Canonical wnt signaling activity in early stages of chick lung development

Wnt signaling pathway is an essential player during vertebrate embryonic development which has been associated with several developmental processes such as gastrulation, body axis formation and morphogenesis of numerous organs, namely the lung. Wnt proteins act through specific transmembrane receptors, which activate intracellular pathways that regulate cellular processes such as cell proliferation, differentiation and death. Morphogenesis of the fetal lung depends on epithelial-mesenchymal interactions that are governed by several growth and transcription factors that regulate cell proliferation, fate, migration and differentiation. This process is controlled by different signaling pathways such as FGF, Shh and Wnt among others. Wnt signaling is recognized as a key molecular player in mammalian pulmonary development but little is known about its function in avian lung development. The present work characterizes, for the first time, the expression pattern of several Wnt signaling members, such as wnt-1, wnt-2b, wnt-3a, wnt-5a, wnt-7b, wnt-8b, wnt-9a, lrp5, lrp6, sfrp1, dkk1, β-catenin and axin2 at early stages of chick lung development. In general, their expression is similar to their mammalian counterparts. By assessing protein expression levels of active/total β-catenin and phospho-LRP6/LRP6 it is revealed that canonical Wnt signaling is active in this embryonic tissue. In vitro inhibition studies were performed in order to evaluate the function of Wnt signaling pathway in lung branching. Lung explants treated with canonical Wnt signaling inhibitors (FH535 and PK115-584) presented an impairment of secondary branch formation after 48 h of culture along with a decrease in axin2 expression levels. Branching analysis confirmed this inhibition. Wnt-FGF crosstalk assessment revealed that this interaction is preserved in the chick lung. This study demonstrates that Wnt signaling is crucial for precise chick lung branching and further supports the avian lung as a good model for branching studies since it recapitulates early mammalian pulmonary development.Rute S. Moura was supported by a grant of ON.2 SR&TD Integrated Program (N-01-01-0124-01-07), ref: UMINHO/BPD/31/2013. The funders had no role in study design, data collection and analysis

The F-box protein FBXO45 promotes the proteasome-dependent degradation of p73

The transcription factor p73, a member of the p53 family, mediates cell-cycle arrest and apoptosis in response to DNA damage-induced cellular stress, acting thus as a proapoptotic gene. Similar to p53, p73 activity is regulated by post-translational modification, including phosphorylation, acetylation and ubiquitylation. In C. elegans, the F-box protein FSN-1 controls germline apoptosis by regulating CEP-1, the single ancestral p53 family member. Here we report that FBXO45, the human ortholog of FSN-1, binds specifically to p73 triggering its proteasome-dependent degradation. Importantly, SCF(FBXO45) ubiquitylates p73 both in vivo and in vitro. Moreover, siRNA-mediated depletion of FBXO45 stabilizes p73 and concomitantly induces cell death in a p53-independent manner. All together, these results show that the orphan F-box protein FBXO45 regulates the stability of p73, highlighting a conserved pathway evolved from nematode to human by which the p53 members are regulated by an SCF-dependent mechanism

β-catenin promotes endothelial survival by regulating eNOS activity and flow-dependent anti-apoptotic gene expression

Increased endothelial cell (EC) apoptosis is associated with the development of atherosclerotic plaques that develop predominantly at sites exposed to disturbed flow (DF). Strategies to promote EC survival may therefore represent a novel therapeutic approach in cardiovascular disease. Nitric oxide (NO) and β-catenin have both been shown to promote cell survival and they interact in ECs as we previously demonstrated. Here we investigated the physiological role of β-catenin as a mediator of NO-induced cell survival in ECs. We found that β-catenin depleted human umbilical vein ECs (HUVEC) stimulated with pharmacological activators of endothelial NO synthase (eNOS) showed a reduction in eNOS phosphorylation (Ser1177) as well as reduced intracellular cyclic guanosine monophosphate levels compared to control cells in static cultures. In addition, β-catenin depletion abrogated the protective effects of the NO donor, Snitroso-N-acetylpenicillamine, during TNFα- and H2O2-induced apoptosis. Using an orbital shaker to generate shear stress, we confirmed eNOS and β-catenin interaction in HUVEC exposed to undisturbed flow and DF and showed that β-catenin depletion reduced eNOS phosphorylation. β-catenin depletion promoted apoptosis exclusively in HUVEC exposed to DF as did inhibition of soluble guanylate cyclase (sGC) or β-catenin transcriptional activity. The expression of the pro-survival genes, Bcl-2 and survivin was also reduced following inhibition of β-catenin transcriptional activity, as was the expression of eNOS. In conclusion, our data demonstrate that β-catenin is a positive regulator of eNOS activity and cell survival in human ECs. sGC activity and β-catenin-dependent transcription of Bcl-2, survivin, BIRC3 and eNOS are essential to maintain cell survival in ECs under DF