Mouse Mammary Epithelial Cells Research Articles

LncRNA Miat knockdown enhances pirarubicin-mediated anticancer sensitivity in breast cancer cells.

Pirarubicin (THP) is a widely used antitumor agent in clinical practice, but its reduced sensitivity during treatment has limited its use. The aim of this study was to investigate the role and mechanism of LncRNA Miat knockdown in improving THP sensitivity. We assessed the role of Miat overexpression/knockdown on THP-mediated 4T1 anticancer activity by CCK8, TUNEL, flow cytometry, wound healing assay, Transwell, Ca2+ , real time quantitative PCR (RT-qPCR) and Western blot. The results showed that Miat expression was higher in 4T1 mouse breast cancer cells than in HC11 mouse mammary epithelial cells, while THP decreased Miat expression in 4T1. Miat knockdown in combination with further reduced cell viability, promoted apoptosis and inhibited migration compared to THP alone. This may be related to the reduction of calcium ions in 4T1. In conclusion, Miat knockdown enhanced the sensitivity of THP to 4T1 by inhibiting calcium channels.

Development of Highly Sensitive Anti-Mouse HER2 Monoclonal Antibodies for Flow Cytometry

Overexpression of human epidermal growth factor receptor 2 (HER2) in breast cancer is an important target of monoclonal antibody (mAb) therapy such as trastuzumab. Due to the development of trastuzumab–deruxtecan, an antibody-drug conjugate, the targetable HER2-positive breast cancer patients have been expanded. To evaluate the developing modalities using anti-HER2 mAbs, reliable preclinical mouse models are required. Therefore, sensitive mAbs against mouse HER2 (mHER2) should be established. This study developed anti-mHER2 mAbs using the Cell-Based Immunization and Screening (CBIS) method. The established anti-mHER2 mAbs, H2Mab-300 (rat IgG2b, kappa) and H2Mab-304 (rat IgG1, kappa), reacted with mHER2-overexpressed Chinese hamster ovary-K1 (CHO/mHER2) and endogenously mHER2-expressed cell line, NMuMG (a mouse mammary gland epithelial cell) via flow cytometry. Furthermore, these mAbs never recognized mHER2-knockout NMuMG cells. The kinetic analysis using flow cytometry indicated that the dissociation constant (KD) values of H2Mab-300 and H2Mab-304 for CHO/mHER2 were 1.2 × 10−9 M and 1.7 × 10−9 M, respectively. The KD values of H2Mab-300 and H2Mab-304 for NMuMG were 4.9 × 10−10 M and 9.0 × 10−10 M, respectively. These results indicated that H2Mab-300 and H2Mab-304 could apply to the detection of mHER2 using flow cytometry and may be useful to obtain the proof of concept in preclinical studies.

Leucine and arginine enhance milk fat and milk protein synthesis via the CaSR/Gi/mTORC1 and CaSR/Gq/mTORC1 pathways.

Amino acids (AAs) not only constitute milk protein but also stimulate milk synthesis through the activation of mTORC1 signaling, but which amino acids that have the greatest impact on milk fat and protein synthesis is still very limited. In this study, we aimed to identify the most critical AAs involved in the regulation of milk synthesis and clarify how these AAs regulate milk synthesis through the G-protein-coupled receptors (GPCRs) signaling pathway. In this study, a mouse mammary epithelial cell line (HC11) and porcine mammary epithelial cells (PMECs) were selected as study subjects. After treatment with different AAs, the amount of milk protein and milk fat synthesis were detected. Activation of mTORC1 and GPCRs signaling induced by AAs was also investigated. In this study, we demonstrate that essential amino acids (EAAs) are crucial to promote lactation by increasing the expression of genes and proteins related to milk synthesis, such as ACACA, FABP4, DGAT1, SREBP1, α-casein, β-casein, and WAP in HC11 cells and PMECs. In addition to activating mTORC1, EAAs uniquely regulate the expression of calcium-sensing receptor (CaSR) among all amino-acid-responsive GPCRs, which indicates a potential link between CaSR and the mTORC1 pathway in mammary gland epithelial cells. Compared with other EAAs, leucine and arginine had the greatest capacity to trigger GPCRs (p-ERK) and mTORC1 (p-S6K1) signaling in HC11 cells. In addition, CaSR and its downstream G proteins Gi, Gq, and Gβγ are involved in the regulation of leucine- and arginine-induced milk synthesis and mTORC1 activation. Taken together, our data suggest that leucine and arginine can efficiently trigger milk synthesis through the CaSR/Gi/mTORC1 and CaSR/Gq/mTORC1 pathways. We found that the G-protein-coupled receptor CaSR is an important amino acid sensor in mammary epithelial cells. Leucine and arginine promote milk synthesis partially through the CaSR/Gi/mTORC1 and CaSR/Gq/mTORC1 signaling systems in mammary gland epithelial cells. Although this mechanism needs further verification, it is foreseeable that this mechanism may provide new insights into the regulation of milk synthesis.

