Detection and regulation of leptin receptor mRNA in ovine mammary epithelial cells during pregnancy and lactation

Epithelial cells within the mammary gland undergo developmental programmes of proliferation and apoptosis during the pregnancy cycle. After weaning, secretory epithelial cells are removed by apoptosis. To determine whether members of the Bcl-2 gene family could be involved in regulating this process, we have examined whether changes in their expression occur during this developmental apoptotic program in vivo. Bax and Bcl-x were evenly expressed throughout development. However, expression of Bak and Bad was increased during late pregnancy and lactation, and the proteins were present during the time of maximal apoptotic involution. Thereafter, their levels declined. In contrast, Bcl-w was expressed in pregnancy and lactation but was downregulated at the onset of apoptosis. Bcl-2 was not detected in lactating or early involuting mammary gland. Thus, the pro-apoptotic proteins Bax, Bak and Bad, as well as the death-suppressors Bcl-x, Bcl-2 and Bcl-w, are synthesised in mouse mammary gland, and dynamic changes in the expression profiles of these proteins occurs during development. To determine if changes in Bak and Bcl-w expression could regulate mammary apoptosis, their effect on cultured mouse mammary epithelial cells was examined in transient transfection assays. Enforced expression of Bak induced rapid mammary apoptosis, which could be suppressed by coexpression of Bcl-w. In extracts of mammary tissue in vivo, Bak heterodimerized with Bcl-x whereas Bax associated with Bcl-w, but Bak/Bcl-w heterodimers were not detected. Thus, Bak and Bcl-w may regulate cell death through independent pathways. These results support a model in which mammary epithelial cells are primed for apoptosis during the transition from pregnancy to lactation by de novo expression of the death effectors Bak and Bad. It is suggested that these proteins are prevented from triggering apoptosis by anti-apoptotic Bcl-2 family proteins until involution, when the levels of Bcl-w decline. Our study provides evidence that regulated changes in the expression of cell death genes may contribute to the developmental control of mammary apoptosis.

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Expression of Prolactin mRNA in Rat Mammary Gland During Pregnancy and Lactation

Journal of Histochemistry & Cytochemistry ◽

10.1177/002215540004800308 ◽

2000 ◽

Vol 48 (3) ◽

pp. 389-395 ◽

Cited By ~ 13

Author(s):

Toshiki Iwasaka ◽

Shinobu Umemura ◽

Kochi Kakimoto ◽

Haruko Koizumi ◽

Yoshiyuki R. Osamura

Keyword(s):

Mammary Gland ◽

Epithelial Cells ◽

Mammary Epithelial Cells ◽

Late Pregnancy ◽

Mammary Epithelial ◽

Rt Pcr ◽

Pregnancy And Lactation ◽

Rat Mammary Gland ◽

Pcr Method

We studied the expression of prolactin (PRL) mRNA in the mammary gland of resting, pregnant, lactating, and weanling rats using in situ and solution reverse transcriptase-polymerase chain reaction (RT-PCR). In mid- to late pregnancy and throughout lactation, PRL mRNA was detected in both in situ and solution RT-PCR. These PRL mRNA signals were clearly identified in the cytoplasm of alveolar and ductal mammary epithelial cells by the in situ RT-PCR method. In mid- to late pregnancy, such as at the initiating point of PRL mRNA expression, we confirmed in some cases a lack of PRL mRNA by solution RT-PCR. In addition, in the early weaning phase, no signals were detected by solution RT-PCR. However, slight focal signals were detected in some poorly vacuolated cytoplasm of regressing acinar cells by in situ RT-PCR. These findings suggest that PRL mRNA in rat mammary gland begins in mid- to late pregnancy in parallel with the development of the mammary gland, continues throughout lactation, and declines in the early phase of weaning, with regression of mammary epithelial cells.

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RagD regulates amino acid mediated-casein synthesis and cell proliferation via mTOR signalling in cow mammary epithelial cells

Journal of Dairy Research ◽

10.1017/s0022029918000146 ◽

2018 ◽

Vol 85 (2) ◽

pp. 204-211 ◽

Cited By ~ 2

Author(s):

Ying Mu ◽

Dongmei Zheng ◽

Cong Wang ◽

Wei Yu ◽

Xiaonan Zhang

Keyword(s):

