Overexpression of Mos, Ras, Src, and Fos inhibits mouse mammary epithelial cell differentiation.

Mammary epithelial cells terminally differentiate in response to lactogenic hormones. We present evidence that oncoprotein overexpression is incompatible with this hormone-inducible differentiation and results in striking cellular morphological changes. In mammary epithelial cells in culture, lactogenic hormones (glucocorticoid and prolactin) activated a transfected beta-casein promoter and endogenous beta-casein gene expression. This response to lactogenic hormone treatment was paralleled by a decrease in cellular AP-1 DNA-binding activity. Expression of the mos, ras, or src (but not myc) oncogene blocked the activation of the beta-casein promoter induced by the lactogenic hormones and was associated with the maintenance of high levels of AP-1. Mos expression also increased c-fos and c-jun mRNA levels. Overexpression of Fos and Jun from transiently transfected constructs resulted in a functional inhibition of the glucocorticoid receptor in these mouse mammary epithelial cells. This finding clearly suggests that glucocorticoid receptor inhibition arising from oncogene expression will contribute to the block in hormonally induced mammary epithelial cell differentiation. Expression of Src resulted in the loss of the normal organization and morphological phenotype of mammary epithelial cells in the epithelial/fibroblastic line IM-2. Activation of a conditional c-fos/estrogen receptor gene encoding an estrogen-dependent Fos/estrogen receptor fusion protein also morphologically transformed mammary epithelial cells and inhibited initiation of mammary epithelial differentiation-associated expression of the beta-casein and WDNM 1 genes. In response to estrogen treatment, the cells displayed a high level of AP-1 DNA-binding activity. Our results demonstrate that high cellular AP-1 levels contribute to blocking the ability of mammary epithelial cells in culture to respond to lactogenic hormones. This and other studies indicate that the oncogene products Mos, Ras, and Src exert their effects, at least in part, by stimulating cellular Fos and probably cellular Jun activity.

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Interaction of two sequence-specific single-stranded DNA-binding proteins with an essential region of the beta-casein gene promoter is regulated by lactogenic hormones

Molecular and Cellular Biology ◽

10.1128/mcb.13.12.7303-7310.1993 ◽

1993 ◽

Vol 13 (12) ◽

pp. 7303-7310

Author(s):

S Altiok ◽

B Groner

Keyword(s):

Dna Binding ◽

Binding Proteins ◽

Mammary Epithelial Cells ◽

Dna Binding Proteins ◽

Binding Activity ◽

Mammary Epithelial ◽

Nuclear Proteins ◽

Single Stranded Dna ◽

Dna Binding Activity ◽

Beta Casein

Transcription of the beta-casein gene in mammary epithelial cells is regulated by the lactogenic hormones insulin, glucocorticoids, and prolactin. The DNA sequence elements in the promoter which confer the action of the hormones on the transcriptional machinery and the nuclear proteins binding to this region have been investigated. We found that 221 nucleotides of promoter sequence 5' of the RNA start site are sufficient to mediate the induction of a chloramphenicol acetyltransferase reporter gene in transfected HC11 mammary epithelial cells. Deletion of 5' sequences to position -183 results in a construct with enhanced basal activity which still retains inducibility. A -170 beta-casein promoter-chloramphenicol acetyltransferase construct has very low transcriptional activity, which indicates the presence of a negative regulatory in the region between -221 and -183 and a positive regulatory element between -183 and -170. Band shift analysis showed that the promoter region between -194 and -163 specifically binds two nuclear proteins. The proteins are sequence-specific, single-stranded DNA-binding proteins which exclusively recognize the upper DNA strand and most likely play a repressing role in transcription. DNA binding activity of these nuclear proteins was observed only in nuclear extracts from mammary glands of mice in late pregnancy and postlactation, not during lactation. Hormonal control of the DNA binding activity of these proteins was also observed in the mammary epithelial cell line HC11. Mixing experiments showed that extracts from mammary tissue of lactating mice and from lactogenic hormone-treated HC11 cells contain an activity which can suppress the DNA binding of the single-stranded DNA-binding proteins.2+ identical specificity to the single-stranded DNA.