EMab-300 Detects Mouse Epidermal Growth Factor Receptor-Expressing Cancer Cell Lines in Flow Cytometry.

Epidermal Growth Factor Receptor (EGFR) overexpression or its mutation mediates the sustaining proliferative signaling, which is an important hallmark of cancer. Human EGFR-targeting monoclonal antibody (mAb) therapy such as cetuximab has been approved for clinical use in patients with colorectal cancers and head and neck squamous cell carcinomas. A reliable preclinical mouse model is essential to further develop the mAb therapy against EGFR. Therefore, sensitive mAbs against mouse EGFR (mEGFR) should be established. In this study, we developed a specific and sensitive mAb for mEGFR using the Cell-Based Immunization and Screening (CBIS) method. The established anti-mEGFR mAb, EMab-300 (rat IgG1, kappa), reacted with mEGFR-overexpressed Chinese hamster ovary-K1 (CHO/mEGFR) and endogenously mEGFR-expressed cell lines, including NMuMG (a mouse mammary gland epithelial cell) and Lewis lung carcinoma cells, using flow cytometry. The kinetic analysis using flow cytometry indicated that the KD of EMab-300 for CHO/mEGFR and NMuMG was 4.3 × 10-8 M and 1.9 × 10-8 M, respectively. These results indicated that EMab-300 applies to the detection of mEGFR using flow cytometry and may be useful to obtain the proof of concept in preclinical studies.

Global Down-regulation of Gene Expression Induced by Mouse Mammary Tumor Virus (MMTV) in Normal Mammary Epithelial Cells.

Mouse mammary tumor virus (MMTV) is a betaretrovirus that causes breast cancer in mice. The mouse mammary epithelial cells are the most permissive cells for MMTV, expressing the highest levels of virus upon infection and being the ones later transformed by the virus due to repeated rounds of infection/superinfection and integration, leading eventually to mammary tumors. The aim of this study was to identify genes and molecular pathways dysregulated by MMTV expression in mammary epithelial cells. Towards this end, mRNAseq was performed on normal mouse mammary epithelial cells stably expressing MMTV, and expression of host genes was analyzed compared with cells in its absence. The identified differentially expressed genes (DEGs) were grouped on the basis of gene ontology and relevant molecular pathways. Bioinformatics analysis identified 12 hub genes, of which 4 were up-regulated (Angp2, Ccl2, Icam, and Myc) and 8 were down-regulated (Acta2, Cd34, Col1a1, Col1a2, Cxcl12, Eln, Igf1, and Itgam) upon MMTV expression. Further screening of these DEGs showed their involvement in many diseases, especially in breast cancer progression when compared with available data. Gene Set Enrichment Analysis (GSEA) identified 31 molecular pathways dysregulated upon MMTV expression, amongst which the PI3-AKT-mTOR was observed to be the central pathway down-regulated by MMTV. Many of the DEGs and 6 of the 12 hub genes identified in this study showed expression profile similar to that observed in the PyMT mouse model of breast cancer, especially during tumor progression. Interestingly, a global down-regulation of gene expression was observed, where nearly 74% of the DEGs in HC11 cells were repressed by MMTV expression, an observation similar to what was observed in the PyMT mouse model during tumor progression, from hyperplasia to adenoma to early and late carcinomas. Comparison of our results with the Wnt1 mouse model revealed further insights into how MMTV expression could lead to activation of the Wnt1 pathway independent of insertional mutagenesis. Thus, the key pathways, DEGs, and hub genes identified in this study can provide important clues to elucidate the molecular mechanisms involved in MMTV replication, escape from cellular anti-viral response, and potential to cause cell transformation. These data also validate the use of the MMTV-infected HC11 cells as an important model to study early transcriptional changes that could lead to mammary cell transformation.

