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Department of Animal Science, University of Zulia, Maracaibo, Venezuela 4005Department of Animal and Poultry Science, University of Guelph, Ontario, N1G 2W1, Canada
This study examined the localization of cellular glutathione peroxidase (GPx1) and extracellular glutathione peroxidase (GPx3) in lactating mammary tissue and in primary cultures of bovine mammary epithelial cells (BMEC). The effect of selenium as selenomethionine (SeMet) on the growth and viability of BMEC and GPx protein expression and activity were also studied. Single mammary epithelial cells were recovered by serial collagenase/hyaluronidase digestion from lactating bovine mammary tissue and cultured in a low-serum collagen gel system enriched with lactogenic hormones and 0, 10, 20, or 50 nM SeMet. Positive immunostaining with anti-cytokeratin and bovine anti-casein confirmed the epithelial nature and differentiated state of BMEC. Addition of SeMet to media facilitated rapid confluence of BMEC and formation of dome structures. Immunohistochemical and immunocytochemical staining revealed that both GPx1 and GPx3 are synthesized by BMEC and localized in the cytoplasm and nucleus. Up to 50 nM SeMet linearly increased BMEC number and viability over 5 d of culture. Bovine mammary epithelial cells cultured in SeMet-supplemented medium also exhibited markedly elevated GPx activity and linear increases in abundance of GPx1 and GPx3 proteins. It is apparent that SeMet degradation to release Se for synthesis of selenoproteins is carried out by BMEC. Results indicate that bovine mammary epithelial cells express GPx1 and GPx3 in vivo and in vitro; SeMet enhances expression of these selenoproteins in vitro and the growth and viability of BMEC.
Selenium is an essential mineral for animal nutrition. Its biological functions are associated with the Se-containing proteins, which possess selenocysteine residues at the active site. In mammals, there are 19 selenoproteins with known functions and all of them are enzymes (
). The glutathione peroxidase (GPx) family of selenoproteins catalyzes the reduction of lipid and hydrogen peroxides to lipid alcohols and water, respectively, with glutathione as a reductant. The GPx family is an essential component of the mammalian antioxidative system protecting membrane lipids and macromolecules from oxidative damage (
). Both cellular GPx (GPx1) and extracellular GPx (GPx3) are expressed at the mRNA and protein level in liver, heart, placenta, gastrointestinal tract, thyroid, kidney, and red blood cells in many species including human, rat, and mouse (
). The final Se-containing member of the family, GPx2, is primarily a gastrointestinal enzyme, and abundance of the protein in mammary tissue is very low (
) and GPx removes ROS. Thus, we hypothesized that improvement of GPx status of mammary epithelial cells would improve their survival in culture. Selenium status can modulate GPx expression and activity (
). A key factor in this effect is the synthesis of selenocysteine on its specific transfer RNA for incorporation into the polypeptide chain of GPx during translation (
Selenium deficiency reduces the abundance of mRNA for Se-dependent glutathione peroxidase 1 by a UGA-dependent mechanism likely to be nonsense codon-mediated decay of cytoplasmic RNA.
Dietary Se exists in both inorganic and organic forms. The preferred form of dietary Se for feeding dairy cows is organic, which is predominantly selenomethionine (SeMet), because it is more effective at enriching the Se content of milk (
). In order for SeMet to stimulate synthesis of selenoproteins such as GPx, the Se must be released by enzymatic degradation and converted to selenophosphate, which is the substrate for cotranslational selenocysteine synthesis (
). To our knowledge, there has been no study of GPx responses to Se status in primary cultures of bovine mammary epithelial cells (BMEC). Given that GPx1 and GPx3 are the main GPx forms expressed in mammary epithelial cells and milk of several species including the bovine (
), we hypothesized that BMEC can express both GPx1 and GPx3, and that an increase in the level of SeMet in culture media would increase absolute GPx activity and cell growth. To test the hypotheses, we localized GPx1 and GPx3 in lactating bovine mammary tissue and BMEC and evaluated the effect of SeMet on BMEC growth and GPx activity.