Cell Proliferation ◽

Amino Acid ◽

Epithelial Cells ◽

Mammary Epithelial Cells ◽

Mammary Epithelial ◽

Mammary Tissue ◽

Lactation Period ◽

Mtor Inhibition ◽

Pregnancy And Lactation ◽

Casein Synthesis

This research paper addresses the hypothesis that RagD is a key signalling factor that regulates amino acid (AA) mediated-casein synthesis and cell proliferation in cow mammary epithelial cells (CMECs). The expression of RagD was analysed at different times during pregnancy and lactation in bovine mammary tissue from dairy cows. We showed that expression of RagD at lactation period was higher (P < 0·05) than that at pregnancy period. When CMECs were treated with methionine (Met) or lysine (Lys), expression of RagD, β-casein (CSN2), mTOR and p-mTOR, and cell proliferation were increased. Further, when CMECs were treated to overexpress RagD, expression of CSN2, mTOR and p-mTOR, and cell proliferation were up-regulated. Furthermore, the increase in expression of CSN2, mTOR and p-mTOR, and cell proliferation in response to Met or Lys supply was inhibited by inhibiting RagD, and those effects were reversed in the overexpression model. When CMECs were treated with RagD overexpression together with mTOR inhibition or conversely with RagD inhibition together with mTOR overexpression, results showed that the increase in expression of CSN2 and cell proliferation in response to RagD overexpression was prevented by inhibiting mTOR, and those effects were reversed by overexpressing mTOR. The interaction of RagD with subunit proteins of mTORC1 was analysed, and the result showed that RagD interacted with Raptor. CMECs were treated with Raptor inhibition, and the result showed that the increase in expression of mTOR and p-mTOR in response to RagD overexpression was inhibited by inhibiting Raptor.In conclusion, our study showed that RagD is an important activation factor of mTORC1 in CMECs, activating AA-mediated casein synthesis and cell proliferation, potentially acting via Raptor.

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Up-regulation of polymeric immunoglobulin receptor mRNA in mammary epithelial cells by IFN-γ

Molecular and Cellular Endocrinology ◽

10.1016/s0303-7207(02)00183-1 ◽

2002 ◽

Vol 194 (1-2) ◽

pp. 95-105 ◽

Cited By ~ 12

Author(s):

A Rincheval-Arnold ◽

L Belair ◽

A Cencic ◽

J Djiane

Keyword(s):

Epithelial Cells ◽

Mammary Epithelial Cells ◽

Mammary Epithelial ◽

Polymeric Immunoglobulin Receptor ◽

Immunoglobulin Receptor ◽

Receptor Mrna ◽

Ifn Γ

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MicroRNA in the ovine mammary gland during early pregnancy: spatial and temporal expression of miR-21, miR-205, and miR-200

Physiological Genomics ◽

10.1152/physiolgenomics.00091.2012 ◽

2013 ◽

Vol 45 (4) ◽

pp. 151-161 ◽

Cited By ~ 31

Author(s):

Laurent Galio ◽

Stéphanie Droineau ◽

Patrick Yeboah ◽

Hania Boudiaf ◽

Stephan Bouet ◽

...

Keyword(s):

Cell Proliferation ◽

Mammary Gland ◽

Epithelial Cells ◽

Early Pregnancy ◽

Mammary Epithelial Cells ◽

Mammary Epithelial ◽

Normal Mammary Gland ◽

Basal Cells ◽

Pregnancy And Lactation ◽

Luminal Cells

The mammary gland undergoes extensive remodeling between the beginning of pregnancy and lactation; this involves cellular processes including cell proliferation, differentiation, and apoptosis, all of which are under the control of numerous regulators. To unravel the role played by miRNA, we describe here 47 new ovine miRNA cloned from mammary gland in early pregnancy displaying strong similarities with those already identified in the cow, human, or mouse. A microarray study of miRNA variations in the adult ovine mammary gland during pregnancy and lactation showed that 100 miRNA are regulated according to three principal patterns of expression: a decrease in early pregnancy, a peak at midpregnancy, or an increase throughout late pregnancy and lactation. One miRNA displaying each pattern (miR-21, miR-205, and miR-200b) was analyzed by qRT-PCR. Variations in expression were confirmed for all three miRNA. Using in situ hybridization, we detected both miR-21 and miR-200 in luminal mammary epithelial cells when expressed, whereas miR-205 was expressed in basal cells during the first half of pregnancy and then in luminal cells during the second half. We therefore conclude that miR-21 is strongly expressed in the luminal cells of the normal mammary gland during early pregnancy when extensive cell proliferation occurs. In addition, we show that miR-205 and miR-200 are coexpressed in luminal cells, but only during the second half of pregnancy. These two miRNA may cooperate to maintain epithelial status by repressing an EMT-like program, to achieve and preserve the secretory phenotype of mammary epithelial cells.

