Xuejun Gao

Leucyl-tRNA Synthetase Regulates Lactation and Cell Proliferation via mTOR Signaling in Dairy Cow Mammary Epithelial Cells

The role of LeuRS, an aminoacyl-tRNA synthetase, as an intracellular l-leucine sensor for the mTORC1 pathway has been the subject of much research recently. Despite this, the association between LeuRS and lactation in dairy cow mammary epithelial cells (DCMECs) remains unknown. In this study, we found that LeuRS expression in mammary gland tissue was significantly higher during lactation than pregnancy. Moreover, our data demonstrates that LeuRS is localized in the cytoplasm. Treatment with leucine increased DCMECs viability and proliferation, as well as mammalian target of rapamycin (mTOR), p-mTOR, ribosomal protein S6 kinase 1 (S6K1), p-S6K1, β-Casein, sterol regulatory element binding protein 1c (SREBP-1c), glucose transporter 1 (GLUT1), and Cyclin D1 mRNA and protein expression. Secretion of lactose and triglyceride were also increased. siRNA-mediated knockdown of LeuRS led to reduction in all of these processes. Based on these data, LeuRS up-regulates the mTOR pathway to promote proliferation and lactation of DCMECs in response to changes in the intracellular leucine concentration.

Epigenetic Regulation of miR‐29s Affects the Lactation Activity of Dairy Cow Mammary Epithelial Cells

Milk is important for human nutrition, and enhanced milk quality has become a major selection criterion for the genetic improvement of livestock. Epigenetic modifications have been shown to be involved in mammary gland development; but the mechanisms underlying their effects remain unknown. MicroRNAs are involved in the regulation of milk synthesis and in mammary gland development. Our study is the first to investigate the roles of miR‐29s and epigenetic regulation in dairy cow mammary epithelial cells (DCMECs). Our results show that miR‐29s regulate the DNA methylation level by inversely targeting both DNMT3A and DNMT3B in DCMECs. The inhibition of miR‐29s caused global DNA hypermethylation and increased the methylation levels of the promoters of important lactation‐related genes, including casein alpha s1 (CSN1S1), E74‐like factor 5 (ElF5), peroxisome proliferator‐activated receptor gamma (PPARγ), sterol regulatory element binding protein‐1 (SREBP1), and glucose transporter 1 (GLUT1). The inhibition of miR‐29s reduced the secretion of lactoprotein, triglycerides (TG) and lactose by DCMECs. Moreover, the treatment of DCMECs with 5‐aza‐2′‐deoxycytidine (5‐Aza‐dC) decreased the methylation levels of the miR‐29b promoter and increased the expression of miR‐29b. The link between miR‐29s and DNMT3A/3B enhances our understanding of the roles of miRNAs in mammary gland function, and our data will inform more experimentally oriented studies to identify new mechanisms of regulating lactation. We present new insights regarding the epigenetic regulation of lactation performance. Improved understanding of the molecular basis of lactation will aid in the development of strategies for optimizing milk quality in dairy cows and modifying the lactation performance of offspring. J. Cell. Physiol. 230: 2152–2163, 2015.

Function of SREBP1 in the Milk Fat Synthesis of Dairy Cow Mammary Epithelial Cells

Sterol regulatory element-binding proteins (SREBPs) belong to a family of nuclear transcription factors. The question of which is the most important positive regulator in milk fat synthesis in dairy cow mammary epithelial cells (DCMECs) between SREBPs or other nuclear transcription factors, such as peroxisome proliferator-activated receptor γ (PPARγ), remains a controversial one. Recent studies have found that mTORC1 (the mammalian target of rapamycin C1) regulates SREBP1 to promote fat synthesis. Thus far, however, the interaction between the SREBP1 and mTOR (the mammalian target of rapamycin) pathways in the regulation of milk fat synthesis remains poorly understood. This study aimed to identify the function of SREBP1 in milk fat synthesis and to characterize the relationship between SREBP1 and mTOR in DCMECs. The effects of SREBP1 overexpression and gene silencing on milk fat synthesis and the effects of stearic acid and serum on SREBP1 expression in the upregulation of milk fat synthesis were investigated in DCMECs using immunostaining, Western blotting, real-time quantitative PCR, lipid droplet staining, and detection kits for triglyceride content. SREBP1 was found to be a positive regulator of milk fat synthesis and was shown to be regulated by stearic acid and serum. These findings indicate that SREBP1 is the key positive regulator in milk fat synthesis.