Influence of Simulated Microgravity on Mammary Epithelial Cells Grown as 2D and 3D Cultures.

During space travel, astronauts will experience a unique environment that includes continuous exposure to microgravity and stressful living conditions. Physiological adaptation to this is a challenge and the effect of microgravity on organ development, architecture, and function is not well understood. How microgravity may impact the growth and development of an organ is an important issue, especially as space flight becomes more commonplace. In this work, we sought to address fundamental questions regarding microgravity using mouse mammary epithelial cells in 2D and 3D tissue cultures exposed to simulated microgravity. Mouse mammary HC11 cells contain a higher proportion of stem cells and were also used to investigate how simulated microgravity may impact mammary stem cell populations. In these studies, we exposed mouse mammary epithelial cells to simulated microgravity in 2D and then assayed for changes in cellular characteristics and damage levels. The microgravity treated cells were also cultured in 3D to form acini structures to define if simulated microgravity affects the cells' ability to organize correctly, a quality that is of key importance for mammary organ development. These studies identify changes occurring during exposure to microgravity that impact cellular characteristics such as cell size, cell cycle profiles, and levels of DNA damage. In addition, changes in the percentage of cells revealing various stem cell profiles were observed following simulated microgravity exposure. In summary, this work suggests microgravity may cause aberrant changes in mammary epithelial cells that lead to an increase in cancer risk.

Comparative transcriptomic analysis of mammary gland tissues reveals the critical role of GPR110 in palmitic acid-stimulated milk protein and fat synthesis.

The G protein-coupled receptors (GPCR) sensing nutritional signals (amino acids, fatty acids, glucose, etc.) are not fully understood. In this research, we used transcriptome sequencing to analyse differentially expressed genes (DEG) in mouse mammary gland tissues at puberty, lactation and involution stages, in which eight GPCR were selected out and verified by qRT-PCR assay. It was further identified the role of GPR110-mediating nutrients including palmitic acid (PA) and methionine (Met) to improve milk synthesis using mouse mammary epithelial cell line HC11. PA but not Met affected GPR110 expression in a dose-dependent manner. GPR110 knockdown decreased milk protein and fat synthesis and cell proliferation and blocked the stimulation of PA on mechanistic target of rapamycin (mTOR) phosphorylation and sterol-regulatory element binding protein 1c (SREBP-1c) expression. In summary, these experimental results disclose DEG related to lactation and reveal that GPR110 mediates PA to activate the mTOR and SREBP-1c pathways to promote milk protein and fat synthesis.

Sodium acetate regulates milk fat synthesis through the activation of GPR41/GPR43 signaling pathway.

Fat is a critical component in milk, which provided energy for the early growth and development of mammals. Milk fat is positively related to the concentration of acetate in the blood, while the underlying mechanism is still unclear. This study is to investigate the effects of sodium acetate (NaAc) on milk fat synthesis in the mammary gland, and explored the underlying mechanism. In vitro experiments were carried out in mouse mammary epithelial cell line (HC11) cells cultured with NaAc to explore the potential pathway of NaAc on milk fat synthesis. Furthermore, 24 pregnant mice (from d 18.5 of gestation to d 7 of lactation, exposed to 200 mM NaAc drinking water) were used as an in vivo model to verify the results. In this study, we found that NaAc promoted milk fat synthesis and the expression of related genes and proteins in HC11 mammary epithelial cells with the activation of GPCR and mTORC1 signaling pathways (p < 0.05). Pretreatment with the mTORC1 inhibitors and G protein inhibitors attenuated the NaAc-induced milk fat synthesis in HC11 mammary epithelial cells (p < 0.05). Importantly, the effect of NaAc on milk synthesis was attenuated in GPR41 and GPR43 knockdown HC11 mammary epithelial cells (p < 0.05). This evidence indicates that NaAc might regulate milk fat synthesis through the GPR41/GPR43-mTORC1 pathway. Consistently, in in vivo experiment, dietary supplementation with NaAc significantly increased milk fat content and fat synthesis-related proteins in mice mammary glands with the activation of mTORC1 and GPCR signaling pathways at peak lactation (p < 0.05). The addition of NaAc promoted the increase of milk fat synthesis in HC11 mammary epithelial cells and mice mammary glands at peak lactation. Mechanistically, NaAc activates GPR41 and GPR43 receptors, leading to the activation of the mTORC1 signaling pathway to promote the synthesis of milk fat.Graphical abstract.

