Biological characteristics of mechanosensitive channels MscS and MscL in Actinobacillus pleuropneumoniae.

Actinobacillus pleuropneumoniae is an important respiratory pathogen that can cause porcine contagious pleuropneumonia (PCP), resulting in significant economic losses in swine industry. Microorganisms are subjected to drastic changes in environmental osmolarity. In order to alleviate the drastic rise or fall of osmolarity, cells activate mechanosensitive channels MscL and MscS through tension changes. MscL not only regulates osmotic pressure but also has been reported to secrete protein and uptake aminoglycoside antibiotic. However, MscL and MscS, as the most common mechanosensitive channels, have not been characterized in A. pleuropneumoniae. In this study, the osmotic shock assay showed that MscL increased sodium adaptation by regulating cell length. The results of MIC showed that deletion of mscL decreased the sensitivity of A. pleuropneumoniae to multiple antibiotics, while deletion of mscS rendered A. pleuropneumoniae hypersensitive to penicillin. Biofilm assay demonstrated that MscL contributed the biofilm formation but MscS did not. The results of animal assay showed that MscL and MscS did not affect virulence in vivo. In conclusion, MscL is essential for sodium hyperosmotic tolerance, biofilm formation, and resistance to chloramphenicol, erythromycin, penicillin, and oxacillin. On the other hand, MscS is only involved in oxacillin resistance.IMPORTANCEBacterial resistance to the external environment is a critical function that ensures the normal growth of bacteria. MscL and MscS play crucial roles in responding to changes in both external and internal environments. However, the function of MscL and MscS in Actinobacillus pleuropneumoniae has not yet been reported. Our study shows that MscL plays a significant role in osmotic adaptation, antibiotic resistance, and biofilm formation of A. pleuropneumoniae, while MscS only plays a role in antibiotic resistance. Our findings provide new insights into the functional characteristics of MscL and MscS in A. pleuropneumoniae. MscL and MscS play a role in antibiotic resistance and contribute to the development of antibiotics for A. pleuropneumoniae.

RNA sequencing screening and gene function analysis uncover G protein-coupled receptor 183 as a key mediator for methionine to stimulate milk synthesis in mouse mammary epithelial cells.

Methionine (Met) can activate the mechanistic target of rapamycin (mTOR) to promote milk synthesis in mammary epithelial cells. However, it is largely unknown which G protein-coupled receptor can mediate the stimulation of Met on mTOR activation. In this study, we employed transcriptome sequencing to analyse which G protein-coupled receptors were associated with the role of Met and further used gene function study approaches to explore the role of G protein-coupled receptor 183 (GPR183) in Met stimulation on mTOR activation in HC11 cells. We identified nine G protein-coupled receptors including GPR183 whose expression levels were upregulated by Met treatment through RNA sequencing and subsequent quantitative real-time PCR analysis. Using GPR183 knockdown and overexpression technology, we demonstrate that GPR183 is a positive regulator of milk protein and fat synthesis and proliferation of HC11 cells. Met affected GPR183 expression in a dose-dependent manner, and GPR183 mediated the stimulation of Met (0·6 mM) on milk protein and fat synthesis, cell proliferation and mTOR phosphorylation and mRNA expression. The inhibition of phosphoinositide 3-kinase blocked the phosphorylation of mTOR and AKT stimulated by GPR183 activation. In summary, through RNA sequencing and gene function study, we uncover that GPR183 is a key mediator for Met to activate the phosphoinositide 3-kinase-mTOR signalling and milk synthesis in mouse mammary epithelial cells.

Testing the Significance of Ranked Gene Sets in Genome-wide Transcriptome Profiling Data Using Weighted Rank Correlation Statistics.

