Involvement of connective tissue growth factor (CTGF) in insulin-like growth factor-I (IGF1) stimulation of proliferation of a bovine mammary epithelial cell line

The objective of this study was to determine the mechanism by which insulin-like growth factor-I (IGF1) stimulates proliferation of mammary epithelial cells, using the bovine mammary epithelial cell line MAC-T as a model. IGF1 significantly up- or down-regulated the expression of 155 genes in MAC-T cells. Among the most significantly suppressed was the gene for connective tissue growth factor (CTGF), a secretory protein that has both proliferative and apoptotic effects and is also a low-affinity binding protein of IGF1. IGF1 inhibited CTGF expression through the PI3K-Akt signaling pathway. Administration of growth hormone (GH), a strong stimulator of IGF1 production in vivo, decreased mammary CTGF mRNA in cattle; however, GH did not affect CTGF expression in MAC-T cells, suggesting that IGF1 may also inhibit CTGF expression in the mammary gland. Added alone CTGF stimulated proliferation of MAC-T cells, but in combination with IGF1 it attenuated IGF1's stimulation of proliferation of MAC-T cells. Excess IGF1 reversed this attenuating effect of CTGF. Despite being an IGF binding protein, CTGF did not affect IGF1-induced phosphorylation of IGF1 receptor (IGF1R) or IGF1R expression in MAC-T cells, indicating that the attenuating effect of CTGF on IGF1 stimulated proliferation of MAC-T cells was not mediated by decreasing IGF1's ability to bind to IGF1R or by decreasing IGF1R expression. Overall, these results suggest a novel biochemical and functional relationship between CTGF and IGF1 in the bovine mammary gland, where IGF1 may inhibit CTGF expression to reduce the attenuating effect of CTGF on IGF1 stimulated proliferation of epithelial cells.     

Growth hormone and lactogenic hormones can reduce the leptin mRNA expression in bovine mammary epithelial cells

Leptin mRNA is expressed in not only adipocytes but also mammary epithelial cells and leptin protein is present in milk. Although milk leptin is thought to influence metabolism or the immune system in neonates, there is little information about the regulation of leptin expression in mammary epithelial cells. We examined the effect of growth hormone (GH) and/or lactogenic hormone complex (DIP; dexamethasone, insulin and prolactin) on leptin mRNA expression in mammary epithelial cells. We used a bovine mammary epithelial cell (BMEC) clonal line, which was established from a 26-day pregnant Holstein heifer. We confirmed that the mRNA was expressed in BMECs and the expression was significantly reduced by GH and/or DIP, when the cells were cultured on both plastic plates and cell culture inserts at days 2 and 7 after stimulation with lactogenic hormones. GH and/or DIP significantly increased level of alpha-casein mRNA in BMECs after 7 days on the cell culture inserts, but no mRNA expression was detected at day 2. GH and DIP significantly stimulated the secretion of alpha-casein from BMEC on cell culture inserts at 3.5 and 7 days. However, neither alpha-casein mRNA expression nor secretion was observed in the BMECs cultured on plastic dishes, even in the presence of GH or/and DIP. These results indicate that GH and DIP can directly reduce leptin mRNA expression in both undifferentiated and functionally differentiated bovine mammary epithelial cell.     

Cytotoxic effects of peroxynitrite, polymorphonuclear neutrophils, free-radical scavengers, inhibitors of myeloperoxidase, and inhibitors of nitric oxide synthase on bovine mammary secretory epithelial cells

