Mammary Epithelial Cells Research Articles

Cytoarchitecture of Breast Cancer Cells under Diabetic Conditions: Role of Regulatory Kinases-Rho Kinase and Focal Adhesion Kinase.

Diabetes greatly reduces the survival rates in breast cancer patients due to chemoresistance and metastasis. Reorganization of the cytoskeleton is crucial to cell migration and metastasis. Regulatory cytoskeletal protein kinases such as the Rho kinase (ROCK) and focal adhesion kinase (FAK) play a key role in cell mobility and have been shown to be affected in cancer. It is hypothesized that diabetes/high-glucose conditions alter the cytoskeletal structure and, thus, the elasticity of breast cancer cells through the ROCK and FAK pathway, which can cause rapid metastasis of cancer. The aim of the study was to investigate the role of potential mediators that affect the morphology of cancer cells in diabetes, thus leading to aggressive cancer. Breast cancer cells (MDA-MB-231 and MCF-7) were treated with 5 mM glucose (low glucose) or 25 mM glucose (high glucose) in the presence of Rho kinase inhibitor (Y-27632, 10 mM) or FAK inhibitor (10 mM). Cell morphology and elasticity were monitored using atomic force microscopy (AFM), and actin staining was performed by fluorescence microscopy. For comparative study, normal mammary breast epithelial cells (MCF-10A) were used. It was observed that high-glucose treatments modified the cytoskeleton of the cells, as observed through AFM and fluorescence microscopy, and significantly reduced the elasticity of the cells. Blocking the ROCK or FAK pathway diminished the high-glucose effects. These changes were more evident in the breast cancer cells as compared to the normal cells. This study improves the knowledge on the cytoarchitecture properties of diabetic breast cancer cells and provides potential pathways that can be targeted to prevent such effects.

Sirtuin 1 Inhibits Fatty Acid Synthesis through Forkhead Box Protein O1-Mediated Adipose Triglyceride Lipase Expression in Goat Mammary Epithelial Cells.

Sirtuin 1 (SIRT1) is a key upstream regulator of lipid metabolism; however, the molecular mechanisms by which SIRT1 regulates milk fat synthesis in dairy goats remain unclear. This study aimed to investigate the regulatory roles of SIRT1 in modulating lipid metabolism in goat mammary epithelial cells (GMECs) and its impact on the adipose triglyceride lipase (ATGL) promoter activity using RNA interference (RNAi) and gene overexpression techniques. The results showed that SIRT1 is significantly upregulated during lactation compared to the dry period. Additionally, SIRT1 knockdown notably increased the expressions of genes related to fatty acid synthesis (SREBP1, SCD1, FASN, ELOVL6), triacylglycerol (TAG) production (DGAT2, AGPAT6), and lipid droplet formation (PLIN2). Consistent with the transcriptional changes, SIRT1 knockdown significantly increased the intracellular contents of TAG and cholesterol and the lipid droplet abundance in the GMECs, while SIRT1 overexpression had the opposite effects. Furthermore, the co-overexpression of SIRT1 and Forkhead box protein O1 (FOXO1) led to a more pronounced increase in ATGL promoter activity, and the ability of SIRT1 to enhance ATGL promoter activity was nearly abolished when the FOXO1 binding sites (FKH1 and FKH2) were mutated, indicating that SIRT1 enhances the transcriptional activity of ATGL via the FKH element in the ATGL promoter. Collectively, our data reveal that SIRT1 enhances the transcriptional activity of ATGL through the FOXO1 binding sites located in the ATGL promoter, thereby regulating lipid metabolism. These findings provide novel insights into the role of SIRT1 in fatty acid metabolism in dairy goats.

403 Impact of lysine, methionine, and histidine deficiencies on gene expression and upstream transcriptional regulators in bovine mammary epithelial cells: An RNA-sequencing analysis

