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Proteomics-based functional studies reveal that galectin-3 plays a protective role in the pathogenesis of intestinal Behcet's disease.

ABSTRACT: The pathogenesis of intestinal Behçet's disease (BD) remains poorly understood. Therefore, we aimed to discover and validate biomarkers using proteomics analysis and subsequent functional studies. After two-dimensional electrophoresis, candidate proteins were identified using matrix-assisted laser desorption/ionization tandem time-of-flight mass spectrometry (MALDI-TOF/TOF MS). We validated these results by evaluating the protein levels and their functions in vitro using HT-29 colorectal cancer cells, colon tissues from patients and mice, and murine bone marrow derived macrophages (BMDMs). Of the 30 proteins differentially expressed in intestinal BD tissues, we identified seven using MALDI-TOF/TOF MS. Focusing on galectin-3, we found that TGF-B and IL-10 expression was significantly lower in shLGALS3-transfected cells. Expression of GRP78 and XBP1s and apoptosis rates were all higher in shLGALS3-transfected cells upon the induction of endoplasmic reticulum stress. In response to lipopolysaccharide stimulation, microtubule-associated protein 1 light chain 3B accumulated and lysosomes decreased in these cells. Finally, Salmonella typhimurium infection induced caspase-1 activation and increased IL-1? production, which facilitated activation of the NLRC4 inflammasome, in Lgals3-/- murine BMDMs compared to wild type BMDMs. Our data suggest that galectin-3 may play a protective role in the pathogenesis of intestinal BD via modulation of ER stress, autophagy, and inflammasome activation.

SUBMITTER: Lee HJ

PROVIDER: S-EPMC6691011 | biostudies-literature | 2019 Aug

REPOSITORIES: biostudies-literature

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Proteomics-based functional studies reveal that galectin-3 plays a protective role in the pathogenesis of intestinal Behçet's disease.

Lee Hyun Jung HJ Kim Jae Hyeon JH Hong Sujeong S Hwang Inhwa I Park Soo Jung SJ Kim Tae Il TI Kim Won Ho WH Yu Je-Wook JW Kim Seung Won SW Cheon Jae Hee JH

Scientific reports 20190812 1

The pathogenesis of intestinal Behçet's disease (BD) remains poorly understood. Therefore, we aimed to discover and validate biomarkers using proteomics analysis and subsequent functional studies. After two-dimensional electrophoresis, candidate proteins were identified using matrix-assisted laser desorption/ionization tandem time-of-flight mass spectrometry (MALDI-TOF/TOF MS). We validated these results by evaluating the protein levels and their functions in vitro using HT-29 colorectal cancer ...[more]

PMID: 31406212

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Project description:Myotonic dystrophy type 1 (DM1, MIM #160900) is an autosomal dominant disorder, clinically characterized by progressive muscular weakness and multisystem degeneration. The broad phenotypes observed in DM1 patients resemble the appearance of a multisystem accelerated aging process. However, the molecular mechanisms underlying these phenotypes remain largely unknown. Transcriptomic analysis of fibroblasts derived from DM1 patients and healthy individuals revealed a decrease in cell cycle activity, cell division, and DNA damage response in DM1, all of which were required for the accumulation of cellular senescence. Serial passage studies in vitro confirmed the accelerated increase in senescence and the acquisition of a senescence-associated secretory phenotype in DM1 fibroblasts. Moreover, functional studies highlighted the impact of BMI-1/p16INK4a pathway dysregulation in DM1-associated cellular phenotypes. Importantly, treatment with the senolytic compounds, quercetin, dasatinib, or navitoclax, reversed the accelerated aging phenotypes in both DM1 fibroblasts in vitro and in fruit flies in vivo. Our results identified the accumulation of senescence-related processes as a major driver of DM1 pathophysiology and therefore, demonstrated the efficacy of senolytic compounds in a pre-clinical setting. This approach warrants further assessment as a novel therapeutic strategy for DM1. Cell isolation and culture: For the isolation of primary fibroblasts from healthy donors and DM1 patients (age range, 34 to 71), punch skin biopsies were cut into 2–3 mm3 fragments and placed on a surface moistened with modified Eagle’s medium, containing 13% newborn calf serum, 0.4% penicillin/streptomycin (Gibco, Waltham, MA, USA) and 2 mM L-glutamine (Gibco). Flasks were incubated vertically for 3–6 h at 37ºC under 5% CO2 and then returned to the horizontal position. Human fibroblasts were cultured in Dulbecco’s modified Eagle medium (DMEM, Gibco) containing 10% fetal bovine serum (Sigma-Aldrich, St Louis, MO, USA), 1% L-glutamine (Gibco), and 1% penicillin–streptomycin (Gibco). Fibroblasts were tested regularly for mycoplasma contamination. Seven cultures from different DM1 patients and five from healthy controls were established. Transcriptomic study and data analysis: RNA was extracted from the fibroblasts from six DM1 patients and four age- and sex-matched controls at an early passage using TRIzol (Life Technologies, Carlsbad, CA, USA). Large-scale gene expression analysis was performed using the Human Clariom D microarray (Affymetrix, Santa Clara, CA, USA). RNA integrity was confirmed using an RNA 6000 Nano kit (Agilent Technologies, Santa Clara, CA, USA). Those samples with an RNA integrity value above 9 were used in the analysis. A total of 300 ng of RNA from each sample was used for microarray analysis, following the manufacturer's instructions. Microarray data analysis: The bead intensities were mapped to gene information using BeadStudio 3.2 (Illumina). Background correction was performed using the Affymetrix Robust Multi-array Analysis (RMA) background correction model. Variance stabilization was performed using the log2 scaling, and gene expression normalization was calculated with the method implemented in the lumi package of R-Bioconductor. Data post-processing and graphics were performed with in-house developed functions in Matlab (MathWorks™). The GO terms were taken from the AMIGO gene ontology database. The significance of the GO terms of the DEGs was addressed calculating the P-values using an enrichment approach based on the hypergeometric distribution. All the sets of GO terms were back-propagated from the final term appearing in the gene annotation to the root term of each GO.

Dataset Information

Proteomics-based functional studies reveal that galectin-3 plays a protective role in the pathogenesis of intestinal Behcet's disease.

Publications

Proteomics-based functional studies reveal that galectin-3 plays a protective role in the pathogenesis of intestinal Behçet's disease.

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