ARID3A plays a key regulatory role in palmitic acid-stimulated milk fat synthesis in mouse mammary epithelial cells.

Palmitic acid (PA) can stimulate milk fat synthesis in mammary gland, but the specific mechanism is still unclear. In our research, we aim to explore the role and corresponding mechanism of AT-rich interaction domain 3A (ARID3A) in milk fat synthesis stimulated by PA. We found that ARID3A protein level in mouse mammary gland tissues during lactation was much higher than that during puberty and involution. ARID3A knockdown and gene activation showed that ARID3A stimulated the synthesis of triglycerides and cholesterol in HC11 cells, secretion of free fatty acids from cells and lipid droplet formation in cells. ARID3A also promoted the expression and maturation of SREBP1 in HC11 cells. PA stimulated ARID3A protein expression and SREBP1 expression and maturation in a dose-dependent manner, and the PI3K specific inhibitor LY294002 blocked the stimulation of PA on ARID3A expression. ARID3A knockdown blocked the stimulation of PA on SREBP1 protein expression and maturation. We further showed that ARID3A was localized in the nucleus and PA stimulated this localization, and ARID3A knockdown blocked the stimulation of PA on the mRNA expression of SREBP1. To sum up, our data reveal that ARID3A is a key mediator for PA to promote SREBP1 mRNA expression and stimulate milk fat synthesis in mammary epithelial cells.

In Vivo Gene Delivery into Mouse Mammary Epithelial Cells Through Mammary Intraductal Injection.

Mouse mammary glands comprise ductal trees, which are lined by epithelial cells and have one opening at the tip of each nipple. The epithelial cells play a major role in mammary gland function and are the origin of most mammary tumors. Introducing genes of interest into mouse mammary epithelial cells is a critical step in evaluating gene function in epithelial cells and generating mouse mammary tumor models. This goal can be accomplished through the intraductal injection of a viral vector carrying the genes of interest into the mouse mammary ductal tree. The injected virus subsequently infects mammary epithelial cells, bringing in the genes of interest. The viral vector can be lentiviral, retroviral, adenoviral, or adenovirus-associated viral (AAV). This study demonstrates how a gene of interest is delivered into mammary epithelial cells through mouse mammary intraductal injection of a viral vector. A lentivirus carrying GFP is used to show stable expression of a delivered gene, and a retrovirus carrying Erbb2 (HER2/Neu) is used to demonstrate oncogene-induced atypical hyperplastic lesions and mammary tumors.

Gut microbiota-mediated secondary bile acid alleviates Staphylococcus aureus-induced mastitis through the TGR5-cAMP-PKA-NF-κB/NLRP3 pathways in mice

Although emerging evidence shows that gut microbiota-mediated metabolic changes regulate intestinal pathogen invasions, little is known about whether and how gut microbiota-mediated metabolites affect pathogen infection in the distal organs. In this study, untargeted metabolomics was performed to identify the metabolic changes in a subacute ruminal acidosis (SARA)-associated mastitis model, a mastitis model with increased susceptibility to Staphylococcus aureus (S. aureus). The results showed that cows with SARA had reduced cholic acid (CA) and deoxycholic acid (DCA) levels compared to healthy cows. Treatment of mice with DCA, but not CA, alleviated S. aureus-induced mastitis by improving inflammation and the blood-milk barrier integrity in mice. DCA inhibited the activation of NF-κB and NLRP3 signatures caused by S. aureus in the mouse mammary epithelial cells, which was involved in the activation of TGR5. DCA-mediated TGR5 activation inhibited the NF-κB and NLRP3 pathways and mastitis caused by S. aureus via activating cAMP and PKA. Moreover, gut-dysbiotic mice had impaired TGR5 activation and aggravated S. aureus-induced mastitis, while restoring TGR5 activation by spore-forming bacteria reversed these changes. Furthermore, supplementation of mice with secondary bile acids producer Clostridium scindens also activated TGR5 and alleviated S. aureus-induced mastitis in mice. These results suggest that impaired secondary bile acid production by gut dysbiosis facilitates the development of S. aureus-induced mastitis and highlight a potential strategy for the intervention of distal infection by regulating gut microbial metabolism.