Abstract Essential (E) amino acids (AA) may alter bovine mammary epithelial cell functions by impacting mRNA expression of genes through their upstream transcription factors. The objective of this research was to study the impact of EAA deficiencies on gene expression and to identify upstream transcriptional factors mediating their effects in bovine mammary epithelial cells. Epithelial cells were collected from the mammary glands of lactating dairy cows and cultured in combined media of DMEM/F12 and Medium 170, differentiated for 5 d, and subsequently treated in triplicate for 60 h with a control medium containing all EAA at normal plasma concentrations for cows (CTL), or a medium with 20 μM Lys (LY), 5 μM Met (LM), or 10 μM His (LH), which are each 25% of normal concentration. RNA was isolated from the cells and sequenced on an Illumina NovaSeq 6000 producing 150 base pair paired-end reads with a minimum sequencing depth of 150 million reads. Reads were cleaned with ‘fastp’ (Chen et al., 2018), aligned to the bovine reference genome (ARS-UCD1.2) using ‘STAR’ aligner (Dobin et al., 2013), and genes counted using ‘featureCounts’ (Liao et al., 2014). Differential expression of genes between treatment pairs was determined using the ‘DESeq2’ package (Love et al., 2014) in R. Genes with an absolute log2 fold change ≥ 1 and a false discovery rate of < 0.05 were considered differentially expressed. The number of differentially expressed genes relative to CTL was 780 for LY, 383 for LH, and 536 for LM. QIAGEN Ingenuity Pathway Analysis (IPA) identified four pathways consistently affected across LY, LM, and LH: kinetochore metaphase signaling, mitotic roles of polo-like kinase, cell cycle: G2/M DNA damage checkpoint regulation, and interferon signaling (Figure 1-3). Notably, the kinetochore metaphase signaling pathway was identified as the most significant across all treatments, suggesting a strong link to cell proliferation and apoptosis during EAA deficiency. The interferon signaling might be related to the Janus kinase 2/signal transducer and activator of transcription 5 (Jak2/STAT5) pathway which can promote the expression of genes encoding milk proteins such as β-casein and β-lactoglobulin. Furthermore, the IPA identified upstream transcription factors related to cell proliferation, apoptosis, autophagy, protein translation, endoplasmic reticulum biogenesis, and amino acid metabolism including NUPR1, FOXM1, MYC, CCND1, TP53, DDIT3, and ATF4 across all treatments. The findings suggest that AA deprivation regulates bovine mammary epithelial cell function by instigating amino acid response pathways and altering secretory cell number.

A comprehensive bioinformatic analysis of the role of TGF-β1-stimulated activating transcription factor 3 by non-coding RNAs during breast cancer progression

A potent growth inhibitor for normal mammary epithelial cells is transforming growth factor beta 1 (TGF-β1). When breast tissues lose the anti-proliferative activity of this factor, invasion and bone metastases increase. Human breast cancer (hBC) cells express more activating transcription factor 3 (ATF3) when exposed to TGF-β1, and this transcription factor is essential for BC development and bone metastases. Non-coding RNAs (ncRNAs), including circular RNAs (circRNAs) and microRNAs (miRNAs), have emerged as key regulators controlling several cellular processes. In hBC cells, TGF-β1 stimulated the expression of hsa-miR-4653–5p that putatively targets ATF3. Bioinformatics analysis predicted that hsa-miR-4653–5p targets several key signaling components and transcription factors, including NFKB1, STAT1, STAT3, NOTCH1, JUN, TCF3, p300, NRF2, SUMO2, and NANOG, suggesting the diversified role of hsa-miR-4653–5p under physiological and pathological conditions. Despite the high abundance of hsa-miR-4653–5p in hBC cells, the ATF3 level remained elevated, indicating other ncRNAs could inhibit hsa-miR-4653–5p’s activity. In silico analysis identified several circRNAs having the binding sites for hsa-miR-4653–5p, indicating the sponging activity of circRNAs towards hsa-miR-4653–5p. The study's findings suggest that TGF-β1 regulates circRNAs and hsa-miR-4653–5p, which in turn affects ATF3 expression, thus influencing BC progression and bone metastasis. Therefore, focusing on the TGF-β1/circRNAs/hsa-miR-4653–5p/ATF3 network could lead to new ways of diagnosing and treating BC.

