A Lentivirus-Mediated Genetic Screen Identifies Dihydrofolate Reductase (DHFR) as a Modulator of β-Catenin/GSK3 Signaling

The multi-protein β-catenin destruction complex tightly regulates β-catenin protein levels by shuttling β-catenin to the proteasome. Glycogen synthase kinase 3β (GSK3β), a key serine/threonine kinase in the destruction complex, is responsible for several phosphorylation events that mark β-catenin for ubiquitination and subsequent degradation. Because modulation of both β-catenin and GSK3β activity may have important implications for treating disease, a complete understanding of the mechanisms that regulate the β-catenin/GSK3β interaction is warranted. We screened an arrayed lentivirus library expressing small hairpin RNAs (shRNAs) targeting 5,201 human druggable genes for silencing events that activate a β-catenin pathway reporter (BAR) in synergy with 6-bromoindirubin-3′oxime (BIO), a specific inhibitor of GSK3β. Top screen hits included shRNAs targeting dihydrofolate reductase (DHFR), the target of the anti-inflammatory compound methotrexate. Exposure of cells to BIO plus methotrexate resulted in potent synergistic activation of BAR activity, reduction of β-catenin phosphorylation at GSK3-specific sites, and accumulation of nuclear β-catenin. Furthermore, the observed synergy correlated with inhibitory phosphorylation of GSK3β and was neutralized upon inhibition of phosphatidyl inositol 3-kinase (PI3K). Linking these observations to inflammation, we also observed synergistic inhibition of lipopolysaccharide (LPS)-induced production of pro-inflammatory cytokines (TNFα, IL-6, and IL-12), and increased production of the anti-inflammatory cytokine IL-10 in peripheral blood mononuclear cells exposed to GSK3 inhibitors and methotrexate. Our data establish DHFR as a novel modulator of β-catenin and GSK3 signaling and raise several implications for clinical use of combined methotrexate and GSK3 inhibitors as treatment for inflammatory disease

Relationships, love and sexuality: what the Filipino teens think and feel

<p>Abstract</p> <p>Background</p> <p>In order to achieve a change among teens' sexual behavior, an important step is to improve our knowledge about their opinions concerning relationships, love and sexuality.</p> <p>Methods</p> <p>A questionnaire including topics on relationships, love and sexuality was distributed to a target population of 4,000 Filipino students from third year high school to third year college. Participants were obtained through multi-stage sampling of clusters of universities and schools. This paper concentrates on teens aged 13 to 18.</p> <p>Results</p> <p>Students reported that they obtained information about love and sexuality mainly from friends. However, they valued parents' opinion more than friends'. They revealed few conversations with their parents on these topics. A majority of them would like to have more information, mainly about emotion-related topics. Almost half of respondents were not aware that condoms are not 100% effective in preventing STIs or pregnancies. More girls, compared to boys, were sensitive and opposed to several types of sexism. After adjusting for sex, age and institution, the belief of 100% condom effectiveness and the approval of pornography and sexism were associated with being sexually experienced.</p> <p>Conclusion</p> <p>There is room for further encouraging parents to talk more with their children about sexuality, specially aspects related to feelings and emotions in order to help them make better sexual choices. Indeed, teens wish to better communicate with their parents on these issues. Condoms are regarded as safer than what they really are by almost half of the participants of this study, and such incorrect knowledge seems to be associated with sexual initiation.</p