Regulation of type II deiodinase expression by EGF and  glucocorticoid in HC11 mouse mammary epithelium

Thyroid hormones are important for mammary gland growth and development. The iodothyronine deiodinases play a key role in thyroid hormone metabolism. We have showed that type II 5′-deiodinase (5′D2) activity and mRNA are present in the mouse mammary gland and that their levels are reduced in the lactating gland. To investigate the regulatory mechanism of mouse 5′D2 gene ( mdio2) expression in mammary epithelium, we employed the HC11 cell line, which is derived from mouse mammary epithelial cells and retains the ability to express differentiated function. HC11 cells were treated with combinations of insulin, glucocorticoid (GC, dexamethasone), prolactin, and epidermal growth factor (EGF), and 5′D2 activity and the D2-to-GAPDH mRNA ratio were measured by125I− release from 125I-labeled thyroxine and semiquantitative RT-PCR, respectively. EGF increased both 5′D2 activity and mRNA levels about twofold. GC reduced both 5′D2 activity and mRNA in a dose-dependent manner, and their levels were decreased to approximately one-tenth and one-fifth, respectively, of control levels. These data demonstrated that mdio2expression in HC11 cells is upregulated by EGF mainly at the pretranslational level and downregulated by GC at both pre- and posttranslational levels. Furthermore, we showed that GC reduced the promoter activity of the 627- bp 5′-upstream region of the mdio2/luciferase chimeric reporter gene, suggesting that GC exerts its effect, at least in part, at the transcriptional level.

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Differential expression of the growth hormone receptor and growth hormone-binding protein in epithelia and stroma of the mouse mammary gland at various physiological stages

Journal of Endocrinology ◽

10.1677/joe.0.1610077 ◽

1999 ◽

Vol 161 (1) ◽

pp. 77-87 ◽

Cited By ~ 16

Author(s):

YN Ilkbahar ◽

G Thordarson ◽

IG Camarillo ◽

F Talamantes

Keyword(s):

Growth Hormone ◽

Mammary Gland ◽

Epithelial Cells ◽

Growth Hormone Receptor ◽

Mammary Epithelial Cells ◽

Late Pregnancy ◽

Mammary Glands ◽

Mouse Mammary Gland ◽

Mammary Epithelial ◽

Mrna Levels

Increasing evidence suggests that GH is important in normal mammary gland development. To investigate this further, we studied the distribution and levels of growth hormone receptor (GHR) and GH-binding protein (GHBP) in the mouse mammary gland. At three weeks of age, the epithelial component of the right fourth inguinal mammary gland of female mice was removed. These animals were then either maintained as virgins until they were killed or they were mated. One group of the mated mice was killed on day 18 of pregnancy and the remaining mated animals were allowed to carry their pups until term and were killed on day 6 of lactation. At the time of death, both the intact left and the de-epithelialized right mammary glands were collected from all three groups. Some of the intact glands served as a source of epithelial cells, free of stroma. The mRNA levels for GHR and GHBP were measured in intact glands, epithelia-cleared fat pads, and isolated mammary epithelial cells. GHR and GHBP mRNAs were expressed in both the mammary epithelium and stroma. However, the levels of both GHR and GHBP mRNAs were significantly higher in the stroma as compared with the epithelium component. This increase for both mRNAs was from 3- to 12-fold at each physiological state examined. In the intact gland, both GHR and GHBP transcripts were highest in virgins, declined during late pregnancy, and the lowest levels were found in the lactating gland. GHBP and GHR protein concentrations were also assessed in intact glands and epithelia-free fat pads. Similar to the mRNAs, GHR and GHBP protein levels (means+/-s.e.m.) in intact glands were highest in virgin mice (0.891+/-0.15 pmoles/mg protein and 0.136+/-0.26 pmoles/mg protein respectively), declined during late pregnancy (0. 354+/-0.111 pmoles/mg protein and 0.178+/-0.039 pmoles/mg protein respectively), and were lowest during lactation (0.096+0.037 pmoles/mg protein and 0.017+0.006 pmoles/mg protein respectively). Immunocytochemistry utilizing specific antisera against mouse (m) GHR and mGHBP revealed that the two proteins are localized to both the stroma and parenchyma of mouse mammary glands, with similar patterns of immunostaining throughout the different physiological stages analyzed. GHR immunolocalized to the plasma membrane and cytosol of mammary epithelial cells and adipocytes, whereas the GHBP immunostaining was nuclear and cytosolic. In conclusion, we report here that GHR and GHBP mRNAs and proteins are expressed in both the epithelium and the stroma of mammary glands of virgin, pregnant, and lactating mice. In intact glands, GHR and GHBP proteins, as well as their transcripts are higher in abundance in virgin relative to lactating mice. At all physiological stages, GHR and GHBP mRNA levels are higher in the stroma compared with the parenchyma. These findings indicate that the actions of GH in the mammary gland are both direct through its binding to the epithelia, and indirect by binding to the stroma and stimulation of IGF-I production which, in turn, affects mammary epithelial development.