Materials and Methods
Animals and Mammary Tissue Sample Collection
Mammary tissue was collected from 8 multiparous lactating Holstein cows at 172 ± 16 DIM producing an average of 31.0 ± 2.1 kg milk/d culled from the Elora Dairy Research Centre herd (Elora, Ontario, Canada) and sent for slaughter at the University of Guelph Meat Laboratory (Guelph, Ontario, Canada). All cows were clinically healthy and free of mammary infection (SCC <150,000 cells/mL milk). After stunning, cows were bled out and their mammary glands were excised, washed with Betadine (Perdue Frederick Co., Norwalk, CT), and rinsed with 70% ethanol. Samples of mammary parenchyma (four 30-g pieces) were aseptically removed from the interior of the gland within 30 min of death and transferred to the laboratory at 37°C in a 500-mL bottle containing sterile Hanks’ balanced salt solution (Sigma Chemical Co., Oakville, Ontario, Canada) supplemented (HBSSs) with antibiotic-antimycotic solution (10,000 U/mL penicillin G, 10 mg/mL streptomycin, and 25 μg/mL amphotericin B; Sigma Chemical Co.), 1.5 mL of 1 M HEPES buffer (Sigma Chemical Co.), and 15 mL of fetal bovine serum (FBS; Invitrogen, Burlington, Ontario, Canada).
Isolation of Bovine Primary Mammary Epithelial Cells and Cell Cultures
Primary mammary epithelial cells were isolated by adaptations and modifications of the methods of
to decrease contamination with erythrocytes. Both methods serve to reduce contamination with fibroblasts and myoepithelial cells. In a laminar flow hood, six 10-g pieces of mammary tissue were washed 3 times in HBSSs, trimmed of all visible nonepithelial parenchyma, and thoroughly minced in 50-mL beakers with scissors. Minced tissues were washed 4 times with HBSSs to remove milk and blood, and transferred to a 100-mL bottle containing 50 mL of HBSSs supplemented with collagenase type III from Clostridium histolyticum (400 U/mL; Worthington Biochemicals, Freehold, NJ), hyaluronidase (100 U/mL; Sigma Chemical Co.), and DNase (2 U/mL; Sigma Chemical Co.) for 2 h at 37°C in a shaking water bath at ∼110 oscillations/min. Every 60 min, the digestion suspension was passed sequentially through nylon mesh of 200- (Spectrum, Los Angeles, CA), 100-, and 40-μm mesh (BD Biosciences, Mississauga, Ontario, Canada), and dispersed cells were collected by centrifugation at 100 × g for 5 min after addition of FBS (about 1/10 vol. The supernatant was decanted and the cell pellet was washed twice in HBSSs to eliminate residual enzyme. The resulting preparation consisted mainly of single and clumped epithelial cells and single erythrocytes. To separate BMEC from erythrocytes, the cell suspension was incubated for 1 h at 37°C in a ThinCert tissue cell culture insert (452.4 mm2 culture surface, 8.0 μm pore size; Greiner Bio-One, Monroe, NC) and washed every 20 min in HBSSs. Erythrocytes filtered through to the bottom of the plate, and BMEC were recovered by inversion and washing of the membrane. After centrifugation at 100 × g for 5 min, HBSSs was decanted and the final cell pellet was resuspended in primary culture medium (PCM) containing 45% RPMI 1640 (Sigma Chemical Co.), 45% Dulbecco's modified Eagle's medium (Sigma Chemical Co.), 5% FBS, 2% antibiotic-antimycotic solution, 1 mM sodium pyruvate (Sigma Chemical Co.), 2 mMl-glutamine (Sigma Chemical Co.), 40 mM HEPES buffer, bovine insulin (5 μg/mL; Sigma Chemical Co.), hydrocortisone (1 μg/mL; Sigma Chemical Co.), sheep prolactin (1 μg/mL; Sigma Chemical Co.), murine epidermal growth factor (5 ng/mL; Sigma Chemical Co.), and bovine transferrin (10 μg/mL; Sigma Chemical Co.); PCM was changed every 24 h. The BMEC were counted on a hemacytometer slide (improved Neubauer chamber; Fisher Scientific, Whitby, Ontario, Canada) and checked for viability by exclusion of 0.4% (wt/vol) trypan blue dye (Sigma Chemical Co.). More than 90% of the single cells were found to be viable and erythrocytes were absent.