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The nuclear factor YY1 participates in repression of the beta-casein gene promoter in mammary epithelial cells and is counteracted by mammary gland factor during lactogenic hormone induction.

Molecular and Cellular Biology ◽

10.1128/mcb.14.1.128 ◽

1994 ◽

Vol 14 (1) ◽

pp. 128-137 ◽

Cited By ~ 93

Author(s):

V S Meier ◽

B Groner

Keyword(s):

Mammary Gland ◽

Dna Binding ◽

Binding Activity ◽

Mammary Epithelial ◽

Nuclear Factor ◽

Dna Binding Activity ◽

Hormonal Induction ◽

Proximal Site ◽

Beta Casein ◽

Lactogenic Hormones

Expression of the beta-casein milk protein gene in the mammary epithelial cell line HC11 is primarily regulated at the transcriptional level. A 338-bp segment of promoter sequence 5' of the transcription start site is sufficient to confer inducibility by the lactogenic hormones insulin, glucocorticoid hormone, and prolactin. Positively and negatively acting promoter elements and specific DNA binding proteins have been identified. The binding of the mammary gland factor MGF to a site between -80 and -100 is indispensable for hormonal induction of transcription. Binding of MGF activity to DNA is greatly enhanced by the action of the lactogenic hormones. Repression of transcription in the uninduced state is mediated by a promoter element located adjacent to the MGF binding site at positions -110 to -150. This repressor element consists of two interacting protein binding sites. A nuclear factor that binds specifically to the proximal site between positions -110 and -120 has been characterized and found to be identical with the nuclear factor YY1 (delta, NF-E1). YY1 does not bind to the distal site. The simultaneous mutation in the proximal and the distal sites results in high, hormone-independent transcription. This finding suggests that YY1 plays a functional role in the repression and acts in conjunction with a second DNA binding protein. Comparison of YY1 DNA binding activity in uninduced and hormone-induced cells showed that relief of repression is not mediated by changes in the concentration or binding affinity of YY1. Infection of HC11 cells with a YY1-expressing recombinant retrovirus resulted in overexpression of YY1 but did not suppress hormonal induction. The addition of purified MGF decreased YY1 binding to its DNA recognition site in vitro. This finding indicates that MGF regulates the DNA binding activity of YY1 and thereby may cause the relief of transcriptional repression.

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Overexpression of the integrin-linked kinase mesenchymally transforms mammary epithelial cells

Journal of Cell Science ◽

10.1242/jcs.114.6.1125 ◽

2001 ◽

Vol 114 (6) ◽

pp. 1125-1136 ◽

Cited By ~ 4

Author(s):

A. Somasiri ◽

A. Howarth ◽

D. Goswami ◽

S. Dedhar ◽

C.D. Roskelley

Keyword(s):

Epithelial Cell ◽

Epithelial Cells ◽

Mammary Epithelial Cell ◽

Mammary Epithelial Cells ◽

Mammary Epithelial ◽

Cell Morphogenesis ◽

Wild Type ◽

Integrin Linked Kinase ◽

E Cadherin ◽

Cell Cell

Signals generated by the interaction of (β)1 integrins with laminin in the basement membrane contribute to mammary epithelial cell morphogenesis and differentiation. The integrin-linked kinase (ILK) is one of the signaling moieties that associates with the cytoplasmic domain of (β)1 integrin subunits with some specificity. Forced expression of a dominant negative, kinase-dead form of ILK subtly altered mouse mammary epithelial cell morphogenesis but it did not prevent differentiative milk protein expression. In contrast, forced overexpression of wild-type ILK strongly inhibited both morphogenesis and differentiation. Overexpression of wild-type ILK also caused the cells to lose the cell-cell adhesion molecule E-cadherin, become invasive, reorganize cortical actin into cytoplasmic stress fibers, and switch from an epithelial cytokeratin to a mesenchymal vimentin intermediate filament phenotype. Forced expression of E-cadherin in the latter mesenchymal cells rescued epithelial cytokeratin expression and it partially restored the ability of the cells to differentiate and undergo morphogenesis. These data demonstrate that ILK, which responds to interactions between cells and the extracellular matrix, induces a mesenchymal transformation in mammary epithelial cells, at least in part, by disrupting cell-cell junctions.