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Progesterone and EGF inhibit mouse mammary gland prolactin receptor and beta-casein gene expression

AJP Cell Physiology ◽

10.1152/ajpcell.1994.267.5.c1467 ◽

1994 ◽

Vol 267 (5) ◽

pp. C1467-C1472 ◽

Cited By ~ 32

Author(s):

S. Nishikawa ◽

R. C. Moore ◽

N. Nonomura ◽

T. Oka

Keyword(s):

Gene Expression ◽

Mammary Gland ◽

Prolactin Receptor ◽

Mammary Epithelium ◽

Mouse Mammary Gland ◽

Mrna Levels ◽

Casein Gene ◽

Beta Casein

Regulation of mouse mammary gland long-form prolactin receptor (PRL-RL) mRNA levels by progesterone and epidermal growth factor (EGF) and the relationship between PRL-RL and beta-casein gene expression were examined in vivo and in vitro. PRL-RL and beta-casein mRNA levels increased approximately 6- and 15-fold from the pregnant to the lactating period, respectively, when normalized to the level of beta-actin mRNA. Ovariectomy of pregnant mice rapidly reduced the serum concentration of progesterone and increased the level of PRL-RL and beta-casein mRNAs approximately three- and fourfold compared with sham-operated animals 24 h after the operation. Injection of progesterone, but not estrogen, inhibited the increase in both mRNA levels. PRL-RL and beta-casein mRNA levels in cultured mammary epithelium increased in response to insulin, hydrocortisone, and prolactin, whereas progesterone or EGF caused inhibition. The combination of EGF and progesterone produced a greater inhibition than either hormone alone. These results indicate that both progesterone and EGF serve as negative regulators of lactogenesis.

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Endotoxin-Induced Tissue Factor in Human Monocytes is Dependent upon Protein Kinase C Activation

Thrombosis and Haemostasis ◽

10.1055/s-0038-1649673 ◽

1993 ◽

Vol 70 (05) ◽

pp. 800-806 ◽

Cited By ~ 29

Author(s):

C Ternisien ◽

M Ramani ◽

V Ollivier ◽

F Khechai ◽

T Vu ◽

...

Keyword(s):

Protein Kinase C ◽

Protein Kinase ◽

Tissue Factor ◽

Factor Ix ◽

Transcriptional Level ◽

Mrna Levels ◽

Dependent Manner ◽

Human Monocytes ◽

Kinase C ◽

Pkc Activation

SummaryTissue factor (TF) is a transmembrane receptor which, in association with factors VII and Vila, activates factor IX and X, thereby activating the coagulation protease cascades. In response to bacterial lipopolysaccharide (LPS) monocytes transcribe, synthesize and express TF on their surface. We investigated whether LPS-induced TF in human monocytes is mediated by protein kinase C (PKC) activation. The PKC agonists phorbol 12- myristate 13-acetate (PMA) and phorbol 12, 13 dibutyrate (PdBu) were both potent inducers of TF in human monocytes, whereas 4 alpha-12, 13 didecanoate (4 a-Pdd) had no such effect. Both LPS- and PMA-induced TF activity were inhibited, in a concentration dependent manner, by three different PKC inhibitors: H7, staurosporine and calphostin C. TF antigen determination confirmed that LPS-induced cell-surface TF protein levels decreased in parallel to TF functional activity under staurosporine treatment. Moreover, Northern blot analysis of total RNA from LPS- or PMA-stimulated monocytes showed a concentration-dependent decrease in TF mRNA levels in response to H7 and staurosporine. The decay rate of LPS-induced TF mRNA evaluated after the arrest of transcription by actinomycin D was not affected by the addition of staurosporine, suggesting that its inhibitory effect occurred at a transcriptional level. We conclude that LPS-induced production of TF and its mRNA by human monocytes are dependent on PKC activation.

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Fine-tuning of Epithelial EGFR signals Supports Coordinated Mammary Gland Development

10.1101/2020.09.10.291872 ◽

2020 ◽

Author(s):

Alexandr Samocha ◽

Hanna M. Doh ◽

Vaishnavi Sitarama ◽

Quy H. Nguyen ◽

Oghenekevwe Gbenedio ◽

...