Background: Popular gene set enrichment analysis approaches assumed that genes in the gene set contributed to the statistics equally. However, the genes in the transcription factors (TFs) derived gene sets, or gene sets constructed by TF targets identified by the ChIP-Seq experiment, have a rank attribute, as each of these genes have been assigned with a p-value which indicates the true or false possibilities of the ownerships of the genes belong to the gene sets. Objectives: Ignoring the rank information during the enrichment analysis will lead to improper statistical inference. We address this issue by developing of new method to test the significance of ranked gene sets in genome-wide transcriptome profiling data. Methods: A method was proposed by first creating ranked gene sets and gene lists and then applying weighted Kendall's tau rank correlation statistics to the test. After introducing top-down weights to the genes in the gene set, a new software called "Flaver" was developed. Results: Theoretical properties of the proposed method were established, and its differences over the GSEA approach were demonstrated when analyzing the transcriptome profiling data across 55 human tissues and 176 human cell-lines. The results indicated that the TFs identified by our method have higher tendency to be differentially expressed across the tissues analyzed than its competitors. It significantly outperforms the well-known gene set enrichment analyzing tools, GOStats (9%) and GSEA (17%), in analyzing well-documented human RNA transcriptome datasets. Conclusions: The method is outstanding in detecting gene sets of which the gene ranks were correlated with the expression levels of the genes in the transcriptome data.

Research on the Interaction Mechanism and Structural Changes in Human Serum Albumin with Hispidin Using Spectroscopy and Molecular Docking.

The interaction between human serum albumin (HSA) and hispidin, a polyketide abundantly present in both edible and therapeutic mushrooms, was explored through multispectral methods, hydrophobic probe assays, location competition trials, and molecular docking simulations. The results of fluorescence quenching analysis showed that hispidin quenched the fluorescence of HSA by binding to it via a static mechanism. The binding of hispidin and HSA was validated further by synchronous fluorescence, three-dimensional fluorescence, and UV/vis spectroscopy analysis. The apparent binding constant (Ka) at different temperatures, the binding site number (n), the quenching constants (Ksv), the dimolecular quenching rate constants (Kq), and the thermodynamic parameters (∆G, ∆H, and ∆S) were calculated. Among these parameters, ∆H and ∆S were determined to be 98.75 kJ/mol and 426.29 J/(mol·K), respectively, both exhibiting positive values. This observation suggested a predominant contribution of hydrophobic forces in the interaction between hispidin and HSA. By employing detergents (SDS and urea) and hydrophobic probes (ANS), it became feasible to quantify alterations in Ka and surface hydrophobicity, respectively. These measurements confirmed the pivotal role of hydrophobic forces in steering the interaction between hispidin and HSA. Site competition experiments showed that there was an interaction between hispidin and HSA molecules at site I, which situates the IIA domains of HSA, which was further confirmed by the molecular docking simulation.

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 âˆ¼ -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.

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.

Identification of upstream transcription factor binding sites in orthologous genes using mixed Student's t-test statistics.

BACKGROUND: Transcription factor (TF) regulates the transcription of DNA to messenger RNA by binding to upstream sequence motifs. Identifying the locations of known motifs in whole genomes is computationally intensive. METHODOLOGY/PRINCIPAL FINDINGS: This study presents a computational tool, named "Grit", for screening TF-binding sites (TFBS) by coordinating transcription factors to their promoter sequences in orthologous genes. This tool employs a newly developed mixed Student's t-test statistical method that detects high-scoring binding sites utilizing conservation information among species. The program performs sequence scanning at a rate of 3.2 Mbp/s on a quad-core Amazon server and has been benchmarked by the well-established ChIP-Seq datasets, putting Grit amongst the top-ranked TFBS predictors. It significantly outperforms the well-known transcription factor motif scanning tools, Pscan (4.8%) and FIMO (17.8%), in analyzing well-documented ChIP-Atlas human genome Chip-Seq datasets. SIGNIFICANCE: Grit is a good alternative to current available motif scanning tools.

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.

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.

Met stimulates ARID1A degradation and activation of the PI3K-SREBP1 signaling to promote milk fat synthesis in bovine mammary epithelial cells.

Methionine (Met) can promote milk fat synthesis in bovine mammary epithelial cells (BMECs), but the potential molecular mechanism is largely unknown. In this report, we aim to explore the role and molecular mechanism of AT-rich interaction domain 1A (ARID1A) in milk fat synthesis stimulated by Met. ARID1A knockdown and activation indicated that ARID1A negatively regulated the synthesis of triglycerides, cholesterol and free fatty acids and the formation of lipid droplets in BMECs. ARID1A also negatively regulated the phosphorylation of PI3K and AKT proteins, as well as the expression and maturation of SREBP1. Met stimulated the phosphorylation of PI3K and AKT proteins, as well as the expression and maturation of SREBP1, while ARID1A gene activation blocked the stimulatory effects of Met. We further found that ARID1A was located in the nucleus of BMECs, and Met reduced the nuclear localization and expression of ARID1A. ARID1A gene activation blocked the stimulation of PI3K and SREBP1 mRNA expression by Met. In summary, our data suggests that ARID1A negatively regulates milk fat synthesis stimulated by Met in BMECs through inhibiting the PI3K-SREBP1 signaling pathway, which may provide some new perspectives for improving milk fat synthesis.

