Slit2–Robo4 signalling promotes vascular stability by blocking Arf6 activity

Slit–Roundabout (Robo) signalling has a well-understood role in axon guidance1–5. Unlike in the nervous system, however, Slitdependent activation of an endothelial-specific Robo, Robo4, does not initiate a guidance program. Instead, Robo4 maintains the barrier function of the mature vascular network by inhibiting neovascular tuft formation and endothelial hyperpermeability induced by pro-angiogenic factors 6. In this study, we used cell biological and biochemical techniques to elucidate the molecular mechanism underlying the maintenance of vascular stability by Robo4. Here, we demonstrate that Robo4 mediates Slit2-dependent suppression of cellular protrusive activity through direct interaction with the intracellular adaptor protein paxillin and its paralogue, Hic-5. Formation of a Robo4–paxillin complex at the cell surface blocks activation of the small GTPase Arf6 and, consequently, Rac by recruitment of Arf-GAPs (ADP-ribosylation factor- directed GTPase-activating proteins) such as GIT1. Consistent with these in vitro studies, inhibition of Arf6 activity in vivo phenocopies Robo4 activation by reducing pathologic angiogenesis in choroidal and retinal vascular disease and VEGF-165 (vascular endothelial growth factor-165)-induced retinal hyperpermeability. These data reveal that a Slit2–Robo4–paxillin–GIT1 network inhibits the cellular protrusive activity underlying neovascularization and vascular leak, and identify a new therapeutic target for ameliorating diseases involving the vascular system

Large expert-curated database for benchmarking document similarity detection in biomedical literature search

Document recommendation systems for locating relevant literature have mostly relied on methods developed a decade ago. This is largely due to the lack of a large offline gold-standard benchmark of relevant documents that cover a variety of research fields such that newly developed literature search techniques can be compared, improved and translated into practice. To overcome this bottleneck, we have established the RElevant LIterature SearcH consortium consisting of more than 1500 scientists from 84 countries, who have collectively annotated the relevance of over 180 000 PubMed-listed articles with regard to their respective seed (input) article/s. The majority of annotations were contributed by highly experienced, original authors of the seed articles. The collected data cover 76% of all unique PubMed Medical Subject Headings descriptors. No systematic biases were observed across different experience levels, research fields or time spent on annotations. More importantly, annotations of the same document pairs contributed by different scientists were highly concordant. We further show that the three representative baseline methods used to generate recommended articles for evaluation (Okapi Best Matching 25, Term Frequency-Inverse Document Frequency and PubMed Related Articles) had similar overall performances. Additionally, we found that these methods each tend to produce distinct collections of recommended articles, suggesting that a hybrid method may be required to completely capture all relevant articles. The established database server located at https://relishdb.ict.griffith.edu.au is freely available for the downloading of annotation data and the blind testing of new methods. We expect that this benchmark will be useful for stimulating the development of new powerful techniques for title and title/abstract-based search engines for relevant articles in biomedical research.Peer reviewe

Co-culture of induced pluripotent stem cells with cardiomyocytes is sufficient to promote their differentiation into cardiomyocytes.

Various types of stem cells and non-stem cells have been shown to differentiate or transdifferentiate into cardiomyocytes by way of co-culture with appropriate inducer cells. However, there is a limited demonstration of a co-culture induction system utilizing stem cell-derived cardiomyocytes as a stimulatory source for cardiac reprogramming (of stem cells or otherwise). In this study, we utilized an inductive co-culture method to show that previously differentiated induced pluripotent stem (iPS) cell-derived cardiomyocytes (iCMs), when co-cultivated with iPS cells, constituted a sufficient stimulatory system to induce cardiac differentiation. To enable tracking of both cell populations, we utilized GFP-labeled iPS cells and non-labeled iCMs pre-differentiated using inhibitors of GSK and Wnt signaling. Successful differentiation was assessed by the exhibition of spontaneous self-contractions, structural organization of α-actinin labeled sarcomeres, and expression of cardiac specific markers cTnT and α-actinin. We found that iCM-iPS cell-cell contact was essential for inductive differentiation, and this required overlaying already adherent iPS cells with iCMs. Importantly, this process was achieved without the exogenous addition of pathway inhibitors and morphogens, suggesting that 'older' iCMs serve as an adequate stimulatory source capable of recapitulating the necessary culture environment for cardiac differentiation

β Integrin Tyrosine Phosphorylation Is a Conserved Mechanism for Regulating Talin-induced Integrin Activation*

Integrins are large membrane-spanning receptors fundamental to cell adhesion and migration. Integrin adhesiveness for the extracellular matrix is activated by the cytoskeletal protein talin via direct binding of its phosphotyrosine-binding-like F3 domain to the cytoplasmic tail of the β integrin subunit. The phosphotyrosine-binding domain of the signaling protein Dok1, on the other hand, has an inactivating effect on integrins, a phenomenon that is modulated by integrin tyrosine phosphorylation. Using full-length tyrosine-phosphorylated 15N-labeled β3, β1A, and β7 integrin tails and an NMR-based protein-protein interaction assay, we show that talin1 binds to the NPXY motif and the membrane-proximal portion of β3, β1A, and β7 tails, and that the affinity of this interaction is decreased by integrin tyrosine phosphorylation. Dok1 only interacts weakly with unphosphorylated tails, but its affinity is greatly increased by integrin tyrosine phosphorylation. The Dok1 interaction remains restricted to the integrin NPXY region, thus phosphorylation inhibits integrin activation by increasing the affinity of β integrin tails for a talin competitor that does not form activating membrane-proximal interactions with the integrin. Key residues governing these specificities were identified by detailed structural analysis, and talin1 was engineered to bind preferentially to phosphorylated integrins by introducing the mutation D372R. As predicted, this mutation affects talin1 localization in live cells in an integrin phosphorylation-specific manner. Together, these results indicate that tyrosine phosphorylation is a common mechanism for regulating integrin activation, despite subtle differences in how these integrins interact with their binding proteins

