Varelas, Xaralabos

Fibroblasts actively respond to changes in extracellular matrix mechanical properties, and positive feedback between matrix stiffness and fibroblast activation may play a pivotal role in fibrosis. Microarray analysis of matrix‐stiffness regulated genes in human lung fibroblasts identified significant overrepresentation of transcriptional targets of YAP (yes‐associated protein) and TAZ (transcriptional coactivator with PDZ‐binding motif), consistent with a prominent role for these factors in fibroblast mechanoresponsiveness. Immunofluorescence imaging demonstrated that YAP and TAZ transitioned from primarily nuclear localization on relatively stiff matrices (25.6 kPa) to cytoplasmic on soft matrices (0.4 kPa). RNAi‐mediated knockdown of YAP/TAZ significantly reduced the cell spreading, focal adhesion length, traction forces, proliferation, pro‐fibrotic gene expression and collagen synthesis observed on stiff matrices, but exerted negligible effects on soft matrices. Conditional overexpression of YAP overcame soft matrix limitations on proliferation, while overexpression of either YAP or TAZ promoted pro‐fibrotic gene expression on both soft and stiff matrices. Taken together, these results support a central role for YAP and TAZ in matrix stiffness‐dependent activation of fibroblasts to a proliferative, contractile and matrix synthetic state implicated in the pathogenesis of fibrosis. Grant Funding Source : Supported by NIH RO1 HL092961

Matrix stiffness is a central regulator of fibroblast function. However, the transcriptional mechanisms linking matrix stiffness to changes in fibroblast phenotype are incompletely understood. Here, we evaluated the effect of matrix stiffness on genome-wide chromatin accessibility in freshly isolated lung fibroblasts using ATAC-seq. We found higher matrix stiffness profoundly increased global chromatin accessibility relative to lower matrix stiffness, and these alterations were in close genomic proximity to known profibrotic gene programs. Motif analysis of these regulated genomic loci identified ZNF416 as a putative mediator of fibroblast stiffness responses. Genome occupancy analysis using ChIP-seq confirmed that ZNF416 occupies a broad range of genes implicated in fibroblast activation and tissue fibrosis, with relatively little overlap in genomic occupancy with other mechanoresponsive and profibrotic transcriptional regulators. Using loss- and gain-of-function studies, we demonstrated that ZNF416 plays a critical role in fibroblast proliferation, extracellular matrix synthesis, and contractile function. Together, these observations identify ZNF416 as novel mechano-activated transcriptional regulator of fibroblast biology.

Pharmacologic stimulation of a G protein–coupled receptor preferentially expressed on fibroblasts reverses experimental lung and liver fibrosis.

ABSTRACT Tissue fibrosis is a chronic disease driven by persistent fibroblast activation that has recently been linked to epigenetic modifications. Here, we screened a small library of epigenetic small-molecule modulators to identify compounds capable of inhibiting or reversing TGFβ-mediated fibroblast activation. We identified pracinostat, an HDAC inhibitor, as a potent attenuator of lung fibroblast activation and confirmed its efficacy in patient-derived fibroblasts isolated from fibrotic lung tissue. Mechanistically, we found that HDAC-dependent transcriptional repression was an early and essential event in TGFβ-mediated fibroblast activation. Treatment of lung fibroblasts with pracinostat broadly attenuated TGFβ-mediated epigenetic repression and promoted fibroblast quiescence. We confirmed a specific role for HDAC-dependent histone deacetylation in the promoter region of the anti-fibrotic gene PPARGC1A (PGC1α) in response to TGFβ stimulation. Finally, we identified HDAC7 as a key factor whose siRNA-mediated knockdown attenuates fibroblast activation without altering global histone acetylation. Together, these results provide novel mechanistic insight into the essential role HDACs play in TGFβ-mediated fibroblast activation via targeted gene repression.

ABT-263 blocks BCL-X L to induce apoptosis in human dermal fibroblasts from patients with scleroderma and in myofibroblasts in mice, reversing dermal fibrosis.