Cell Number Estimates
To quantify the effect of SeMet on cell count and viability, BMEC were seeded at an approximate density of 3 × 104 cells/well, from 1 mL of culture medium, onto 24-well plates of BioCoat Cellware coated with collagen type I from rat tail tendon (BD Biosciences) and incubated in an atmosphere of 5% CO2 in air at 37°C for 5 d. One plate and 3 parallel wells for each treatment were seeded from each cow. dl-Selenomethionine (C5H11NO2Se; Fisher Scientific) was added to the medium at concentrations of 0, 10, 20, and 50 nM. Cells were harvested every 24 h for 5 d by trypsinization and were resuspended in Hanks’ balanced salt solution containing 50% FBS. Cells were counted on a hemacytometer, and DNA was quantified in cell suspensions washed twice with ice-cold PBS and lysed in CelLytic MT Cell Lysis Reagent (Sigma Chemical Co.) containing protease inhibitor cocktail (Sigma Chemical Co.). Supernatants resulting from a 10-min centrifugation of the lysate at 4°C and 13,000 × g were assayed for DNA by fluorescent Hoechst-33258 dye binding (DNAQF kit, Sigma Chemical Co.) in a Wallac Victor 1420 Multilabel Counter (Perkin-Elmer, Wellesley, MA) with excitation and emission wavelengths set at 355 and 460 nm, respectively (
). Morphology of cultured cells was evaluated under a Leica DM IL inverted microscope with phase contrast (Wetzlar, Germany).
GPx Enzymatic Activity Assay
To quantify effects of SeMet on GPx activity, 6 parallel wells of BMEC were plated for each SeMet concentration of 0, 10, 20, or 50 nM for each cow. Cells were harvested every 24 h for 5 d by mechanical scraping, and lysates were prepared as described previously. Glutathione peroxidase activity was assayed by an enzyme-coupled spectrophotometric procedure (CGP1 kit, Sigma Chemical Co.) according to
. Protein concentrations of cell lysates were quantified using the bicinchoninic acid assay (Pierce, Rockford, IL) using BSA as a standard.
Gel Electrophoresis and Western Blotting of GPx1 and GPx3
To examine the effect of SeMet on GPx1 and GPx3 protein abundances in BMEC, secretory cells were plated into 60-mm culture dishes of BioCoat Cellware (BD Biosciences) at a density of 2 × 106 cells/dish and treated with 0, 10, 20, or 50 nM SeMet for 5 d. Cell lysates containing 35 μg of protein were boiled in an equal volume of 2× sample loading buffer [4% SDS, 20% glycerol, 10% β-mercaptoethanol, 0.125 M Tris HCl (pH 6.8), and 0.004% bromophenol blue; Sigma Chemical Co.] at 95°C for 5 min and separated on 12.5% SDS-PAGE with a 5% stacking gel in SDS-Tris-glycine running buffer. Proteins were electrotransferred (Mini Trans-Blot Cell, Bio-Rad Laboratories Inc., Mississauga, Ontario, Canada) to a polyvinylidene fluorine membrane (Millipore, Mississauga, Canada) in Tris-glycine-methanol buffer at 100 V for 60 min. The membranes were then dried for 30 min at room temperature and cut into 2 pieces, horizontally, along the 35 kDa size marker to allow primary detection of GPx1 (22 kDa) or GPx3 (25 kDa) and β-actin (42 kDa) from the same blot. Membranes were blocked with blocking buffer [5% (wt/vol) nonfat dry milk in 0.36% (wt/vol) Tris base (pH 7.5), 0.1% (vol/vol) Tween 20, and 1.16% (wt/vol) NaCl] overnight at 4°C. The upper molecular weight portions were incubated with primary mouse monoclonal anti-β actin (42 kDa; 1:5,000; Abcam, Cambrige, MA), and the lower portions were incubated with polyclonal rabbit anti-GPx1 (1 μg/mL; Abcam) or monoclonal mouse anti-GPx3 (1:1,000; Abcam) overnight at 4°C. After washing 6 times with blocking buffer, membranes were incubated with goat anti-rabbit IgG (1:4,000; Abcam) or rabbit anti-mouse IgG (1:4,000; Abcam) labeled with horseradish peroxidase for 45 min at room temperature. After washing 6 times with 0.36% (wt/vol) Tris base (pH 7.5), 0.1% (vol/vol) Tween 20, and 1.16% (wt/vol) NaCl, proteins were developed by autoradiography utilizing Enhancer ChemiLuminescence (Amersham, Arlington Heights, IL). Finally, images from radiographic film were scanned and the integrated density was determined by ImageJ software (http://rbs.info.nih.gov/ij). Relative density was quantified by normalization of the integrated density of each blot to that of the corresponding β-actin.
