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 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 ErbB4 Splice Variants Cyt1 and Cyt2 Differ by 16 Amino Acids and Exert Opposing Effects on the Mammary Epithelium In Vivo

2009 ◽  
Vol 29 (18) ◽  
pp. 4935-4948 ◽  
Author(s):  
Rebecca S. Muraoka-Cook ◽  
Melissa A. Sandahl ◽  
Karen E. Strunk ◽  
Leah C. Miraglia ◽  
Carty Husted ◽  
...  
Keyword(s):  
Amino Acids ◽  
Mammary Epithelial Cells ◽  
Splice Variants ◽  
Three Dimensional ◽  
Mammary Epithelium ◽  
Mammary Epithelial ◽  
Lumen Formation ◽  
Transgenic Expression ◽  
Opposing Effects

ABSTRACT Data concerning the prognostic value of ErbB4 in breast cancer and effects on cell growth have varied in published reports, perhaps due to the unknown signaling consequences of expression of the intracellular proteolytic ErbB4 s80HER4 fragment or due to differing signaling capabilities of alternatively spliced ErbB4 isoforms. One isoform (Cyt1) contains a 16-residue intracellular sequence that is absent from the other (Cyt2). We expressed s80Cyt1 and s80Cyt2 in HC11 mammary epithelial cells, finding diametrically opposed effects on the growth and organization of colonies in three-dimensional matrices. Whereas expression of s80Cyt1 decreased growth and increased the rate of three-dimensional lumen formation, that of s80Cyt2 increased proliferation without promoting lumen formation. These results were recapitulated in vivo, using doxycycline-inducible, mouse breast-transgenic expression of s80Cyt1 amd s80Cyt2. Expression of s80Cyt1 decreased growth of the mammary ductal epithelium, caused precocious STAT5a activation and lactogenic differentiation, and increased cell surface E-cadherin levels. Remarkably, ductal growth inhibition by s80Cyt1 occurred simultaneously with lobuloalveolar growth that was unimpeded by s80Cyt1, suggesting that the response to ErbB4 may be influenced by the epithelial subtype. In contrast, expression of s80Cyt2 caused epithelial hyperplasia, increased Wnt and nuclear β-catenin expression, and elevated expression of c-myc and cyclin D1 in the mammary epithelium. These results demonstrate that the Cyt1 and Cyt2 ErbB4 isoforms, differing by only 16 amino acids, exhibit markedly opposing effects on mammary epithelium growth and differentiation.

scholarly journals Deletion of IGF-1 Receptors in Cardiomyocytes Attenuates Cardiac Aging in Male Mice

Endocrinology ◽  
2016 ◽  
Vol 157 (1) ◽  
pp. 336-345 ◽  
Author(s):  
Sangmi Ock ◽  
Wang Soo Lee ◽  
Jihyun Ahn ◽  
Hyun Min Kim ◽  
Hyun Kang ◽  
...  
Keyword(s):  
Kinase Inhibitor ◽  
Nuclear Factor Κb ◽  
Nuclear Factor ◽  
Wild Type ◽  
Hypertrophic Response ◽  
Age Related ◽  
Receptor Activator ◽  
Phosphoinositide 3 Kinase ◽  
Cultured Cardiomyocytes

Abstract IGF-1 receptor (IGF-1R) signaling is implicated in cardiac hypertrophy and longevity. However, the role of IGF-1R in age-related cardiac remodeling is only partially understood. We therefore sought to determine whether the deletion of the IGF-1R in cardiomyocytes might delay the development of aging-associated myocardial pathologies by examining 2-year-old male cardiomyocyte-specific IGF-1R knockout (CIGF1RKO) mice. Aging was associated with the induction of IGF-1R expression in hearts. Cardiomyocytes hypertrophied with age in wild-type (WT) mice. In contrast, the cardiac hypertrophic response associated with aging was blunted in CIGF1RKO mice. Concomitantly, fibrosis was reduced in aged CIGF1RKO compared with aged WT hearts. Expression of proinflammatory cytokines such as IL-1α, IL-1β, IL-6, and receptor activator of nuclear factor-κB ligand was increased in aged WT hearts, but this increase was attenuated in aged CIGF1RKO hearts. Phosphorylation of Akt was increased in aged WT, but not in aged CIGF1RKO, hearts. In cultured cardiomyocytes, IGF-1 induced senescence as demonstrated by increased senescence-associated β-galactosidase staining, and a phosphoinositide 3-kinase inhibitor inhibited this effect. Furthermore, inhibition of phosphoinositide 3-kinase significantly prevented the increase in IL-1α, IL-1β, receptor activator of nuclear factor-κB ligand, and p21 protein expression by IGF-1. These data reveal an essential role for the IGF-1-IGF-1R-Akt pathway in mediating cardiomyocyte senescence.

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