PU.1 Controls Fibroblast Polarization and Tissue Fibrosis

Thomas Wohlfahrt , Simon Rauber , Steffen Uebe , Markus Luber , Alina Soare , Arif Ekici , Stefanie Weber , Alexandru-Emil Matei , Chih-Wei Chen , Christiane Maier , Emmanuel Karouzakis , Hans P Kiener , Elena Pachera , Clara Dees , Christian Beyer , Christoph Daniel , Kolja Gelse , Andreas E Kremer , Elisabeth Naschberger , Michael Stürzl , Falk Butter , Michael Sticherling , Susetta Finotto , Alexander Kreuter , Mark H Kaplan , Astrid Jüngel, Steffen Gay , Stephen L Nutt , David W Boykin , Gregory M K Poon , Oliver Distler , Georg Schett , Jörg H W Distler , Andreas Ramming 
Source: Nature
Publication Date: (2019)
Issue: 566 (7744), : 344-349
Research Area:
Dermatology/Tissue Engineering
Cells used in publication:
Fibroblast, dermal(NHDF-Ad), human adult
Species: human
Tissue Origin: dermal
4D-Nucleofector® X-Unit

As indicated, fibroblasts (from patients with fibrosis or arthritis or healthy persons) were transfected with 0.1 µg of either pUNO1 empty vector (control) or pUNO1-hSPI1a (both from InvivoGen) plasmids using the 4D-Nucleofector (Lonza). Gene silencing was achieved using either 3 µg control CRISPR–Cas9 plasmid or 3 µg of PU.1 CRISPR–Cas9 KO plasmid (h2) (Santa Cruz Biotechnology). miR-155 silencing was performed using the anti-hsa-miR-155-5p miScript miRNA inhibitor or the miScript negative control inhibitor (1,500 ng)
(both from QIAGEN).
Reporter assays. Human fibroblasts were transfected with a COL1A1 luciferase reporter plasmid (Active Motif) using the 4D-Nucleofector. Luciferase assays were performed using the Renilla Luciferase Assay System according to the manufacturer’s instructions (Progema). Relative light units were obtained with a Luminoskan Ascent instrument with automated well-wise injection (Thermo Fisher Scientific). Relative light units were normalized to the protein concentration, as determined by a Bradford protein assay according to the manufacturer’s protocol (Bio-Rad).


Fibroblasts are polymorphic cells with pleiotropic roles in organ morphogenesis, tissue homeostasis and immune responses. In fibrotic diseases, fibroblasts synthesize abundant amounts of extracellular matrix, which induces scarring and organ failure. By contrast, a hallmark feature of fibroblasts in arthritis is degradation of the extracellular matrix because of the release of metalloproteinases and degrading enzymes, and subsequent tissue destruction. The mechanisms that drive these functionally opposing pro-fibrotic and pro-inflammatory phenotypes of fibroblasts remain unknown. Here we identify the transcription factor PU.1 as an essential regulator of the pro-fibrotic gene expression program. The interplay between transcriptional and post-transcriptional mechanisms that normally control the expression of PU.1 expression is perturbed in various fibrotic diseases, resulting in the upregulation of PU.1, induction of fibrosis-associated gene sets and a phenotypic switch in extracellular matrix-producing pro-fibrotic fibroblasts. By contrast, pharmacological and genetic inactivation of PU.1 disrupts the fibrotic network and enables reprogramming of fibrotic fibroblasts into resting fibroblasts, leading to regression of fibrosis in several organs.