nsc-74859 and Inflammation

Immunoglobulin A nephropathy (IgAN) is one of the most common glomerular diseases worldwide. Various studies have identified a host of microRNAs (miRNAs) abnormally expressed in IgAN and might affect the pathogenesis and progression of IgAN. However, miR-200bc/429 cluster in the pathopoiesis of IgAN remains poorly understood. For this study, we found that miR-200bc/429 cluster is downregulated in IgAN tissues and IgAN podocytes and HK2 cells compared with their matched controls respectively. In addition, overexpression of miR-200bc/429 cluster in IgAN podocytes and HK2 cells could attenuate the release of inflammatory cytokines MCP-1, IL-6 and RANTES. Moreover, the 3' untranslated region (UTR) of TNF-like weak inducer of apoptosis (TWEAK) was identified to be a direct target of miR-200bc/429 cluster. Furthermore, our results showed that miR-200bc/429 cluster can inhibit TWEAK mediated NF-κB pathway activation in IgAN. Overall, our findings revealed that miR-200bc/429 cluster alleviates inflammation in IgAN through TWEAK/Fn14 system and might serve as a biomarker as well as a promising therapeutic target for IgAN.

Topics: Aminosalicylic Acids; Animals; Apoptosis; Benzenesulfonates; Butadienes; Cell Line; Cytokine TWEAK; Glomerulonephritis, IGA; Glycation End Products, Advanced; Inflammation; Interleukin-6; MAP Kinase Signaling System; Mice; MicroRNAs; Nitriles; Osteocytes; p38 Mitogen-Activated Protein Kinases; STAT3 Transcription Factor; Vascular Endothelial Growth Factor A

Signal transducer and activator of transcription 3 (STAT3) is phosphorylated by various kinases, several of which have been implicated in aberrant fibroblast activation in fibrotic diseases including systemic sclerosis (SSc). Here we show that profibrotic signals converge on STAT3 and that STAT3 may be an important molecular checkpoint for tissue fibrosis. STAT3 signaling is hyperactivated in SSc in a TGFβ-dependent manner. Expression profiling and functional studies in vitro and in vivo demonstrate that STAT3 activation is mediated by the combined action of JAK, SRC, c-ABL, and JNK kinases. STAT3-deficient fibroblasts are less sensitive to the pro-fibrotic effects of TGFβ. Fibroblast-specific knockout of STAT3, or its pharmacological inhibition, ameliorate skin fibrosis in experimental mouse models. STAT3 thus integrates several profibrotic signals and might be a core mediator of fibrosis. Considering that several STAT3 inhibitors are currently tested in clinical trials, STAT3 might be a candidate for molecular targeted therapies of SSc.

Topics: Adolescent; Adult; Aged; Aminosalicylic Acids; Animals; Benzenesulfonates; Biopsy; Bleomycin; Collagen; Enzyme Activation; Female; Fibroblasts; Fibrosis; Humans; Inflammation; Male; Mice; Microscopy, Confocal; Middle Aged; Phosphorylation; Receptors, Transforming Growth Factor beta; Scleroderma, Systemic; Signal Transduction; Skin; STAT3 Transcription Factor; Transforming Growth Factor beta; Young Adult

This protocol describes microsphere-based protease assays for use in flow cytometry and high-throughput screening. This platform measures a loss of fluorescence from the surface of a microsphere due to the cleavage of an attached fluorescent protease substrate by a suitable protease enzyme. The assay format can be adapted to any site or protein-specific protease of interest and results can be measured in both real time and as endpoint fluorescence assays on a flow cytometer. Endpoint assays are easily adapted to microplate format for flow cytometry high-throughput analysis and inhibitor screening.

Topics: Animals; Biotinylation; Flow Cytometry; Fluorescence Resonance Energy Transfer; Green Fluorescent Proteins; High-Throughput Screening Assays; Humans; Inflammation; Kinetics; Microspheres; Peptide Hydrolases; Peptides; Reproducibility of Results; Temperature