USP11 deubiquitinates E-cadherin and maintains the luminal fate of mammary tumor cells to suppress breast cancer

Basal-like breast cancer may originate from luminal epithelial or cancerous cells. Inadequately repaired DNA damage impairs luminal differentiation and promotes aberrant luminal to basal trans-differentiation in mammary epithelial cells (MECs). Ubiquitin-specific peptidase 11 (USP11), a deubiquitinase, plays a critical role in DNA damage repair. The role of USP11 in controlling mammary cell differentiation and tumorigenesis remains poorly understood. We generated Usp11 knockout mice and breast cancer cell lines expressing wild-type (WT) and mutant form of USP11. By using these mutant mice, cell lines, and human USP11-deficient and -proficient breast cancer tissues, we tested how USP11 controls mammary cell fate. We generated Usp11 knock-out mice and found that deletion of Usp11 reduced the expression of E-cadherin and promoted DNA damage in MECs. Overexpression of WT USP11, but not a deubiquitinase-inactive mutant form of USP11, promoted luminal differentiation, enhanced DNA damage repair, and suppressed tumorigenesis in mice. Mechanistically, we found that USP11 enhanced the protein expression of E-cadherin dependent on its deubiquitinase activity, and that USP11 deubiquitinated E-cadherin at K738. We discovered that USP11 bound to E-cadherin through its C-terminal region. In human breast cancers, expression of USP11 was positively correlated with that of E-cadherin, and high USP11 predicted better recurrence-free survival. Our findings provide compelling genetic and biochemical evidence that USP11 not only promotes DNA damage repair but also deubiquitinates E-cadherin and maintains the luminal feature of mammary tumor cells, thereby suppressing luminal breast cancer.

Milk Lipid Regulation in Dairy Goats: A Comprehensive Review.

The growth, development, and milk production traits of dairy goats, which are important sources of high-quality animal protein, are significantly influenced by a combination of genetic and environmental factors. It is imperative to identify key genetic loci that govern economically valuable traits in order to enhance breeding programs. Despite advancements in genomic technologies, there are still gaps in knowledge regarding the interplay between genetic factors and environmental influences, particularly in relation to the regulation of milk production and quality. Therefore, the aim of this paper was to synthesize advancements in the genetic and environmental factors affecting milk production and quality in dairy goats and identify key regulatory mechanisms. This review summarizes the recent progress on the identification of genes associated with milk production traits using whole-genome resequencing, the use of transcriptomic profiling to identify genes linked to milk production, the exploration of regulatory mechanisms of lipid metabolism in goat mammary epithelial cells, and the evaluation of the influence of nutritional factors on milk quality. A comprehensive understanding of these interactions is essential for enhancing breeding strategies and ensuring the sustainability of dairy goat farming. Future research should incorporate multi-omics approaches to unravel the intricate regulatory processes governing milk production and adapt practices to meet global demand while upholding economic and environmental sustainability.

The chromatin remodeler DEK promotes proliferation of mammary epithelium and is associated with H3K27me3 epigenetic modifications.

The DEK chromatin remodeling protein was previously shown to confer oncogenic phenotypes to human and mouse mammary epithelial cells using in vitro and knockout mouse models. However, its functional role in normal mammary gland epithelium remained unexplored. We developed two novel mouse models to study the role of Dek in normal mammary gland biology in vivo . Mammary gland-specific Dek over-expression in mice resulted in hyperproliferation of cells that visually resembled alveolar cells, and a transcriptional profile that indicated increased expression of cell cycle, mammary stem/progenitor, and lactation-associated genes. Conversely, Dek knockout mice exhibited an alveologenesis or lactation defect, resulting in dramatically reduced pup survival. Analysis of previously published single-cell RNA-sequencing of mouse mammary glands revealed that Dek is most highly expressed in mammary stem cells and alveolar progenitor cells, and to a lesser extent in basal epithelial cells, supporting the observed phenotypes. Mechanistically, we discovered that Dek is a modifier of Ezh2 methyltransferase activity, upregulating the levels of histone H3 trimethylation on lysine 27 (H3K27me3) to control gene transcription. Combined, this work indicates that Dek promotes proliferation of mammary epithelial cells via cell cycle deregulation. Furthermore, we report a novel function for Dek in alveologenesis and histone H3 K27 trimethylation.

Overexpression of PER2 Promotes De Novo Fatty Acid Synthesis, Fatty Acid Desaturation, and Triglyceride Accumulation in Bovine Mammary Epithelial Cells.