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The nuclear factor YY1 participates in repression of the beta-casein gene promoter in mammary epithelial cells and is counteracted by mammary gland factor during lactogenic hormone induction

Molecular and Cellular Biology ◽

10.1128/mcb.14.1.128-137.1994 ◽

1994 ◽

Vol 14 (1) ◽

pp. 128-137

Author(s):

V S Meier ◽

B Groner

Keyword(s):

Mammary Gland ◽

Dna Binding ◽

Binding Activity ◽

Mammary Epithelial ◽

Nuclear Factor ◽

Dna Binding Activity ◽

Hormonal Induction ◽

Proximal Site ◽

Beta Casein ◽

Lactogenic Hormones

Expression of the beta-casein milk protein gene in the mammary epithelial cell line HC11 is primarily regulated at the transcriptional level. A 338-bp segment of promoter sequence 5' of the transcription start site is sufficient to confer inducibility by the lactogenic hormones insulin, glucocorticoid hormone, and prolactin. Positively and negatively acting promoter elements and specific DNA binding proteins have been identified. The binding of the mammary gland factor MGF to a site between -80 and -100 is indispensable for hormonal induction of transcription. Binding of MGF activity to DNA is greatly enhanced by the action of the lactogenic hormones. Repression of transcription in the uninduced state is mediated by a promoter element located adjacent to the MGF binding site at positions -110 to -150. This repressor element consists of two interacting protein binding sites. A nuclear factor that binds specifically to the proximal site between positions -110 and -120 has been characterized and found to be identical with the nuclear factor YY1 (delta, NF-E1). YY1 does not bind to the distal site. The simultaneous mutation in the proximal and the distal sites results in high, hormone-independent transcription. This finding suggests that YY1 plays a functional role in the repression and acts in conjunction with a second DNA binding protein. Comparison of YY1 DNA binding activity in uninduced and hormone-induced cells showed that relief of repression is not mediated by changes in the concentration or binding affinity of YY1. Infection of HC11 cells with a YY1-expressing recombinant retrovirus resulted in overexpression of YY1 but did not suppress hormonal induction. The addition of purified MGF decreased YY1 binding to its DNA recognition site in vitro. This finding indicates that MGF regulates the DNA binding activity of YY1 and thereby may cause the relief of transcriptional repression.

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Endocytosis and intracellular transport of transferrin across the lactating rabbit mammary epithelial cell.

Journal of Histochemistry & Cytochemistry ◽

10.1177/40.10.1527373 ◽

1992 ◽

Vol 40 (10) ◽

pp. 1501-1510 ◽

Cited By ~ 7

Author(s):

T Seddiki ◽

S Delpal ◽

M Ollivier-Bousquet

Keyword(s):

Epithelial Cell ◽

Epithelial Cells ◽

Mammary Epithelial Cell ◽

Intracellular Transport ◽

Mammary Epithelial Cells ◽

Basal Membrane ◽

Mammary Epithelial ◽

Casein Micelles ◽

Human Transferrin ◽

Rabbit Milk

To study the transcytosis and segregation of ligand in the mammary epithelial cell, endocytosis and intracellular transit of human blood transferrin were followed in lactating rabbit mammary epithelial cells. Human transferrin labeled with biotin added to an incubation medium was bound to the basal membrane of mammary epithelial cells and carried across the cell to the lumen of the acini within 5-60 min. At the same time, biotinylated human transferrin accumulated at the apex of the cell. After incubation with human transferrin labeled with colloidal gold, label was detected inside endosome-like structures, vesicles and saccules of the Golgi apparatus, and inside the lumen within 2-5 min. A significant label accumulated at the apex of the cell after 30-60 min. Biotin labeling did not modify the time of transit of human transferrin, as attested by comparison with the time of transit of native transferrin. Human transferrin was never detected inside vesicles containing casein micelles. In contrast, rabbit milk transferrin was immunocytochemically detected inside vesicles containing casein micelles. These results indicate that transcytosis of human transferrin follows a pathway different from vesicles that carry casein micelles.

