Transport proteins in mammary epithelial cells

Transporters localized in membranes of secreting mammary epithelial cells are involved in delivery of essential nutrients into milk. However, drugs and other xenobiotics may be substrates of these transporters and thus be actively secreted into milk, which may pose a health threat to breastfed infants and dairy consumers. Aims of the thesis were to determine expressions of drug transporters in mammary gland tissue and to assess mammary cell models for studies of these proteins. Gene expressions of members of the ABC (BCRP/ABCG2, MDR1/ABCB1, MRP1/ABCC1) and SLC (OATP1A2/SLCO1A2, OCTN1/SLC22A4, OCT1/SLC22A1) families were measured in murine and bovine mammary tissue and in murine (HC11) and bovine (BME-UV) mammary epithelial cell lines. BCRP function was assessed by transport experiments with mitoxantrone (MX) in HC11 cells. Effects of the imidazole fungicide prochloraz on transporter expression and function in HC11 and BME-UV cells were examined. Expressions of BCRP and OCT1 in murine mammary glands were increased during gestation and lactation, whereas MDR1, MRP1, OATP1A2 and OCTN1 were decreased, compared to expression in virgins. All transporters measured in mammary glands of mice were detected in bovine mammary tissue. All transporters investigated in vivo were also detected in HC11 cells, while only MDR1 and MRP1 were detected in BME-UV cells. Differentiation of HC11 cells resulted in increased BCRP protein expression, while MDR1 expression was reduced. The BCRP inhibitor elacridar reduced secretion and increased accumulation of MX in both undifferentiated and differentiated HC11 cells. An increased accumulation of MX was observed in BCRP gene silenced HC11 cells. Prochloraz treatment induced MDR1 gene expression and protein function in both differentiated HC11 and BME-UV cells, resulting in decreased accumulation of the MDR1 substrate digoxin. In conclusion, we demonstrated that the HC11 and BME-UV mammary cell models are valuable tools for identifying substrates, inhibitors and inducers of transport proteins expressed in the mammary epithelium during lactation. The models can be used both to examine if chemical compounds are actively transported into milk and if they disrupt the normal function of transporters which may result in a disturbed delivery of essential nutrients into milk

Transporter protein expression and localization in murine mammary epithelial HC11 cells

Milk is the sole food for breast-fed infants and a significant source of nutrition for consumers of dairy products. It has a very complex composition containing both macronutrients and micronutrients needed for growth and development. Drugs, carcinogens and environmental toxins can be secreted in milk posing a potential health risk for breast-fed infants and milk consumers. Active transport by membrane proteins in secretory mammary epithelial cells can explain this process. Several membrane protein transporters from different protein families (Solute carrier (SLC) and ATP-binding cassette (ABC) super families) mediate transport of a wide range of different compounds. The major purpose of this project was to investigate transporter protein expression of mouse Bcrp, Mdr1, Octn1 and Oatp3 (Oatp1a5) by using a murine mammary epithelial HC11 cell line. Differentiated HC11 cells featuring a milk protein synthesizing and secreting phenotype were used in western blot experiments to detect the proteins of interest. In this cell model it was possible to detect upregulation of membrane transporters mouse Bcrp and Octn1 as a result of the differentiation. In addition, immunohistochemistry on lactating mouse mammary gland tissues was performed to detect the localization of mouse Octn1. Octn1 was localized at the apical membrane of mammary epithelial cells supporting the theory that it plays a role in the transport of carnitine into milk. The impact of lipopolysaccharide (LPS) treatment, mimicking mammary inflammation, on protein expression of transporters in secreting HC11 cells were examined by western blotting. However, at the LPS concentration used no effects were detected. This study provides basic tools for understanding the nature of secretion of nutrients and nonnutrients to milk by membrane transporters. Further investigations are required to understand the correlation between protein expression and function of membrane transporters in secreting HC11 cells

A model of secreting murine mammary epithelial HC11 cells comprising endogenous Bcrp/Abcg2 expression and function

Breast cancer resistance protein (Bcrp/Abcg2) and multidrug transporter 1 (Mdr1/Abcb1) are efflux proteins located in the apical membrane of mammary epithelial cells (MEC). Bcrp is induced in MEC during gestation and lactation, while Mdr1 is down-regulated during lactation. Numerous drugs and toxic compounds are known to be actively secreted into milk by Bcrp, but most chemicals have not been investigated in this respect, emphasizing the need for functional Bcrp studies in an established cell line with secreting mammary epithelial cells. The present study was undertaken to examine expressions of Bcrp and Mdr1 in mammary epithelial HC11 cells, derived from a mid-gestational murine mammary gland. In addition, Bcrp function was assessed by transport experiments with mitoxantrone (MX) in undifferentiated HC11 cells, in HC11 cells subjected to Bcrp RNA interference (RNAi) as well as in HC11 cells stimulated to differentiate by treatment with lactogenic hormones. Differentiated HC11 cells organized into alveolar-resembling structures and gene expression of the major milk protein B-casein was induced, whereas undifferentiated cells formed monolayers with lower B-casein expression. Bcrp and Mdr1 gene and protein were expressed in both undifferentiated and differentiated HC11 cells. Differentiation of HC11 cells resulted in increased Bcrp protein expression, while Mdr1 gene and protein expressions were reduced. The Bcrp inhibitor elacridar (GF120918) reduced secretion and increased accumulation of MX in both undifferentiated and differentiated HC11 cells. Silencing of the Bcrp gene caused an increased accumulation of MX. The results indicate that the HC11 cell model provides a promising tool to investigate transport of potential Bcrp substrates in mammary epithelial cells

Staphylococcus aureus and Lipopolysaccharide Modulate Gene Expressions of Drug Transporters in Mouse Mammary Epithelial Cells Correlation to Inflammatory Biomarkers.

