دورية أكاديمية
Targeting alternative splicing of fibronectin in human renal proximal tubule epithelial cells with antisense oligonucleotides to reduce EDA+ fibronectin production and block an autocrine loop that drives renal fibrosis.
| العنوان: | Targeting alternative splicing of fibronectin in human renal proximal tubule epithelial cells with antisense oligonucleotides to reduce EDA+ fibronectin production and block an autocrine loop that drives renal fibrosis. |
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| المؤلفون: | Phanish MK; SWT Institute for Renal Research, Renal Unit, St Helier Hospital, Epsom and St Helier University Hospitals NHS Trust, London, UK; St Georges' University of London, London, UK. Electronic address: m.phanish@nhs.net., Heidebrecht F; SWT Institute for Renal Research, Renal Unit, St Helier Hospital, Epsom and St Helier University Hospitals NHS Trust, London, UK., Jackson M; IONIS Pharmaceuticals, 2855, Gazelle Ct, Carlsbad, CA 92010, USA., Rigo F; IONIS Pharmaceuticals, 2855, Gazelle Ct, Carlsbad, CA 92010, USA., Dockrell MEC; SWT Institute for Renal Research, Renal Unit, St Helier Hospital, Epsom and St Helier University Hospitals NHS Trust, London, UK; St Georges' University of London, London, UK. Electronic address: mark.dockrell@nhs.net. |
| المصدر: | Experimental cell research [Exp Cell Res] 2024 Sep 01; Vol. 442 (1), pp. 114186. Date of Electronic Publication: 2024 Aug 02. |
| نوع المنشور: | Journal Article |
| اللغة: | English |
| بيانات الدورية: | Publisher: Academic Press Country of Publication: United States NLM ID: 0373226 Publication Model: Print-Electronic Cited Medium: Internet ISSN: 1090-2422 (Electronic) Linking ISSN: 00144827 NLM ISO Abbreviation: Exp Cell Res Subsets: MEDLINE |
| أسماء مطبوعة: | Publication: Orlando Fl : Academic Press Original Publication: New York, Academic Press. |
| مواضيع طبية MeSH: | Fibronectins*/metabolism , Fibronectins*/genetics , Kidney Tubules, Proximal*/metabolism , Kidney Tubules, Proximal*/pathology , Kidney Tubules, Proximal*/cytology , Oligonucleotides, Antisense*/pharmacology , Oligonucleotides, Antisense*/genetics , Fibrosis*/metabolism , Alternative Splicing*/genetics , Transforming Growth Factor beta1*/metabolism , Epithelial Cells*/metabolism , Epithelial Cells*/pathology , Epithelial Cells*/drug effects, Humans ; Cells, Cultured ; Autocrine Communication ; Matrix Metalloproteinase 2/metabolism ; Matrix Metalloproteinase 2/genetics ; Matrix Metalloproteinase 9/metabolism ; Matrix Metalloproteinase 9/genetics |
| مستخلص: | TGFβ1 is a powerful regulator of fibrosis; secreted in a latent form, it becomes active after release from the latent complex. During tissue fibrosis, the EDA + isoform of cellular fibronectin is overexpressed. In pulmonary fibrosis it has been proposed that the fibronectin splice variant including an EDA domain (FN EDA+) activates latent TGFβ. Our work investigates the potential of blocking the 'splicing in' of EDA with antisense oligonucleotides to inhibit TGFβ1-induced EDA + fibronectin and to prevent the cascade of events initiated by TGFβ1 in human renal proximal tubule cells (PTEC). Human primary PTEC were treated with TGFβ1 for 48 h, medium removed and the cells transfected with RNase H-independent antisense oligonucleotides (ASO) designed to block EDA exon inclusion (ASO5). The efficacy of ASO to block EDA exon inclusion was assessed by EDA + fibronectin RNA and protein expression; the expression of TGFβ, αSMA (α smooth muscle actin), MMP2 (matrix metalloproteinse-2), MMP9 (matrix metalloproteinse-9), Collagen I, K Cadherin and connexin 43 was analysed. Targeting antisense oligonucleotides designed to block EDA exon inclusion in fibronectin pre mRNA were effective in reducing the amount of TGFβ1 -induced cellular EDA + fibronectin RNA and secreted EDA + fibronectin protein (assessed by western immunoblotting and immunocytochemistry) in human proximal tubule cells in an in vitro cell culture model. The effect was selective for EDA + exon with no effect on EDB + fibronectin RNA and total fibronectin mRNA. Exogenous TGFβ1 induced endogenous TGFβ, αSMA, MMP2, MMP9 and Col I mRNA. TGFβ1 treatment for 48h reduced the expression of K-Cadherin and increased the expression of connexin-43. These TGFβ1-induced pro-fibrotic changes were attenuated by ASO5 treatment. 48 h after the removal of exogenous TGFβ, further increases in αSMA, MMP2, MMP9 was observed; ASO5 significantly inhibited this subsequent increase. ASO5 treatment also significantly inhibited ability of the cell culture medium harvested at the end of the experiment (96h) to stimulate SMAD3 reporter cells. The role of endogenous TGFβ1 was confirmed by the use of a TGFβ receptor inhibitor. Our results demonstrate a critical role of FN EDA+ in a cycle of TGFβ driven pro-fibrotic responses in human PTEC and blocking its production with ASO technology offers a potential therapy to interrupt this vicious circle and hence limit the progression of renal fibrosis. Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.) |
| فهرسة مساهمة: | Keywords: Antisense oligonucleotides; EDA+ fibronectin; RNA splicing; Renal tubulointerstitial fibrosis; TGFβ1 signalling |
| المشرفين على المادة: | 0 (Fibronectins) 0 (Oligonucleotides, Antisense) 0 (Transforming Growth Factor beta1) EC 3.4.24.24 (Matrix Metalloproteinase 2) 0 (TGFB1 protein, human) EC 3.4.24.35 (Matrix Metalloproteinase 9) |
| تواريخ الأحداث: | Date Created: 20240804 Date Completed: 20240910 Latest Revision: 20240910 |
| رمز التحديث: | 20240911 |
| DOI: | 10.1016/j.yexcr.2024.114186 |
| PMID: | 39098465 |
| قاعدة البيانات: | MEDLINE |
Full Text Finder | تدمد: | 1090-2422 |
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| DOI: | 10.1016/j.yexcr.2024.114186 |