Immune-related effects of compound astragalus polysaccharide and sulfated epimedium polysaccharide on newborn piglets.

This study aimed to evaluate the immune effects of compound astragalus polysaccharide and sulfated epimedium polysaccharide (APS-sEPS) on the peripheral blood lymphocyte and intestinal mucosa in newborn piglets. A total of 40 newborn piglets were randomly divided into four groups during a 25-day experiment, including APS-sEPS, APS, sEPS and control group. The results showed that supplementation with APS-sEPS to newborn piglets remarkably increased the physiological parameters, especially the WBC. In peripheral blood, piglets that received APS-sEPS showed the highest proliferation of T lymphocytes, the percentage of CD3 + CD4+ and CD3 + CD8+ cells were the highest on days 15 and 25 (p < 0.05). The serum concentrations of IFN-γ on days 7 and 15, and IL-4, IL-10, sIgA on days 7, 15 and 25 in APS-sEPS group were significantly higher than those in the control group (p < 0.05). Furthermore, the villus length and the ratio of villus length to crypt depth in APS-sEPS group were both significantly increased compared to that of control group (p < 0.05). In the duodenum, jejunum and illume, the concentrations of IFN-γ, IL-10, total IgG and sIgA in APS-sEPS group were all significantly higher than that in control group (p < 0.05). In intestinal mucosa, APS-sEPS significantly increased the expression of NF-κB and IRF-3 mRNA in each section of small intestine of piglets. Nevertheless, in the illume segment, the effect of APS-sEPS was more significant than that of APS and sEPS (p < 0.05). The expression of TLR4 was more significant than that of control group in duodenum only. The results from the present research provide evidence that the suckling piglets administered with APS-sEPS supplement exhibited enhanced immune function of peripheral blood lymphocyte and expression of specific antibodies, and ameliorated intestinal morphological development and increased activities of humoral immune response in the small intestine, which would be related to the activation of the TLR4-NF-κB signaling pathway and IRF3.

Splicing mutations in AMELX and ENAM cause amelogenesis imperfecta.

BACKGROUND: Amelogenesis imperfecta (AI) is a developmental enamel defect affecting the structure of enamel, esthetic appearance, and the tooth masticatory function. Gene mutations are reported to be relevant to AI. However, the mechanism underlying AI caused by different mutations is still unclear. This study aimed to reveal the molecular pathogenesis in AI families with 2 novel pre-mRNA splicing mutations. METHODS: Two Chinese families with AI were recruited. Whole-exome sequencing and Sanger sequencing were performed to identify mutations in candidate genes. Minigene splicing assays were performed to analyze the mutation effects on mRNA splicing alteration. Furthermore, three-dimensional structures of mutant proteins were predicted by AlphaFold2 to evaluate the detrimental effect. RESULTS: The affected enamel in family 1 was thin, rough, and stained, which was diagnosed as hypoplastic-hypomature AI. Genomic analysis revealed a novel splicing mutation (NM_001142.2: c.570 + 1G > A) in the intron 6 of amelogenin (AMELX) gene in family 1, resulting in a partial intron 6 retention effect. The proband in family 2 exhibited a typical hypoplastic AI, and the splicing mutation (NM_031889.2: c.123 + 4 A > G) in the intron 4 of enamelin (ENAM) gene was observed in the proband and her father. This mutation led to exon 4 skipping. The predicted structures showed that there were obvious differences in the mutation proteins compared with wild type, leading to impaired function of mutant proteins. CONCLUSIONS: In this study, we identified two new splicing mutations in AMELX and ENAM genes, which cause hypoplastic-hypomature and hypoplastic AI, respectively. These results expand the spectrum of genes causing AI and broaden our understanding of molecular genetic pathology of enamel formation.

Role of macrophage polarisation in skin wound healing.

