DNAJA1 positively regulates amino acid-stimulated milk protein and fat synthesis in bovine mammary epithelial cells.

Several cellular processes, including the recovery of misfolded proteins, the folding of polypeptide chains, transit of polypeptides across the membrane, construction and disassembly of protein complexes, and modulation of protein control, are carried out by DnaJ homolog subfamily A member 1 (DNAJA1), which belongs to the DnaJ heat-shock protein family. It is unknown if DNAJA1 regulates the production of milk in bovine mammary epithelium cells (BMECs). Methionine and leucine increased DNAJA1 expression and nuclear location, as seen by us. In contrast to DNAJA1 knockdown, overexpression of DNAJA1 boosted the production of milk proteins and fats as well as mammalian target of rapamycin (mTOR) and sterol regulatory element binding protein-1c (SREBP-1c). As a result of amino acids, mTOR and SREBP-1c gene expression are stimulated, and DNAJA1 is a positive regulator of BMECs' amino acid-induced controlled milk protein and fat production.

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.

NCOA5 is a master regulator of amino acid-induced mTOR activation and ß-casein synthesis in bovine mammary epithelial cells.

The nuclear receptor co-activator 5 (NCOA5) is known as a co-activator or co-repressor that influences gene expression and cellular physiology, but its roles and detailed molecular mechanism is still largely unknown. In this study, we explored the role and molecular mechanism of NCOA5 in amino acid-induced activation of the mechanistic target of rapamycin (mTOR) and milk protein synthesis in bovine mammary epithelial cells (BMECs). Methionine (Met) and leucine (Leu) significantly up-regulated the expression of NCOA5. NCOA5 overexpression increased mTOR phosphorylation and ß-casein synthesis, whereas its knockdown exhibited the opposite effects. Furthermore, inhibition of phosphatidylinositol 3-kinase (PI3K) completely abolished the stimulatory effects of Met and Leu on NCOA5 expression. ChIP-qPCR analysis detected that NCOA5 bound to the mTOR promoter, and this interaction was enhanced by the stimulation of Met and Leu. These above data reveal that NCOA5 is a key regulator of amino acid-induced PI3K-mediated mTOR activation and ß-casein synthesis in BMECs.

Cyclase-associated protein 1 is a key negative regulator of milk synthesis and proliferation of bovine mammary epithelial cells.

Adenylyl cyclase-associated protein (CAP) is a highly conserved protein. Previous reports have suggested that CAP1 may be a negative regulator of cellular proliferation, migration, and adhesion and the development of cell carcinomas. The molecular mechanism of CAP1 regulation of downstream pathways, as well as how CAP1 is regulated by environmental stimuli and upstream signalling, is not well understood. In this present study, we assessed the role of CAP1 in milk synthesis and proliferation of bovine mammary epithelial cells. Using gene overexpression and silencing methods, CAP1 was found to negatively regulate milk synthesis and proliferation of cells via the PI3K-mTOR/SREBP-1c/Cyclin D1 signalling pathway. Hormones, such as prolactin and oestrogen, and amino acids, such as methionine and leucine, stimulate MMP9 expression and trigger CAP1 degradation, and thus, abrogate its inhibition of synthesis of milk protein, fat, and lactose by and proliferation of bovine mammary epithelial cells. The results of our study help deepen our understanding of the regulatory mechanisms underlying milk synthesis and aid in characterizing the molecular mechanisms of CAP1. Previous reports have suggested that CAP1 is a negative regulator of cellular proliferation and anabolism, but the molecular mechanisms are largely unknown. In this present study, we identified CAP1 as a negative regulator of milk synthesis and proliferation of bovine mammary epithelial cells. Our results will deepen our understanding of the regulatory mechanisms underlying milk synthesis and aid in exploring the molecular mechanisms of CAP1.

NUCKS1 is a novel regulator of milk synthesis in and proliferation of mammary epithelial cells via the mTOR signaling pathway.

Nuclear ubiquitous casein and cyclin-dependent kinase substrate 1 (NUCKS1) is a highly phosphorylated nuclear protein ubiquitously expressed in vertebrates. NUCKS1 has been reported to be a key chromatin modifier and transcriptional regulator of a number of signaling pathways, but the physiological role and detailed mechanism are still limited. In this study, we assessed the role of NUCKS1 on milk synthesis in and proliferation of mammary epithelial cells from a dairy cow. NUCKS1 was located in the nucleus of mammary epithelial cells, and the expression of NUCKS1 was stimulated by amino acids (Met and Leu) and hormones (estrogen and prolactin). Gene function study approaches detected that NUCKS1 positively regulated milk protein, milk fat, and lactose synthesis, and also increased the cell number, cell viability, and cell cycle progression. NUCKS1 mediated the stimulation of amino acids and hormones on the messenger RNA expression of the mechanistic target of rapamycin (mTOR), SREBP-1c, and Cyclin D1. The expression of NUCKS1 is dramatically higher in mouse mammary tissue of lactating period, compared with that in puberty and dry period. Taken together, these results reveal that NUCKS1 is a new mediator of milk synthesis in and proliferation of mammary epithelial cells via regulating the mTOR signaling pathway.

The phosphorylation of Tudor-SN mediated by JNK is involved in the regulation of milk protein synthesis induced by prolactin in BMECs.

