RNAi screening identifies a mechanosensitive ROCK-JAK2-STAT3 network central to myofibroblast activation

Abstract

Myofibroblasts play key roles in wound healing and pathological fibrosis. Here, we used an RNAi screen to characterize myofibroblast regulatory genes, using a high-content imaging approach to quantify α-smooth muscle actin stress fibers in cultured human fibroblasts. Screen hits were validated on physiological compliance hydrogels, and selected hits tested in primary fibroblasts from patients with idiopathic pulmonary fibrosis. Our RNAi screen led to the identification of STAT3 as an essential mediator of myofibroblast activation and function. Strikingly, we found that STAT3 phosphorylation, while responsive to exogenous ligands on both soft and stiff matrices, is innately active on a stiff matrix in a ligand/receptor-independent, but ROCK- and JAK2-dependent fashion. These results demonstrate how a cytokine-inducible signal can become persistently activated by pathological matrix stiffening. Consistent with a pivotal role for this pathway in driving persistent fibrosis, a STAT3 inhibitor attenuated murine pulmonary fibrosis when administered in a therapeutic fashion after bleomycin injury. Our results identify novel genes essential for the myofibroblast phenotype, and point to STAT3 as an important target in pulmonary fibrosis and other fibrotic diseases.

TGFβ-induced fibroblast activation requires persistent and targeted HDAC-mediated gene repression

“…4A). Many of these genes are strongly linked to myofibroblast activation and fibrotic tissue remodeling (Bhattacharyya et al, 2016;Estany et al, 2014;Henderson and Sheppard, 2013;Hung et al, 2013;Le et al, 2014;Oh et al, 2018;Schafer et al, 2017;Trojanowska, 2008;Tschumperlin et al, 2018), consistent with our prior functional assays demonstrating that pracinostat reduces pro-fibrotic cellular functions. GO analysis using DAVIDv6.8 revealed that pracinostat blocked the TGFβ induction of pro-fibrotic pathways, such as wound healing, positive regulation of MAPK cascade and ECM organization ( Fig.…”

“…Specifically, diseased lung fibroblasts have been shown to exhibit causative alterations in DNA methylation, histone acetylation and micro (mi)RNAs when compared to their non-diseased counterparts (Coward et al, 2018(Coward et al, , 2014(Coward et al, , 2009Huang et al, 2014;Khalil et al, 2015;Lino Cardenas et al, 2013;Liu et al, 2010;Miao et al, 2018;Sanders et al, 2012Sanders et al, , 2017Tang et al, 2013;Xiao et al, 2016;Yang et al, 2014Yang et al, , 2013. Recently, we identified several epigenetic regulators in an siRNA screen of fibroblast activation (Oh et al, 2018). Taken together, these studies demonstrate that fibroblast activation is a complex phenomenon regulated by multiple epigenetic mechanisms.…”

“…To identify epigenetic regulators of fibroblast activation, we developed an image-based screening assay using α-smooth muscle actin (αSMA) immunofluorescence intensity as a primary outcome. Cytoskeletal αSMA is critical for contractility, and has previously been shown to be an effective metric for evaluating fibroblast activation (Hinz et al, 2001;Oh et al, 2018;Tatler and Jenkins, 2012). Using IMR90 human fetal lung fibroblasts, we validated the range and sensitivity of this assay by quantifying αSMA intensity as a dose response to TGFβ stimulation (Fig.…”

High-throughput screening discovers antifibrotic properties of haloperidol by hindering myofibroblast activation

“…Consistent with this hypothesis, Sigmar1 upregulation was reported upon cell treatment with other ER stress inducers, such as glucose, heat shock, and thapsigargin (72). Also, previous genetic screenings identified either the noncanonical TGF-β pathway or calcium as a major regulator of myofibroblast activation (23)(24)(25). For instance, TRPC6-mediated calcium signaling induced myofibroblast differentiation, although this channel essentially controls calcium influx from the extracellular space and not its release from the ER, as in our model (24).…”

“…Genetic screening has been used over the last several years to discover relevant pathways involved in myofibroblast activation (23)(24)(25). Here, we report the results of a high-content (HC), fluorescence-microscopy-based high-throughput screening (HTS), that led to the discovery of haloperidol, a common antipsychotic drug, as a potent inhibitor of myofibroblast activation.…”