OBJECTIVE: To determine cytotoxic effects of activated polymorphonuclear neutrophils (PMN) and peroxynitrite on bovine mammary secretory epithelial cells before and after addition of nitric oxide synthase inhibitors, myeloperoxidase (MPO) inhibitors, and free-radical scavengers. SAMPLE POPULATION: Polymorphonuclear neutrophils from 3 lactating cows. PROCEDURE: Cells from the bovine mammary epithelial cell line MAC-T were cultured. Monolayers were treated with activated bovine PMN, lipopolysaccharide (LPS), phorbol 12-myristate 13-acetate (PMA), 3-morpholino-sydnonimine (SIN-1), 4-amino-benzoic acid hydrazide (ABAH), NG-monomethyl-L-arginine, histidine, and superoxide dismutase (SOD). At 24 hours, activity of lactate dehydrogenase in culture medium was used as a relative index of cell death. Tyrosine nitration of proteins in MAC-T cell lysates was determined by visual examination of immunoblots. RESULTS: Lipopolysaccharide, PMA, and < or = 0.1 mM SIN-1 were not toxic to MAC-T cells. Activated PMN, > or = 6 mg of histidine/ml, and 0.5 mM SIN-1 were toxic. Together, histidine and 500,000 activated PMN/ml also were toxic. NG-monomethyl-L-arginine did not have an effect, but ABAH decreased PMN-mediated cytotoxicity. Ten and 50 U of SOD/ml protected MAC-T cells from cytotoxic effects of 0.5 mM SIN-1. Compared with control samples, nitration of MAC-T tyrosine residues decreased after addition of 500,000 PMN/ml or > or = 6 mg of histidine/ml. Superoxide dismutase increased and SIN-1 decreased tyrosine nitration of MAC-T cell proteins in a dose-responsive manner. CONCLUSIONS AND CLINICAL RELEVANCE: Peroxynitrite, MPO, and histidine are toxic to mammary secretory epithelial cells. Superoxide dismutase and inhibition of MPO activity mitigate these effects. Nitration of MAC-T cell tyrosine residues may be positively associated with viability.     

Evaluation of a replacement method for mammary gland biopsies by comparing gene expression in udder tissue and mammary epithelial cells isolated from milk

Somatic cells isolated from milk offer an attractive non-invasive replacement of invasive udder biopsies for monitoring bovine mammary gland metabolism. However, for metabolic gene expression studies the mammary gland epithelial cells (MEC) isolated from milk have to be purified from the non-epithelial leukocyte fraction in milk samples. In our study, enrichment of MEC by using anti-cytokeratin peptide 18 (KRT18) antibody coated magnetic beads was evaluated. MEC showed a substantially increased expression of the epithelial-cell-specific KRT18 gene compared to udder tissue. The expression levels of genes specific for mammary gland epithelial cells (CSN3 and LALBA) showed a significant positive correlation in MEC and also in udder tissue. However, no significant correlation of the expression of a specific gene was found between udder and MEC samples. Therefore, MEC isolated from total milk samples via KRT18 antibodies probably do not reflect the true metabolic situation of the bovine udder. Thus, quantitative gene expression profiling of MEC isolated via KRT18 antibodies has to be interpreted carefully with respect to the situation in the udder.     

IGF-1 stimulates protein synthesis by enhanced signaling through mTORC1 in bovine mammary epithelial cells

Using the MAC-T cell line as a model, the effects of insulin-like growth factor (IGF)-1 on the regulation of protein synthesis through the mammalian target of rapamycin complex 1 (mTORC1) signaling in bovine mammary epithelial cells were evaluated. Global rates of protein synthesis increased by 47% within 30 min of IGF-1 treatment. The effect of IGF-1 on protein synthesis was associated with enhanced association of the eukaryotic initiation factor (eIF) 4E with eIF4G and a concomitant reduction of eIF4E association with eIF4E-binding protein-1 (4E-BP1). There was a progressive increase in the phosphorylation state of ribosomal protein S6 kinase-1, a downstream target of mTORC1 in response to IGF-1. In addition, IGF-1 stimulated mTORC1 kinase activity toward 4E-BP1 in vitro. Phosphorylation on Ser473 of Akt was induced by IGF-1 within 5 min and remained elevated throughout a 30-min time course. The effect of IGF-1 on Akt phosphorylation was also concentration dependent. Activation of Akt by IGF-1 led to increased phosphorylation of tuberous sclerosis complex 2 on Thr1426, without any change in its association with tuberous sclerosis complex 1. Phosphorylation of proline-rich Akt substrate of 40-kDa (PRAS40) at Thr246 was stimulated by IGF-1. The amount of PRAS40 associated with mTORC1 decreased in response to IGF-1, and PRAS40 binding to mTORC1 was inversely related to its phosphorylation level. Overall, these results suggest that activation of the PI3K-Akt pathway by IGF-1 stimulated global protein synthesis in bovine mammary epithelial cells through changes in the phosphorylation and association state of components of the mTORC1 signaling pathway.     