Immunohistochemical Staining and Microscopy Procedure
Immunohistochemical localization of GPx1 and GPx3 in normal lactating bovine mammary tissue was performed similarly to
Immunohistochemical detection of human gastrointestinal glutathione peroxidase in normal tissue and cultured cells with novel mouse monoclonal antibodies.
. Samples collected at slaughter were fixed in 10% (vol/vol) neutral buffered formalin for 24 h at room temperature, embedded in paraffin wax, and sectioned into 5.0-μm sections. After deparaffinization, sections were heated at 400 W in a microwave oven for 7.5 min in 10 mM citrate buffer (pH 6.0) for antigen retrieval. Nonspecific binding sites were blocked by incubation in 3% (vol/vol) normal goat serum (Sigma Chemical Co.) in PBS for 30 min at room temperature. Sections were incubated with polyclonal rabbit anti-GPx1 (1:200; Abcam) and monoclonal mouse anti-GPx3 (1:200; Abcam) overnight in a humidified chamber at 4°C. For negative controls, PBS was used instead of primary antibody. Staining of GPx1 and GPx3 was revealed by incubation with fluorescein isothiocyanate (FITC)-labeled goat anti-rabbit or goat anti-mouse for 1 h. Vectashield mounting medium with 0.15% (wt/vol) 4′,6 diamidino-2-phenylindole (DAPI; Vector Laboratories, Burlingame, Canada) was used to counterstain the nucleus and to preserve fluorescence. Images were obtained on a Leica DMR microscope equipped with epifluorescence illumination and a CoolSNAP camera (RS Photometrics, Tucson, AZ) interfaced with digital imaging analysis software (OpenLab 2.2; Improvision, Boston, MA). Filter sets utilized were 360/440 nm for DAPI and 450/515 nm for FITC.
Immunocytochemical Staining and Microscopy Procedure
Immunocytochemical localization of GPx1, GPx3, cytokeratin, and caseins in BMEC was performed by a modification of the method of
. Bovine mammary epithelial cells (1 × 105) were grown to near confluency or confluency on collagen type I-coated 22-mm diameter glass coverslips (BD Biosciences) that fit into a 6-well plate. Medium was supplemented with 4 levels of SeMet (0, 10, 20, and 50 nM) and replaced every 48 h. By d 5, monolayers were fixed in 2.0% (wt/vol) paraformaldehyde (Sigma Chemical Co.) in PBS (pH 7.2) for 1 h at 4°C and washed in washing buffer (PBS, 0.1% wt/vol sodium azide, 2% vol/vol FBS, pH 7.2). Cell membranes were permeabilized with 0.2% Tween 20 solution in PBS for 15 min at 37°C in the water bath, washed in washing buffer, and blocked in washing buffer containing 10% (vol/vol) nonimmune goat serum for 30 min. Coverslips were incubated at 4°C overnight in a humid chamber with either monoclonal mouse antibody against cytokeratin AE1/AE3 (1:100; Dako, Mississauga, Ontario, Canada), polyclonal rabbit anti-GPx1 (1:200; Abcam), monoclonal mouse anti-GPx3 (1:200; Abcam), or polyclonal rabbit antibody against bovine milk caseins (1:300; Immunology Consultants Laboratory Inc., Newberg, OR). Cells were washed twice in washing buffer for 10 min each and subsequently incubated for 1 h in the dark with goat FITC-labeled anti-rabbit (Abcam), goat FITC-labeled anti-mouse (Abcam), or goat Texas Red-labeled anti-rabbit (Abcam). After 2 more washes, cells were counterstained and fluorescence preserved with Vectashield mounting medium containing 0.15% (wt/vol) DAPI (Vector Laboratories). Images were obtained as described above with the addition of a 560/675 nm filter set for Texas Red.
Se Content of Culture Media and FBS
Samples of PCM and FBS were analyzed for Se content by inductively coupled plasma mass spectrometry (
). The media and FBS used in these experiments contained 4.2 ± 2.2 and 56 ± 8.6 nM Se, respectively (n = 6; mean ± SD).
Statistical Analysis
Results are presented as means ± SEM. The effects of 4 SeMet levels (0, 10, 20, or 50 nM) on BMEC growth, viability, and GPx activity in BMEC over 5 d of incubation were analyzed by a mixed model in SAS (SAS Institute Inc., Cary, NC) with repeated measures over time according to
where μ = overall mean, Si = fixed effect of SeMet level (i = 1 to 4), Tj = repeated time effect (j = 1 to 5), STij = SeMet × time interaction effect, and errork(ij) = experimental error (k = 1 to 3). Covariance in repeated measures on the same cow was accommodated with a compound symmetry structure. Linear and quadratic orthogonal contrasts of SeMet level were determined with coefficients calculated in PROC IML of SAS (SAS Institute Inc.) to match the SeMet level.