The core clock gene Period2 (PER2) is associated with mammary gland development and lipid synthesis in rodents and has recently been found to have a diurnal variation in the process of lactation, but has not yet been demonstrated in bovine mammary epithelial cells (BMECs). To explore the regulatory function of PER2 on milk fat synthesis in bovine mammary epithelial cells, we initially assessed the expression of clock genes and milk fat metabolism genes for 24 h using real-time quantitative PCR and fitted the data to a cosine function curve. Subsequently, we overexpressed the PER2 in BMECs using plasmid vector (pcDNA3.1-PER2), with empty vector pcDNA3.1-myc as the control. After transfecting BMECs for 48 h, we assessed the protein abundance related to milk fat synthesis by Western blot, the expression of genes coding for these proteins using real time-quantitative PCR, the production of triacylglycerol, and the fatty acid profile. The findings indicated that a total of nine clock genes (PER1/2, CRY1/2, REV-ERBα, BMAL1, NCOR1, NR2F2, FBXW11), seven fatty acid metabolism genes (CD36, ACSS2, ACACA, SCD, FADS1, DGAT1, ADFP), and six nuclear receptor-related genes (INSIG1, SCAP, SREBF1, C/EBP, PPARG, LXR) exhibited oscillation with a period close to 24 h in non-transfected BMECs (R2 ≥ 0.7). Compared to the control group (transfected with empty pcDNA3.1-myc), the triglyceride content significantly increased in the PER2 overexpression group (p < 0.05). The lipogenic genes for fatty acid transport and triglyceride synthesis (ACACA, SCD, LPIN1, DGAT1, and SREBF1) were upregulated after PER2 overexpression, along with the upregulation of related protein abundance (p < 0.05). The contents and ratios of palmitic acid (C16:0), oleic acid (C18:1n9c), and trans-oleic acid (C18:1n9t) were significantly increased in the overexpression group (p < 0.05). Overall, the data supported that PER2 participated in the process of milk fat metabolism and is potentially involved in the de novo synthesis and desaturation of fatty acid in bovine mammary epithelial cells.

Three-dimensional Culture of Buffalo (Bubalus bubalis) Mammary Epithelial Cell Line

Background: The mammary gland is a crucial organ in dairy animals, responsible for milk production, regulated by intricate hormonal and cellular interactions. Traditional two-dimensional (2D) cell culture systems used in mammary gland research fail to replicate the complex in vivo environment, limiting the understanding of mammary gland biology. The study explores the advantages of three-dimensional (3D) culture systems for Buffalo Mammary Epithelial Cells (BuMECs), aiming to enhance morphological and functional differentiation compared to 2D cultures. Methods: BuMECs were cultured in Dulbecco’s Modified Eagle’s Medium (DMEM/F12) supplemented with 10% FBS and specific growth factors. For 2D culture, BuMECs were grown on a plastic substratum. For 3D culture, cells were cultured on top of and embedded in growth factor-reduced (GFR) Matrigel. Morphological differentiation was observed under phase-contrast microscopy. Total RNA was extracted and cDNA was synthesized to measure β-casein gene expression using quantitative real-time PCR (qPCR). Protein expression was analyzed by Western blotting, targeting total casein. Result: BuMECs grown in 3D cultures exhibited enhanced morphological differentiation, forming alveolar and ductal structures, unlike the monolayer formation observed in 2D cultures. Rapid formation of duct-like structures was noted within 24 hours in 3D cultures. Functional differentiation was significantly improved, evidenced by a five-fold increase in β-casein mRNA expression and higher protein levels of α, β and κ-casein in 3D cultured cells compared to 2D cultures. Western blot analysis confirmed the elevated casein protein expression in 3D cultures.

Salinity and prolactin regulate anoctamin 1 in the model teleost, Fundulus heteroclitus.