Keyword(s):

Mammary Gland ◽

Mammary Epithelial Cells ◽

Mammary Epithelium ◽

Gene Signature ◽

Mammary Epithelial ◽

Fine Tuning ◽

Basal Cells ◽

Stem And Progenitor Cells

SummaryDuring puberty, robust morphogenesis occurs in the mammary gland; stem- and progenitor-cells develop into mature basal- and luminal-cells to form the ductal tree. The receptor signals that govern this process in mammary epithelial cells (MECs) are incompletely understood. The EGFR has been implicated and here we focused on EGFR’s downstream pathway component Rasgrp1. We find that Rasgrp1 dampens EGF-triggered signals in MECs. Biochemically and in vitro, Rasgrp1 perturbation results in increased EGFR-Ras-PI3K-AKT and mTORC1-S6 kinase signals, increased EGF-induced proliferation, and aberrant branching-capacity in 3D cultures. However, in vivo, Rasgrp1 perturbation results in delayed ductal tree maturation with shortened branches and reduced cellularity. Rasgrp1-deficient MEC organoids revealed lower frequencies of basal cells, the compartment that incorporates stem cells. Molecularly, EGF effectively counteracts Wnt signal-driven stem cell gene signature in organoids. Collectively, these studies demonstrate the need for fine-tuning of EGFR signals to properly instruct mammary epithelium during puberty.

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Riboflavin uptake transporter Slc52a2 (RFVT2) is upregulated in the mouse mammary gland during lactation

AJP Regulatory Integrative and Comparative Physiology ◽

10.1152/ajpregu.00507.2015 ◽

2016 ◽

Vol 310 (7) ◽

pp. R578-R585 ◽

Cited By ~ 8

Author(s):

Alex Man Lai Wu ◽

Liana Dedina ◽

Pooja Dalvi ◽

Mingdong Yang ◽

John Leon-Cheon ◽

...

Keyword(s):

Mammary Gland ◽

Breast Milk ◽

Milk Fat ◽

Fluorescent Protein ◽

Mammary Epithelial Cells ◽

Mdck Cells ◽

Basolateral Membrane ◽

Mouse Mammary Gland ◽

Transport Systems ◽

Human Breast Milk

While it is well recognized that riboflavin accumulates in breast milk as an essential vitamin for neonates, transport mechanisms for its milk excretion are not well characterized. The multidrug efflux transporter ABCG2 in the apical membrane of milk-producing mammary epithelial cells (MECs) is involved with riboflavin excretion. However, it is not clear whether MECs possess other riboflavin transport systems, which may facilitate its basolateral uptake into MECs. We report here that transcripts encoding the second ( SLC52A2) and third ( SLC52A3) member of the recently discovered family of SLC52A riboflavin uptake transporters are expressed in milk fat globules from human breast milk. Furthermore, Slc52a2 and Slc52a3 mRNA are upregulated in the mouse mammary gland during lactation. Importantly, the induction of Slc52a2, which was the major Slc52a riboflavin transporter in the lactating mammary gland, was also observed at the protein level. Subcellular localization studies showed that green fluorescent protein-tagged mouse SLC52A2 mainly localized to the cell membrane, with no preferential distribution to the apical or basolateral membrane in polarized kidney MDCK cells. These results strongly implicate a potential role for SLC52A2 in riboflavin uptake by milk-producing MECs, a critical step in the transfer of riboflavin into breast milk.

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Selective Response to Insulin Versus Insulin-Like Growth Factor-I and -II and Up-Regulation of Insulin Receptor Splice Variant B in the Differentiated Mouse Mammary Epithelium

Endocrinology ◽

10.1210/en.2008-0668 ◽

2009 ◽

Vol 150 (6) ◽

pp. 2924-2933 ◽

Cited By ~ 29

Author(s):

Chiara Berlato ◽

Wolfgang Doppler

Keyword(s):

