Proliferation Of Bovine Mammary Epithelial Cells Research Articles

Alfalfa xeno-miR159a regulates bovine mammary epithelial cell proliferation and milk protein synthesis by targeting PTPRF.

Milk protein content is an important index to evaluate the quality and nutrition of milk. Accumulating evidence suggests that microRNAs (miRNAs) play important roles in bovine lactation, but little is known regarding the cross-kingdom regulatory roles of plant-derived exogenous miRNAs (xeno-miRNAs) in milk protein synthesis, particularly the underlying molecular mechanisms. The purpose of this study was to explore the regulatory mechanism of alfalfa-derived xeno-miRNAs on proliferation and milk protein synthesis in bovine mammary epithelial cells (BMECs). Our previous study showed that alfalfa miR159a (mtr-miR159a, xeno-miR159a) was highly expressed in alfalfa, and the abundance of mtr-miR159a was significantly lower in serum and whey from high-protein-milk dairy cows compared with low-protein-milk dairy cows. In this study, mRNA expression was detected by real-time quantitative PCR (qRT-PCR), and casein content was evaluated by enzyme-linked immunosorbent assay (ELISA). Cell proliferation and apoptosis were detected using the cell counting kit 8 (CCK-8) assay, 5-ethynyl-2'-deoxyuridine (EdU) staining, western blot, and flow cytometry. A dual-luciferase reporter assay was used to determine the regulation of Protein Tyrosine Phosphatase Receptor Type F (PTPRF) by xeno-miR159a. We found that xeno-miR159a overexpression inhibited proliferation of BMEC and promoted cell apoptosis. Besides, xeno-miR159a overexpression decreased β-casein abundance, and increased α-casein and κ-casein abundance in BMECs. Dual-luciferase reporter assay result confirmed that PTPRF is a target gene of xeno-miR159a. These results provide new insights into the mechanism by which alfalfa-derived miRNAs regulate BMECs proliferation and milk protein synthesis.

Bta-miR-106b Regulates Bovine Mammary Epithelial Cell Proliferation, Cell Cycle, and Milk Protein Synthesis by Targeting the CDKN1A Gene

Our previous studies found that bta-miR-106b and its corresponding target gene, CDKN1A, were differentially expressed between the mammary epithelium of lactating Holstein cows with extremely high and low milk protein and fat percentage, implying the potential role of bta-miR-106b in milk composition synthesis. In this study, with luciferase assay experiment, bta-miR-106b was validated to target the 3'-untranslated region (UTR) of bovine CDKN1A, thereby regulating its expression. Moreover, in bovine mammary epithelial cells (BMECs), over-expression of bta-miR-106b significantly down-regulated the CDKN1A expression at both mRNA and protein levels, and inhibitors of bta-miR-106b increased CDKN1A expression. Of note, we observed that bta-miR-106b accelerated cell proliferation and cell cycle, and changed the expressions of protein synthesis related pathways such as JAK-STAT and PI3K/AKT/mTOR through regulating CDKN1A expression. Our findings highlight the important regulatory role of bta-miR-106b in milk protein synthesis by targeting CDKN1A in dairy cattle.

LncRNA MPFAST Promotes Proliferation and Fatty Acid Synthesis of Bovine Mammary Epithelial Cell by Sponging miR-103 Regulating PI3K-AKT Pathway.

Long noncoding RNAs (lncRNAs) have an essential role in mammary gland development and lactation. Our earlier study showed that the lncRNA mammary proliferation and fatty acid synthesis-associated transcript (MPFAST) is highly expressed in the Holstein cow mammary gland during the middle lactation period compared to the dry period, which indicates its potential role in lactation. Therefore, gain- and loss-of-function experiments were performed on bovine mammary epithelial cells (BMECs) by cell counting kit 8 (CCK8), 5-ethynyl-2'-deoxyuridine (EdU), real-time quantitative polymerase chain reaction (RT-qPCR), and western blot. The results indicated that MPFAST promoted the viability and proliferation of BMECs. The oil red O staining and cellular triglyceride assay further showed that MPFAST promoted the number of lipid droplets and cellular triglyceride synthesis in BMECs. Bioinformatics analysis showed that MPFAST could act as a molecular sponge for miR-103, and PIK3R1 was a potential target of miR-103, which was further confirmed by the dual-luciferase reporter assay, RT-qPCR, and western blot. The overexpression of MPFAST promoted the expression of PIK3R1 at mRNA and protein levels. It also significantly increased the mRNA relative expression levels of AKT, mTOR, and SREBP1, and the protein relative expression levels of AKT and p-AKT in the PI3K-AKT signaling pathway. In contrast, the inhibition of MPFAST resulted in the downregulation of the PI3K-AKT signaling pathway genes. These results indicated that MPFAST regulates the expression of the genes in the PI3K-AKT signaling pathway through sponging miR-103 and promotes the proliferation and synthesis of fatty acids of BMECs. Our results would provide a new direction for further exploring the regulatory mechanism of lncRNA in the mammary gland.