To maintain internal ion balance in marine environments, teleost fishes leverage seawater (SW)-type ionocytes to actively secrete Na+ and Cl- into the environment. It is well established that SW-type ionocytes use apically expressed cystic fibrosis transmembrane conductance regulator 1 (Cftr1) as a conduit for Cl- to exit the gill. Here, we investigated whether the Ca2+-activated Cl- channel, anoctamin 1 (Ano1), provides an additional path for Cl--secretion in euryhaline mummichogs (Fundulus heteroclitus). Two ano1 gene isoforms, denoted ano1.1a and -1.1b, exhibited higher expression in the gill and opercular epithelium of mummichogs long-term acclimated to SW versus fresh water (FW). Branchial ano1.1b and cftr1 expression was increased in mummichogs sampled 24 h after transfer from FW to SW; ano1.1a and -1.1b were upregulated in the gill and opercular epithelium following transfer from SW to hypersaline SW. Alternatively, the expression of ano1.1a, -1.1b, and cftr1 in the gill and opercular epithelium was markedly decreased after transfer from SW to FW. Given its role in attenuating ion secretion, we probed whether prolactin downregulates ano1 isoforms. In addition to attenuating cftr1 expression, a prolactin injection reduced branchial ano1.1a and -1.1b levels. Given how Ano1 mediates Cl- secretion by mammalian epithelial cells, the salinity- and prolactin-sensitive nature of ano1 expression reported here indicates that Ano1 may constitute a novel Cl--secretion pathway in ionocytes. This study encourages a wider evaluation of this putative Cl--secretion pathway and its regulation by hormones in teleost fishes.NEW & NOTEWORTHY In this study, we provide evidence in a teleost fish that the Ca2+-activated Cl- channel, anoctamin 1 may provide an additional path for Cl- secretion by seawater-type ionocytes. Not only is this the first report of a Cftr-independent Cl--secreting pathway conferring survival in seawater but also the first description of its regulation by the pituitary hormone prolactin.

Tunable Hybrid Hydrogels of Alginate and Cell-Derived dECM to Study the Impact of Matrix Alterations on Epithelial-to-Mesenchymal Transition.

Epithelial-to-mesenchymal transition (EMT) is crucial for tumor progression, being linked to alterations in the extracellular matrix (ECM). Understanding the ECM's role in EMT can uncover new therapeutic targets, yet replicating these interactions in vitro remains challenging. It is shown that hybrid hydrogels of alginate (ALG) and cell-derived decellularized ECM (dECM), with independently tunable composition and stiffness, are useful 3D-models to explore the impact of the breast tumor matrix on EMT. Soft RGD-ALG hydrogels (200Pa), used as neutral bulk material, supported mammary epithelial cells morphogenesis without spontaneous EMT, allowing to define the gene, protein, and biochemical profiles of cells at different TGFβ1-induced EMT states. To mimic the breast tumor composition, dECM from TGFβ1-activated fibroblasts (adECM) are generated, which shows upregulation of tumor-associated proteins compared to ndECM from normal fibroblasts. Using hybrid adECM-ALG hydrogels, it is shown that the presence of adECM induces partial EMT in normal epithelial cells, and amplifes TGF-β1 effects compared to ALG and ndECM-ALG. Increasing the hydrogel stiffness to tumor-like levels (2.5kPa) have a synergistic effect, promoting a more evident EMT. These findings shed light on the complex interplay between matrix composition and stiffness in EMT, underscoring the utility of dECM-ALG hydrogels as a valuable in vitro platform for cancer research.

Minthostachys verticillata essential oil modulates cytokine synthesis and Staphylococcus aureus internalization in MAC-T cells at least through TLR4/MyD88/NFkB pathway.

The aim of this study was to evaluate the activation pathway(s) triggered by Minthostachys verticillata essential oil (EO) in bovine mammary epithelial cells (MAC-T) challenged with a strain of bovine Staphylococcus aureus. MAC-T cells were stimulated with EO, S. aureus or pre-treated with EO and then challenged with S. aureus. Cytokine's release was measured by ELISA. The mRNA for TLR2, TLR4, NOD2, MyD88 and NFκB was quantified by RT-qPCR. S. aureus adherence and internalization was also evaluated. MAC-T cells stimulated with S. aureus synthesized high levels of IL-1ß and IL-6 were kept up to 48 h, while IL-4 levels were not altered.Cells pre-treated with EO for 2 and 6h and then challenged with S. aureus showed a significant increase of IL-1β and IL-6. However, in these cells, a decrease in IL-1ß and IL-6 levels and an increase of IL-4 values was observed from 24 h. No significant increase in the expression levels of TLR2 or NOD2 was detected in all stimulated cells. However, the expression of TLR4, MyD88 and NFκB was increased in cells stimulated with S. aureus at 2 and 6 h as well as in cells pre-treated with EO between 2 and 6h and then challenged with S. aureus. The NFκB expression levels was similar to control at 24h in all stimulated cells, although pro-inflammatory cytokine levels and TLR4 and MyD88 expression levels remained high in cells stimulated with S. aureus. This results suggested the activation of other pathways independent of MyD88 by the pathogen that involucrated the activation of others transcription factors. Pre-treatment with EO during 2, 6 and 24h did not affect S. aureus adherence but decreased its internalization. In conclusion, pre-treatment with EO increased the IL-1β and IL-6 synthesis during the first hours post-challenged with S. aureus up-regulating TLR4/MyD88/NFκB pathway. Furthermore, EO increased the IL-4 levels from 6 to 24 h down-regulating the NFκB and possibly other transcription factors activated by the pathogen, which decreased its internalization into MAC-T cells.