Skin wound healing is a complex pathophysiological change that is driven by macrophages and their secreted related factors. Depending on the stimuli, macrophages can be polarised into two subtypes of macrophages with completely different phenotypes and functions, namely M1 and M2. The aim of this study was to explore the role of M1 and M2 macrophages in skin healing in order to develop new drugs for the treatment of refractory wounds. Primary bone marrow-derived macrophages (BMDMs) were isolated from rats and expanded in vitro using macrophage colony stimulating factor. In addition, the BMDMs were polarised into the M1 and M2 subtypes using lipopolysaccharides (LPS) and interleukin-4 (IL-4), respectively. Cytokine levels in the culture supernatants were measured by an enzyme linked immunosorbent assay. Epidermal wounds were made on the dorsal surface of rats, and treated with M1 or M2 cell suspensions or phosphate buffered saline. Wound healing was recorded on days 1, 3, 7, 10, and 14 after stamping, and the wound healing rate was measured by haematoxylin-eosin and Masson staining. A total of 3 to 4 × 107 bone marrow cells were extracted from each rat femur. The BMDM culture had 87.1% CD45+ cells, 89.2% CD68+ cells, and 86.5% CD45+ CD68+ cells. Furthermore, IL-12 (P < .05) and IL-10 (P ≥ .05) levels, respectively, increased and decreased in the culture supernatants of the M1 cells after LPS stimulation compared with those in the M0 (unstimulated) group. Likewise, IL-4 stimulation led to a significant increase in IL-10 levels (P < .01) in the conditioned media of M2 cells, while that of IL-12 decreased slightly (P ≥ .05). In the rat model, the infusion of M2 cells accelerated wound healing and tissue regeneration, whereas the M1 cells delayed the recruitment of inflammatory cells, granulation growth, and collagen deposition, which impaired wound healing. Macrophage polarisation and activation are critical for skin wound healing. While exogenous M1 cell infusion delayed wound healing, the M2 cells promoted wound healing in a rat model.

METTL3 is a key regulator of milk synthesis in mammary epithelial cells.

The enzyme m6 A methyltransferase-like 3 (METTL3) catalyzes N6 -methyladenosine (m6 A) modification in eukaryotic messenger RNAs (mRNAs). However, the physiological function and molecular mechanism of METTL3 in mammalian cells have not been fully understood. Here we showed that METTL3 was highly expressed in mouse mammary gland of the lactation period. METTL3 was located in the nucleus of bovine mammary epithelial cells (MECs), and methionine (Met) and ß-estrodial (E2) upregulated METTL3 protein level. METTL3 knockdown decreased milk protein and fat synthesis, whereas its overexpression had the opposite effects. METTL3 overexpression stimulated mRNA expression and protein phosphorylation of the mechanistic target of rapamycin (mTOR) and mRNA and protein expression of sterol regulatory element binding protein 1 (SREBP1), whereas METTL3 knockdown blocked the stimulatory effects of Met and E2 on these processes. Furthermore, METTL3 overexpression led to increased mRNA m6 A methylation of mTOR and SREBP1, whereas METTL3 knockdown suppressed the stimulatory effects of Met and E2 on these processes. The interaction between METTL3 and glycyl-tRNA synthetase (GlyRS) was confirmed by Co-immunoprecipitation and fluorescence resonance energy transfer approaches, and colocalization observation further showed that Met and E2 treatment increased this interaction. GlyRS knockdown abolished METTL3 protein levels upregulated by Met and E2, and METTL3 knockdown markedly decreased the effects of GlyRS overexpression on mTOR expression and phosphorylation and SREBP1 expression. In summary, we demonstrate that METTL3 is a key positive regulator of Met and E2-stimulated and GlyRS-mediated mTOR and SREBP1 signaling pathways and milk protein and fat synthesis in mammary epithelial cells.

GPRC6A is a key mediator of palmitic acid regulation of lipid synthesis in bovine mammary epithelial cells.