SV40T/E6E7-Induced Proliferation Is Involved in the Activity of E2F3 in Bovine Mammary Epithelial Cells.

Simple SummaryA previous study demonstrated that SV40T does not require E2F1, E2F2, and E2F3 activators to induce proliferation in mouse embryo fibroblasts (MEFs). Our results showed that, at an early stage, primary bovine mammary epithelial cells (BMECs) lacking the E2F1 expression have the capacity to proliferate and show E2F2 and E2F3 slight protein levels. At a late stage, primary BMECs deficient for E2F3 completely abolish any proliferative ability and exhibit a severe cell senescence signal, although the E2F2 can be expressed at a late stage of primary BMECs. Compared with the late stage of primary BMECs, the BMECs immortalized by SV40T and E6E7 restored the protein level of E2F3. In conclusion, this study revealed a molecular mechanism where E2F3 controls the BMECs’ proliferation and senescence.The E2F family of transcription factor is divided into activators and repressors that control cell proliferation. Bovine mammary epithelial cells (BMECs) can be immortalized using human papillomavirus 16 E6E7 (HPV16 E6E7) and simian vacuolating virus 40 large T antigen (SV40T). In addition, SV40T does not require E2F1, E2F2, and E2F3 activators to induce proliferation in mouse embryo fibroblasts (MEFs). However, we report that E2F3 activator is required to induce the proliferation of BMECs. Our results showed that, at an early stage, primary BMECs lacking the E2F1 expression have the capacity to proliferate and show E2F2 and E2F3 slight protein levels. At a late stage, primary BMECs deficient for E2F3 completely abolish any proliferative ability and exhibit a severe cell senescence signal, although the E2F2 can be expressed at a late stage of primary BMECs. Compared with the late stage of primary BMECs, the BMECs immortalized by SV40T and E6E7 restored the protein level of E2F3 and enhanced the CDK4, CDK6, cyclin D3, and CDK2 protein level, leading to proliferating robustly. Surprisingly, it was found that p53, p21Cip1, and p27Kip1 were upregulated in SV40T and E6E7-immortalized BMECs, relatively to primary BMECs. Notably, Cdc2 was almost expressed in primary BMECs. However, Cdc2 was elevated in BMECs immortalized by SV40T and E6E7. In conclusion, this study revealed a molecular mechanism where E2F3 controls the BMECs’ proliferation and senescence.

Circadian Gene PER2 Silencing Downregulates PPARG and SREBF1 and Suppresses Lipid Synthesis in Bovine Mammary Epithelial Cells.

