ABSTRACT
Pulmonary arterial hypertension (PAH) is a fatal condition for which there is no cure. Dimethyl Fumarate (DMF) is an FDA approved anti-oxidative and anti-inflammatory agent with a favorable safety record. The goal of this study was to assess the effectiveness of DMF as a therapy for PAH using patient-derived cells and murine models. We show that DMF treatment is effective in reversing hemodynamic changes, reducing inflammation, oxidative damage, and fibrosis in the experimental models of PAH and lung fibrosis. Our findings indicate that effects of DMF are facilitated by inhibiting pro-inflammatory NFκB, STAT3 and cJUN signaling, as well as ßTRCP-dependent degradation of the pro-fibrogenic mediators Sp1, TAZ and ß-catenin. These results provide a novel insight into the mechanism of its action. Collectively, preclinical results demonstrate beneficial effects of DMF on key molecular pathways contributing to PAH, and support its testing in PAH treatment in patients.
Subject(s)
Dimethyl Fumarate/pharmacology , Hypertension, Pulmonary/metabolism , Pulmonary Fibrosis/metabolism , Signal Transduction/drug effects , Animals , Biomarkers , Bleomycin/adverse effects , Disease Models, Animal , Endothelial Cells/drug effects , Endothelial Cells/metabolism , Fibroblasts/metabolism , Gene Expression Regulation , Hypertension, Pulmonary/etiology , Hypertension, Pulmonary/physiopathology , Hypoxia/metabolism , Inflammation/complications , Inflammation/metabolism , Mice , Myocytes, Smooth Muscle/drug effects , Myocytes, Smooth Muscle/metabolism , NF-kappa B/metabolism , Oxidative Stress/drug effects , Proteasome Endopeptidase Complex/metabolism , Proteolysis , Proto-Oncogene Proteins c-jun/metabolism , Pulmonary Fibrosis/etiology , Pulmonary Fibrosis/pathology , STAT3 Transcription Factor/metabolismABSTRACT
The evolutionarily conserved small actin-monomer binding protein profilin is believed to be a housekeeping factor that maintains a general pool of unassembled actin. However, despite similar primary sequences, structural folds, and affinities for G-actin and poly-L-proline, budding yeast profilin ScPFY fails to complement fission yeast profilin SpPRF temperature-sensitive mutant cdc3-124 cells. To identify profilin's essential properties, we built a combinatorial library of ScPFY variants containing either WT or SpPRF residues at multiple positions and carried out a genetic selection to isolate variants that support life in fission yeast. We subsequently engineered ScPFY(9-Mut), a variant containing nine substitutions in the actin-binding region, which complements cdc3-124 cells. ScPFY(9-Mut), but not WT ScPFY, suppresses severe cytokinesis defects in cdc3-124 cells. Furthermore, the major activity rescued by ScPFY(9-Mut) is the ability to enhance cytokinesis formin Cdc12-mediated actin assembly in vitro, which allows cells to assemble functional contractile rings. Therefore an essential role of profilin is to specifically facilitate formin-mediated actin assembly for cytokinesis in fission yeast.
Subject(s)
Actins/metabolism , Cytoskeletal Proteins/metabolism , Profilins/metabolism , Schizosaccharomyces pombe Proteins/metabolism , Schizosaccharomyces/metabolism , Actins/chemistry , Actins/genetics , Cell Cycle Proteins/chemistry , Cell Cycle Proteins/genetics , Cell Cycle Proteins/metabolism , Cytokinesis/genetics , Cytoskeletal Proteins/genetics , Genetic Complementation Test , Immunoblotting , Microscopy, Fluorescence , Models, Molecular , Mutation , Profilins/chemistry , Profilins/genetics , Proline/chemistry , Proline/genetics , Proline/metabolism , Protein Binding , Protein Structure, Secondary , Protein Structure, Tertiary , Saccharomyces cerevisiae/genetics , Saccharomyces cerevisiae/growth & development , Saccharomyces cerevisiae/metabolism , Schizosaccharomyces/genetics , Schizosaccharomyces/growth & development , Schizosaccharomyces pombe Proteins/chemistry , Schizosaccharomyces pombe Proteins/genetics , Time-Lapse Imaging/methodsABSTRACT
Many eukaryotes accomplish cell division by building and constricting a medial actomyosin-based cytokinetic ring (CR). In Schizosaccharomyces pombe, a Hippo-related signaling pathway termed the septation initiation network (SIN) controls CR formation, maintenance, and constriction. However, how the SIN regulates integral CR components was unknown. Here, we identify the essential cytokinetic formin Cdc12 as a key CR substrate of SIN kinase Sid2. Eliminating Sid2-mediated Cdc12 phosphorylation leads to persistent Cdc12 clustering, which prevents CR assembly in the absence of anillin-like Mid1 and causes CRs to collapse when cytokinesis is delayed. Molecularly, Sid2 phosphorylation of Cdc12 abrogates multimerization of a previously unrecognized Cdc12 domain that confers F-actin bundling activity. Taken together, our findings identify a SIN-triggered oligomeric switch that modulates cytokinetic formin function, revealing a novel mechanism of actin cytoskeleton regulation during cell division.