Integrin profle of circulating tumour cells in breast cancer patients

Background. Integrins, as adhesion molecules, play a key role in the interaction of cells with the basal membrane and intercellular matrix. Numerous studies demonstrate evidence of increased expression of integrins on tumor cells in different types of cancer. Thus, β3 and αV integrins are associated with stem-like features of tumor cells, and β4 integrin as α6β4 heterodimer provides anchorage-independent survival of malignant mammary epithelial cells. However, all the described functions of integrins have been investigated exclusively on primary tumor cells. The functional significance and expression pattern of integrins on circulating tumor cells (CTCs) remains unclear. The aim of the study was to evaluate the β3, β4 and αvβ5 integrin expression on CTCs and its association with molecular subtype, stage and lymph node metastasis in breast cancer patients Material and Methods. The study included 22 patients with T1–4N0–3M0 invasive ductal breast carcinoma. Venous blood was taken from patients without neoadjuvant chemotherapy (group 1) and after neoadjuvant chemotherapy (group 2) in the volume of 12 ml into vacuum tubes with EDTA. The expression of CTC integrins including stemness features CD44/CD24, CD133 and ALDH1, and epithelial-mesenchymal transition (EMT) (N-cadherin) was evaluated by flow cytometry. Results. CTCs with β3+β4-αvβ5- and β3-β4+αvβ5+ phenotypes and stemness properties were associated with larger tumor size (T4) in breast cancer patients. The β3 integrin expression was associated with more aggressive molecular subtypes of breast cancer. Administration of neoadjuvant chemotherapy did not affect the expression pattern of β3, β4 and αvβ5 integrins in CTCs. Conclusion. In breast cancer, most CTCs expressed β3, β4 and αvβ5 integrins despite the lack of attachment to the basal membrane and intercellular matrix. The expression of the above integrins on CTCs was associated with breast cancer molecular subtype, stage and lymph node metastasis, and therefore its evaluation can be considered as one of the objectives of liquid biopsy study.

MiR-206 Suppresses Triacylglycerol Accumulation via Fatty Acid Elongase 6 in Dairy Cow Mammary Epithelial Cells.

Cow milk possesses high nutritional value due to its rich array of beneficial fatty acids. It is important to understand the mechanisms involved in lipid metabolism in dairy cows. These mechanisms are driven by a complex molecular regulatory network. In addition, there are many regulatory factors involved in the process of fatty acid metabolism, including transcription factors and non-coding RNAs, amongst others. MicroRNAs (miRNAs) can regulate the expression of target genes and modulate various biological processes, including lipid metabolism. Specifically, miR-206 has been reported to impair lipid accumulation in nonruminant hepatocytes. However, the effects and regulatory mechanisms of miR-206 on lipid metabolism in bovine mammary cells remain unclear. In the present study, we investigated the effects of miR-206 on lipid-related genes and TAG accumulation. The direct downstream gene of miR-206 was subsequently determined via a dual-luciferase assay. Finally, the fatty acid content of bovine mammary epithelial cells (BMECs) upon ELOVL6 inhibition was examined. The results revealed that miR-206 overexpression significantly decreased triacylglycerol (TAG) concentration and abundances of the following: acetyl-coenzyme A carboxylase alpha (ACACA); fatty acid synthase (FASN); sterol regulatory element binding transcription factor 1 (SREBF1); diacylglycerol acyltransferase 1 (DGAT1); 1-acylglycerol-3-phosphate O-acyltransferase 6 (AGPAT6); lipin 1 (LPIN1); and fatty acid elongase 6 (ELOVL6). Overexpression of miR-206 was also associated with an increase in patatin-like phospholipase domain-containing 2 (PNPLA2), while inhibition of miR-206 promoted milk fat metabolism in vitro. In addition, we found that ELOVL6 is a direct target gene of miR-206 through mutation of the binding site. Furthermore, ELOVL6 intervention significantly decreased the TAG levels and elongation indexes of C16:0 and C16:1n-7 in BMECs. Finally, ELOVL6 siRNA partially alleviated the increased TAG accumulation caused by miR-206 inhibition. In summary, we found that miR-206 inhibits milk fatty acid synthesis and lipid accumulation by targeting ELOVL6 in BMECs. The results presented in this paper may contribute to the development of strategies for enhancing the quality of cow milk and its beneficial fatty acids, from the perspective of miRNA-mRNA networks.