Simple SummaryThe present study was constructed to determine the effects of the core circadian clock gene, Period 2 (PER2), on lipid synthesis in bovine mammary epithelial cells (BMECs). Data revealed that PER2-regulated genes were involved in fatty acid de novo synthesis, desaturation, TAG accumulation, and lipid droplet secretion in primary BMECs, partly by inhibiting PPARG and SREBF1. Our overall data suggests that PER2 in bovine mammary cells plays a role in regulating milk fat synthesis directly, or via the activation of the transcription regulators PPARG and SREBF1. This study provides molecular evidence underscoring a link between the circadian clock and lipid metabolism in bovines.PER2, a circadian clock gene, is associated with mammary gland development and lipid synthesis in rodents, partly via regulating peroxisome proliferator-activated receptor gamma (PPARG). Whether such a type of molecular link existed in bovines was unclear. We hypothesized that PER2 was associated with lipid metabolism and regulated cell cycles and apoptosis in bovine mammary epithelial cells (BMECs). To test this hypothesis, BMECs isolated from three mid-lactation (average 110 d postpartum) cows were used. The transient transfection of small interfering RNA (siRNA) was used to inhibit PER2 transcription in primary BMECs. The silencing of PER2 led to lower concentrations of cellular lipid droplets and triacylglycerol along with the downregulation of lipogenic-related genes such as ACACA, FASN, LPIN1, and SCD, suggesting an overall inhibition of lipogenesis and desaturation. The downregulation of PPARG and SREBF1 in response to PER2 silencing underscored the importance of circadian clock signaling and the transcriptional regulation of lipogenesis. Although the proliferation of BMECs was not influenced by PER2 silencing, the number of cells in the G2/GM phase was upregulated. PER2 silencing did not affect cell apoptosis. Overall, the data provided evidence that PER2 participated in the coordination of mammary lipid metabolism and was potentially a component of the control of lipid droplets and TAG synthesis in ruminant mammary cells. The present data suggested that such an effect could occur through direct effects on transcriptional regulators.

Effects of Peptidoglycan, Lipoteichoic Acid and Lipopolysaccharide on Inflammation, Proliferation and Milk Fat Synthesis in Bovine Mammary Epithelial Cells.

The mammary gland of the cow is particularly susceptible to infections of a wide range of pathogenic bacteria, including both Gram-positive and Gram-negative bacteria. The endotoxins of these pathogenic bacteria include peptidoglycan (PGN), lipoteichoic acid (LTA) and lipopolysaccharide (LPS), and they are the pathogen-associated molecular patterns (PAMPs) to induce mastitis. LPS can directly inhibit proliferation and milk fat synthesis of bovine mammary epithelial cells (BMECs) while inducing mastitis, but it is unclear whether PGN and LTA also have such effects. Furthermore, since the three PAMPs usually appear simultaneously in the udder of cows with mastitis, their synergistic effects on proliferation and milk fat synthesis of BMECs are worth investigating. The immortalized BMECs (MAC-T cells) were stimulated for 24 h using various concentrations of PGN, LTA and LPS, respectively, to determine the doses that could effectively cause inflammatory responses. Next, the cells were stimulated for 24 h with no endotoxins (CON), PGN, LTA, LPS, PGN + LTA, and PGN + LTA + LPS, respectively, with the predetermined doses to analyze their effects on proliferation and milk fat synthesis of BMECs. PGN, LTA and LPS successfully induced inflammatory responses of BMECs with doses of 30, 30 and 0.1 μg/mL, respectively. Although the proliferation of BMECs was significantly inhibited in the following order: LTA < PGN + LTA < PGN + LTA + LPS, there was no change in cell morphology and cell death. LTA significantly promoted the expression of fatty acid synthesis-related genes but did not change the content of intracellular triglyceride (TG), compared with the CON group. The mRNA expression of fatty acid synthesis-related genes in the LPS group was the lowest among all the groups. Meanwhile, LPS significantly decreased the content of intracellular non-esterified fatty acids (NEFAs) and TG, compared with the CON group. PGN had no effects on milk fat synthesis. Co-stimulation with PGN, LTA and LPS significantly increased the expression of fat acid synthesis-related genes and the intracellular NEFAs, but decreased intracellular TG, compared with sole LPS stimulation. Collectively, PGN, LTA and LPS showed an additive effect on inhibiting proliferation of BMECs. The promoting role of LTA in fatty acid synthesis might offset the negative effects of LPS in this regard, but co-stimulation with PGN, LTA and LPS significantly decreased intracellular TG content.

