, metabolism of nutrients, and proliferation are involved in the differentiation of mammary epithelial cells. Using mouse mammary epithelial cells as a model system, we investigated the remodeling of zinc homeostasis during differentiation induced by treatment with the lactogenic hormones cortisol and prolactin. RNA-Seq at different stages of differentiation revealed changes in global gene expression, including genes encoding zinc-dependent proteins and regulators of zinc homeostasis. Increases in mRNA levels of three zinc homeostasis genes, Slc39a14 (ZIP14) and metallothioneins (MTs) I and II were induced by cortisol but not by prolactin. The cortisol-induced increase was partially mediated by the nuclear glucocorticoid receptor signaling pathway. An increase in the cytosolic labile Zn2+ pool was also detected in lactating mammary cells, consistent with upregulation of MTs. We found that the zinc transporter ZIP14 was important for the expression of a major milk protein, whey acid protein (WAP), as knockdown of ZIP14 dramatically decreased WAP mRNA levels. In summary, our study demonstrated remodeling of zinc homeostasis upon differentiation of mammary epithelial cells resulting in changes in cytosolic Zn2+ and differential expression of zinc homeostasis genes, and these changes are important for establishing the lactation phenotype.

Short communication: Characterization of the milk protein expression profiles in dairy buffaloes with and without subclinical mastitis

The aim of this study was to characterize the proteins present in milk whey from buffaloes with and without subclinical mastitis using a proteomic approach to identify differentially expressed proteins as potential biomarkers for this disease. Whey from Murrah buffaloes with subclinical mastitis was compared with whey from healthy animals using liquid chromatography-tandem mass spectrometry. The annotated protein databases for Bubalus bubalis and Bos taurus were used in the analysis, and the gene annotations from the buffalo and bovine reference assemblies were also used. After integrating gene annotations from both buffaloes and bovines, a total of 1,033 proteins were identified, of which 156 were differentially expressed. Eighteen biological processes were annotated with Gene Ontology. Cathelicidin-3 was identified as a potential biomarker for subclinical mastitis. These results are important to the characterization of mastitis in the buffalo mammary gland and may aid in the development of tools for early diagnosis.

Camellia (Camellia oleifera Abel.) seed oil promotes milk fat and protein synthesis-related gene expression in bovine mammary epithelial cells.

Camellia (Camellia oleifera Abel.) seed oil is a commonly used edible oil of China. In ancient Chinese literature, it is mentioned to be helpful for postpartum repair and lactation in women. Research on camellia seed oil (CO) as a feed additive for dairy cattle is less. We investigated the effect of CO on the expression of milk fat and protein syntheses‐related genes in differentiated bovine mammary epithelial cells (MAC‐T) using soybean oil (SO) as the control. The results showed that CO increased the expression of genes related to de novo synthesis of fatty acids including sterol regulatory element‐binding protein 1 (SREBP1), acetyl‐CoA carboxylase 1 (ACC), fatty acid synthase (FASN), lipoprotein lipase (LPL), and stearoyl‐CoA desaturase (SCD) (p < .05). Among the milk protein genes analyzed, CO increased β‐casein mRNA expression (p < .05) and decreased αS1‐casein mRNA expression (p < .05) in MAC‐T cells. CO upregulated the pathways related to milk protein synthesis with increased mRNA levels of phosphoinositide 3‐kinase (PI3K), RAC‐alpha serine/threonine‐protein kinase (AKT1), and mammalian target of rapamycin (mTOR) (p < .05) in MAC‐T cells. Ribosomal protein S6 kinase beta‐1 (S6K1) gene was upregulated, and eukaryotic initiation factor 4E (eIF4E) gene (p < .05) was downregulated with CO treatment. The mRNA expression levels of janus kinase 2 (JAK2), activator of transcription 5‐β (STAT5‐β), and E74‐like factor 5 (ELF5) were elevated in MAC‐T cells treated with CO (p < .05). Meanwhile, the protein expression levels of S6K1, STAT5‐β, phosphorylated mTOR (p‐mTOR), p‐S6K1, and p‐STAT5‐β increased in MAC‐T cells treated with CO (p < .05). In summary, CO promoted β‐casein synthesis by regulating PI3K‐mTOR‐S6K1 and JAK2‐STAT5 signaling pathways and influenced fatty acid synthesis by regulating SREBP1‐related genes in MAC‐T cells. We need to further confirm the function of CO using in vivo models.

The microstructure of laminin-111 compensates for dystroglycan loss in mammary epithelial cells in downstream expression of milk proteins

Laminin-111 (Ln-1), an extracellular matrix (ECM) glycoprotein found in the basement membrane of mammary gland epithelia, is essential for lactation. In mammary epithelial cells (MECs), dystroglycan (Dg) is believed to be necessary for polymerization of laminin-111 into networks., thus we asked whether correct polymerization could compensate for Dg loss. Artificially polymerized laminin-111 and the laminin-glycoprotein mix Matrigel, both formed branching, spread networks with fractal dimensions from 1.7 to 1.8, whereas laminin-111 in neutral buffers formed small aggregates without fractal properties (a fractal dimension of 2). In Dg knockout cells, either polymerized laminin-111 or Matrigel readily attached to the cell surface, whereas aggregated laminin-111 did not. In contrast, polymerized and aggregated laminin-111 bound similarly to Dg knock-ins. Both polymerized laminin-111 and Matrigel promoted cell rounding, clustering, formation of tight junctions, and expression of milk proteins, whereas aggregated Ln-1 did not attach to cells or promote functional differentiation. These findings support that the microstructure of Ln-1 networks in the basement membrane regulates mammary epithelial cell function.