MiR-486 Regulates Lactation and Targets the PTEN Gene in Cow Mammary Glands

Mammary gland development is controlled by several genes. Although miRNAs have been implicated in mammary gland function, the mechanism by which miR-486 regulates mammary gland development and lactation remains unclear. We investigated miR-486 expression in cow mammary gland using qRT-PCR and ISH and show that miR-486 expression was higher during the high-quality lactation period. We found that miR-486 targets phosphoinositide signaling in the cow mammary gland by directly downregulating PTEN gene expression and by altering the expression of downstream genes that are important for the function of the mammary gland, such as AKT, mTOR. We analyzed the effect of β-casein, lactose and triglyceride secretion in bovine mammary gland epithelial cells (BMECs) transfected by an inhibitor and by mimics of miR-486. Our results identify miR-486 as a downstream regulator of PTEN that is required for the development of the cow mammary gland.

Molecular network including eIF1AX, RPS7, and 14-3-3γ regulates protein translation and cell proliferation in bovine mammary epithelial cells

14-3-3γ, an isoform of the 14-3-3 protein family, was proved to be a positive regulator of mTOR pathway. Here, we analyzed the function of 14-3-3γ in protein synthesis using bovine mammary epithelial cells (BMECs). We found that 14-3-3γ interacted with eIF1AX and RPS7 by 14-3-3γ coimmunoprecipitation (CoIP) and matrix-assisted laser desorption/ionization-time-of-flight/time-of-flight (MALDI-TOF/TOF) peptide mass fingerprinting analysis. These interactions of 14-3-3γ with eIF1AX and RPS7 were further confirmed by colocalization and fluorescence resonance energy transfer (FRET) analysis. We also found that methionine could promote protein synthesis and trigger the protein expression levels of 14-3-3γ, eIF1AX and RPS7. Analysis of overexpression and inhibition of 14-3-3γ confirmed that it positively affected the protein expression levels of eIF1AX, RPS7, Stat5 and mTOR pathway to promote protein synthesis and cell proliferation in BMECs. We further showed that overexpression of eIF1AX and RPS7 also triggered protein translation and cell proliferation. From these results, we conclude that molecular network including eIF1AX, RPS7, and 14-3-3γ regulates protein translation and cell proliferation in BMECs.

Influence on Cellular Signal Transduction Pathway in Dairy Cow Mammary Gland Epithelial Cells by Galactopoietic Compound Isolated from Vaccariae segetalis

Abstract The galactopoietic mechanism of Vaccaria segetalis is still unknown. Understanding dibutyl phthalate (DBP) separated from Vaccaria segetalis on the expression of lactation signal transduction genes of mammary gland epithelial cells, including prlr, era, akt1, socs2, pparγ and elf5, will be helpful to reveal the molecular mechanism. Western blot and qRT-PCR were used to study the change of prlr, era, akt, socs2, pparγ, and elf5 expression at mRNA and protein level. Co-localization expression of prolactin receptor (PRLR) and estrogen receptor α (ERα) was observed by immunofluorescence; the expression changes of miRNAs (21, 125b, 143, and 195) and the secretion of β-casein and lactose were detected by qRT-PCR and RP-HPLC. The results showed that Vaccaria segetalis active compound had similar fuctions as estrogen and/or prolactin (PRL) in dairy cow mammary gland epithelial cells (DCMECs), increased the expressions of prlr, era, akt1, and elf5 genes, while repressed pparγ expressions. DBP promoted socs2 mRNA expression, but its protein expressions were repressed. Furthermore, both DBP and PRL could repress the expressions of miRNA-125b, miRNA-143 and miRNA-195 in DCMECs. DBP could repress the expression of miRNA-21, while the influence of PRL on miRNA-21 was not certain. DBP could promote the lactation ability of DCMECs by regulating the ER and PRLR cellular signal transduction pathway.

Metabolic Regulation of Mammary Gland Epithelial Cells of Dairy Cow by Galactopoietic Compound Isolated from Vaccariae segetalis

Abstract In previous experiment, we isolated a compound dibutyl phthalate (DBP) from Vaccaria segetalis which had galactopoietic function on mammary gland epithelial cells of dairy cow (DCMECs). In this experiment, we ascertained the metabolic regulation function of DBP on DCMECs. Many genes related to lactation including Stat5, AMPK, β-casein, Glutl, SREBP-1, PEPCK, and ACC were detected by real-time PCR. Furthermore, Stat5 and AMPK were detected by Western blot and immunofluorescence co-localization, respectively. The results showed that DBP stimulates the expression of Stat5 and p-Stat5, thus activates Stat5 cell signal transduction pathway and stimulates β-casein synthesis. DBP also raises the activities of Glutl and AMPK to stimulate glucose uptake and glycometabolism and activates the expression of AMPK downstream target genes PEPCK and ACC and expression of SREBP-1 to stimulate milk fat synthesis. In addition, the activities of HK, G-6-PDH, ICDH, ATPase, and energy charges were stimulated by DBP to increase the energy metabolism level of DCMECs. The results showed DBP stimulates energy metabolism related to galactopoietic function in DCMECs.