The protective roles of tea tree oil extracts in bovine mammary epithelial cells and polymorphonuclear leukocytes

BackgroundTea tree oil (TTO) plays an important role in antibacterial activity and alleviating the inflammatory responses. Bovine mammary epithelium and polymorphonuclear leukocytes (PMNL) can actively respond to bovine mastitis infection. However, regulatory effects of TTO extracts on the innate immune response of bovine mammary epithelial cells (BMECs) and PMNL remain not reported. Therefore, aim of the study was to evaluate the effects of TTO extracts on the mRNA levels of the genes involved in the innate immune response of BMECs and PMNL.ResultsOur results demonstrated that addition of 0.025% and 0.05% TTO increased the proliferation of BMECs, and significantly enhanced (P < 0.05) the viability of BMECs exposed to Staphylococcus aureus (S. aureus). An inhibitory effect was observed against the growth of S. aureus by TTO incubation. The 0.05% TTO reduced S. aureus biofilm formation, association and invasion of S. aureus to BMECs, and changed the morphological and structural features of S. aureus. The proinflammatory cytokines IL-1β, IL-6, and TNF-α were decreased (P < 0.001) by the incubation of TTO. Interestingly, the expression of IL-8 known for PMNL chemotactic function was elevated (P < 0.05) by 0.05% TTO treatment. Consistently, 0.05% TTO increased the migration of PMNL in S. aureus-exposed BMECs when compared with S. aureus treatment alone (P < 0.05). In addition, PMNL incubated with 0.05% TTO decreased the levels of NFKB inhibitor alpha (NFKBIA) and TNF-α.ConclusionsOur results indicate that use of TTO can relieve the BMECs pro-inflammatory response caused by S. aureus and promote the migration of PMNL to mount the innate immune responses, and it may be novel strategy for the treatment of bovine mastitis caused by S. aureus.

Kp-10 promotes bovine mammary epithelial cell proliferation by activating GPR54 and its downstream signaling pathways.

It has been reported that the proliferation and apoptosis of mammary epithelial cells affect milk production. Therefore, ensuring adequate mammary epithelial cells is expected to enhance milk production. This study is devoted to studying the effects of kisspeptin-10 (Kp-10), a peptide hormone composed of 10 amino acids, on bovine mammary epithelial cell (bMEC) proliferation and exploring the underlying mechanism of its action. bMECs were treated with various concentrations of Kp-10 (1, 10, 100, and 1,000 nM), and 100 nM Kp-10 promoted the proliferation of the bMECs. Kp-10 promoted the cell cycle transition from G1 to the S and G2 phases, increased the protein levels of Cyclin D1 and Cyclin D3, and reduced the expression levels of the p21 gene. This study also showed that inhibition of G protein-coupled receptor 54 (GPR54), AKT, mTOR, and ERK1/2 reduced the proliferation of the bMECs that had been induced by Kp-10. In addition, Kp-10 decreased the complexes formed by Rb and E2F1 and increased the expression levels of the E2F1 target genes. These results indicate that Kp-10 promotes bMEC proliferation by activating GPR54 and its downstream signaling pathways.

Transcription factor E2F4 is a positive regulator of milk biosynthesis and proliferation of bovine mammary epithelial cells.

The transcription factor E2F4 is a key determinant of cell differentiation and cell-cycle progression, but its function and regulatory mechanism are not completely understood. Here, we report that E2F4 acts as a positive regulator of the biosynthesis of milk components and proliferation of bovine mammary epithelial cells (BMECs). Overexpression of E2F4 in BMECs resulted in the upregulation of β-casein, triglyceride, and lactose levels and increased cell proliferation, whereas E2F4 knockdown by small interfering RNA had the opposite effects. We further detected that overexpression of E2F4 significantly increased the messenger RNA expression of mTOR, SREBP-1c, and Cyclin D1, and increased protein levels of SREBP-1c, and Cyclin D1, and the ratio of p-mTOR/mTOR, whereas E2F4 knockdown had the opposite effects. E2F4 was almost entirely located in the nucleus, and we further identified, via ChIP-qPCR analysis, that mTOR, SREBP-1c, and Cyclin D1 were E2F4 target genes, and exogenous administration of methionine, leucine, β-estradiol, and prolactin markedly increased the protein levels of E2F4 and its binding to the promoters of these three genes. In summary, our data reveal that E2F4 responds to extracellular stimuli and regulates the expression of mTOR, SREBP-1c, and Cyclin D1 for milk biosynthesis and proliferation of BMECs.

U2AF65 enhances milk synthesis and growth of bovine mammary epithelial cells by positively regulating the mTOR-SREBP-1c signalling pathway.

