Pathways to understanding the genomic aetiology of osteoarthritis.

Osteoarthritis is a common, complex disease with no curative therapy. In this review, we summarize current knowledge on disease aetiopathogenesis and outline genetics and genomics approaches that are helping catalyse a much-needed improved understanding of the biological underpinning of disease development and progression.

The ChEMBL database in 2017.

ChEMBL is an open large-scale bioactivity database (https://www.ebi.ac.uk/chembl), previously described in the 2012 and 2014 Nucleic Acids Research Database Issues. Since then, alongside the continued extraction of data from the medicinal chemistry literature, new sources of bioactivity data have also been added to the database. These include: deposited data sets from neglected disease screening; crop protection data; drug metabolism and disposition data and bioactivity data from patents. A number of improvements and new features have also been incorporated. These include the annotation of assays and targets using ontologies, the inclusion of targets and indications for clinical candidates, addition of metabolic pathways for drugs and calculation of structural alerts. The ChEMBL data can be accessed via a web-interface, RDF distribution, data downloads and RESTful web-services.

PI4K2ß/AP-1-based TGN-endosomal sorting regulates Wnt signaling.

Endosomal membrane traffic serves crucial roles in cell physiology, signaling, and development. Sorting between endosomes and the trans-Golgi network (TGN) is regulated among other factors by the adaptor AP-1, an essential component of multicellular organisms. Membrane recruitment of AP-1 requires phosphatidylinositol 4-phosphate [PI(4)P], though the precise mechanisms and PI4 kinase isozyme (or isozymes) involved in generation of this PI(4)P pool remain unclear. The Wnt pathway is a major developmental signaling cascade and depends on endosomal sorting in Wnt-sending cells. Whether TGN/endosomal sorting modulates signaling downstream of Frizzled (Fz) receptors in Wnt-receiving cells is unknown. Here, we identify PI4-kinase type 2ß (PI4K2ß) as a regulator of TGN/endosomal sorting and Wnt signaling. PI4K2ß and AP-1 interact directly and are required for efficient sorting between endosomes and the TGN. Zebrafish embryos depleted of PI4K2ß or AP-1 lack pectoral fins due to defective Wnt signaling. Rescue experiments demonstrate requirements for PI4K2ß-AP-1 complex formation and PI4K2ß-mediated PI(4)P synthesis. Furthermore, PI4K2ß binds to the Fz-associated component Dishevelled (Dvl) and regulates endosomal recycling of Fz receptors and Wnt target gene expression. These data reveal an evolutionarily conserved role for PI4K2ß and AP-1 in coupling phosphoinositide metabolism to AP-1-mediated sorting and Wnt signaling.

In vivo conditions to identify Prkci phosphorylation targets using the analog-sensitive kinase method in zebrafish.

Protein kinase C iota is required for various cell biological processes including epithelial tissue polarity and organ morphogenesis. To gain mechanistic insight into different roles of this kinase, it is essential to identify specific substrate proteins in their cellular context. The analog-sensitive kinase method provides a powerful tool for the identification of kinase substrates under in vivo conditions. However, it has remained a major challenge to establish screens based on this method in multicellular model organisms. Here, we report the methodology for in vivo conditions using the analog-sensitive kinase method in a genetically-tractable vertebrate model organism, the zebrafish. With this approach, kinase substrates can uniquely be labeled in the developing zebrafish embryo using bulky ATPγS analogs which results in the thiophosphorylation of substrates. The labeling of kinase substrates with a thiophosphoester epitope differs from phosphoesters that are generated by all other kinases and allows for an enrichment of thiophosphopeptides by immunoaffinity purification. This study provides the foundation for using the analog-sensitive kinase method in the context of complex vertebrate development, physiology, or disease.

Gene expression dynamics after murine pancreatitis unveils novel roles for Hnf1α in acinar cell homeostasis.