Proteomic analysis to unravel the effect of heat stress on gene expression and milk synthesis in bovine mammary epithelial cells

Heat stress can play a negative effect on milk yield and composition of dairy cattle, leading to immeasurable economic loss. The basic components of the mammary gland are the alveoli; these alveolar mammary epithelial cells reflect the milk producing ability of dairy cows. In this study, we exposed bovine mammary epithelial cells to heat stress and compared them to a control group using isobaric tags for relative and absolute quantitation combined with liquid chromatography coupled with tandem mass spectrometry. Compared with a control group, 104 differentially elevated proteins (>1.3‐fold) and 167 decreased proteins (<0.77‐fold) were identified in the heat treatment group. Gene Ontology analysis identified a majority of the differentially expressed proteins are associated in cell‐substrate junction assembly, catabolic processes and metabolic processes. Some of these significantly regulated proteins were related to the synthesis and secretion of milk, such as milk protein and fat. This finding was further supported by the results obtained from the reduced β‐casein expression through the system of plasminogen activator – plasminogen – plasmin and decreased fatty acid synthase could partly explain why milk fat synthesis ability of dairy cows decreased under heat stress. Our results highlight the effects of heat stress on synthesis of milk protein and fat, thus providing additional clues for further studies of heat stress on dairy milk production.     

Lactogenic hormones stimulate expression of lipogenic genes but not glucose transporters in bovine mammary gland

During the onset of lactation, there is a dramatic increase in the expression of glucose transporters (GLUT) and a group of enzymes involved in milk fat synthesis in the bovine mammary gland. The objective of this study was to investigate whether the lactogenic hormones mediate both of these increases. Bovine mammary explants were cultured for 48, 72, or 96 h with the following hormone treatments: no hormone (control), IGF-I, insulin (Ins), Ins + hydrocortisone + ovine prolactin (InsHPrl), or Ins + hydrocortisone + prolactin + 17β-estradiol (InsHPrlE). The relative expression of β-casein, α-lactalbumin, sterol regulatory element binding factor 1 (SREBF1), fatty acid synthase (FASN), acetyl-CoA carboxylase α (ACACA), stearyol-CoA desaturase (SCD), GLUT1, GLUT8, and GLUT12 were measured by real-time PCR. Exposure to the lactogenic hormone combinations InsHPrl and InsHPrlE for 96 h stimulated expression of β-casein and α-lactalbumin mRNA by several hundred-fold and also increased the expression of SREBF1, FASN, ACACA, and SCD genes in mammary explants (P < 0.01). However, those hormone combinations had no effect on GLUT1 or GLUT8 expression and inhibited GLUT12 expression by 50% after 72 h of treatment (P < 0.05). In separate experiments, the expression of GLUTs in the mouse mammary epithelial cell line HC11 or in bovine primary mammary epithelial cells was not increased by lactogenic hormone treatments. Moreover, treatment of dairy cows with bovine prolactin had no effect on GLUT expression in the mammary gland. In conclusion, lactogenic hormones clearly stimulate expression of milk protein and lipogenic genes, but they do not appear to mediate the marked up-regulation of GLUT expression in the mammary gland during the onset of lactation.     

Role of extracellular matrix and prolactin in functional differentiation of bovine BME-UV1 mammary epithelial cells

Interactions between extracellular matrix (ECM) and epithelial cells are necessary for proper organisation and function of the epithelium. In the present study we show that bovine mammary epithelial cell line BME-UV1 cultured on ECM components, commercially available as Matrigel, constitutes a good model for studying mechanisms controlling functional differentiation of the bovine mammary gland. In contact with Matrigel BME-UV1 cells induce apicobasal polarity, and within 16 days form three dimensional (3D) acinar structures with a centrally localized hollow lumen, which structurally resemble mammary alveoli present in the functionally active mammary gland. We have shown that the 3D culture system enables a high expression and proper localisation of integrin receptors and tight junction proteins in BME-UV1 cells to be induced. This effect was not obtained in cells grown in the classical 2D culture system on plastic. Moreover, ECM highly stimulated the synthesis of one of the major milk proteins, beta-casein, even in the absence of prolactin. Our results show that contact with ECM plays an important role in the lactogenic activity of bovine MECs, however, prolactin is necessary for the efficient secretion of milk proteins.     

