Metabolomics,Unknown,Transcriptomics,Genomics,Proteomics

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RNA-seq of murine mesenchymal stem cells during differentiation to osteoblasts, treated with tofacitinib or baricitinib against vehicle control

ABSTRACT: Rheumatoid arthritis (RA) is a chronic inflammatory autoimmune disease, characterized by synovial hyperplasia and progressive joint destruction. For treating inflammatory diseases, such as RA, the Janus kinase (JAK)-mediated signaling pathway has emerged as a therapeutic target. Therefore, JAK inhibitors, such as tofacitinib and baricitinib, are approved for the treatment of RA. While the immunomodulatory characteristics of JAK inhibition (JAKi) are well defined, the current knowledge of how JAKi affects bone homeostasis is limited. Addressing this question becomes increasingly relevant, as current therapeutic options can slow down RA progression, but joint damage and especially perarticular bone erosion remain. Therefore, the impact of JAKi on bone homeostasis requires further elucidation. For in vitro analysis, the impact of JAK inhibitors tofacitinib and baricitinib on bone-forming osteoblasts was analyzed with respect to their mineralization capability. JAKi, by both tofacitinib and baricitinib, increased mineralization function in mesenchymal stem cells (MSCs)-derived osteoblasts. Having observed increased mineralization capability in mesenchymal stem cells (MSCs)-derived osteoblasts, RNA-seq was performed to shed light on potential signaling pathways and mediators causing this effect. For that, MSCs were plated at 6,000 cells per well in a 24-well-plate in alpha-MEM, supplemented with 10% heat-inactivated FCS and 1% P/S. After one day medium was changed to alpha-MEM, 10% heat-inactivated FCS, 1% P/S and 568 mikromolar L-Ascorbic acid 2-phosphate 351 sesquimagnesium salt hydrate, containing either tofacitinib (500 nM), baricitinib (300 nM) or DMSO as vehicle control. After osteogenic induction, RNA was isolated via phenol-chloroform extraction. At least 3 mikrogram high quality RNA were used for RNA-seq analysis with the Illumina TrueSeq Stranded mRNA Kit and sequenced on an Illumina HiSeq 2500 platform.

INSTRUMENT(S): Illumina HiSeq 2500

ORGANISM(S): Mus musculus

SUBMITTER: Silke Frey

PROVIDER: E-MTAB-8536 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Project description:Rheumatoid arthritis (RA) fibroblast‐like synoviocytes (FLS) derived from hip and knee have distinctive DNA methylation and transcriptome patterns in interleukin (IL)‐6 signaling and Janus kinase (JAK)–signal transducers and activators of transcription (STAT) pathways. To determine the functional effects of these joint‐specific signatures, we evaluated how RA hip and knee FLS differ in their response to IL‐6.Hip or knee RA FLS were obtained after arthroplasty. Previously published datasets on epigenetic landscape of FLS were mined to identify joint‐specific IL‐6–related epigenomic differences. RNA sequencing was performed on five RA hip and five knee FLS treated with or without IL‐6. Differential gene expression was determined using edgeR software. STAT3 phosphorylation was measured using bead assays. Sensitivity to tofacitinib was evaluated by measuring CCL2 inhibition using quantitative polymerase chain reaction. Assay for Transposase‐Accessible Chromatin sequencing and histone chromatin immunoprecipitation sequencing datasets from RA FLS were analyzed to identify epigenomic differences between hip and knee. Differential chromatin accessibility was associated with IL‐6, IL‐6R, and JAK1 genes. H3K27ac was also differentially marked at other JAK‐STAT–related genes, including STAT3‐STAT5A region. Principal component analysis of RNA sequencing data confirmed segregation between RA hip and knee FLS under basal conditions, that persisted following IL‐6 treatment. STAT3 phosphorylation after IL‐6 was significantly higher in knee than hip FLS and was highly correlated with JAK1 protein levels. Knee FLS were less sensitive to the JAK inhibitor tofacitinib than hip FLS. RA hip and knee FLS have distinct transcriptomes, epigenetic marks, and STAT3 activation patterns in the IL‐6 pathway. These joint‐specific differences might contribute to a differential clinical response in individual joints to targeted therapies such as JAK inhibitors.

Dataset Information

RNA-seq of murine mesenchymal stem cells during differentiation to osteoblasts, treated with tofacitinib or baricitinib against vehicle control

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