Western blotting data were analyzed with PROC GLM of SAS (SAS Institute Inc.). Linear and quadratic contrasts of SeMet level were calculated. Significance was declared at P < 0.05.
Results
Immunohistochemical Localization of GPx1 and GPx3 in Bovine Mammary Tissue
Polyclonal anti-GPx1 and monoclonal anti-GPx3 primary antibodies were used to detect GPx1 and GPx3 proteins, respectively. These primary antibodies were demonstrated to be non-cross-reactive by Western blotting analysis of bovine plasma and erythrocyte hemolysate. The anti-GPx1 specifically detected the 22-kDa GPx1 protein in erythrocyte hemolysate (Figure 1A) but did not detect the 25-kDa GPx3 protein that was recognized in plasma by anti-GPx3 (Figure 1B). Bovine mammary epithelial cells showed positive staining with both primary antibodies. In epithelial and red blood cells, anti-GPx1 produced prominent cytoplasmic staining with low nuclear staining (Figure 1A). Immunoreactivity to GPx3 was found in the cytoplasm of epithelial cells with reduced or absent nuclear staining (Figure 1B). A strong staining pattern from anti-GPx3 was seen in the interstitial space, consistent with its localization in the extracellular fluid. Cell nucleus was stained with DAPI, which revealed mononucleate cells (Figure 1C). Conversely, when PBS was used instead of primary antibody as a negative control, staining was not detected (Figure 1D).
Figure 1Immunohistochemical detection of cellular glutathione peroxidase (GPx1) and extracellular glutathione peroxidase (GPx3) in normal lactating bovine mammary tissue. Cross-reactivity of anti-GPx1 and anti-GPx3 in bovine plasma (2 μL) and bovine erythrocyte lysate (10 μg) were resolved by Western blotting analysis. Immunohistochemistry was performed on formalin-fixed, paraffin-embedded lactating bovine mammary tissue and immunostained with anti-GPx1 or anti-GPx3 by fluorescein isothiocyanate-labeled method. A) In the lactating bovine mammary gland, GPx1 localization is clearly seen in the cytoplasm of mammary epithelial cells (arrow) and red blood cells (arrowhead), with no staining in the extracellular space; B) GPx3 is immunolocalized in the cytoplasm of mammary epithelial cells (arrow) and extracellular fluid (arrowhead); C) 4′,6 diamidino-2-phenylindole (DAPI) was used as a nuclear marker (arrow); and D) PBS was used instead of primary antibody as control.
Cells collected after 2 h of digestion with collagenase/hyaluronidase were predominantly viable single BMEC and a few epithelial cell clumps. The major cellular contaminants were erythrocytes, which were removed using tissue cell culture inserts. Phase-contrast microscopy of BMEC revealed a prominent nucleus and cytoplasmic vacuoles (Figure 2A), which have been observed in normal mammary secretory epithelial cells (
). The BMEC and epithelial cell clumps attached to the collagen gel within 12 to 24 h and spread out to monolayers with a typical cobblestone organization in all treatments (Figure 2B). By d 2, cells had flattened with a prominent nucleus, but cytoplasmic vacuoles were reduced in size and numbers. The BMEC cultured in 10, 20, or 50 nM SeMet reached 90% confluency with dome-like structures by d 5 (Figure 2C). In contrast, BMEC with 0 nM SeMet remained subconfluent without dome-like structures and failed to become confluent even if kept for up to 12 d (Figure 2D).
Figure 2Phase-contrast photomicrographs showing the effect of selenomethionine (SeMet) on bovine mammary epithelial cell (BMEC) culture. A) Minutes after plating, numerous cytoplasmic vacuoles (arrows) were detected in the 4 treatments; B) between 12 and 24 h after plating, cells formed monolayer aggregates with cobblestone morphology, which was found in all treatments; C) a representative micrograph of 7-d-old culture with 50 nM of SeMet added showing confluency and dome-like structures (arrow); D) 12-d-old culture without SeMet added, showing subconfluency and absence of dome-like structures; E) immunofluorescent staining of BMEC on collagen type I-coated glass coverslips with polyclonal antibody against bovine caseins (α, β, and kappa) found in the different treatments; F) Immunofluorescent staining of BMEC on collagen type I-coated glass coverslips with monoclonal antibody against cytokeratin AE1/AE3 5 d postculture with evidence of tonofilament among cells found in all treatments.
