1α,25-Dihydroxyvitamin D3 Stimulates Activator Protein 1 DNA-Binding Activity by a Phosphatidylinositol 3-Kinase/Ras/MEK/Extracellular Signal Regulated Kinase 1/2 and c-Jun N-Terminal Kinase 1-Dependent Increase in c-Fos, Fra1, and c-Jun Expression in Human Keratinocytes

1α,25-Dihydroxyvitamin D3 added to human keratinocytes increases differentiation through an activation of the transcription factor activator protein 1. We have previously reported that the 1α,25-dihydroxyvitamin D3-induced increase of activator protein 1 DNA binding activity is mediated by a protein kinase C-independent mechanism. The purpose of this study was to investigate further the mechanisms by which 1α,25-dihydroxyvitamin D3 modulates activator protein 1 DNA binding activity in cultured normal human keratinocytes. Western blotting experiments revealed that 1α,25-dihydroxyvitamin D3 caused a rapid and transient activation of the mitogen-activated protein kinases, extracellular signal regulated kinase 1/2 and c-Jun N-terminal kinase 1. 1α,25-Dihydroxyvitamin D3 also enhanced the expression of the activator protein 1 subunits, c-Fos, Fra1, and c-Jun as determined by northern and western blotting. The 1α,25-dihydroxyvitamin D3-induced activator protein 1 DNA binding activity was completely blocked by the MEK inhibitor PD 98059 indicating that the MEK/extracellular signal regulated kinase pathway is involved in the activation of activator protein 1. Transfection experiments showed that 1α,25-dihydroxyvitamin D3 also increased the activator protein 1-dependent transactivation, which was completely blocked by expression of a dominant negative Ras, suggesting that the 1α,25-dihydroxyvitamin D3-induced activator protein 1 activity involves Ras-dependent signaling. Furthermore, preincubation of the keratinocytes with the specific phosphatidylinositol 3-kinase inhibitors, Wortmannin and LY294002, demonstrated that the 1α,25-dihydroxyvitamin D3-induced activation of extracellular signal regulated kinase 1/2 and c-Jun N-terminal kinase 1 required phosphatidylinositol 3-kinase activity. Finally, preincubation of keratinocytes with a polyclonal antibody against the membrane receptor annexin II, blocked the 1α,25-dihydroxyvitamin D3-induced activation of extracellular signal regulated kinase 1/2 and c-Jun N-terminal kinase 1. Taken together, our results indicate that 1α,25-dihydroxyvitamin D3, via binding to the membrane receptor annexin II, induces activation of the phos-phatidylinositol 3-kinase/Ras/MEK/extracellular signal regulated kinase 1/2 and c-Jun N-terminal kinase 1 signal transduction pathway resulting in increased expression of c-Fos, Fra1, and c-Jun, and subsequently increased activator protein 1 DNA binding activity and gene transcription

STRAW-b (STRings for Absorption length in Water-b): the second pathfinder mission for the Pacific Ocean Neutrino Experiment

Since 2018, the potential for a high-energy neutrino telescope, named the Pacific Ocean Neutrino Experiment (P-ONE), has been thoroughly examined by two pathfinder missions, STRAW and STRAW-b, short for short for Strings for Absorption Length in Water. The P-ONE project seeks to install a neutrino detector with a one cubic kilometer volume in the Cascadia Basin's deep marine surroundings, situated near the western shores of Vancouver Island, Canada. To assess the environmental conditions and feasibility of constructing a neutrino detector of that scale, the pathfinder missions, STRAW and STRAW-b, have been deployed at a depth of 2.7 km within the designated site for P-ONE and were connected to the NEPTUNE observatory, operated by Ocean Networks Canada (ONC). While STRAW focused on analyzing the optical properties of water in the Cascadia Basin, \ac{strawb} employed cameras and spectrometers to investigate the characteristics of bioluminescence in the deep-sea environment. This report introduces the STRAW-b concept, covering its scientific objectives and the instrumentation used. Furthermore, it discusses the design considerations implemented to guarantee a secure and dependable deployment process of STRAW-b. Additionally, it showcases the data collected by battery-powered loggers, which monitored the mechanical stress on the equipment throughout the deployment. The report also offers an overview of STRAW-b's operation, with a specific emphasis on the notable advancements achieved in the data acquisition (DAQ) system and its successful integration with the server infrastructure of ONC.Comment: 20 pages, 11 figures, 2 table

StUbEx:Stable Tagged Ubiquitin Exchange System for the Global Investigation of Cellular Ubiquitination