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Interaction of two sequence-specific single-stranded DNA-binding proteins with an essential region of the beta-casein gene promoter is regulated by lactogenic hormones.

Molecular and Cellular Biology ◽

10.1128/mcb.13.12.7303 ◽

1993 ◽

Vol 13 (12) ◽

pp. 7303-7310 ◽

Cited By ~ 31

Author(s):

S Altiok ◽

B Groner

Keyword(s):

Dna Binding ◽

Binding Proteins ◽

Mammary Epithelial Cells ◽

Dna Binding Proteins ◽

Binding Activity ◽

Mammary Epithelial ◽

Nuclear Proteins ◽

Single Stranded Dna ◽

Dna Binding Activity ◽

Beta Casein

Transcription of the beta-casein gene in mammary epithelial cells is regulated by the lactogenic hormones insulin, glucocorticoids, and prolactin. The DNA sequence elements in the promoter which confer the action of the hormones on the transcriptional machinery and the nuclear proteins binding to this region have been investigated. We found that 221 nucleotides of promoter sequence 5' of the RNA start site are sufficient to mediate the induction of a chloramphenicol acetyltransferase reporter gene in transfected HC11 mammary epithelial cells. Deletion of 5' sequences to position -183 results in a construct with enhanced basal activity which still retains inducibility. A -170 beta-casein promoter-chloramphenicol acetyltransferase construct has very low transcriptional activity, which indicates the presence of a negative regulatory in the region between -221 and -183 and a positive regulatory element between -183 and -170. Band shift analysis showed that the promoter region between -194 and -163 specifically binds two nuclear proteins. The proteins are sequence-specific, single-stranded DNA-binding proteins which exclusively recognize the upper DNA strand and most likely play a repressing role in transcription. DNA binding activity of these nuclear proteins was observed only in nuclear extracts from mammary glands of mice in late pregnancy and postlactation, not during lactation. Hormonal control of the DNA binding activity of these proteins was also observed in the mammary epithelial cell line HC11. Mixing experiments showed that extracts from mammary tissue of lactating mice and from lactogenic hormone-treated HC11 cells contain an activity which can suppress the DNA binding of the single-stranded DNA-binding proteins.2+ identical specificity to the single-stranded DNA.

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Zip3 plays a major role in zinc uptake into mammary epithelial cells and is regulated by prolactin

AJP Cell Physiology ◽

10.1152/ajpcell.00471.2004 ◽

2005 ◽

Vol 288 (5) ◽

pp. C1042-C1047 ◽

Cited By ~ 57

Author(s):

Shannon L. Kelleher ◽

Bo Lönnerdal

Keyword(s):

Mammary Gland ◽

Epithelial Cell ◽

Epithelial Cells ◽

Mammary Epithelial Cell ◽

Mammary Epithelial Cells ◽

Cell Model ◽

Mammary Epithelial ◽

Maternal Circulation ◽

Physiological Explanation ◽

Secretory Phenotype

During lactation, a substantial amount of Zn2+ is transferred by the mammary gland from the maternal circulation into milk; thus secretory mammary epithelial cells must tightly regulate Zn2+ transport to ensure optimal Zn2+ transfer to the suckling neonate. To date, six Zn2+ import proteins (Zip1–6) have been identified; however, Zip3 expression is restricted to tissues with unique requirements for Zn2+, such as the mammary gland, which suggests that it may play a specialized role in this tissue. In the present study, we have used a unique mammary epithelial cell model (HC11) to characterize the role of Zip3 in mammary epithelial cell Zn2+ transport. Confocal microscopy demonstrated that Zip3 is localized to the cell surface in mammary epithelial cells and transiently relocalized to an intracellular compartment in cells with a secretory phenotype. Total 65Zn transport was higher in secreting cells, while gene silencing of Zip3 decreased 65Zn uptake into mammary epithelial cells, particularly in those with a secretory phenotype. Finally, reduced expression of Zip3 ultimately resulted in cell death, indicating that mammary epithelial cells have a unique requirement for Zip3-mediated Zn2+ import, which may reflect the unique requirement for Zn2+ of this highly specialized cell type and thus provides a physiological explanation for the restricted tissue distribution of this Zn2+ importer.