Tudor staphylococcal nuclease (Tudor-SN) is a multifunctional protein involved in a variety of cellular processes and plays a critical role in the regulation of gene expression. Recently, Tudor-SN was found to be upregulated in mammary epithelial cells during lactation in response to prolactin, which further to regulate milk protein synthesis. However, the detailed regulatory mechanism of Tudor-SN to milk protein still remains to be elucidated. In our study, we observed that the levels of Tudor-SN and phosphor-Tudor-SN (Thr103) were both enhanced upon prolactin stimulation. Immunofluorescence assays demonstrated that prolactin treatment facilitated the nuclear transport of Tudor-SN. Further study revealed that the phosphorylation of Tudor-SN was depended on activated JNK. Coimmunoprecipitation assays disclosed that Tudor-SN might be phosphorylated directly by JNK. Using gene mutation assays, we further discovered that mutation of Thr to Ala at site of 103 prevented the nuclear transport of Tudor-SN. Thus, these results suggested the essential mechanism of the activated Tudor-SN in milk protein regulation in response to prolactin, which may provide some new sights into improve milk protein production.

Annexin A2 positively regulates milk synthesis and proliferation of bovine mammary epithelial cells through the mTOR signaling pathway.

Annexin A2 (AnxA2) has been shown to play multiple roles in growth, development, and metabolism, but the functions of AnxA2 and the signaling pathways associated with AnxA2 are still not fully understood. In this study, we aim to reveal whether and how AnxA2 could be involved in milk synthesis and proliferation of bovine mammary epithelial cells (BMECs). Using gene function study approaches, we found that AnxA2 positively regulates PIP3 level, phosphorylation of mTOR, and protein levels of SREBP-1c and Cyclin D1 leading to milk synthesis and cell proliferation. We further observed that both AnxA2-36 kD phosphorylated form and AnxA2-33 kD protein could be induced from AnxA2-36 kD protein in BMECs under methionine, leucine, estrogen or prolactin stimulation. These above results strongly demonstrate that AnxA2 functions as a critical regulator for amino acid or hormone-induced milk synthesis and cell proliferation via the PI3K-mTOR-SREBP-1c/Cyclin D1 signaling pathway.

MiR-139 suppresses ß-casein synthesis and proliferation in bovine mammary epithelial cells by targeting the GHR and IGF1R signaling pathways.

BACKGROUND: MicroRNAs have important roles in many biological processes. However, the role of miR-139 in healthy mammary gland remains unclear. The objective of this study was to investigate the effects of miR-139 on lactation in dairy cows. RESULTS: Here, we found that miR-139 was down-regulated in mid-lactation dairy cow mammary tissues compared with mid-pregnancy tissues. Then, we prioritized two of potential target genes of miR-139 in cow, growth hormone receptor (GHR) and type I insulin-like growth factor receptor (IGF1R) for further functional studies based on their roles in lactation processes. Dual luciferase reporter assays validated direct binding of miR-139 to the 3'- untranslated region (UTR) of GHR and IGF1R. Moreover, over-expression or silencing of miR-139 affected mRNA levels of GHR and IGF1R in cultured bovine mammary epithelial cells (BMECs). Furthermore, over-expression of miR-139 decreased protein levels of ß-casein, proliferation in mammary epithelial cell, and the protein levels of IGF1R and key members of the GHR or IGF1R pathways as well, whereas silencing miR-139 produced the opposite result. Among these signal molecules, signal transducer and activator of transcription-5 (STAT5), protein kinase B (also known as AKT1), mammalian target of rapamycin (mTOR), and p70-S6 Kinase (p70S6K) are involed in ß-casein synthesis, and Cyclin D1 is involved in cell proliferation. In addition, silencing GHR decreased protein levels of ß-casein, IGF1R, and key members of the IGF1R pathway, whereas co-silencing miR-139 and GHR rescued the expression of GHR and reversed GHR silencing effects. CONCLUSIONS: Our results demonstrate that GHR and IGF1R are target genes of miR-139 in dairy cow. MiR-139 suppresses ß-casein synthesis and proliferation in BMECs by targeting the GHR and IGF1R signaling pathways.

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 affects mTOR pathway and regulates lactogenesis in dairy cow mammary epithelial cells.

14-3-3 proteins are an acidic protein family that is highly conserved and widely distributed in eukaryotic cells. Recent studies have found that 14-3-3 proteins play critical roles in cell signal transductions, cell growth and differentiation, and protein synthesis. 14-3-3γ is an important member of 14-3-3 protein family. In our previous study, we found that 14-3-3γ was upregulated by estrogen in dairy cow mammary epithelial cell (DCMEC), but the function and mechanism of 14-3-3γ is not known. In this experiment, we first cultured and purified the primary DCMEC and found 14-3-3γ located both in the cytoplasm and nucleus by using immunofluorescence assay. Methionine, lysine, estrogen, and prolactin could upregulate the expression of 14-3-3γ, stimulate the secretion of ß-casein and triglyceride, and raise the cell viability of DCMEC. We constructed a stable 14-3-3γ overexpression cell line of DCMEC and found that the expressions of mTOR and p-mTOR, the secretion of triglyceride and ß-casein (CSN2), and the cell viability of DCMEC were all upregulated. We also observed the effects of 14-3-3γ gene silencing and gained consistent results with 14-3-3γ overexpression. These findings reveal that 14-3-3γ affects the mTOR pathway and regulates lactogenesis in DCMECs.