Fatty acids (FAs) can promote lipid synthesis in the mammary gland via stimulating lipogenic gene expression, but the underlying molecular mechanism is still not fully understood. Here, we showed the dose-dependent effects of palmitic acid (PA) on lipid synthesis in primary bovine mammary epithelial cells (BMECs) and explored the corresponding molecular mechanism. BMECs were treated with PA (0, 50, 100, 150, and 200 µM), and the 100 µM treatment had the best stimulatory effect on lipid synthesis and expression and maturation of sterol regulatory element-binding protein 1c (SREBP-1c) in cells. Inhibition of phosphatidylinositol 3-kinase (PI3K) almost totally blocked the stimulation of PA on SREBP-1c expression, whereas protein kinase Cα (PKCα) knockdown only partially decreased the stimulation of PA on SREBP-1c expression but abolished the stimulation of PA on its maturation. Knockdown of GPR120 did not change the stimulation of PA on the SREBP-1c signaling. G protein-coupled receptor family C group 6 member A  (GPRC6A) knockdown almost totally blocked the stimulation of FA on PI3K and PKCα phosphorylation as well as SREBP-1c expression and maturation. Furthermore, PA dose-dependently promoted GPRC6A expression and plasma membrane localization. Together, these above results reveal that GPRC6A is a key mediator of PA signaling to lipid synthesis in BMECs via the PI3K/PKCα-SREBP-1c pathways.

Taurine stimulates protein synthesis and proliferation of C2C12 myoblast cells through the PI3K-ARID4B-mTOR pathway.

Taurine (Tau) has many profound physiological functions, but its role and molecular mechanism in muscle cells are still not fully understood. In this study, we investigated the role and underlying molecular mechanism of Tau on protein synthesis and proliferation of C2C12 myoblast cells. Cells were treated with Tau (0, 60, 120, 180 and 240 µM) for 24 h. Tau dose-dependently promoted protein synthesis, cell proliferation, mechanistic target of rapamycin protein (mTOR) phosphorylation and also AT-rich interaction domain 4B (ARID4B) expression, with the best stimulatory effects at 120 µM. LY 294002 treatment showed that Tau promoted ARID4B expression in a phosphoinositide 3-kinase (PI3K)-dependent manner. ARID4B knockdown (by small interfering RNA transfection for 24 h) prevented Tau from stimulating protein synthesis and cell proliferation, whereas ARID4B gene activation (using the CRISPR/dCas9 technology) had stimulatory effects. ARID4B knockdown abolished Tau signalling to mRNA expression and protein phosphorylation of mTOR, whereas ARID4B gene activation had stimulatory effects. Chromatin immunoprecipitation (ChIP)-PCR identified that all of ARID4B, H3K27ac and H3K27me3 bound to the -4368 to -4591 bp site in the mTOR promoter, and ChIP-quantitative PCR (qPCR) further detected that Tau stimulated ARID4B binding to this site. ARID4B knockdown or gene activation did not affect H3K27me3 binding to the mTOR promoter but decreased or increased H3K27ac binding, respectively. Furthermore, ARID4B knockdown abolished the stimulation of Tau on H3K27ac binding to the mTOR promoter. In summary, these data uncover that Tau promotes protein synthesis and proliferation of C2C12 myoblast cells through the PI3K-ARID4B-mTOR pathway, providing a deep understanding of how Tau regulates anabolism in muscle cells.

Isoleucine stimulates mTOR and SREBP-1c gene expression for milk synthesis in mammary epithelial cells through BRG1-mediated chromatin remodelling.

Several amino acids can stimulate milk synthesis in mammary epithelial cells (MEC); however, the regulatory role of isoleucine (Ile) and underlying molecular mechanism remain poorly understood. In this study, we aimed to evaluate the regulatory effects of Ile on milk protein and fat synthesis in MEC and reveal the mediation mechanism of Brahma-related gene 1 (BRG1) on this regulation. Ile dose dependently affected milk protein and fat synthesis, mechanistic target of rapamycin (mTOR) phosphorylation, sterol regulatory element binding protein 1c (SREBP-1c) expression and maturation, and BRG1 protein expression in bovine MEC. Phosphatidylinositol 3 kinase (PI3K) inhibition by LY294002 treatment blocked the stimulation of Ile on BRG1 expression. BRG1 knockdown and gene activation experiments showed that it mediated the stimulation of Ile on milk protein and fat synthesis, mTOR phosphorylation, and SREBP-1c expression and maturation in MEC. ChIP-PCR analysis detected that BRG1 bound to the promoters of mTOR and SREBP-1c, and ChIP-qPCR further detected that these bindings were increased by Ile stimulation. In addition, BRG1 positively regulated the binding of H3K27ac to these two promoters, while it negatively affected the binding of H3K27me3 to these promoters. BRG1 knockdown blocked the stimulation of Ile on these two gene expressions. The expression of BRG1 was higher in mouse mammary gland in the lactating period, compared with that in the puberty or dry period. Taken together, these experimental data reveal that Ile stimulates milk protein and fat synthesis in MEC via the PI3K-BRG1-mTOR/SREBP-1c pathway.