Functional analysis of FABP3 in the milk fat synthesis signaling pathway of dairy cow mammary epithelial cells

Milk fat is the major energy component of milk, and regulation of its production relies on transcription factors sterol regulatory element-binding protein 1 (SREBP1) and peroxisome proliferator-activated receptor gamma (PPARγ). As one of the target genes of SREBP1 and PPARγ, fatty acid-binding protein 3(FABP3) is the main protein allowing for rapid diffusion and selective targeting of long-chain fatty acids toward specific organelles for metabolism. Whether FABP3 plays an important role in milk fat synthesis signaling pathway is largely unknown. In this study, we observed the effects of FABP3 overexpression and gene silencing in dairy cow mammary epithelial cells, as well as the effects of oleic acid, stearic acid, and palmitic acid on the expressions of FABP3 and lipid droplet formation, by using quantitative reverse transcriptase (qRT)-PCR, Western blotting, and fluorescent immunostaining techniques. FABP3 upregulated the expression of SREBP1 and PPARγ to increase lipid droplet accumulation. Oleic acid, stearic acid, and palmitic acid also increased lipid droplet accumulation by affecting expression of FABP3. These findings shed new insights for understanding the mechanism of FABP3 in regulating milk fat synthesis.

Tudor-SN Regulates Milk Synthesis and Proliferation of Bovine Mammary Epithelial Cells

Tudor staphylococcal nuclease (Tudor-SN) is a highly conserved and ubiquitously expressed multifunctional protein, related to multiple and diverse cell type- and species-specific cellular processes. Studies have shown that Tudor-SN is mainly expressed in secretory cells, however knowledge of its role is limited. In our previous work, we found that the protein level of Tudor-SN was upregulated in the nucleus of bovine mammary epithelial cells (BMEC). In this study, we assessed the role of Tudor-SN in milk synthesis and cell proliferation of BMEC. We exploited gene overexpression and silencing methods, and found that Tudor-SN positively regulates milk synthesis and proliferation via Stat5a activation. Both amino acids (methionine) and estrogen triggered NFκB1 to bind to the gene promoters of Tudor-SN and Stat5a, and this enhanced the protein level and nuclear localization of Tudor-SN and p-Stat5a. Taken together, these results suggest the key role of Tudor-SN in the transcriptional regulation of milk synthesis and proliferation of BMEC under the stimulation of amino acids and hormones.

14-3-3γ Regulates Lipopolysaccharide-Induced Inflammatory Responses and Lactation in Dairy Cow Mammary Epithelial Cells by Inhibiting NF-κB and MAPKs and Up-Regulating mTOR Signaling.

As a protective factor for lipopolysaccharide (LPS)-induced injury, 14-3-3γ has been the subject of recent research. Nevertheless, whether 14-3-3γ can regulate lactation in dairy cow mammary epithelial cells (DCMECs) induced by LPS remains unknown. Here, the anti-inflammatory effect and lactation regulating ability of 14-3-3γ in LPS-induced DCMECs are investigated for the first time, and the molecular mechanisms responsible for their effects are explored. The results of qRT-PCR showed that 14-3-3γ overexpression significantly inhibited the mRNA expression of tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6), interleukin-1β (IL-1β) and inducible nitric oxide synthase (iNOS). Enzyme-linked immunosorbent assay (ELISA) analysis revealed that 14-3-3γ overexpression also suppressed the production of TNF-α and IL-6 in cell culture supernatants. Meanwhile, CASY-TT Analyser System showed that 14-3-3γ overexpression clearly increased the viability and proliferation of cells. The results of kit methods and western blot analysis showed that 14-3-3γ overexpression promoted the secretion of triglycerides and lactose and the synthesis of β-casein. Furthermore, the expression of genes relevant to nuclear factor-κB (NF-κB) and mitogen-activated protein kinase (MAPKs) and lactation-associated proteins were assessed by western blot, and the results suggested that 14-3-3γ overexpression inactivated the NF-κB and MAPK signaling pathways by down-regulating extracellular signal regulated protein kinase (ERK), p38 mitogen-activated protein kinase (p38MAPK) and inhibitor of NF-κB (IκB) phosphorylation levels, as well as by inhibiting NF-κB translocation. Meanwhile, 14-3-3γ overexpression enhanced the expression levels of β-casein, mammalian target of rapamycin (mTOR), ribosomal protein S6 kinase 1 (S6K1), serine/threonine protein kinase Akt 1 (AKT1), sterol regulatory element binding protein 1 (SREBP1) and peroxisome proliferator-activated receptor gamma (PPARγ). These results suggest that 14-3-3γ was able to attenuate the LPS-induced inflammatory responses and promote proliferation and lactation in LPS-induced DCMECs by inhibiting the activation of the NF-κB and MAPK signaling pathways and up-regulating mTOR signaling pathways to protect against LPS-induced injury.