Functional differentiation of BMEC was confirmed in the different treatments by positive staining for bovine caseins in coverslip monolayers prepared from BMEC (Figure 2E). The epithelial nature of BMEC was confirmed with a monoclonal antibody against cytokeratin AE1/AE3. After 7 d of culture, cells were identified as more than 95% epithelial in each treatment by positive staining of cytoplasm and characteristic tonofilament connections between cells, which are important to intracellular communication and cell polarity (Figure 2F).
Effect of SeMet on BMEC Growth and Viability
The BMEC were cultured for 5 d to evaluate treatment effects on cell growth and viability. Both cell number (Figure 3A) and DNA (Figure 3B) increased linearly (P < 0.001) and quadratically (P < 0.001) with the level of SeMet added to media. Numbers of BMEC showed as much as a 5-fold increase in 5 d with SeMet added to the media, compared with 3-fold for cells without SeMet.
Figure 3Influence of selenomethionine (SeMet) on bovine mammary epithelial cell number: A) counted by hemacytometer; B) DNA assay. Data are expressed as mean ± SEM of 3 independent experiments. *Values are statistically different (P < 0.01) within each period.
Viability of BMEC cultured in media without added SeMet decreased (P < 0.05) from 90 to 70% during the first 24 h of incubation and remained at 70% thereafter (Figure 4). The addition of SeMet caused linear (P < 0.001) and quadratic (P < 0.001) increases in the capacity of BMEC to maintain viability during 5 d of culture.
Figure 4Influence of selenomethionine (SeMet) on the viability of bovine mammary epithelial cell measured by trypan blue exclusion. Data are expressed as mean ± SEM of 3 independent experiments. *Values are statistically different (P < 0.05) within each period.
There was a linear (P < 0.001) and quadratic (P = 0.003) increase of GPx activity in BMEC cultured with increasing levels of SeMet as early as 24 h after the start of incubation (Figure 5). This effect of SeMet persisted until 120 h of incubation. There was no effect of time on GPx activity.
Figure 5Influence of 5 d of culture with selenomethionine (SeMet) on the glutathione peroxidase (GPx) activity of bovine mammary epithelial cells. Cells were harvested and lysed every 24 h up to 5 d. The GPx activity was measured in the cell lysis and the means ± SEM of 3 independent experiments are shown. One unit of GPx was defined as the amount of enzyme that caused the oxidation of 1.0 μmol of NADPH to NADP+ per minute at pH 8.0 at 25°C in a coupled reaction with reduced glutathione, glutathione reductase, and tert-butyl hydroperoxides. Enzyme activity was expressed using a molar extinction coefficient of NADPH = 6.22 ɛmM. *P < 0.03; **P < 0.05; ***P < 0.01: values are statistically different within each period.
To determine whether GPx1 or GPx3 abundance in BMEC is regulated by SeMet, cell lysates from 5 d cultures were probed by Western blotting analysis (Figure 6A and Figure 7A). Both linear (P < 0.001) and quadratic (P = 0.037) increases in GPx1 abundance due to SeMet addition were apparent (Figure 6B). Although immunoblots for GPx3 were less intense than for GPx1, SeMet added to media linearly (P < 0.001) and quadratically (P = 0.002) increased the protein expression of GPx3 in BMEC (Figure 7B). The highest expression of GPx1 and GPx3 proteins was found in BMEC cultured in 50 nM SeMet.
Figure 6Effect of 5 d of culture with selenomethionine (SeMet) on cellular glutathione peroxidase (GPx1) protein expression in bovine mammary epithelial cells. A) A representative Western blot of cell lysates is shown; B) densitometric analysis of GPx1 protein expression relative to the corresponding β-actin control. Data are representative of 3 independent experiments. Asterisks above histograms show differences among treatments (* vs. **, ***P < 0.01; ** vs. ***P < 0.05).