ARID1B blocks methionine-stimulated mTOR activation to inhibit milk fat and protein synthesis in and proliferation of mouse mammary epithelial cells

Met can function through the mTOR signaling pathway, but the molecular mechanism is not fully understood. Here we investigated the role of ARID1B in this regulatory process. ARID1B knockdown promoted milk fat and protein synthesis in and cell proliferation of HC11 cells and increased mTOR mRNA expression and protein phosphorylation, whereas ARID1B gene activation had the opposite effects. ARID1B gene activation totally blocked Met's stimulation on mTOR mRNA expression. ARID1B bound to one region of the mTOR promoter, and Met reduced the binding of ARID1B on this promoter. LY294002 blocked Met-induced reduction of ARID1B mRNA and protein level. Cycloheximide treatment did not affect the decrease of ARID1B by Met. MG132 but not chloroquine restored ARID1B degradation induced by Met. Our data reveal that ARID1B is a key negative regulator of milk fat and protein synthesis in and proliferation of HC11 cells, and blocks Met-stimulated mTOR gene transcription.

Niacin/β-hydroxybutyrate regulates milk fat and milk protein synthesis via the GPR109A/Gi/mTORC1 pathway.

As a crucial receptor of BHBA and niacin, GPR109A is largely expressed in the mammary gland. However, the role of GPR109A in milk synthesis and its underlying mechanism is still largely unknown. In this study, we first investigated the effect of GPR109A agonists (niacin/BHBA) on milk fat and milk protein synthesis in a mouse mammary epithelial cell line (HC11) and PMECs (porcine mammary epithelial cells). The results showed that both niacin and BHBA promote milk fat and milk protein synthesis with the activation of mTORC1 signaling. Importantly, knockdown GPR109A attenuated the niacin-induced increase of milk fat and protein synthesis and the niacin-induced activation of mTORC1 signaling. Furthermore, we found that GPR109A downstream G protein-Gαi and -Gβγ participated in the regulation of milk synthesis and the activation of mTORC1 signaling. Consistent with the finding in vitro, dietary supplementation with niacin increases milk fat and protein synthesis in mice with the activation of GPR109A-mTORC1 signaling. Collectively, GPR109A agonists promote the synthesis of milk fat and milk protein through the GPR109A/Gi/mTORC1 signaling pathway.

Chromatin remodeler BRM is a key mediator of leucine-stimulated mTOR gene transcription in mouse mammary epithelial cells

Brahma (BRM) is one of the core ATPase subunits of SWI/SNF chromatin remodeling complex, and participates in various important cellular regulatory processes. However, the role of BRM in regulating gene expression of the mechanistic target of rapamycin (mTOR) still remains unknown. In this study, we explored the effects and the corresponding molecular mechanisms of BRM on Leucine (Leu)-stimulated mTOR activation in and proliferation of a mouse mammary epithelial cell (MEC) line (HC11 cell). Initially, we found that the abundance of BRM protein in mammary gland tissue during lactation was significantly higher than that during puberty and involution. BRM knockdown inhibited HC11 cell proliferation, mRNA expression of mTOR and subsequent protein phosphorylation, whereas BRM gene activation had the opposite effect. Leu affected the level of BRM protein and mTOR phospphorylation in a dose-dependent manner, and BRM knockdown totally blocked the stimulation of Leu on mTOR mRNA expression and protein phospphorylation. ChIP-PCR detected that BRM was bound to the −4368 ∼ −4591 bp site of the mTOR promoter, and ChIP-qPCR further detected that Leu stimulated BRM to bind to this site. In conclusion, these data reveal that BRM is a positive regulator of HC11 cell proliferation and mediates Leu's stimulation on mTOR gene transcription and protein phosphorylation. Our data provide a new theoretical basis for the involvement of BRM in cell proliferation and regulation of the mTOR signaling pathway.

