scholarly journals NFκB1/p50 Is Not Required for Tumor Necrosis Factor-Stimulated Growth of Primary Mammary Epithelial Cells: Implications for NFκB2/p52 and RelB

Endocrinology ◽  
2007 ◽  
Vol 148 (1) ◽  
pp. 268-278 ◽  
Author(s):  
Jiping Zhang ◽  
Mary Ann Warren ◽  
Suzanne F. Shoemaker ◽  
Margot M. Ip
Keyword(s):  
Dna Binding ◽  
Epithelial Cells ◽  
Cyclin D1 ◽  
Mammary Epithelial Cells ◽  
Three Dimensional ◽  
Nuclear Factor Κb ◽  
Mammary Epithelial ◽  
Wild Type ◽  
Nuclear Extracts ◽  
Dna Binding Complexes

Nuclear factor κB (NFκB) plays an important role in mammary gland development and breast cancer. We previously demonstrated that TNF stimulates growth of mammary epithelial cells (MEC) in a physiologically relevant three-dimensional primary culture system, accompanied by enhanced DNA-binding of the NFκB p50 homodimer. To further understand the mechanism of TNF-stimulated growth of primary MEC, the requirement for NFκB1/p50, and the role of cyclin D1 in TNF-stimulated growth were examined. TNF induced the formation of DNA-binding complexes of p50 and p52 with their coactivator bcl3 in MEC nuclear extracts. Concomitantly, TNF increased the binding of NFκB proteins to the κB site on the cyclin D1 promoter, and increased expression of cyclin D1 mRNA and protein. Using MEC from p50 null mice, we found that p50 was not required for TNF-induced growth nor for up-regulation of cyclin D1. However, TNF induced a p52/RelB NFκB DNA-binding complex in p50 null MEC nuclear extracts. In addition, we found that in wild-type MEC, TNF stimulated the occupancy of p52 and RelB on the cyclin D1 promoter κB site, whereas p50 was present constitutively. These data suggest that in wild-type MEC, TNF stimulates the interaction of bcl3 with p50 and p52, and the binding of p52, as well as RelB, to cyclin D1 promoter κB sites, and as a consequence, stimulates the growth of MEC. In the absence of p50, p52 and RelB can compensate for p50 in TNF-stimulated growth and cyclin D1 induction in MEC.

scholarly journals A Role for Site-Specific Phosphorylation of Mouse Progesterone Receptor at Serine 191 in Vivo

2014 ◽  
Vol 28 (12) ◽  
pp. 2025-2037 ◽  
Author(s):  
Sandra L. Grimm ◽  
Robert D. Ward ◽  
Alison E. Obr ◽  
Heather L. Franco ◽  
Rodrigo Fernandez-Valdivia ◽  
...  
Keyword(s):  
Mammary Epithelial Cells ◽  
Phosphorylation Site ◽  
Three Dimensional ◽  
Progesterone Receptors ◽  
Nuclear Factor Κb ◽  
Mammary Epithelial ◽  
Nuclear Factor ◽  
Wild Type ◽  
Receptor Activator

Progesterone receptors (PRs) are phosphorylated on multiple sites, and a variety of roles for phosphorylation have been suggested by cell-based studies. Previous studies using PR-null mice have shown that PR plays an important role in female fertility, regulation of uterine growth, the uterine decidualization response, and proliferation as well as ductal side-branching and alveologenesis in the mammary gland. To study the role of PR phosphorylation in vivo, a mouse was engineered with homozygous replacement of PR with a PR serine-to-alanine mutation at amino acid 191. No overt phenotypes were observed in the mammary glands or uteri of PR S191A treated with progesterone (P4). In contrast, although PR S191A mice were fertile, litters were 19% smaller than wild type and the estrous cycle was lengthened slightly. Moreover, P4-dependent gene regulation in primary mammary epithelial cells (MECs) was altered in a gene-selective manner. MECs derived from wild type and PR S191A mice were grown in a three-dimensional culture. Both formed acinar structures that were morphologically similar, and proliferation was stimulated equally by P4. However, P4 induction of receptor activator of nuclear factor-κB ligand and calcitonin was selectively reduced in S191A cultures. These differences were confirmed in freshly isolated MECs. Chromatin immunoprecipitation analysis showed that the binding of S191A PR to some of the receptor activator of nuclear factor-κB ligand enhancers and a calcitonin enhancer was substantially reduced. Thus, the elimination of a single phosphorylation site is sufficient to modulate PR activity in vivo. PR contains many phosphorylation sites, and the coordinate regulation of multiple sites is a potential mechanism for selective modulation of PR function.

