Session Type: Poster Session (Monday)
Session Time: 9:00AM-11:00AM
Background/Purpose: Systemic sclerosis (SSc) is a connective tissue disease characterized by fibrosis of the skin and internal organs. Previous studies have shown that dermal fibroblast in patients with SSc frequently shows decreased levels of Fli1 due to hypermethylation. We aimed to clarify the mechanisms underlying the regulation of Fli1 gene of fibrosis using Fli1 deficient cells and mice generated by the CRISPR/Cas9-mediated gene edition.
Methods: Fli1-deficient fibroblast cell line (Fli1ΔNIH3T3 cells) and mice (Fli1+/- mice) were generated by CRISPR/Cas9 system using paired guide RNAs specific for mouse Fli1 and a nickase Cas9 that reduces off-target problem. NIH3T3 cells were transfected with a lentiCRISPR v2 vector and then selected by puromycin. We evaluated collagen and profibrotic cytokine production in the absence of Fli1 by mRNA level. To reveal the mechanism for induction of collagen in Fli1ΔNIH3T3 cells, we treated the cells with various antibodies and inhibitors and performed RNA-sequencing. In order to estimate in vivo efficacy of LY294002 derivative on fibrotic disease, we utilized bleomycin-induced lung fibrosis with Fli1+/- mice and treated with LY294002 derivative by intra-peritoneal injection 3 times and evaluated lung fibrosis by histology, collagen content, and Ashcraft scores. We also evaluated the ex vivo effect of candidate drug using skin fibroblasts from patients with SSc.
Results: Fli1ΔNIH3T3 cells were found to have pro-fibrotic characteristics such as increased expression of COL1A1, COL1A2 as well as increased expression of TGF-b1, CTGF, IL-6, FN, and ACTA2. Antibody neutralization of TGF-β1 and IL-6 didn’t inhibited collagen synthesis in Fli1ΔNIH3T3 cells. In addition, whereas MAPK inhibitors of Erk U0126, JNK SP600125, p38 SB20358 failed to suppress the increased collagen production, Nintedanib, a triple kinase inhibitor of VEGFR, FGFR, and PDGFR partially inhibited collagen synthesis. Surprisingly, a phosphoinositide 3-kinases (PI3K) inhibitor, LY294002, which also has inhibitory activity against bromodomain-containing protein (BRD) 2,3 and 4, had a major inhibitory effect on COL1A2 mRNA expression, suggesting the PI3K-Akt and bromodomain pathways have a major pro-fibrotic role in Fli1ΔNIH3T3 cells. This correlated with the fact that, p-Akt expression was increased in Fli1ΔNIH3T3 cells. In addition, a low toxicity LY294002 derivative also inhibited collagen synthesis in Fli1ΔNIH3T3, confirming PI3K and BRD4 dual inhibition was most effective to reduce COL1A2 production. Furthermore, analyses of RNA-seq revealed several molecules to induce collagen via PI3K-Akt pathway. Finally, LY294002 derivative treatment significantly ameliorated lung fibrosis as evaluated by Masson Trichrome staining, decreased collagen accumulation in the lung and Ashcroft clinical scores. In both normal and SSc fibroblasts, COL1A2 mRNA were significantly inhibited by LY294002 derivative.
Conclusion: Lack of Fli1 expression activates the molecule that induces collagen accumulation through PI3K-Akt and bromodomain pathway. PI3K and BRD4 dual inhibitor showed therapeutic potential in treating fibrosis.
To cite this abstract in AMA style:Ota Y, Kitani A, Strober W. PI3K-Akt Pathway Plays a Crucial Role in Production of Collagen in Fli1 Deficient Condition and Its Inhibitor Has the Therapeutic Potential in Treating Fibrosis [abstract]. Arthritis Rheumatol. 2019; 71 (suppl 10). https://acrabstracts.org/abstract/pi3k-akt-pathway-plays-a-crucial-role-in-production-of-collagen-in-fli1-deficient-condition-and-its-inhibitor-has-the-therapeutic-potential-in-treating-fibrosis/. Accessed October 24, 2020.
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ACR Meeting Abstracts - https://acrabstracts.org/abstract/pi3k-akt-pathway-plays-a-crucial-role-in-production-of-collagen-in-fli1-deficient-condition-and-its-inhibitor-has-the-therapeutic-potential-in-treating-fibrosis/