Daidzein stimulates fatty acid-induced fat deposition in C2C12 myoblast cells via the G protein-coupled receptor 30 pathway.

Fat deposition in skeletal muscle is an important aspect of improving meat quality. Isoflavones can promote animal anabolism, but whether and how they regulate muscle fat deposition remain largely unclear. In this study, we explored the role and corresponding molecular mechanism of one of the major isoflavones, daidzein, in fat deposition in C2C12 myoblast cells. In the absence of fatty acids (FAs), daidzein did not promote triglyceride synthesis and lipid droplet formation in cells but increased sterol regulatory element-binding protein 1c (SREBP-1c) expression and maturation. In the presence of FAs, daidzein enhanced FAs-induced fat deposition and the SREBP-1c signaling. Daidzein promoted FAs-induced nuclear factor κB1 (NFκB1) phosphorylation and activated the SREBP-1c signaling in a PI3K-dependent manner. G protein-coupled receptor 30 (GPR30) knockdown but not estrogen receptor α (ERα) knockdown blocked the stimulation of daidzein on the PI3K-NFκB1-SREBP-1c signaling pathway, while both knockdown did not affect the stimulation of FAs on this signaling. qRT-PCR and ChIP-qPCR further detected that daidzein stimulated NFκB1-targeted SREBP-1c transcription. Daidzein did not affect ERα expression in cells, but it stimulated GPR30 expression and cytoplasmic localization. These results reveal that daidzein promotes FAs-induced fat deposition through the GPR30 signaling in C2C12 myoblast cells.

Daidzein promotes milk synthesis and proliferation of mammary epithelial cells via the estrogen receptor α-dependent NFκB1 activation.

Isoflavones possess a wide range of physiological effects. However, it is still unclear whether isoflavones can promote milk synthesis in mammary gland. This study aimed to determine the effects of a main soy isoflavone, daidzein, on milk synthesis and proliferation of mammary epithelial cells (MECs) and reveal the underlying molecular mechanism. Primary bovine MECs were treated with different concentrations of daidzein (0, 5, 10, 20, 40, and 80 µM). Daidzein dose-dependently promoted α- and ß-casein and lipid synthesis, cell cycle transition, and cell amount, with the best stimulatory effect at 20 µM. Daidzein also stimulated mTOR activation and Cyclin D1 and SREBP-1c expression. Daidzein induced the expression and nuclear localization of estrogen receptor α (ERα), and ERα knockdown blocked the stimulation of daidzein on these above signaling pathways. ERα knockdown also abolished the stimulation of daidzein on NFκB1 expression and phosphorylation, and NFκB1 was required for daidzein to enhance the mTOR, Cyclin D1 and SREBP-1c signaling pathways. In summary, our findings reveal that daidzein stimulates milk synthesis and proliferation of MECs via the ERα-dependent NFκB1 activation.

Phospho-Tudor-SN coordinates with STAT5 to regulate prolactin-stimulated milk protein synthesis and proliferation of bovine mammary epithelial cells.

Tudor staphylococcal nuclease (Tudor-SN) participates in milk synthesis and cell proliferation in response to prolactin (PRL) and plays a regulatory role on mTOR phosphorylation. However, the complicated molecular mechanism of Tudor-SN regulating milk protein synthesis and cell proliferation still remains to be illustrated. In present study, we observed that the proteins level of phosphorylated Tudor-SN and phosphorylated STAT5 were simultaneously enhanced upon PRL treatment in bovine mammary epithelial cells (BMECs). Tudor-SN overexpression and knockdown experiment showed that Tudor-SN positively regulated the synthesis of milk protein, cell proliferation and the phosphorylation of STAT5, which was dependent on Tudor-SN phosphorylation. STAT5 knockdown experiment showed that Tudor-SN stimulated mTOR pathway through regulating STAT5 activation, which was required for PRL to activate the mTOR pathway. Thus, these results demonstrate the primary mechanism of Tudor-SN coordinating with STAT5 to regulate milk protein synthesis and cell proliferation under stimulation of PRL in BMECs, which may provide some new perspectives for increasing milk production.