scholarly journals PIN1 Is an E2F Target Gene Essential for Neu/Ras-Induced Transformation of Mammary Epithelial Cells

2002 ◽  
Vol 22 (15) ◽  
pp. 5281-5295 ◽  
Author(s):  
Akihide Ryo ◽  
Yih-Cherng Liou ◽  
Gerburg Wulf ◽  
Masafumi Nakamura ◽  
Sam W. Lee ◽  
...  
Keyword(s):  
Epithelial Cells ◽  
Cyclin D1 ◽  
Transcriptional Activation ◽  
Target Gene ◽  
Mammary Epithelial Cells ◽  
Cell Transformation ◽  
Mammary Tumorigenesis ◽  
Mammary Epithelial ◽  
Oncogenic Signaling ◽  
And Function

ABSTRACT Oncogenes Neu/HER2/ErbB2 and Ras can induce mammary tumorigenesis via upregulation of cyclin D1. One major regulatory mechanism in these oncogenic signaling pathways is phosphorylation of serines or threonines preceding proline (pSer/Thr-Pro). Interestingly, the pSer/Thr-Pro motifs in proteins exist in two completely distinct cis and trans conformations, whose conversion is catalyzed specifically by the essential prolyl isomerase Pin1. By isomerizing pSer/Thr-Pro bonds, Pin1 can regulate the conformation and function of certain phosphorylated proteins. We have previously shown that Pin1 is overexpressed in breast tumors and positively regulates cyclin D1 by transcriptional activation and posttranslational stabilization. Moreover, in Pin1 knockout mice, mammary epithelial cells fail to undergo massive proliferation during pregnancy, as is the case in cyclin D1 null mice. These results indicate that Pin1 is upregulated in breast cancer and may be involved in mammary tumors. However, the mechanism of Pin1 overexpression in cancer and its significance in cell transformation remain largely unknown. Here we demonstrate that PIN1 expression is mediated by the transcription factor E2F and enhanced by c-Neu and Ha-Ras via E2F. Furthermore, overexpression of Pin1 not only confers transforming properties on mammary epithelial cells but also enhances the transformed phenotypes of Neu/Ras-transformed mammary epithelial cells. In contrast, inhibition of Pin1 suppresses Neu- and Ras-induced transformed phenotypes, which can be fully rescued by overexpression of a constitutively active cyclin D1 mutant that is refractory to the Pin1 inhibition. Thus, Pin1 is an E2F target gene that is essential for the Neu/Ras-induced transformation of mammary epithelial cells through activation of cyclin D1.

scholarly journals MMTV Env encodes an ITAM responsible for transformation of mammary epithelial cells in three-dimensional culture

2005 ◽  
Vol 201 (3) ◽  
pp. 431-439 ◽  
Author(s):  
Elad Katz ◽  
Mohamed H. Lareef ◽  
John C. Rassa ◽  
Shannon M. Grande ◽  
Leslie B. King ◽  
...  
Keyword(s):  
Epithelial Cells ◽  
Tyrosine Kinases ◽  
Mammary Epithelial Cells ◽  
Cell Transformation ◽  
Three Dimensional ◽  
Soft Agar ◽  
Mammary Epithelial ◽  
Matrigel Invasion ◽  
Tumor Virus