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P2Y receptor regulation of sodium transport in human mammary epithelial cells

AJP Cell Physiology ◽

10.1152/ajpcell.00068.2007 ◽

2007 ◽

Vol 293 (5) ◽

pp. C1472-C1480 ◽

Cited By ~ 19

Author(s):

So Yeong Lee ◽

Melissa L. Palmer ◽

Peter J. Maniak ◽

Soo Hwa Jang ◽

Pan Dong Ryu ◽

...

Keyword(s):

Epithelial Cells ◽

Mammary Epithelial Cells ◽

Mammary Epithelial ◽

Receptor Subtypes ◽

P2y Receptor ◽

Rt Pcr ◽

Human Mammary Epithelial Cells ◽

Receptor Mrna ◽

Human Mammary Epithelial ◽

Immortalized Cells

Primary human mammary epithelial (HME) cells were immortalized by stable, constitutive expression of the catalytic subunit of human telomerase. Purinergic receptors were identified by RT-PCR and quantitative RT-PCR from mRNA isolated from primary and immortalized cells grown to confluence on membrane filters. Several subtypes of P2Y receptor mRNA were identified including P2Y1, P2Y2, P2Y4, and P2Y6 receptors. RT-PCR experiments also revealed expression of A2b adenosine receptor mRNA in primary and immortalized cells. Confluent monolayers of HME cells exhibited a basal short-circuit current ( Isc) that was abolished by amiloride and benzamil. When monolayers were cultured in the presence of hydrocortisone, mRNA expression of Na+ channel (ENaC) α-, β-, and γ-subunits increased approximately threefold compared with that in cells grown without hydrocortisone. In addition, basal benzamil-sensitive Na+ transport was nearly twofold greater in hydrocortisone-treated monolayers. Stimulation with UTP, UDP, or adenosine 5′- O-(3-thiotriphosphate) (ATPγS) produced increases in intracellular calcium concentration that were significantly reduced following pretreatment with the calcium-chelating agent BAPTA-AM. Concentration-response relationships indicated that the rank order of potency for these agonists was UTP > UDP > ATPγS. Basolateral stimulation with UTP produced a rapid but transient increase in Isc that was significantly reduced if cells were pretreated with BAPTA-AM or benzamil. Moreover, basolateral treatment with either charybdotoxin or clotrimazole significantly inhibited the initial UTP-dependent increase in Isc and eliminated the sustained current response. These results indicate that human mammary epithelial cells express multiple P2 receptor subtypes and that Ca2+ mobilization evoked by P2Y receptor agonists stimulates Na+ absorption by increasing the activity of Ca2+-activated K+ channels located in the basolateral membrane.

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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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Metabolic cooperativity between epithelial cells and adipocytes of mice

AJP Cell Physiology ◽

10.1152/ajpcell.1981.241.5.c204 ◽

1981 ◽

Vol 241 (5) ◽

pp. C204-C208 ◽

Cited By ~ 19

Author(s):

J. C. Bartley ◽

J. T. Emerman ◽

M. J. Bissell

Keyword(s):

Epithelial Cells ◽

Mammary Epithelial Cells ◽

Glycogen Synthase ◽

Lipid Synthesis ◽

Abdominal Fat ◽

Mammary Epithelial ◽

Early Lactation ◽

Systemic Factors ◽

Pregnancy And Lactation ◽

Fat Pads

We have demonstrated that glycogen and lipid synthesis in adipocytes is modulated by the lactational state and that this modulation in mammary adipocytes requires the presence of the adjacent epithelial cells. Glycogen and lipid synthesis from [14C]glucose was measured in mammary fat pads cleared of epithelium, in abdominal fat pads, and in adipocytes from both sources and from intact mammary gland of mature virgin, pregnant, and lactating mice. Accumulation of glycogen, the activity of glycogen synthase, and the lipogenic rate in abdominal and mammary adipocytes remained high during pregnancy but decreased to insignificant levels by early lactation. The depressant effects of lactation were observed solely in those mammary adipocytes isolated from intact glands. The presence of mammary epithelial cells was also required to effect the stimulated lipogenesis in mammary adipocytes during pregnancy, We conclude that the metabolic activity of adipocytes is modulated both during pregnancy and lactation to channel nutrients to the mammary epithelial cell. The fact that the changes occur in mammary adipocytes only when epithelial cells are present indicates that local as well as systemic factors are operating in these modulations.

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