U2 snRNP auxiliary factor 65kDa (U2AF65) is a splicing factor that promotes prespliceosome assembly. The function of U2AF65 in alternative splicing has been identified; however, the essential physiological role of U2AF65 remains poorly understood. In this study, we investigated the regulatory role of U2AF65 in milk synthesis and growth of bovine mammary epithelial cells (BMECs). Our results showed that U2AF65 localizes in the nucleus. Treatment with amino acids (Met and Leu) and hormones (prolactin and β-estradiol) upregulated the expression of U2AF65 in these cells. U2AF65 overexpression increased the synthesis of β-casein, triglycerides, and lactose; increased cell viability; and promoted proliferation of BMECs. Furthermore, our results showed that U2AF65 positively regulated mTOR phosphorylation and expression of mature mRNA of mTOR and SREBP-1c. Collectively, our findings demonstrate that U2AF65 regulates the mRNA expression of signalling molecules (mTOR and SREBP-1c) involved in milk synthesis and growth of BMECs, possibly via controlling the splicing and maturation of these mRNAs. U2 snRNP auxiliary factor 65kDa (U2AF65) is a splicing factor that promotes prespliceosome assembly. The essential physiological role of U2AF65 remains poorly understood. In the present study, we confirmed that U2AF65 functions as a positive regulator of milk synthesis in and proliferation of bovine mammary epithelial cells via the mTOR-SREBP-1c signalling pathway. Therefore, our study uncovers the regulatory role of U2AF65 in milk synthesis and cell proliferation.

VA inhibits LPS-induced oxidative stress via modulating Nrf2/NF-κB-signalling pathways in bovine mammary epithelial cells

The current study was conducted to examine the pre-protection effects of vitamin A (VA) on lipopolysaccharide (LPS)-induced oxidative damage in bovine mammary epithelial cells (BMECs) and to explore the antioxidant mechanisms of VA via nuclear factor erythroid 2-related factor (Nrf2)/nuclear factor kappa-B (NF-κB)-signalling pathways. The BMECs were divided into 10 treatment groups with six replicates per treatment and were cultured with dimethyl sulphoxide (DMSO, vehicle negative control) or 0, 0.05, 0.1, 0.2, 0.5, 1, 2, 3, or 4 μg/mL of VA for 24 h and then incubated in the absence or presence of LPS (1 μg/mL) and VA for an additional 6 h. The results showed that exposure to LPS alone decreased the cell proliferation compared with the control. The addition of VA (from 0.05 to 4 μg/mL) promoted the proliferation of BMECs, and had positive effect on activities of glutathione peroxidase and its gene or protein expression but decreased NO and IL-1 production in a quadratic manner. Furthermore, VA significantly inhibited LPS-induced NF-κB activation and had a positive effect on Nrf2 activation in a quadratic dose-response manner and VA at a concentration of 1 μg/mL exhibited the strongest effect. The protective effect of VA dose greater than 2 μg/mL was weakened or even had no protective effect. These results suggest that VA pre-treatment protects BMECs from LPS-induced oxidative stress is due to Nrf2-signalling pathway activation and NF-κB-signalling pathway inhibition.HighlightsVA protected against LPS-induced inflammatory cytokines IL-1β production on dose dependent manner quadraticlly, and the concentration of 1 μg/mL exhibited the strongest effect.VA significantly inhibited LPS-induced NF-κB activation and had a positive effect on Nrf2 activation in a quadratic dose-response manner.VA might be a valuable agent for the treatment of anti-inflammatory and antioxidant.

Interferon-γ regulates cell malignant growth via the c-Abl/HDAC2 signaling pathway in mammary epithelial cells.

