Progesterone Receptor Activates Msx2 Expression by Downregulating TNAP/Akp2 and Activating the Bmp Pathway in EpH4 Mouse Mammary Epithelial Cells

Previously we demonstrated that EpH4 mouse mammary epithelial cells induced the homeobox transcription factor Msx2 either when transfected with the progesterone receptor (PR) or when treated with Bmp2/4. Msx2 upregulation was unaffected by Wnt inhibitors s-FRP or Dkk1, but was inhibited by the Bmp antagonist Noggin. We therefore hypothesized that PR signaling to Msx2 acts through the Bmp receptor pathway. Herein, we confirm that transcripts for Alk2/ActR1A, a non-canonical BmpR Type I, are upregulated in mammary epithelial cells overexpressing PR (EpH4-PR). Increased phosphorylation of Smads 1,5, 8, known substrates for Alk2 and other BmpR Type I proteins, was observed as was their translocation to the nucleus in EpH4-PR cells. Analysis also showed that Tissue Non-Specific Alkaline Phosphatase (TNAP/Akp2) was also found to be downregulated in EpH4-PR cells. When an Akp2 promoter-reporter construct containing a ½PRE site was transfected into EpH4-PR cells, its expression was downregulated. Moreover, siRNA mediated knockdown of Akp2 increased both Alk2 and Msx2 expression. Collectively these data suggest that PR inhibition of Akp2 results in increased Alk2 activity, increased phosphorylation of Smads 1,5,8, and ultimately upregulation of Msx2. These studies imply that re-activation of the Akp2 gene could be helpful in downregulating aberrant Msx2 expression in PR+ breast cancers

Characterization of ductal and lobular breast carcinomas using novel prolactin receptor isoform specific antibodies

<p>Abstract</p> <p>Background</p> <p>Prolactin is a polypeptide hormone responsible for proliferation and differentiation of the mammary gland. More recently, prolactin's role in mammary carcinogenesis has been studied with greater interest. Studies from our laboratory and from others have demonstrated that three specific isoforms of the prolactin receptor (PRLR) are expressed in both normal and cancerous breast cells and tissues. Until now, reliable isoform specific antibodies have been lacking. We have prepared and characterized polyclonal antibodies against each of the human PRLR isoforms that can effectively be used to characterize human breast cancers.</p> <p>Methods</p> <p>Rabbits were immunized with synthetic peptides of isoform unique regions and immune sera affinity purified prior to validation by Western blot and immunohistochemical analyses. Sections of ductal and lobular carcinomas were stained with each affinity purified isoform specific antibody to determine expression patterns in breast cancer subclasses.</p> <p>Results</p> <p>We show that the rabbit antibodies have high titer and could specifically recognize each isoform of PRLR. Differences in PRLR isoform expression levels were observed and quantified using histosections from xenografts of established human breast cancer cells lines, and ductal and lobular carcinoma human biopsy specimens. In addition, these results were verified by real-time PCR with isoform specific primers. While nearly all tumors contained LF and SF1b, the majority (76%) of ductal carcinoma biopsies expressed SF1a while the majority of lobular carcinomas lacked SF1a staining (72%) and 27% had only low levels of expression.</p> <p>Conclusions</p> <p>Differences in the receptor isoform expression profiles may be critical to understanding the role of PRL in mammary tumorigenesis. Since these antibodies are specifically directed against each PRLR isoform, they are valuable tools for the evaluation of breast cancer PRLR content and have potential clinical importance in treatment of this disease by providing new reagents to study the protein expression of the human PRLR.</p

Tyrosine Phosphorylation of the UDP-Glucose Dehydrogenase of Escherichia coli Is at the Crossroads of Colanic Acid Synthesis and Polymyxin Resistance

BACKGROUND:In recent years, an idiosyncratic new class of bacterial enzymes, named BY-kinases, has been shown to catalyze protein-tyrosine phosphorylation. These enzymes share no structural and functional similarities with their eukaryotic counterparts and, to date, only few substrates of BY-kinases have been characterized. BY-kinases have been shown to participate in various physiological processes. Nevertheless, we are at a very early stage of defining their importance in the bacterial cell. In Escherichia coli, two BY-kinases, Wzc and Etk, have been characterized biochemically. Wzc has been shown to phosphorylate the UDP-glucose dehydrogenase Ugd in vitro. Not only is Ugd involved in the biosynthesis of extracellular polysaccharides, but also in the production of UDP-4-amino-4-deoxy-L-arabinose, a compound that renders E. coli resistant to cationic antimicrobial peptides. METHODOLOGY/PRINCIPAL FINDINGS:Here, we studied the role of Ugd phosphorylation. We first confirmed in vivo the phosphorylation of Ugd by Wzc and we demonstrated that Ugd is also phosphorylated by Etk, the other BY-kinase identified in E. coli. Tyrosine 71 (Tyr71) was characterized as the Ugd site phosphorylated by both Wzc and Etk. The regulatory role of Tyr71 phosphorylation on Ugd activity was then assessed and Tyr71 mutation was found to prevent Ugd activation by phosphorylation. Further, Ugd phosphorylation by Wzc or Etk was shown to serve distinct physiological purposes. Phosphorylation of Ugd by Wzc was found to participate in the regulation of the amount of the exopolysaccharide colanic acid, whereas Etk-mediated Ugd phosphorylation appeared to participate in the resistance of E. coli to the antibiotic polymyxin. CONCLUSIONS/SIGNIFICANCE:Ugd phosphorylation seems to be at the junction between two distinct biosynthetic pathways, illustrating the regulatory potential of tyrosine phosphorylation in bacterial physiology

Climate-sensitive health priorities in Nunatsiavut, Canada

Background: This exploratory study used participatory methods to identify, characterize, and rank climate-sensitive health priorities in Nunatsiavut, Labrador, Canada. Methods: A mixed method study design was used and involved collecting both qualitative and quantitative data at regional, community, and individual levels. In-depth interviews with regional health representatives were conducted throughout Nunatsiavut (n = 11). In addition, three PhotoVoice workshops were held with Rigolet community members (n = 11), where participants took photos of areas, items, or concepts that expressed how climate change is impacting their health. The workshop groups shared their photographs, discussed the stories and messages behind them, and then grouped photos into re-occurring themes. Two community surveys were administered in Rigolet to capture data on observed climatic and environmental changes in the area, and perceived impacts on health, wellbeing, and lifestyles (n = 187). Results: Climate-sensitive health pathways were described in terms of inter-relationships between environmental and social determinants of Inuit health. The climate-sensitive health priorities for the region included food security, water security, mental health and wellbeing, new hazards and safety concerns, and health services and delivery. Conclusions: The results highlight several climate-sensitive health priorities that are specific to the Nunatsiavut region, and suggest approaching health research and adaptation planning from an EcoHealth perspective