UID-Dual Transcriptome Sequencing Analysis of the Molecular Interactions between Streptococcus agalactiae ATCC 27956 and Mammary Epithelial Cells.

Streptococcus agalactiae ATCC 27956 is a highly contagious Gram-positive bacterium that causes mastitis, has a high infectivity for mammary epithelial cells, and becomes challenging to treat. However, the molecular interactions between it and mammary epithelial cells remain poorly understood. This study analyzed differential gene expression in mammary epithelial cells with varying levels of S. agalactiae infection using UID-Dual transcriptome sequencing and bioinformatics tools. This study identified 211 differentially expressed mRNAs (DEmRNAs) and 452 differentially expressed lncRNAs (DElncRNAs) in host cells, primarily enriched in anti-inflammatory responses, immune responses, and cancer-related processes. Additionally, 854 pathogen differentially expressed mRNAs (pDEmRNAs) were identified, mainly enriched in protein metabolism, gene expression, and biosynthesis processes. Mammary epithelial cells activate pathways, such as the ERK1/2 pathway, to produce reactive oxygen species (ROS) to eliminate bacteria. The bacteria disrupt the host's innate immune mechanisms by interfering with the alternative splicing processes of mammary epithelial cells. Specifically, the bacterial genes of tsf, prfB, and infC can interfere with lncRNAs targeting RUNX1 and BCL2L11 in mammary epithelial cells, affecting the alternative splicing of target genes and altering normal molecular regulation.

Inflammatory Signaling via PEIZO1 Engages and Enhances the LPS-Mediated Apoptosis during Clinical Mastitis.

Bovine clinical mastitis is characterized by inflammation and immune responses, with apoptosis of mammary epithelial cells as a cellular reaction to infection. PIEZO1, identified as a mechanotransduction effector channel in nonruminant animals and sensitive to both mechanical stimuli or inflammatory signals like lipopolysaccharide (LPS). However, its role in inflammatory processes in cattle has not been well-documented. The aim of this study was to elucidate the in situ expression of PIEZO1 in bovine mammary gland and its potential involvement in clinical mastitis. We observed widespread distribution and upregulation of PIEZO1 in mammary epithelial cells in clinical mastitis cows and LPS-induced mouse models, indicating a conserved role across species. In vitro studies using mammary epithelial cells (MAC-T) revealed that LPS upregulates PIEZO1. Notably, the effects of PIEZO1 artificial activator Yoda1 increased apoptosis and NLRP3 expression, effects mitigated by PIEZO1 silencing or NLRP3 inhibition. In conclusion, the activation of the PIEZO1-NLRP3 pathway induces abnormal apoptosis in mammary epithelial cells, potentially serving as a regulatory mechanism to combat inflammatory responses to abnormal stimuli.

Selenomethionine Promotes Milk Protein and Fat Synthesis and Proliferation of Mammary Epithelial Cells through the GPR37-mTOR-S6K1 Signaling.

Selenomethionine (SeMet) is an important nutrient, but its role in milk synthesis and the GPCR related to SeMet sensing is still largely unknown. Here, we determined the dose-dependent role of SeMet on milk protein and fat synthesis and proliferation of mammary epithelial cells (MECs), and we also uncovered the GPCR-mediating SeMet function. At 24 h postdelivery, lactating mother mice were fed a maintenance diet supplemented with 0, 5, 10, 20, 40, and 80 mg/kg SeMet, and the feeding process lasted for 18 days. The 10 mg/kg group had the best increase in milk production, weight gain of offspring mice, and mammary gland weight and acinar size, whereas a higher concentration of SeMet gradually decreased the weight gain of the offspring mice and showed toxic effects. Transcriptome sequencing was performed to find the differentially expressed genes (DEGs) between the mammary gland tissues of mother mice in the 10 mg/kg SeMet treatment group and the control group. A total of 258 DEGs were screened out, including 82 highly expressed genes including GPR37 and 176 lowly expressed genes. SeMet increased milk protein and fat synthesis in HC11 cells and cell proliferation, mTOR and S6K1 phosphorylation, and expression of GPR37 in a dose-dependent manner. GPR37 knockdown decreased milk protein and fat synthesis in HC11 cells and cell proliferation and blocked SeMet stimulation on mTOR and S6K1 phosphorylation. Taken together, our data demonstrate that SeMet can promote milk protein and fat synthesis and proliferation of MECs and functions through the GPR37-mTOR-S6K1 signaling pathway.