Interferon-γ (IFN-γ) has been used to control cancers in clinical treatment. However, an increasing number of reports have suggested that in some cases effectiveness declines after a long treatment period, the reason being unclear. We have reported previously that long-term IFN-γ treatment induces malignant transformation of healthy lactating bovine mammary epithelial cells (BMECs) in vitro. In this study, we investigated the mechanisms underlying the malignant proliferation of BMECs under IFN-γ treatment. The primary BMECs used in this study were stimulated by IFN-γ (10 ng/mL) for a long term to promote malignancy. We observed that IFN-γ could promote malignant cell proliferation, increase the expression of cyclin D1/cyclin-dependent kinase 4 (CDK4), decrease the expression of p21, and upregulate the expression of cellular-abelsongene (c-Abl) and histone deacetylase 2 (HDAC2). The HDAC2 inhibitor, valproate (VPA) and the c-Abl inhibitor, imatinib, lowered the expression level of cyclin D1/CDK4, and increased the expression level of p21, leading to an inhibitory effect on IFN-γ-induced malignant cell growth. When c-Abl was downregulated, the HDAC2 level was also decreased by promoted proteasome degradation. These data suggest that IFN-γ promotes the growth of malignant BMECs through the c-Abl/HDAC2 signaling pathway. Our findings suggest that long-term application of IFN-γ may be closely associated with the promotion of cell growth and even the carcinogenesis of breast cancer.

Vitamin A pretreatment protects NO-induced bovine mammary epithelial cells from oxidative stress by modulating Nrf2 and NF-κB signaling pathways.

It is known that physiological overproduction of nitric oxide (NO) contributes to oxidative stress and inflammation. Our published studies indicated that vitamin A (VA) reduces NO-induced oxidative stress in bovine mammary epithelial cells (BMECs) by increasing antioxidant enzyme activities. However, the precise mechanism is unclear. The present study was conducted to examine the protective effects of VA on NO-induced damage to BMECs in vitro using diethylenetriamine nitric oxide (DETA-NO) as the NO donor and to explore the intracellular signaling mechanisms of VA that involve nuclear factor erythroid 2-related factor (Nrf2) and nuclear factor kappa-B (NF-κB). Subconfluent BMECs were divided into 10 treatment groups with 6 replicates per treatment and were cultured with dimethyl sulfoxide (DMSO, vehicle negative control) or 0, 0.05, 0.1, 0.2, 0.5, 1, 2, 3, or 4 μg/mL of VA for 24 h and then incubated in the absence or presence of DETA-NO (1,000 μmol/liter) and VA for an additional 6 h. The results showed that exposure to DETA alone decreased cell proliferation compared with the negative control. Pretreatment with VA promoted the proliferation of BMECs, increased the activities of antioxidative enzymes including selenoprotein glutathione peroxidase (GPx) and thioredoxin reductase (TrxR) and their gene and protein expression but decreased NO and interleukin 1 (IL-1) contents in a quadratic manner (P < 0.05). In addition, the expression of mRNA and protein of factors that are related to NF-κB or Nrf2 signaling pathways in BMECs were regulated by VA in a quadratic dose-dependent manner; VA at a concentration of 1 μg/mL exhibited the strongest effect. Together, these results suggest that VA promotes antioxidant functions of BMECs by regulating the synthesis of selenoproteins including GPx and TrxR and by reducing concentrations of IL-1 and NO in vitro by modulating Nrf2 and NF-κB signaling pathways.

Twinfilin 1 enhances milk bio-synthesis and proliferation of bovine mammary epithelial cells via the mTOR signaling pathway

Twinfilin1 (TWF1) is an actin monomer-binding protein, which biological function has not yet been fully uncovered. In our previous study, we found by mass spectrometry analysis that TWF1 might be one of the major proteins responsible for milk bio-synthesis and proliferation of bovine mammary epithelial cells (BMECs). The purpose of this study was to explore the possible mechanism by which TWF1 regulates signaling pathways that enhance milk bio-synthesis and proliferation of BMECs. We first explored the effects of TWF1 on milk bio-synthesis and cell proliferation, and analyzed the role of TWF1 on the protein levels of signaling molecules (mTOR, SREBP-1c and Cyclin D1) related to milk bio-synthesis and cell proliferation. Then we determinate the impacts of amino acids (methionine and leucine) and hormones (estrogen and prolactin) on the expressions of TWF1. These results reveal that TWF1 is highly induced by the stimulation of amino acids and hormones and involved in regulation of milk bio-synthesis and cell proliferation via the mTOR pathway in BMECs.

DEAD-box helicase 6 (DDX6) is a new negative regulator for milk synthesis and proliferation of bovine mammary epithelial cells.