Use of an immortalized bovine mammary epithelial cell line (MAC-T) to measure the mitogenic activity of extracts from heifer mammary tissue: effects of nutrition and ovariectomy

The objectives of the experiment were (1) to determine whether MAC-T cells would accurately mimic the previously observed proliferative responses of primary mammary epithelial cells (MEC) to mammary tissue extracts from high and low-fed heifers and (2) to determine whether mammary tissue extracts from ovariectomized (OVX) heifers would have lower mitogenic activity than intact controls. Addition of mammary tissue extracts to cell culture media of MAC-T cells plated on plastic or collagen-coated plastic to a range of concentrations between 1 and 8% resulted in dose-dependent increases in cell proliferation. Furthermore, mammary tissue extracts from low-fed prepubertal heifers aged 9 months, stimulated significantly more proliferation of MAC-T cells, as measured by 3H-thymidine incorporation into DNA than mammary tissue extracts from high-fed heifers (40.6 cpm x 10(3) per well versus 21.9+/-1.8 cpm x 10(3) per well). These observations suggested that MAC-T cells would be a suitable alternative to primary MECs for measuring the mitogenic activity of mammary tissue extracts. Conversely, no difference was observed in the mitogenic activity of mammary tissue extracts from OVX or control heifers. Possibly, MAC-T cells provide a good model for nutrition- but not ovarian-induced changes in mammary growth. Alternatively, that reduction of in vivo mammary development following OVX did not result in reduced mitogenic activity of the mammary tissue extracts emphasizes that heifer mammary development is the result of complex interactions between local growth factors and systemic hormones.     

丁酸钠通过AMPK通路调控LPS造成牛乳腺上皮细胞脂代谢紊乱的作用机制

通过向脂多糖（LPS）诱导牛乳腺上皮细胞系（MAC-T）脂代谢紊乱模型中添加不同浓度（2、8、16 μmol·L^-1）的丁酸钠,探讨其对细胞脂代谢的调控机理及炎症损伤的修复作用。分别用1 000 ng·mL^-1 LPS刺激MAC-T细胞9 h后,检测细胞脂滴面积及三酰甘油（TG）含量;用不同浓度的丁酸钠刺激MAC-T细胞12 h后,流式细胞术检测细胞凋亡率;试验共分为5组：对照组、LPS处理组、2 μmol·L^-1丁酸钠+LPS处理组、8 μmol·L^-1丁酸钠+LPS处理组和16 μmol·L^-1丁酸钠+LPS处理组,分别对细胞TG含量、AMPK信号通路蛋白、脂代谢关键基因以及相关炎症因子进行检测。结果显示：LPS会造成MAC-T细胞总脂滴面积显著下降（P<0.05）,TG含量极显著下降（P<0.01）;不同浓度的丁酸钠对MAC-T细胞凋亡率没有影响;与对照组相比,LPS处理组TG含量极显著下降（P<0.01）、P-AMPK表达水平显著上升（P<0.05）、脂合成代谢相关基因ACC、SCD-1以及FAS mRNA表达水平均显著（P<0.05）或极显著（P<0.01）下降、脂分解代谢相关基因CPT-1、CPT-2以及ACO mRNA表达水平均显著上升（P<0.05）、炎症因子TNF-α和IL-6含量显著上升（P<0.05）;与LPS处理组相比,（2、8、16 μmol·L^-1）丁酸钠+LPS处理组TG含量有一定程度上升,P-AMPK表达水平下降,脂合成代谢相关基因表达水平上升,脂分解代谢相关基因表达水平有一定程度下降,炎症因子TNF-α和IL-6含量下降。本研究表明丁酸钠会通过AMPK通路激活脂合成代谢,调控TG的合成,并且对MAC-T细胞的炎症损伤具有一定的缓解作用。     

Exfoliation rate of mammary epithelial cells in milk on bovine mastitis caused by Staphylococcus aureus is associated with bacterial load

The exfoliation rate of mammary epithelial cells (MECs) in milk is affected by physiological, breeding and environmental factors. Little is known about the relationship between the MEC exfoliation into milk and mammary‐infected Staphylococcus aureus (S. aureus) load on bovine mastitis caused by S. aureus. The aim of this study was to investigate the relationship between S. aureus load and the proportion of MEC exfoliation in milk using five substantial bovine mastitis models. In 64 randomly extracted milk samples from udders at 3–21 days after S. aureus infusion, there were various samples with different numbers of S. aureus counts and somatic cell counts. No significant correlations were found between the S. aureus counts and somatic cell count (r = 0.338). In contrast, a significant correlation was noted between S. aureus counts and the proportion of cytokeratin‐positive cells in the milk from the infused udders (r = 0.734, P < 0.01). In conclusion, the increasing MEC exfoliation rate in milk from mastitis udders caused by S. aureus may contribute to reduced milk yield.