title: MiR-29a reduces TIMP-1 production by dermal fibroblasts via targeting TGF-β activated kinase 1 binding protein 1, implications for systemic sclerosis.
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.
  
  
  OBJECTIVE
  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-β 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-β 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.
authors.affiliation: Newcastle University, Musculoskeletal Research Group, Institute of Cellular Medicine, Newcastle upon Tyne, United Kingdom; L. Hirszferd Institute of Immunology and Experimental Therapy, Polish Academy of Science, Wroclaw, Poland.
authors.forename: Marzena
authors.initials: M
authors.lastname: Ciechomska
authors.affiliation: Newcastle University, Musculoskeletal Research Group, Institute of Cellular Medicine, Newcastle upon Tyne, United Kingdom.
authors.forename: Steven
authors.initials: S
authors.lastname: O'Reilly
authors.affiliation: Newcastle University, Fibrosis Research Group, Institute of Cellular Medicine, Newcastle upon Tyne, United Kingdom.
authors.forename: Monika
authors.initials: M
authors.lastname: Suwara
authors.affiliation: L. Hirszferd Institute of Immunology and Experimental Therapy, Polish Academy of Science, Wroclaw, Poland.
authors.forename: Katarzyna
authors.initials: K
authors.lastname: Bogunia-Kubik
authors.affiliation: Newcastle University, Musculoskeletal Research Group, Institute of Cellular Medicine, Newcastle upon Tyne, United Kingdom; University Medical Center Utrecht, Department of Rheumatology & Clinical Immunology, Utrecht, the Netherlands.
authors.forename: Jacob M
authors.initials: JM
authors.lastname: van Laar
mesh_terms: D020413:3' Untranslated Regions / Q000235:genetics; D048868:Adaptor Proteins, Signal Transducing / Q000096:biosynthesis* / Q000235:genetics; D020405:Dermis / Q000378:metabolism* / Q000473:pathology; D016326:Extracellular Matrix Proteins / Q000096:biosynthesis / Q000235:genetics; D005260:Female; D005347:Fibroblasts / Q000378:metabolism* / Q000473:pathology; D005786:Gene Expression Regulation; D006367:HeLa Cells; D006801:Humans; D008297:Male; D035683:MicroRNAs / Q000096:biosynthesis* / Q000235:genetics; D059748:Proteolysis; D012595:Scleroderma, Systemic / Q000235:genetics / Q000378:metabolism* / Q000473:pathology; D019715:Tissue Inhibitor of Metalloproteinase-1 / Q000096:biosynthesis* / Q000235:genetics
chemical_list: D020413:3' Untranslated Regions; D048868:Adaptor Proteins, Signal Transducing; D016326:Extracellular Matrix Proteins; C532045:MIRN29a microRNA, human; D035683:MicroRNAs; C486300:TAB1 protein, human; C560822:TIMP1 protein, human; D019715:Tissue Inhibitor of Metalloproteinase-1