Expression of immunoreceptor tyrosine-based activation motif (ITAM)-containing signaling proteins is normally restricted to hematopoietic tissues. The basal activity of ITAM-containing proteins is mediated through negative regulation by coreceptors restricted to hematopoietic tissues. We have identified an ITAM signaling domain encoded within the env gene of murine mammary tumor virus (MMTV). Three-dimensional structures derived in vitro from murine cells stably transfected with MMTV env display a depolarized morphology in comparison with control mammary epithelial cells. This effect is abolished by Y>F substitution within the Env ITAM, as well as inhibitors of Syk and Src protein tyrosine kinases. Env-expressing cells bear hallmarks of cell transformation such as sensitivity to apoptosis induced by tumor necrosis factor (TNF)–related apoptosis-inducing ligand (TRAIL) or TNFα, as well as down-regulation of E-cadherin and Keratin-18. Human normal mammary epithelial cells expressing MMTV Env also develop transformed phenotype, as typified by growth in soft agar and Matrigel invasion. These disruptions are abrogated by Y>F substitutions. We conclude that ITAM-dependent signals are generated through MMTV Env and trigger early hallmarks of transformation of mouse and human mammary epithelial cells. Therefore, these data suggest a heretofore unappreciated potential mechanism for the initiation of breast cancer and identify MMTV Env and ITAM-containing proteins in human breast tumors as probable oncoproteins.

scholarly journals Cyclic AMP regulates formation of mammary epithelial acini in vitro

2012 ◽  
Vol 23 (15) ◽  
pp. 2973-2981 ◽  
Author(s):  
Pavel I. Nedvetsky ◽  
Sang-Ho Kwon ◽  
Jayanta Debnath ◽  
Keith E. Mostov
Keyword(s):  
Epithelial Cells ◽  
Cyclic Amp ◽  
Mammary Epithelial Cells ◽  
Three Dimensional ◽  
Mammary Epithelial ◽  
Inhibitory Antibody ◽  
Proapoptotic Protein ◽  
Dependent Polarization ◽  
Dependent Protein

Epithelial cells form tubular and acinar structures notable for a hollow lumen. In three-dimensional culture utilizing MCF10A mammary epithelial cells, acini form due to integrin-dependent polarization and survival of cells contacting extracellular matrix (ECM), and the apoptosis of inner cells of acini lacking contact with the ECM. In this paper, we report that cyclic AMP (cAMP)-dependent protein kinase A (PKA) promotes acinus formation via two mechanisms. First, cAMP accelerates redistribution of α6-integrin to the periphery of the acinus and thus facilitates the polarization of outer acinar cells. Blocking of α6-integrin function by inhibitory antibody prevents cAMP-dependent polarization. Second, cAMP promotes the death of inner cells occupying the lumen. In the absence of cAMP, apoptosis is delayed, resulting in perturbed luminal clearance. cAMP-dependent apoptosis is accompanied by a posttranscriptional PKA-dependent increase in the proapoptotic protein Bcl-2 interacting mediator of cell death. These data demonstrate that cAMP regulates lumen formation in mammary epithelial cells in vitro, both through acceleration of polarization of outer cells and apoptosis of inner cells of the acinus.

scholarly journals The Ras oncogene signals centrosome amplification in mammary epithelial cells through cyclin D1/Cdk4 and Nek2

Oncogene ◽  
2010 ◽  
Vol 29 (36) ◽  
pp. 5103-5112 ◽  
Author(s):  
X Zeng ◽  
F Y Shaikh ◽  
M K Harrison ◽  
A M Adon ◽  
A J Trimboli ◽  
...  
Keyword(s):  
Epithelial Cells ◽  
Cyclin D1 ◽  
Mammary Epithelial Cells ◽  
Centrosome Amplification ◽  
Mammary Epithelial ◽  
Ras Oncogene
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