Figure 7Effect of 5 d of culture with selenomethionine (SeMet) on extracellular glutathione peroxidase (GPx3) protein expression in bovine mammary epithelial cells. A) A representative Western blot of cell lysates is shown; B) densitometric analysis of GPx3 protein expression relative to the corresponding β-actin control. Data are representative of 3 independent experiments. Asterisks above histograms show differences among treatments (* vs. **, ***P < 0.01; ** vs. ***P < 0.05).
Immunocytochemistry of cells grown on coverslips revealed strong cytoplasmic staining, with weak or absent nuclear staining for GPx1 and GPx3, respectively (Figure 8A and Figure 8B). The control showed no staining (Figure 8C). These results confirm that BMEC express both selenoproteins after 5 d of culture.
Figure 8Immunocytochemical detection of cellular glutathione peroxidase (GPx1) and extracellular glutathione peroxidase (GPx3) in bovine mammary epithelial cells grown on collagen type I-coated glass coverslips for 5 d to near confluency. Cells show strong cytoplasmic immunostaining with both A) GPx1 and B) GPx3, but moderate to weak immunostaining in nucleus; C) PBS was used instead of primary antibody as control.
GPx1 and GPx3 Are Both Synthesized by Bovine Mammary Epithelial Cells
This study demonstrates that both GPx1 and GPx3 are synthesized by lactating bovine mammary epithelial cells. First, GPx1 and GPx3 were localized cytoplasmically by immunofluorescent staining of mammary tissue collected at slaughter, extending the findings of
that both selenoproteins are expressed at the mRNA level. However, the origin of GPx3 was unclear. It is presumed that GPx3 is produced and secreted mainly by the liver and the kidney, from where it is transported through blood plasma to other tissues, including the mammary glands (
). Indeed, immunohistochemistry revealed the presence of GPx3 in the interstitial space and this observation does not preclude the possibility that GPx3 found inside mammary epithelial cells was of extracellular origin. Therefore, we isolated individual BMEC by enzymatic digestion of mammary parenchyma, taking care to remove erythrocytes that could be an extra-epithelial source of GPx1, and cultured them for 5 d on collagen type I. After 5 d of culture, by which time GPx in the original plated cells would have disappeared, GPx1 and GPx3 were detected inside cells by immunocytochemistry. The absence of nuclear GPx3 and the presence of cytoplasmic GPx1 were as expected from previous studies in the human glioma PU118-9 cell (
). Our finding that GPx3 is expressed by cultured BMEC lends support to the hypothesis that GPx3 in bovine milk is synthesized and secreted by mammary epithelial cells. Although the biological functions of GPx3 in alveolar milk are unclear, the occurrence of GPx3 at this level may be associated with external protection of mammary secretory cell apical membranes from lipid or hydrogen peroxides, which can be produced by at least 44 enzymes found in milk such as superoxide dismutase, xanthine oxidase, and sulfhydryl oxidase (
SeMet Helps to Maintain the Differentiated State of BMEC
Primary cultures of BMEC were used to observe cell behavior under conditions that mimic physiological conditions relevant to lactation. For example, the use of primary cultures allowed us to study the effect of SeMet on BMEC without the influence of other mammary cell types, tissues of the body, or the circulatory and immune systems. A good in vitro culture model should permit normal cell growth and maintenance of cell differentiation, which underlies the biochemical and morphological characteristics typical of mammary epithelial cells in vivo. Two common strategies to induce mammary differentiation are incubation with the lactogenic hormones insulin, hydrocortisone, and prolactin, and seeding on extracellular collagen, both of which we used. Casein secretion in culture is a marker of the differentiated state (
). In our cultures, the positive staining of BMEC with anti-casein antibody suggests that cells grown with or without SeMet retained the ability to synthesize casein during the experimental period. Cytokeratin expression has also been used to distinguish between epithelial and nonepithelial cells in tissues and cultures (
). The epithelial nature of our BMEC was confirmed by positive staining with anti-cytokeratin cocktail antibody (AE1/AE3) after 7 d in culture. The immunostaining patterns against caseins and cytokeratins are consistent with previous studies (
). Additionally, the outgrowth pattern of attached BMEC, observed 48 h after seeding by phase contrast microscopy, was notably affected by SeMet addition to the media. Cells cultured in SeMet-supplemented media grew into a confluent layer with dome formation by 5 d, whereas this outgrowth pattern was not seen without added SeMet. The in vivo arrangement of milk secretory cells is a hollow, spherical alveolus with tight contact between adjacent cells. Cell-cell contact is essential for normal mammary tissue development, mediated by cadherins (
), and dome formation in vitro is recognized as a manifestation of the alveolus-forming behavior of milk secretory cells that facilitates polar secretion of milk components (
). The Se concentration of 4.2 ± 2.2 nM in complete media was apparently insufficient to support cell proliferation or survival necessary for rapid confluency and formation of epithelial domes, thus supplementation was required to maintain the differentiated state of these BMEC in vitro.