The Aqueous Extract of Hemerocallis citrina Baroni Improves the Lactation-Promoting Effect in Bovine Mammary Epithelial Cells through the PI3K-AKT Signaling Pathway.

Insufficient milk supply is a widespread issue faced by women globally and associated with a higher risk of health problems in infants and mothers. Hemerocallis citrina Baron, commonly known as daylily, is a perennial edible plant often used in traditional Asian cuisine to promote lactation. However, the active compound(s) and mechanism of its lactation-promoting effect remain unclear. This study aimed to confirm the traditional use of daylily in promoting lactation and investigate its potential active components and underlying molecular mechanisms. Our results showed that the aqueous extracts of H. citrina Baroni (HAE) significantly enhanced milk production, and the serum levels of lactation-related hormones, and promoted mammary gland development in lactating rats, as well as increased the levels of milk components in bovine mammary epithelial cells (BMECs) (p < 0.05). UHPLC-Q-Exactive Orbitrap-MS analysis revealed that hexamethylquercetin (HQ) is the representative flavonoid component in HAE, accounting for 42.66% of the total flavonoids. An integrated network pharmacology and molecular docking analysis suggested that HQ may be the potential active flavonoid in HAE that promotes lactation, possibly supporting lactation by binding to key target proteins such as STAT5A, PIK3CA, IGF1R, TP53, CCND1, BCL2, INS, AR, and DLD. Cell experiments further demonstrated that HQ could promote cell proliferation and the synthesis of milk proteins, lactose, and milk fat in BMECs. Transcriptomic analysis combined with a quantitative reverse transcription polymerase chain reaction (RT-qPCR) revealed that both HAE and HQ exert a lactation-promoting function mainly through regulating the expression of key genes in the PI3K-Akt signaling pathway.

Syndecan-3 inhibits LPS-induced Inflammation of Bovine Mammary Epithelial Cells through the NF-κB Signal Transduction Pathway

In mastitis, excessive inflammation caused by lipopolysaccharide (LPS) is an important factor leading to mammary tissue damage. Therefore, exploring the regulatory factors that can inhibit the widespread inflammation caused by LPS is crucial. Syndecan-3 (SDC3) has been found to play an active role in anti-inflammatory infection by inhibiting leukocyte adhesion, reducing the accumulation of inflammatory products, such as reactive oxygen species, and competing with chemokines; however, the role and regulatory mechanism of SDC3 in mastitis remains unknown. Therefore, this study aimed to reveal the effect of SDC3 on LPS-induced inflammation in bovine mammary epithelial cells (BMECs) and explore its possible molecular mechanisms. First, we constructed a BMEC inflammatory model. It was found that cells stimulated with 10 μg/mL LPS for 24 h strongly induced the expression of inflammatory cytokines and had no toxic effect on cells, which was the best condition to simulate the BMECs inflammatory response in vitro. Subsequently, we used overexpression and RNAi interference, Real Time Quantitative PCR (RT-qPCR), and Western blot assays to explore the effects of SDC3 on LPS-induced inflammatory factors and their mechanisms. The results showed that overexpression of SDC3 could inhibit the transcriptional levels of inflammatory cytokines IL-6, IL-1β, and TNFα induced by LPS and inhibit the activation of the NF-κB inflammatory pathway by inhibiting the expression of NF-κB p50 and p-IκBα and promoting the expression of IκBα. Our results suggest that SDC3 inhibits the LPS-induced inflammatory response of BMECs through the NF-κB pathway, in which NF-κB p50 may be an important target of SDC3. These findings lay the foundation for elucidating the molecular regulatory mechanisms of dairy cow mastitis.

Effects of L-leucine and sodium acetate on milk protein synthesis under heat stress conditions in bovine mammary epithelial cells in vitro

Effects of L-leucine and sodium acetate on milk protein synthesis under heat stress conditions in bovine mammary epithelial cells in vitro