The ubiquitin-proteasome pathway regulates claudin 5 degradation

The tight junctions (TJs) form continuous intracellular contacts, which help create selective barriers in epithelial and endothelial cell layers. The structures created by the TJs are very dynamic and can be rapidly remodeled in response to physiological and pathological signals. Claudin 5 is a membranal TJ protein which plays a critical role in determining the permeability of endothelial barriers. We describe the regulation of claudin 5 degradation by the ubiquitin-proteasome system (UPS). Our results indicate that claudin 5 has a relatively short half-life and can be polyubiquitinated on lysine 199. This ubiquitination appears to trigger the proteasome-dependent degradation of claudin 5. Other mechanisms also seem to be involved in the post-translational regulation of claudin 5, including a ubiquitin-independent and probably indirect lysosomal-dependent pathway. These findings provide evidence for the involvement of the UPS in the regulation of claudin 5 levels, and set the stage for further research to determine the involvement of this pathway in the modulation of the properties of TJs and cell-layer barriers.     

Involvement of ubiquitin-proteasome system in icariin-induced cardiomyocyte differentiation of embryonic stem cells using two-dimensional gel electrophoresis

Icariin has been shown to significantly facilitate the differentiation of embryonic stem (ES) cells into cardiomyocytes in vitro. However, the mechanism underlying the icariin-induced cardiomyocyte differentiation is still not fully understood. In the present study, 52 differentially displayed proteins selected from two-dimensional electrophoresis gels were identified by MALDI-TOF mass spectrometry analysis. More than half of proteins could be assigned to six main categories: (1) protein synthesis, metabolism, processing and degradation, (2) stress response, (3) cytoskeleton proteins, (4) energy metabolism, (5) carbohydrate metabolism/transport, and (6) RNA/other nucleic acids metabolisms and transport, nuclear proteins. MALDI-TOF/MS showed that icariin treatment resulted in the induction of five ubiquitin-proteasome system (UPS)-related proteins, such as ubiquitin carboxy-terminal hydrolase L1 (UCH-L1), ubiquitin-conjugating enzyme E2N, proteasome 26S, proteasome subunit-alpha type 6, and proteasome subunit-alpha type 2 in the differentiated cardiomyocytes. These results implied that UPS might play an important role in the control of cardiomyocyte differentiation. Epoxomicin (a proteasome inhibitor) significantly reduced the cardiomyocyte differentiation rate of ES cells and proteasome activities, as well as inhibited NF-κB translocation into the nucleus, which were evidently reversed by presence of icariin. Meanwhile, icariin could significantly reverse the reduction of four proteins (proteasome subunit-alpha type 6, proteasome subunit-alpha type 2, UCH-L1, and ubiquitin-conjugating enzyme E2N) expressions owing to application of epoxomicin. These suggest UPS could be a means by which icariin may regulate expressions of key proteins that control cardiomyocyte differentiation. Taken together, these results indicated that UPS played an important role in ES cell differentiate into cardiomyocytes induced by icariin.     

Hypoxia Impairs Muscle Function and Reduces Myotube Size in Tissue Engineered Skeletal Muscle

Contemporary tissue engineered skeletal muscle models display a high degree of physiological accuracy compared with native tissue, and therefore may be excellent platforms to understand how various pathologies affect skeletal muscle. Chronic obstructive pulmonary disease (COPD) is a lung disease which causes tissue hypoxia and is characterized by muscle fiber atrophy and impaired muscle function. In the present study we exposed engineered skeletal muscle to varying levels of oxygen (O₂; 21–1%) for 24 h in order to see if a COPD like muscle phenotype could be recreated in vitro, and if so, at what degree of hypoxia this occurred. Maximal contractile force was attenuated in hypoxia compared to 21% O₂; with culture at 5% and 1% O₂ causing the most pronounced effects with 62% and 56% decrements in force, respectively. Furthermore at these levels of O₂, myotubes within the engineered muscles displayed significant atrophy which was not seen at higher O₂ levels. At the molecular level we observed increases in mRNA expression of MuRF‐1 only at 1% O₂ whereas MAFbx expression was elevated at 10%, 5%, and 1% O₂. In addition, p70S6 kinase phosphorylation (a downstream effector of mTORC1) was reduced when engineered muscle was cultured at 1% O₂, with no significant changes seen above this O₂ level. Overall, these data suggest that engineered muscle exposed to O₂ levels of ≤5% adapts in a manner similar to that seen in COPD patients, and thus may provide a novel model for further understanding muscle wasting associated with tissue hypoxia. J. Cell. Biochem. 118: 2599–2605, 2017. © 2017 The Authors. Journal of Cellular Biochemistry Published by Wiley Periodicals, Inc.