ST3GAL5‐catalyzed gangliosides inhibit TGF‐β‐induced epithelial‐mesenchymal transition via TβRI degradation

Epithelial‐mesenchymal transition (EMT) is pivotal in the initiation and development of cancer cell metastasis. We observed that the abundance of glycosphingolipids (GSLs), especially ganglioside subtypes, decreased significantly during TGF‐β‐induced EMT in NMuMG mouse mammary epithelial cells and A549 human lung adenocarcinoma cells. Transcriptional profiling showed that TGF‐β/SMAD response genes and EMT signatures were strongly enriched in NMuMG cells, along with depletion of UDP‐glucose ceramide glucosyltransferase (UGCG), the enzyme that catalyzes the initial step in GSL biosynthesis. Consistent with this finding, genetic or pharmacological inhibition of UGCG promoted TGF‐β signaling and TGF‐β‐induced EMT. UGCG inhibition promoted A549 cell migration, extravasation in the zebrafish xenograft model, and metastasis in mice. Mechanistically, GSLs inhibited TGF‐β signaling by promoting lipid raft localization of the TGF‐β type I receptor (TβRI) and by increasing TβRI ubiquitination and degradation. Importantly, we identified ST3GAL5‐synthesized a‐series gangliosides as the main GSL subtype involved in inhibition of TGF‐β signaling and TGF‐β‐induced EMT in A549 cells. Notably, ST3GAL5 is weakly expressed in lung cancer tissues compared to adjacent nonmalignant tissues, and its expression correlates with good prognosis.

Cul5 mediates taurine-stimulated mTOR mRNA expression and proliferation of mouse mammary epithelial cells.

Cullin5 (Cul5) protein can regulate multiple signaling pathways; however, it is still largely unknown the role and molecule mechanism of Cul5 in regulation of the mTOR signaling. In this study, we determined the effect of Cul5 on the proliferation of HC11 cells, a mouse mammary epithelial cell line, and explored the corresponding molecular mechanism. We found that Cul5 was highly expressed in mammary gland tissues in the lactation stage compared with that in puberty and involution. Using gene knockdown and activation methods, we showed that Cul5 promoted proliferation of HC11 cells, mRNA expression and protein phosphorylation of mTOR. Taurine (Tau) affected Cul5 mRNA and protein levels in a dose-dependent manner. Cul5 localized to the nucleus and knockdown of Cul5 almost totally blocked the stimulation of Tau on mTOR mRNA expression and protein phosphorylation. PI3K inhibition almost totally abolished the stimulation of Tau on Cul5 expression. In summary, our data uncover that Cul5 is a positive regulator of proliferation of HC11 cells, and mediates the stimulation of Tau on mRNA expression and subsequent protein phosphorylation of mTOR. Our data lay a new theoretical foundation for regulating mammary cell proliferation and promoting milk yield.

Modulation of Milk and Lipid Synthesis and Secretion in a3-Dimensional Mouse Mammary Epithelial Cell Culture Model: Effects of Palmitate and Orlistat.

Human milk synthesis is impacted by maternal diet, serum composition, and substrate uptake and synthesis by mammary epithelial cells (MECs). The milk of obese/high-fat-diet women has an increased fat content, which promote excess infant weight gain and the risk of childhood/adult obesity. Yet, the knowledge of milk synthesis regulation is limited, and there are no established approaches to modulate human milk composition. We established a 3-dimensional mouse MEC primary culture that recreates the milk production pathway and tested the effects of the major saturated fatty acid in human milk (palmitate) and a lipoprotein lipase inhibitor (orlistat) on triglyceride production. Positive immunostaining confirmed the presence of milk protein and intracellular lipid including milk globules in the cytoplasm and extracellular space. The treatment with palmitate activated "milk" production by MECs (β-casein) and the lipid pathway (as evident by increased protein and mRNA expression). Consistent with these cellular changes, there was increased secretion of milk protein and triglyceride in MEC "milk". The treatment with orlistat suppressed milk triglyceride production. Palmitate increased milk and lipid synthesis, partly via lipoprotein lipase activation. These findings demonstrate the ability to examine MEC pathways of milk production via both protein and mRNA and to modulate select pathways regulating milk composition in MEC culture.