OBJECTIVES: During pancreatitis, specific transcriptional programmes govern functional regeneration after injury. The objective of this study was to analyse the dynamic regulation of pancreatic genes and the role of transcriptional regulators during recovery from pancreatitis. DESIGN: Wild-type and genetically modified mice (Hnf1α(-/-) and Ptf1a(+/-)) were used. After caerulein or L-arginine induced pancreatitis, blood or pancreata were processed for enzymatic assays, ELISA, histology, immunohistochemistry, western blotting and quantitative reverse transcriptase-PCR. Nr5a2 promoter reporter and chromatin immunoprecipitation assays for Hnf1α were also performed. RESULTS: After caerulein pancreatic injury, expression of acinar and endocrine genes rapidly decreased, but eventually recovered, depicting distinct cell-type-specific patterns. Pdx1 and Hnf1α mRNAs underwent marked downregulation, matching endocrine/exocrine gene expression profiles. Ptf1a, Pdx1 and Hnf1α protein levels were also reduced and recovered gradually. These changes were associated with transient impairment of exocrine and endocrine function, including abnormal glucose tolerance. On l-arginine pancreatitis, changes in Ptf1a, Pdx1 and Hnf1α gene and protein expression were recapitulated. Reduced Hnf1α and Ptf1a levels after pancreatitis coincided with increased acinar cell proliferation, both in Hnf1α(-/-) and Ptf1a(+/-) mice. Moreover, Hnf1α(-/-) mice had reduced Ptf1a protein as well as transcripts for Ptf1a and digestive enzymes. Dispersed acini from Hnf1α(-/-) mice showed suboptimal secretory responses to caerulein. Bioinformatics analysis did not support a role for Hnf1α as a direct regulator of digestive enzyme genes. Instead, it was found that Hnf1α binds to, and regulates, the promoter of Nr5a2, coding an orphan nuclear receptor that regulates acinar gene expression. CONCLUSIONS: Dynamic changes in gene expression occur on pancreatitis induction, determining altered exocrine and endocrine function. This analysis uncovers roles for Hnf1α in the regulation of acinar cell determination and function. This effect may be mediated, in part, through direct regulation of Nr5a2.

Essential role of TM V and VI for binding the C-terminal sequences of Des-Arg-kinins.

In the kallikrein-kinin and renin-angiotensin systems the main receptors, B1 and B2 (kinin receptors) and AT1 and AT2 (angiotensin receptors) respectively, are seven-transmembrane domain G-protein-coupled receptors. Considering that the B1 agonists Des-Arg9-BK (Arg-Pro-Pro-Gly-Phe-Ser-Pro-Phe), Lys-desArg9-BK or Des-Arg10-KD (Lys-Arg-Pro-Pro-Gly-Phe-Ser-Pro-Phe) and the AT1 agonist (Asp-Arg-Val-Tyr-Ile-His-Pro-Phe) have the same two residues at the C-terminal region (i.e. Pro-Phe), we hypothesized that TM V and TM VI of the B1 receptor could play an essential role in agonist binding and activity, being these regions receptor sites for binding the C-terminal sequences of Des-Arg-kinins similarly to that observed to AT1 receptor. To investigate this hypothesis, we replaced Arg212 for Ala at the top of the TM V and the sequence 274-282 (CPYHFFAFL) in TM VI of the rat kinin B1 receptor by the B2 receptor homologous sequence, 289-297 (FPFQISTFL) and subsequently analyzed the consequences of these mutations by competition binding and functional assays. Despite correct expression, observed at the mRNA and protein level by RT-PCR and confocal microscopy, respectively, no agonist binding and function was verified for the mutated receptors. Therefore, our results suggest an important role for Arg212 in the TM V and a region of TM VI of rat B1 receptor in the interaction with the C-terminal residues of Des-Arg-kinins, similar to that observed with AngII.

Involvement of zebrafish Na+,K+ ATPase in myocardial cell junction maintenance.

Na(+),K(+) ATPase is an essential ion pump involved in regulating ionic concentrations within epithelial cells. The zebrafish heart and mind (had) mutation, which disrupts the alpha1B1 subunit of Na(+),K(+) ATPase, causes heart tube elongation defects and other developmental abnormalities that are reminiscent of several epithelial cell polarity mutants, including nagie oko (nok). We demonstrate genetic interactions between had and nok in maintaining Zonula occludens-1 (ZO-1)-positive junction belts within myocardial cells. Functional tests and pharmacological inhibition experiments demonstrate that Na(+),K(+) ATPase activity is positively regulated via an N-terminal phosphorylation site that is necessary for correct heart morphogenesis to occur, and that maintenance of ZO-1 junction belts requires ion pump activity. These findings suggest that the correct ionic balance of myocardial cells is essential for the maintenance of epithelial integrity during heart morphogenesis.