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MiR-29a Reduces TIMP-1 Production by Dermal Fibroblasts via Targeting TGF- β Activated Kinase 1 Binding Protein 1, Implications for Systemic Sclerosis

Lookup NU author(s): Dr Marzena Ciechomska, Dr Steven O'Reilly, Monika Suwara, Professor Jaap Van Laar

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This work is licensed under a Creative Commons Attribution 4.0 International License (CC BY 4.0).


Abstract

Background: Systemic sclerosis (SSc) is an autoimmune connective tissue disease characterised by skin and internal organs fibrosis due to accumulation of extra cellular matrix (ECM) proteins. Tissue inhibitor of metalloproteinases 1 (TIMP-1) plays a key role in ECM deposition.Aim: To investigate the role of miR-29a in regulation of TAB1-mediated TIMP-1 production in dermal fibroblasts in systemic sclerosis.Methods: Healthy control (HC) and SSc fibroblasts were cultured from skin biopsies. The expression of TIMP-1, MMP-1 and TGF-beta activated kinase 1 binding protein 1 (TAB1) was measured following miR-29a transfection using ELISA, qRT-PCR, and Western Blotting. The functional effect of miR-29a on dermal fibroblasts was assessed in collagen gel assay. In addition, HeLa cells were transfected with 3'UTR of TAB1 plasmid cloned downstream of firefly luciferase gene to assess TAB1 activity. HC fibroblasts and HeLa cells were also transfected with Target protectors in order to block the endogenous miR-29a activity.Results: We found that TAB1 is a novel target gene of miR-29a, also regulating downstream TIMP-1 production. TAB1 is involved in TGF-beta signal transduction, a key cytokine triggering TIMP-1 production. To confirm that TAB1 is a bona fide target gene of miR-29a, we used a TAB1 3'UTR luciferase assay and Target protector system. We showed that miR-29a not only reduced TIMP-1 secretion via TAB1 repression, but also increased functional MMP-1 production resulting in collagen degradation. Blocking TAB1 activity by pharmacological inhibition or TAB1 knockdown resulted in TIMP-1 reduction, confirming TAB1-dependent TIMP-1 regulation. Enhanced expression of miR-29a was able to reverse the profibrotic phenotype of SSc fibroblasts via downregulation of collagen and TIMP-1.Conclusions: miR-29a repressed TAB1-mediated TIMP-1 production in dermal fibroblasts, demonstrating that miR-29a may be a therapeutic target in SSc.


Publication metadata

Author(s): Ciechomska M, O'Reilly S, Suwara M, Bogunia-Kubik K, van Laar JM

Publication type: Article

Publication status: Published

Journal: PLoS One

Year: 2014

Volume: 9

Issue: 12

Online publication date: 30/12/2014

Acceptance date: 28/11/2014

Date deposited: 30/04/2015

ISSN (electronic): 1932-6203

Publisher: Public Library of Science

URL: http://dx.doi.org/10.1371/journal.pone.0115596

DOI: 10.1371/journal.pone.0115596


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