Sanguinarine Enhances the Integrity of the Blood-Milk Barrier and Inhibits Oxidative Stress in Lipopolysaccharide-Stimulated Mastitis.

Mastitis is a common clinical disease which threatens the welfare and health of dairy cows and causes huge economic losses. Sanguinarine (SG) is a plant-derived alkaloid which has many biological functions, including antibacterial and antioxidant properties. The present study attempted to evaluate the effect of SG on lipopolysaccharide (LPS)-induced oxidative stress reactions and explore its potential mechanisms. The expression profile of SG was analyzed by network pharmacology, and it was found that differentially expressed genes were mainly involved in the Wnt signaling pathway and oxidative stress through GO and KEGG enrichment. In in vitro experiments, the dosage of SG was non-toxic to mouse mammary epithelial cells (mMECs) (p &gt; 0.05). SG not only inhibited the increase in ROS induced by LPS, but also enhanced the activity of antioxidant enzymes (p &lt; 0.05). Moreover, the results of the in vivo experiments showed that SG alleviated LPS-induced inflammatory damage of mouse mammary glands and enhanced the integrity of the blood-milk barrier (p &lt; 0.05). Further studies suggested that SG promoted Nrf2 expression and suppressed the activation of the Wnt signaling pathway (p &lt; 0.05). Conclusively, this study clarified the protective effect of SG on mastitis and provided evidence for new potential mechanisms. SG exerted its antioxidant function through activating Nrf2 and inhibiting the Wnt/β-catenin pathway, repairing the blood-milk barrier.

Early effects of lipoteichoic acid from Staphylococcus aureus on milk production-related signaling pathways in mouse mammary epithelial cells

Staphylococcus aureus causes subclinical mastitis; lipoteichoic acid (LTA) from S. aureus causes mastitis-like adverse effects on milk production by mammary epithelial cells (MECs). Here, we investigated the early effects of LTA from S. aureus on mouse MECs using a culture model, in which MECs produced milk components and formed less permeable tight junctions (TJs). In MECs of this model, Toll-like receptor 2 (receptor for LTA), was localized on the apical membrane, similar to MECs in lactating mammary glands. LTA weakened the TJ barrier within 1 h, concurrently with localization changes of claudin 4. LTA treatment for 24 h increased αS1-casein and decreased β-casein levels. In MECs exposed to LTA, the activation level of signal transducer and activator of transcription 5 (major transcriptional factor for milk production) was low. LTA activated signaling pathways related to cell survival (extracellular signal‐regulated kinase, heat shock protein 27, and Akt) and inflammation (p38, c-Jun N-terminal kinase, and nuclear factor κB). Thus, LTA caused abnormalities in casein production and weakened the TJs by affecting multiple signaling pathways in MECs. LTA-induced changes in signaling pathways were not uniform in all MECs. Such complex and semi-negative actions of LTA may contribute to subclinical mastitis caused by S. aureus.

Identification of lysophosphatidic acid in serum as a factor that promotes epithelial apical junctional complex organization

The apical junctional complex (AJC) consists of adherens junctions (AJs) and tight junctions and regulates epithelial integrity and remodeling. However, it is unclear how AJC organization is regulated based on environmental cues. We found here using cultured EpH4 mouse mammary epithelial cells that fetal bovine serum (FBS) in a culture medium showed an activity to promote AJC organization and that FBS showed an activity to promote tight junction formation even in the absence of AJ proteins, such as E-cadherin, αE-catenin, and afadin. Furthermore, we purified the individual factor responsible for these functions from FBS and identified this molecule as lysophosphatidic acid (LPA). In validation experiments, purified LPA elicited the same activity as FBS. In addition, we found that the AJC organization–promoting activity of LPA was mediated through the LPA receptor 1/5 via diacylglycerol–novel PKC and Rho–ROCK pathway activation in a mutually independent, but complementary, manner. We demonstrated that the Rho–ROCK pathway activation–mediated AJC organization was independent of myosin II-induced actomyosin contraction, although this signaling pathway was previously shown to induce myosin II activation. These findings are in contrast to the literature, as previous results suggested an AJC organization–disrupting activity of LPA. The present results indicate that LPA in serum has an AJC organization–promoting activity in a manner dependent on or independent of AJ proteins.