Mammary Gland ◽

Insulin Receptor ◽

Lactate Production ◽

Terminal Differentiation ◽

Mammary Epithelium ◽

Epithelial Cell Line ◽

Human Form ◽

Igf Binding Proteins ◽

Stage Of Development ◽

Hc11 Cells

The terminal differentiation of the mouse mammary gland epithelium during lactation has been shown to require IGFs and/or superphysiological levels of insulin. It has been suggested that IGF receptor I (IGF-IR), in addition to its well-established role in the mammary gland during puberty and pregnancy, serves as the principal mediator of IGFs at this stage of development. However, our analysis of the expression levels of IGF-IR and the two insulin receptor (IR) splice variants, IR-A and IR-B, has revealed a 3- to 4-fold up-regulation of IR-B transcripts and a 6-fold down-regulation of IGF-IR transcripts and protein during terminal differentiation in the developing mammary gland. IR-B expression was also more than 10-fold up-regulated in murine mammary epithelial cell line HC11 during differentiation in vitro. As already described for the human form, murine IR-B cloned from HC11 exhibited selectivity for insulin as compared with IGFs. When differentiated HC11 cells were stimulated by 10 nm insulin, a concentration that is unable to activate IGF-IR, induction of milk protein and lipid synthetic enzyme gene expression, lactate production, and phosphorylation of Akt were observed. In contrast, on differentiated HC11 cells 10 nm IGF-I or 10 nm IGF-II were able to exert growth-promoting effects only. The lack of response of differentiated cells to low levels of IGFs could not be explained by inactivation of IGFs by IGF binding proteins. Our results suggest a previously unrecognized predominant role for IR-B in the differentiated mammary epithelium.

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Expression and function of CCAAT/enhancer binding protein? (C/EBP?) LAP and LIP isoforms in mouse mammary gland, tumors and cultured mammary epithelial cells

Journal of Cellular Biochemistry ◽

10.1002/jcb.1167 ◽

2001 ◽

Vol 82 (3) ◽

pp. 357-370 ◽

Cited By ~ 28

Author(s):

L.R. Dearth ◽

J. Hutt ◽

A. Sattler ◽

A. Gigliotti ◽

J. DeWille

Keyword(s):

Mammary Gland ◽

Epithelial Cells ◽

Protein C ◽

Mammary Epithelial Cells ◽

Binding Protein ◽

Mouse Mammary Gland ◽

Mammary Epithelial ◽

Mammary Gland Tumors ◽

Enhancer Binding Protein ◽

And Function

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Cigarette smoke and its component acrolein augment IL-8/CXCL8 mRNA stability via p38 MAPK/MK2 signaling in human pulmonary cells

AJP Lung Cellular and Molecular Physiology ◽

10.1152/ajplung.00046.2012 ◽

2012 ◽

Vol 303 (10) ◽

pp. L929-L938 ◽

Cited By ~ 51

Author(s):

Nadia Moretto ◽

Serena Bertolini ◽

Claudia Iadicicco ◽

Gessica Marchini ◽

Manminder Kaur ◽

...

Keyword(s):

Epithelial Cells ◽

Cigarette Smoke ◽

P38 Mapk ◽

Mrna Stability ◽

Airway Epithelial Cells ◽

Transcriptional Level ◽

Mrna Levels ◽

Dependent Manner ◽

Pharmacological Inhibition ◽

Pulmonary Cells

Interleukin-8 (IL-8/CXCL8) is an important neutrophil chemoattractant known to be elevated in the airways of cigarette smokers and in patients with chronic obstructive pulmonary disease (COPD). We examined the acute effect of aqueous cigarette smoke extract (CSE) on IL-8 expression in primary human pulmonary cells, in particular in normal human bronchial smooth muscle cells (HBSMCs). IL-8 mRNA levels increased upon CSE exposure in a concentration- and time-dependent manner, and such an effect was accompanied by IL-8 secretion. CSE-evoked elevation of IL-8 mRNA was mimicked by its component acrolein. Both CSE and acrolein induced p38 mitogen-activated protein kinase (MAPK) phosphorylation, accompanied by the phosphorylation of MAPK-activated kinase 2 (MK2), a known downstream substrate of the p38 MAPK, both in HBSMCs and in human airway epithelial cells. Furthermore, pharmacological inhibition of p38 MAPK or MK2 strongly accelerated the decay of IL-8 mRNA levels upon stimulation with CSE or acrolein and subsequent blockade of mRNA neosynthesis with actinomycin D in pulmonary structural cells (HBSMCs and airways epithelial cells) as well as in human alveolar macrophages. Conversely, pharmacological inhibition of ERK1/2 signaling inhibited CSE-induced steady-state levels of IL-8 mRNA without affecting mRNA stability, thus suggesting inhibition at the transcriptional level. In sum, p38 MAPK/MK2 signaling is an important posttranscriptional mechanism underlying upregulation of IL-8 mRNA levels elicited by CSE and acrolein. Given the pivotal role of IL-8 in neutrophil chemotaxis and activation, our results shed light on the mechanisms through which cigarette smoke can initiate inflammation in the lung.

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