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Autocrine Growth Hormone Prevents Lactogenic Differentiation of Mouse Mammary Epithelial Cells

Endocrinology ◽

10.1210/en.2005-1082 ◽

2006 ◽

Vol 147 (4) ◽

pp. 1819-1829 ◽

Cited By ~ 15

Author(s):

Svetlana Mukhina ◽

DongXu Liu ◽

Ke Guo ◽

Mireille Raccurt ◽

Sahra Borges-Bendris ◽

...

Keyword(s):

Epithelial Cell ◽

Epithelial Cells ◽

Mammary Epithelial Cell ◽

Mammary Epithelial Cells ◽

Three Dimensional ◽

Late Pregnancy ◽

Mammary Epithelial ◽

Postnatal Ontogeny ◽

Lactogenic Differentiation ◽

Pregnancy And Lactation

We have examined the expression, postnatal ontogeny, and localization of mouse GH (mGH) and its relative expression during pregnancy, lactation, and weaning in the mouse. mGH mRNA and protein was expressed predominantly in the epithelial component of the mammary gland, and maximal expression was observed during the pubertal period. Autocrine mGH expression dramatically decreased during late pregnancy and lactation. Concordantly, autocrine mGH expression is repressed during forced differentiation of mouse HC11 mammary epithelial cells in culture. Forced expression of mGH in HC11 cells abrogated lactogenic differentiation as indicated by reduced expression of β-casein and reduced expression and loss of lateral epithelial localization of E-cadherin. Forced expression of mGH in mouse mammary epithelial cells increased cell survival and proliferation and consequently increased the size of mammary acinar-like structures formed in three-dimensional Matrigel. Thus, autocrine mGH expression in the mouse mammary epithelial cell is maximal at puberty and prevents mammary epithelial cell differentiation. Autocrine GH will therefore participate in mammary morphogenic processes at puberty.

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Effects of Daidzein or Genistein on Proliferation and Antioxidation of Mammary Epithelial Cell of Dairy Cow In Vitro

Advanced Materials Research ◽

10.4028/www.scientific.net/amr.343-344.649 ◽

2011 ◽

Vol 343-344 ◽

pp. 649-654

Author(s):

Chun Long Liu ◽

Zhong Qiu Li ◽

Xing Jun Feng

Keyword(s):

Epithelial Cell ◽

Epithelial Cells ◽

Dairy Cow ◽

Mammary Epithelial Cell ◽

Mammary Epithelial Cells ◽

Mammary Epithelial ◽

Logarithmic Growth Phase ◽

Mda Content ◽

Mammary Epithelial Cell Proliferation

Effects of Daidzein or Genistein on the proliferation and antioxidation of mammary epithelial cells of dairy cow were investigated in vitro. 10 groups were assigned including blank control, estradiol (E2) group(10 ng/mL), different concentrations of Daidzein (1, 10, 100, 1 000 ng/mL) and Genistein (1, 10, 100, 1 000 ng/mL) groups. The MMT method was used to determine the proliferation effect of Genistein or Daidzein, and the results showed that Genistein at the concentration of 10 and 100 ng/mL, and Daidzein at the concentration of 100 and 1000 ng/mL significantly improved dairy cow mammary epithelial cell proliferation (P<0.05) , while significantly weaker than E2 group(P<0.05). In the antioxidation experiment, the T-SOD and GSH-PX activity, MDA and NO content of the mammary epithelial cells at the logarithmic growth phase treated with Daidzein or Genistein for 24 h were measured and the results showed that 100, 1000 ng/mL Daidzein, and 100, 1000 ng/ mL Genistein significantly increased the T-SOD activities and decreased MDA content (P<0.05).1 000 ng/ mL Daidzein and 100, 1 000 ng/mL Genistein significantly increased the GSH-PX activites (P<0.05). The results showed that proper levels of daidzein and genistein can improv the proliferation and antioxidation function of mammary epithelial cell of dairy cows.

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