Milk synthesis of bovine mammary gland is a complex biological process that is regulated by hormones and nutrients, but the mechanism of these regulations still needs further research. DEAD-box helicase 6 (DDX6) is an important member of the RNA helicase family, involved in the regulation of mRNA storage and translation in different systems, but its physiological role and mechanism are largely unclear. In this study, we describe DDX6 as a potentially novel negative regulator for milk synthesis and proliferation of bovine mammary epithelial cells (BMECs). Treatment of BMECs with amino acids (methionine or leucine) or hormones (estrogen or prolactin) decreased the expression of DDX6. DDX6 expression was lower in mammary tissues of lactation period than in mammary tissues of puberty and dry period. Notably, overexpressing DDX6 in BMECs significantly decreased milk synthesis, cell proliferation, and protein levels of p-mTOR, SREBP-1c, and cyclin D1, while inhibiting DDX6 had the opposite effect. Taken together, these results reveal that DDX6 is a new negative regulator to control milk synthesis and proliferation of BMECs.

Effects of retinoic acid on the synthesis of selenoprotein and the antioxidative indices of bovine mammary epithelial cells in vitro

The present study was conducted to examine the effects of retinoic acid (RA) on the synthesis of selenoprotein and the antioxidative indices of bovine mammary epithelial cells (BMEC) in vitro and to explore the antioxidative mechanisms of RA in the BMEC. The subconfluenced BMEC were divided into six treatments with six replicates per treatment and cultured in a Dulbecco's Modified Eagle's Medium/F12 media (10% fetal bovine serum, 5 µg/ml ovine prolactin, 10 ng/ml epidermal growth factor, 1 g/ml hydrocortisone, 0.5% insulin-transferrin-selenium) containing different levels of RA (0 (control), 0.05, 0.1, 0.2, 1 or 2 mg/ml) for 24 h. Addition of RA promoted the proliferation of BMEC, increased the activities of catalase, superoxide dismutase, total antioxidant capacity, glutathione peroxidase (GPX), thioredoxin reductase (TRXR), and the content of selenoprotein P (SELP) in a dose-dependent manner (P < 0.05). The optimal RA dose was 1 μg/ml. However, positive effect of RA tended to be suppressed when RA was increased to 2 μg/ml. The expressions of mRNA and protein of GPX in BMEC were up-regulated by RA in a quadratic dose-response relationship (P < 0.01), and the addition of 1 μg/ml RA showed the best effect. The mRNA expressions of TRXR1 and SELP as well as the protein expression of TRXR1 were higher at 1-2 μg/ml RA. These results suggested that RA promoted antioxidant function of BMEC by regulating the synthesis of selenoprotein including GPX, TRXR, and SELP in vitro.

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.

Effect of growth factors and lactogenic hormones on expression of plasminogen activator-related genes and cell proliferation in a bovine mammary epithelial cell line

There is conflicting evidence in the literature as to whether up-regulation of urokinase plasminogen activator (u-PA) expression is related to bovine mammary epithelial cell growth. The role of u-PA receptor (u-PAR) and that of the plasminogen activator inhibitors type 1 and type 2 (PAI-1 and PAI-2) in bovine mammary epithelial cell proliferation is not known. The effect of growth factors and various hormones known to affect mammary function on expression of u-PA, u-PAR, PAI-1, PAI-2 and cell proliferation using the BME-UV1 bovine mammary epithelial cell line was examined. Cell proliferation was measured using the MTT assay and direct cell enumeration. Results showed that both IGF-1 and EGF increased cell proliferation but EGF was a more potent mitogen than IGF-1. Furthermore, IGF-1 increased by 2-fold expression of both u-PA and u-PAR while EGF increased by 3·8-fold the expression of only u-PAR. Both growth factors had no effect on expression of PAI-1 and PAI-2. In a manner consistent with changes in gene expression, EGF and to a lesser extent IGF-1 up-regulated total cell associated, membrane-bound and secreted u-PA activity. Thus, a strong correlation exists between u-PAR gene expression along with the activity of u-PA present on cell membranes and cell proliferation. Dexamethasone, prolactin and surprisingly insulin had no effect on cell proliferation. Dexamethasone alone and when combined with insulin or prolactin up-regulated gene expression of both PAI- and PAI-2 but not that of u-PA and u-PAR. Decreased total cell-associated, membrane-bound and secreted u-PA activity was detected in cells cultured in the presence of dexamethasone when combined with insulin or prolactin. However no such effect was observed in the presence of dexamethasone alone. Thus, dexamethasone acting synergistically with prolactin or insulin inhibits the activation of the plasmin-plasminogen system but this inhibition is not correlated with any changes in cell proliferation.