Post-translational modification of proteins with the small polypeptide ubiquitin plays a pivotal role in many cellular processes, altering protein lifespan, location, and function and regulating protein–protein interactions. Ubiquitination exerts its diverse functions through complex mechanisms by formation of different polymeric chains and subsequent recognition of the ubiquitin signal by specific protein interaction domains. Despite some recent advances in the analytical tools for the analysis of ubiquitination by mass spectrometry, there is still a need for additional strategies suitable for investigation of cellular ubiquitination at the proteome level. Here, we present a stable tagged ubiquitin exchange (StUbEx) cellular system in which endogenous ubiquitin is replaced with an epitope-tagged version, thereby allowing specific and efficient affinity purification of ubiquitinated proteins for global analyses of protein ubiquitination. Importantly, the overall level of ubiquitin in the cell remains virtually unchanged, thus avoiding ubiquitination artifacts associated with overexpression. The efficiency and reproducibility of the method were assessed through unbiased analysis of epidermal growth factor (EGF) signaling by quantitative mass spectrometry, covering over 3400 potential ubiquitinated proteins. The StUbEx system is applicable to virtually any cell line and can be readily adapted to any of the ubiquitin-like post-translational modifications

Cellular Proteome Dynamics during Differentiation of Human Primary Myoblasts

Muscle stem cells, or satellite cells, play an important role in the maintenance and repair of muscle tissue and have the capacity to proliferate and differentiate in response to physiological or environmental changes. Although they have been extensively studied, the key regulatory steps and the complex temporal protein dynamics accompanying the differentiation of primary human muscle cells remain poorly understood. Here, we demonstrate the advantages of applying a MS-based quantitative approach, stable isotope labeling by amino acids in cell culture (SILAC), for studying human myogenesis <i>in vitro</i> and characterize the fine-tuned changes in protein expression underlying the dramatic phenotypic conversion of primary mononucleated human muscle cells during <i>in vitro</i> differentiation to form multinucleated myotubes. Using an exclusively optimized triple encoding SILAC procedure, we generated dynamic expression profiles during the course of myogenic differentiation and quantified 2240 proteins, 243 of which were regulated. These changes in protein expression occurred in sequential waves and underlined vast reprogramming in key processes governing cell fate decisions, i.e., cell cycle withdrawal, RNA metabolism, cell adhesion, proteolysis, and cytoskeletal organization. <i>In silico</i> transcription factor target analysis demonstrated that the observed dynamic changes in the proteome could be attributed to a cascade of transcriptional events involving key myogenic regulatory factors as well as additional regulators not yet known to act on muscle differentiation. In addition, we created of a dynamic map of the developing myofibril, providing valuable insights into the formation and maturation of the contractile apparatus <i>in vitro</i>. Finally, our SILAC-based quantitative approach offered the possibility to follow the expression profiles of several muscle disease-associated proteins simultaneously and therefore could be a valuable resource for future studies investigating pathogenesis of degenerative muscle disorders as well as assessing new therapeutic strategies

Overexpression of cyclooxygenase-2 in adipocytes reduces fat accumulation in inguinal white adipose tissue and hepatic steatosis in high-fat fed mice

Cyclooxygenases are known as important regulators of metabolism and immune processes via conversion of C20 fatty acids into various regulatory lipid mediators, and cyclooxygenase activity has been implicated in browning of white adipose tissues. We generated transgenic (TG) C57BL/6 mice expressing the Ptgs2 gene encoding cyclooxygenase-2 (COX-2) in mature adipocytes. TG mice fed a high-fat diet displayed marginally lower weight gain with less hepatic steatosis and a slight improvement in insulin sensitivity, but no difference in glucose tolerance. Compared to littermate wildtype mice, TG mice selectively reduced inguinal white adipose tissue (iWAT) mass and fat cell size, whereas the epididymal (eWAT) fat depot remained unchanged. The changes in iWAT were accompanied by increased levels of specific COX-derived lipid mediators and increased mRNA levels of interleukin-33, interleukin-4 and arginase-1, but not increased expression of uncoupling protein 1 or increased energy expenditure. Epididymal WAT (eWAT) in TG mice exhibited few changes except from increased infiltration with eosinophils. Our findings suggest a role for COX-2-derived lipid mediators from adipocytes in mediating type 2 immunity cues in subcutaneous WAT associated with decreased hepatic steatosis, but with no accompanying induction of browning and increased energy expenditure.ISSN:2045-232