Inflammation in the mammary gland (mastitis) is the most common disease in dairy herds worldwide, often caused by the pathogens Staphylococcus aureus (S. aureus) and Escherichia coli (E. coli). Little is known about the effects of mastitis on drug transporters and the impact on transporter-mediated excretion of drugs into milk. We used murine mammary epithelial HC11 cells, after lactogenic differentiation into a secreting phenotype, and studied gene expressions of ABC- and SLC- transporters after treatment of cells with S. aureus and lipopolysaccharide, an endotoxin secreted by E. coli. The studied transporters were Bcrp, Mdr1, Mrp1, Oatp1a5, Octn1 and Oct1. In addition, Csn2, the gene encoding β-casein, was analyzed. As biomarkers of the inflammatory response, gene expressions of the cytokines Il6 and Tnfα and the chemokine Cxcl2 were determined. Our results show that S. aureus and LPS treatment of cells, at non-cytotoxic concentrations, induced an up-regulation of Mdr1 and of the inflammatory biomarkers, except that Tnfα was not affected by lipopolysaccharide. By simple regression analysis we could demonstrate statistically significant positive correlations between each of the transporters with each of the inflammatory biomarkers in cells treated with S. aureus. The coefficients of determination (R2) were 0.7-0.9 for all but one correlation. After treatment of cells with lipopolysaccharide, statistically significant correlations were only found between Mdr1 and the two parameters Cxcl2 and Il6. The expression of Csn2 was up-regulated in cells treated with S. aureus, indicating that the secretory function of the cells was not impaired. The strong correlation in gene expressions between transporters and inflammatory biomarkers may suggest a co-regulation and that the transporters have a role in the transport of cytokines and chemokines. Our results demonstrate that transporters in mammary cells can be affected by infection, which may have an impact on transport of essential compounds and contaminants into milk

Regression plots with individual data from 2 hours treatment of HC11 cells with <i>S</i>. <i>aureus</i>.

<p>Data from Figs <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0161346#pone.0161346.g002" target="_blank">2</a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0161346#pone.0161346.g003" target="_blank">3</a> were analysed by simple linear regression analysis of dependent variable gene expression of transporters (<i>Bcrp</i>, <i>Mdr1</i>, <i>Mrp1</i>, <i>Oatp1a5</i>, <i>Octn1</i> and <i>Oct1</i>) or <i>Csn2</i> and independent variable gene expression of inflammatory biomarkers (<i>Cxcl2</i>, <i>Il6 and Tnfα</i>). p < 0.001 in all regressions.</p

Relative gene expression of <i>Csn2</i> and transporters in HC11 cells treated with LPS.

<p>Differentiated HC11 cells were treated with LPS for 24 hours. Expressions of the genes encoding β-casein, <i>Csn2</i>, and the transporters <i>Bcrp</i>, <i>Mdr1</i>, <i>Mrp1</i>, <i>Oatp1a5</i>, <i>Octn1</i> and <i>Oct1</i> were determined. Normalized gene expressions are presented as means ± SD; n = 6. Statistically significant differences compared to vehicle controls **p < 0.01.</p

Relative gene expression of inflammatory biomarkers in HC11 cells treated with LPS.

<p>Differentiated HC11 cells were treated with LPS for 24 hours. Gene expressions of the inflammatory biomarkers <i>Cxcl2</i>, <i>Il6 and Tnfα</i> were determined. Normalized gene expressions are presented as means ± SD; n = 6. Statistically significant differences compared to vehicle controls *p < 0.05; **p < 0.01.</p

Relative gene expression of transporters in BME-UV cells without (control) and with lactogenic hormone stimulation (prolactin treated).

<p>Normalized gene expressions are presented as means ± SD; n = 6). Experiment repeated twice and data shown from one representative experiment.</p

"I like the Pilsners after all." Fridolín Macháček, Pilsner historian, archivist, museologist and cultural activist. Images of life and friendship in the mirror of his correspondence with Václav Vojtíšek

Život a dílo Fridolína Macháčka a Václava Vojtíška nebylo dosud monograficky zpracováno, proto tato práce přináší nové poznatky na základě dosud nevyužitých pramenů. Pestrá mozaika odborné i zájmové činnosti, ale také soukromého života, byla sestavena s ohledem na obsah vzájemné korespondence z let 1905-1954, která je v kritické edici přílohou této práce. Úkolem bylo též vystopovat, jak se jejich dílo odrazilo v této korespondenci v širším kontextu vývoje československého archivnictví. Je až neuvěřitelné, jak se oba přátelé dokázali naplno a rychle zapojit do odborné práce ve svých archivech i v dalších institucích. K plnému rozmachu jejich činnosti došlo v období první republiky, kdy platili za přední archivní odborníky a zasloužili se o rozvoj mnoha "venkovských" archivů. Tehdy také publikovali své nejzásadnější vědecké práce, založené na dlouholetém archivním výzkumu. Tento rozvoj byl násilím přerušen okupací a následnými životními zkouškami. Prošli jimi nezlomeni a pracovali dále v rámci možností. Po osvobození už můžeme sledovat rozdílné cesty obou přátel: Vojtíšek se znovu plně zapojil nejen do archivní práce, ale také do výuky pomocných věd historických na pražské univerzitě. Byl přijat mezi akademiky nově ustavené ČSAV a sbíral oficiální ocenění své práce. Macháček naopak, kromě činnosti ve vedení..