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Cytohesin-2/ARNO: A Novel Bridge Between Cell Migration and Immunoregulation in Synovial Fibroblasts.

ABSTRACT: The guanine nucleotide exchange factor cytohesin-2 (ARNO) is a major activator of the small GTPase ARF6 that has been shown to play an important role(s) in cell adhesion, migration and cytoskeleton reorganization in various cell types and models of disease. Interestingly, dysregulated cell migration, in tandem with hyper-inflammatory responses, is one of the hallmarks associated with activated synovial fibroblasts (SFs) during chronic inflammatory joint diseases, like rheumatoid arthritis. The role of ARNO in this process has previously been unexplored but we hypothesized that the pro-inflammatory milieu of inflamed joints locally induces activation of ARNO-mediated pathways in SFs, promoting an invasive cell phenotype that ultimately leads to bone and cartilage damage. Thus, we used small interference RNA to investigate the impact of ARNO on the pathological migration and inflammatory responses of murine SFs, revealing a fully functional ARNO-ARF6 pathway which can be rapidly activated by IL-1β. Such signalling promotes cell migration and formation of focal adhesions. Unexpectedly, ARNO was also shown to modulate SF-inflammatory responses, dictating their precise cytokine and chemokine expression profile. Our results uncover a novel role for ARNO in SF-dependent inflammation, that potentially links pathogenic migration with initiation of local joint inflammation, offering new approaches for targeting the fibroblast compartment in chronic arthritis and joint disease.

SUBMITTER: Wang Y

PROVIDER: S-EPMC8790574 | biostudies-literature |

REPOSITORIES: biostudies-literature

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ARL4D recruits cytohesin-2/ARNO to modulate actin remodeling.

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RNA-Seq approaches to understand the role of ARNO/Cyth2 on IL-1beta-stimulated synovial fibroblasts.

Project description:The aim of this study was to explore the role of ARNO, also known as Cyth2, in synovial fibroblasts using NGS-derived transcriptome analysis (RNA-seq). Synovial fibroblasts were isolated from mouse synovium, and stimulated with IL-1beta for 12 hours in healthy cells and cells where Cyth2 expression was knocked-down by silencing RNA. Methods: Synovial fibroblasts were extracted from the synovium of healthy mice and expanded in vitro, RNA was extracted from naïve, IL-1β stimulated and IL-1β stimulated ARNO knock-down synovial fibroblast. Methods: Whole Transcriptome Profiling of synovial fibroblasts were generated by deep sequencing, in triplicate, using Illumina NextSeq™ 500 platform. Libraries were prepared using polyA selection (TruSeq stranded mRNA kit). Methods: The sequence reads that passed quality filters were aligned to mouse reference genome (GRCM38) using Hisat2 version 2.1.0. we mapped about 30 million sequence reads per sample (75 bp, paired-end) Methods: Featurecounts version 1.4.6 was used to quantify reads counts. Data quality control, non-expressed gene filtering, median ratio normalization (MRN) implemented in DESeq2 package and identification of differentially expressed (DE) genes were done using the R bioconductor project DEbrowser. Results: Firstly, We detected differentially expressed (DE) genes among three conditions, which pass the threshold of >4 fold, adjp <0.01. Then, we detected DE genes in IL-1β stimulated ARNO knock-down synovial fibroblast compared to IL-1β stimulated synovial fibroblast, which pass the threshold of >2 fold, adjp <0.01. DE genes reflect decreased cellular inflammatory response after ARNO knockdown Conclusions: Our results reflect an ARNO-mediated inflammatory response in synovial fibroblast, provides new opportunitties for targeting fibroblast in chronic arthritis and joint disease.

2021-12-25 | GSE192488 | GEO

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| S-EPMC4672287 | biostudies-literature

ARF-GEF cytohesin-2/ARNO regulates R-Ras and ?5-integrin recycling through an EHD1-positive compartment.

Project description:When expressed in epithelial cells, cytohesin-2/ARNO, a guanine nucleotide exchange factor (GEF) for ARF small GTPases, causes a robust migration response. Recent evidence suggests that cytohesin-2/ARNO acts downstream of small the GTPase R-Ras to promote spreading and migration. We hypothesized that cytohesin-2/ARNO could transmit R-Ras signals by regulating the recycling of R-Ras through ARF activation. We found that Eps15-homology domain 1 (EHD1), a protein that associates with the endocytic recycling compartment (ERC), colocalizes with active R-Ras in transiently expressed HeLa cells. In addition, we show that EHD1-positive recycling endosomes are a novel compartment for cytohesin-2/ARNO. Knockdown or expression of GEF-inactive (E156K) cytohesin-2/ARNO causes R-Ras to accumulate on recycling endosomes containing EHD1 and inhibits cell spreading. E156K-ARNO also causes a reduction in focal adhesion size and number. Finally, we demonstrate that R-Ras/ARNO signaling is required for recycling of ?5-integrin and R-Ras to the plasma membrane. These data establish a role for cytohesin-2/ARNO as a regulator of R-Ras and integrin recycling and suggest that ARF-regulated trafficking of R-Ras is required for R-Ras-dependent effects on spreading and adhesion formation.

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