SeMet Improves Growth of BMEC
A linear relationship was detected between SeMet addition to culture media and growth in numbers of BMEC over 5 d. These results are consistent with those reported for HL-60 cells by
and represent the first demonstration that Se as SeMet is an essential trace nutrient for growth of BMEC. The mechanism of enhancement of cell growth induced by SeMet, however, remains unclear. Proliferation and death of cells are the 2 processes that will affect cell number. A Se-induced decrease in apoptosis has been observed in several experimental situations. For example, cardiac cell death due to oxidative stress during recovery from a heart attack is mitigated with Se (
). Effects of Se on cell proliferation are less clear and have mostly been studied at supranutritional concentrations that inhibit cell proliferation (
), characteristics that instill Se with an anticarcinogenic property. However, growth of human leukemia HL-60 cells in serum-free media in vitro was stimulated by 250 nM SeMet, during which there was a stimulation of cell cycle progression to the M phase, mRNA expression of proliferating cell nuclear antigen and several cyclins and cyclin kinases was elevated, and phosphorylation state of cellular protein was increased (
). These results suggest that Se status of cells may be detected and signaled to proliferation machinery by phosphorylation cascades.
SeMet Stimulates Expression of GPx1 and GPx3 in Bovine Mammary Epithelial Cells
It is well established that Se in many forms induces the expression and activity of the GPx enzyme family in the body of Se-deficient animals and in several cell lines in vitro (
). However, there has been no previous demonstration of this effect in BMEC. In order for SeMet to stimulate GPx expression, the Se must be released as H2Se by the sequential actions of methionine β-lyase and a demethylase, or via the trans-sulfuration (trans-selenation) pathway to produce selenocysteine that is acted on by selenocysteine β-lyase (
), inclusion of [35S]-methionine in the perfusate of lactating sheep mammary glands resulted in a small incorporation of label into cysteine residues of casein (
found a low level, compared with liver, of β-lyase activity in extracts of rat mammary tissue that was able to release monomethylated Se from Se-methylselenocysteine. Our observation that GPx1 and GPx3 expression and GPx activity were progressively increased by nanomolar additions of SeMet to cultures indicates that one or the other SeMet-degrading pathway is present in BMEC.
Glutathione peroxidases are just one of many selenoproteins that protect cells against oxidative damage by converting ROS to harmless molecules such as water. Increased expression of selenoproteins may have been responsible for the observed increase in growth of BMEC in vitro. Addition of 70 to 7,000 nM selenite to cultures of bovine corpora lutea decreased concentrations of lipid hydroperoxides and increased cell numbers over 8 to 14 d of culture (
). Lipid-soluble antioxidants like α-tocopherol prevented both the increase in intracellular concentrations of ROS and death of leukemia cells induced in vitro by 3 d of Se deficiency (
). Thus, enzymatic activity of selenoproteins can account for the growth-stimulating effect of Se. If the decline in milk production of the dairy cow during mid and late lactation is due in part to apoptotic signaling from intracellular ROS (
), the GPx-promoting effects of Se supplementation of mammary epithelial cells may serve to improve the persistency of lactation. Indeed, dairy cows supplemented with Se have shown an increase in milk production (
The results presented demonstrate expression of GPx1 and GPx3 proteins in mammary epithelial cells from the lactating dairy cow. Using primary cell cultures, we also show that addition of SeMet to cell culture media increases the protein expression of both selenoenzymes and absolute GPx activity in BMEC. In addition, SeMet at nanomolar concentrations promotes growth and viability of BMEC. Finally, our data confirm that SeMet as a principal source of Se represents an essential nutrient for BMEC culture.
Acknowledgments
This study was supported by grants from NSERC Canada, the Ontario Ministry of Agriculture, Food and Rural Affairs, and University of Zulia, Venezuela. We would like to thank Helen Coates, Brian McDougall, Sam Leo, Qiumei You, and Linda Trouten-Radford (University of Guelph) for their skillful technical assistance. Special thanks are extended to Brian McBride (University of Guelph), who provided a critical review of this study.
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