Hedgehog Signaling as a Therapeutic Target for Airway Remodeling and Inflammation in Allergic Asthma

Genome-wide association studies (GWAS) have shown that variants of patched homolog 1 (PTCH1) are associated with lung function abnormalities in the general population. It has also been shown that sonic hedgehog (SHH), an important ligand for PTCH1, is upregulated in the airway epithelium of patients with asthma and is suggested to be involved in airway remodeling. The contribution of hedgehog signaling to airway remodeling and inflammation in asthma is poorly described. To determine the biological role of hedgehog signaling-associated genes in asthma, gene silencing, over-expression, and pharmacologic inhibition studies were conducted after stimulating human airway epithelial cells or not with transforming growth factor β1 (TGFβ1), an important fibrotic mediator in asthmatic airway remodeling that also interacts with SHH pathway. TGFβ1 increased hedgehog-signaling-related gene expression including SHH, GLI1 and GLI2. Knockdown of PTCH1 or SMO with siRNA, or use of hedgehog signaling inhibitors, consistently attenuated COL1A1 expression induced by TGFβ1 stimulation. In contrast, Ptch1 over-expression augmented TGFβ1-induced an increase in COL1A1 and MMP2 gene expression. We also showed an increase in hedgehog-signaling-related gene expression in primary airway epithelial cells from controls and asthmatics at different stages of cellular differentiation. GANT61, an inhibitor of GLI1/2, attenuated TGFβ1-induced increase in COL1A1 protein expression in primary airway epithelial cells differentiated in air–liquid interface. Finally, to model airway tissue remodeling in vivo, C57BL/6 wildtype (WT) and Ptch1+/− mice were intranasally challenged with house dust mite (HDM) or phosphate-buffered saline (PBS) control. Ptch1+/− mice showed reduced sub-epithelial collagen expression and serum inflammatory proteins compared to WT mice in response to HDM challenge. In conclusion, TGFβ1-induced airway remodeling is partially mediated through the hedgehog signaling pathway via the PTCH1-SMO-GLI axis. The Hedgehog signaling pathway is a promising new potential therapeutic target to alleviate airway tissue remodeling in patients with allergic airways disease

Integrin-mediated Protein Kinase A Activation at the Leading Edge of Migrating Cells

cAMP-dependent protein kinase A (PKA) is important in processes requiring localized cell protrusion, such as cell migration and axonal path finding. Here, we used a membrane-targeted PKA biosensor to reveal activation of PKA at the leading edge of migrating cells. Previous studies show that PKA activity promotes protrusion and efficient cell migration. In live migrating cells, membrane-associated PKA activity was highest at the leading edge and required ligation of integrins such as α4β1 or α5β1 and an intact actin cytoskeleton. α4 integrins are type I PKA-specific A-kinase anchoring proteins, and we now find that type I PKA is important for localization of α4β1 integrin-mediated PKA activation at the leading edge. Accumulation of 3′ phosphorylated phosphoinositides [PtdIns(3,4,5)P3] products of phosphatidylinositol 3-kinase (PI3-kinase) is an early event in establishing the directionality of migration; however, polarized PKA activation did not require PI3-kinase activity. Conversely, inhibition of PKA blocked accumulation of a PtdIns(3,4,5)P3-binding protein, the AKT-pleckstrin homology (PH) domain, at the leading edge; hence, PKA is involved in maintaining cell polarity during migration. In sum, we have visualized compartment-specific PKA activation in migrating cells and used it to reveal that adhesion-mediated localized activation of PKA is an early step in directional cell migration

Slit2–Robo4 signalling promotes vascular stability by blocking Arf6 activity

Slit–Roundabout (Robo) signalling has a well-understood role in axon guidance1–5. Unlike in the nervous system, however, Slitdependent activation of an endothelial-specific Robo, Robo4, does not initiate a guidance program. Instead, Robo4 maintains the barrier function of the mature vascular network by inhibiting neovascular tuft formation and endothelial hyperpermeability induced by pro-angiogenic factors 6. In this study, we used cell biological and biochemical techniques to elucidate the molecular mechanism underlying the maintenance of vascular stability by Robo4. Here, we demonstrate that Robo4 mediates Slit2-dependent suppression of cellular protrusive activity through direct interaction with the intracellular adaptor protein paxillin and its paralogue, Hic-5. Formation of a Robo4–paxillin complex at the cell surface blocks activation of the small GTPase Arf6 and, consequently, Rac by recruitment of Arf-GAPs (ADP-ribosylation factor- directed GTPase-activating proteins) such as GIT1. Consistent with these in vitro studies, inhibition of Arf6 activity in vivo phenocopies Robo4 activation by reducing pathologic angiogenesis in choroidal and retinal vascular disease and VEGF-165 (vascular endothelial growth factor-165)-induced retinal hyperpermeability. These data reveal that a Slit2–Robo4–paxillin–GIT1 network inhibits the cellular protrusive activity underlying neovascularization and vascular leak, and identify a new therapeutic target for ameliorating diseases involving the vascular system