Effects of retinoids on proliferation and plasminogen activator expression in a bovine mammary epithelial cell line.

Effects of two natural (retinol and retinoic acid, RA) and one synthetic N-(4-hydroxyphenyl) retinamide (4-HPR) retinoids on proliferation and expression of urokinase-plasminogen activator (u-PA) by bovine mammary epithelial cells were examined. The BME-UV1 established bovine mammary epithelial cell line was used as a model system. All retinoids tested (retinol, RA and 4-HPR) were effective inhibitors of cell proliferation. When cells were cultured in the absence of fetal bovine calf serum (FBCS), inhibition occurred at concentrations as low as 1 nM for all retinoids tested. The effect of retinoids on cell proliferation was not dose-related when cells were cultured in the absence of FBCS. All retinoids (retinol, RA, 4-HPR), when used in the range 1 nM-10 microM (noncytotoxic concentrations), were equally effective and had identical inhibition patterns. Inhibition of cell proliferation by RA was apparent by 6 h and was higher after 24 h in culture. In contrast, when cells were cultured in the presence of FBCS, the effect of RA and retinol on cell proliferation was dose-related. RA and retinol inhibited cell proliferation (P<0.01) when added to the culture medium in concentrations as low as 10 nM and 100 nM, respectively. 4-HPR was inhibitory (P<0.01) in concentrations as low as 1 nM. Higher concentrations of 4-HPR in the range 1 nM-1 microM had no further effect on cell proliferation. None of the retinoids tested, when added to cultures in the presence or absence of FBCS, could completely arrest cell proliferation at noncytotoxic concentrations. RA at 1 microM inhibited (P<0.05) insulin or IGF-I-induced cell proliferation but had no effect (P>0.05) on u-PA mRNA levels or u-PA activity. Furthermore, RA inhibited cell proliferation in the presence of FBCS but had no effect (P>0.05) on u-PA mRNA levels. Thus, retinoids are effective inhibitors of bovine mammary epithelial cell proliferation and this growth inhibition does not seem to correlate with any changes in u-PA mRNA or u-PA activity.

Differential effects of retinoids on proliferation of bovine mammary epithelial cells in collagen gel culture.

The effects of increasing concentrations of retinol, retinal and retinoic acid on proliferation of bovine mammary epithelial cells were investigated in collagen gel cultures. All retinoids significantly inhibited proliferation of mammary epithelial cells. The relative inhibitory potency of the retinoids was: retinoic acid > retinal > retinol. Maximal inhibition at 10 microg/ml corresponded to a 75-95% inhibition of proliferation obtained in basal medium. Retinol, retinal and retinoic acid also inhibited proliferation of cells growth-stimulated with insulin-like growth factor-I (IGF-I). Retinoids in highest concentrations (10 microg/ml) inhibited 68-85% of proliferation of cells obtained in culture medium containing 25 ng IGF-I/ml. Retinol and retinoic acid also inhibited proliferation of cells growth-stimulated by insulin and other growth factors from the IGF growth factor family (des(1-3)IGF-I and IGF-II), as well as growth factors from the epidermal growth factor family (EGF and TGF-alpha), with retinoic acid being more effective than retinol. At a concentration of 100 ng/ml retinol and retinoic acid inhibited respectively 24-38 and 44-52% of mammary cell proliferation stimulated by growth factors of the IGF family, and at 10,000 ng/ml, 61-71% of cell proliferation was inhibited. The growth-stimulating effect of insulin, EGF and TGF-alpha was inhibited 42-64% by retinol and retinoic acid at 100 ng/ml, and 64-84% at 10,000 ng/ml. The present results show that retinol, retinal and retinoic acid are potent inhibitors of bovine mammary epithelial cell proliferation. It is suggested that retinoids may have concentration-dependent roles in regulation of pubertal mammary growth and development, indicating that the milk yield potential of heifers may be affected by vitamin A status.