title: Development of a high throughput luciferase reporter gene system for screening activators and repressors of human collagen Iα2 gene expression.
abstract: Fibrosis, which is characterized by the excessive production of matrix proteins, occurs in multiple tissues and is associated with increased morbidity and mortality. Despite its significant negative impact on patient outcomes, therapies targeted to treat fibrosis are currently lacking. Screening for inhibitors of the expression of collagen, the primary component of fibrotic lesions, represents an option for the identification of novel lead compounds for therapeutic development with potentially fewer off-target effects compared with the targeting of multifunctional cell signaling pathways. Here we report on the generation of a stable luciferase reporter system using a fibroblast cell line, which can be used for rapidly screening both activators and repressors of human collagen COL1A2 gene transcription in a high throughput setting. This in vitro screening tool was validated using known agonists (scleraxis, TGF-β, angiotensin II, CTGF) and antagonists (TNF-α, pirfenidone) of COL1A2 gene expression. The COL1A2-luc NIH-3T3 fibroblast system provides a useful and effective screen for potential lead compounds with pro- or anti-fibrotic properties.
authors.affiliation: a Institute of Cardiovascular Sciences, St. Boniface General Hospital Research Centre, 351 Tache Avenue, Winnipeg, MB R2H 2A6, Canada.|b Department of Physiology and Pathophysiology, University of Manitoba, St. Boniface Research Centre, 351 Tache Avenue, Winnipeg, MB R2H 2A6, Canada.
authors.forename: Rushita A
authors.initials: RA
authors.lastname: Bagchi
authors.affiliation: a Institute of Cardiovascular Sciences, St. Boniface General Hospital Research Centre, 351 Tache Avenue, Winnipeg, MB R2H 2A6, Canada.
authors.forename: Viktoriya
authors.initials: V
authors.lastname: Mozolevska
authors.affiliation: a Institute of Cardiovascular Sciences, St. Boniface General Hospital Research Centre, 351 Tache Avenue, Winnipeg, MB R2H 2A6, Canada.
authors.forename: Bernard
authors.initials: B
authors.lastname: Abrenica
authors.affiliation: a Institute of Cardiovascular Sciences, St. Boniface General Hospital Research Centre, 351 Tache Avenue, Winnipeg, MB R2H 2A6, Canada.|b Department of Physiology and Pathophysiology, University of Manitoba, St. Boniface Research Centre, 351 Tache Avenue, Winnipeg, MB R2H 2A6, Canada.
authors.forename: Michael P
authors.initials: MP
authors.lastname: Czubryt
mesh_terms: D000804:Angiotensin II / Q000494:pharmacology; D000818:Animals; D003001:Cloning, Molecular; D024042:Collagen Type I / Q000235:genetics*; D055513:Connective Tissue Growth Factor / Q000494:pharmacology; D005347:Fibroblasts / Q000187:drug effects* / Q000378:metabolism; D015870:Gene Expression / Q000187:drug effects*; D017930:Genes, Reporter / Q000187:drug effects; D057166:High-Throughput Screening Assays / Q000379:methods*; D006801:Humans; D008156:Luciferases / Q000235:genetics; D051379:Mice; D041681:NIH 3T3 Cells; D011728:Pyridones / Q000494:pharmacology; D012680:Sensitivity and Specificity; D014158:Transcription, Genetic / Q000187:drug effects; D014162:Transfection; D053773:Transforming Growth Factor beta1 / Q000494:pharmacology; D014409:Tumor Necrosis Factor-alpha / Q000494:pharmacology
chemical_list: C000601279:COL1A2 protein, human; D024042:Collagen Type I; D011728:Pyridones; D053773:Transforming Growth Factor beta1; D014409:Tumor Necrosis Factor-alpha; D000804:Angiotensin II; D055513:Connective Tissue Growth Factor; C093844:pirfenidone; D008156:Luciferases
keywords: collagen; collagène; fibroblast; fibroblaste; fibrose; fibrosis; high-throughput assay; in vitro screening assay; lignée cellulaire stable; luciferase reporter; rapporteur luciférase; stable cell line; test de criblage in vitro; test à haut-débit