Pulmonary arterial stiffness is an independent risk factor for mortality in pulmonary hypertension (PH) and plays a critical role in PH pathophysiology. Our laboratory has recently demonstrated arterial stiffening early in experimental PH, along with evidence for a mechanobiological feedback loop by which arterial stiffening promotes further cellular remodeling behaviors (Liu F, Haeger CM, Dieffenbach PB, Sicard D, Chrobak I, Coronata AM, Suárez Velandia MM, Vitali S, Colas RA, Norris PC, Marinković A, Liu X, Ma J, Rose CD, Lee SJ, Comhair SA, Erzurum SC, McDonald JD, Serhan CN, Walsh SR, Tschumperlin DJ, Fredenburgh LE. JCI Insight 1: e86987, 2016). Cyclooxygenase-2 (COX-2) and prostaglandin signaling have been implicated in stiffness-mediated regulation, with prostaglandin activity inversely correlated to matrix stiffness and remodeling behaviors in vitro, as well as to disease progression in rodent PH models. The mechanism by which mechanical signaling translates to reduced COX-2 activity in pulmonary vascular cells is unknown. The present work investigated the transcriptional regulators Yes-associated protein (YAP) and WW domain-containing transcription regulator 1 (WWTR1, a.k.a., TAZ), which are known drivers of downstream mechanical signaling, in mediating stiffness-induced changes in COX-2 and prostaglandin activity in pulmonary artery smooth muscle cells (PASMCs). We found that YAP/TAZ activity is increased in PAH PASMCs and experimental PH and is necessary for the development of stiffness-dependent remodeling phenotypes. Knockdown of YAP and TAZ markedly induces COX-2 expression and downstream prostaglandin production by approximately threefold, whereas overexpression of YAP or TAZ reduces COX-2 expression and prostaglandin production to near undetectable levels. Together, our findings demonstrate a stiffness-dependent YAP/TAZ-mediated positive feedback loop that drives remodeling phenotypes in PASMCs via reduced COX-2 and prostaglandin activity. The ability to interrupt this critical mechanobiological feedback loop and enhance local prostaglandin activity via manipulation of YAP/TAZ signaling presents a highly attractive novel strategy for the treatment of PH.

Recent studies have implicated the Hippo pathway and its transcriptional effectors YAP and TAZ as necessary for fibroblast activation and tissue fibrosis. To test the specific and sufficient roles for TAZ in driving autonomous fibroblast activation, we cultured NIH3T3 fibroblasts expressing a doxycycline-inducible nuclear-localized mutant of TAZ (TAZ4SA) in scaffold-free 3D hanging drop spheroids, or on matrices of specified mechanical rigidity. Control NIH3T3 fibroblasts formed spheroids in hanging drop culture that remained stable and neither increased nor decreased in size significantly over 15 days. In contrast, TAZ4SA-transduced fibroblasts grew robustly in spheroid culture, and expressed enhanced levels of genes encoding profibrotic soluble factors connective tissue growth factor (CTGF), endothelin-1 (Et-1), and plasminogen activator inhibitor 1 (PAI-1). However, TAZ4SA expression was unable to enhance expression of extracellular matrix (ECM)-encoding genes Col1a1, Col1a2, Col3a1, or Fn1 in spheroid culture. Micromechanical testing indicated that spheroids composed of either control or TAZ4SA-expressing cells were highly compliant and indistinguishable in mechanical properties. In fibroblasts cultured on 2D matrices of compliance similar to spheroids, TAZ4SA expression was able to enhance contractile force generation, but was unable to enhance ECM gene expression. In contrast, culture on stiff hydrogels potentiated TAZ4SA enhancement of ECM expression. TAZ4SA enhancement of Col1a1 expression on soft matrices was potentiated by TGF-β1, while on stiff matrices it was abrogated by inhibition of myocardin-related transcription factor, demonstrating context-dependent crosstalk of TAZ with these pathways. These findings demonstrate sufficiency of TAZ activation for driving fibroblast proliferation, contraction, and soluble profibrotic factor expression, and mechanical context-dependent crosstalk of TAZ with other pathways in regulating Col1a1 expression.

Pathological fibrosis is driven by a feedback loop in which the fibrotic extracellular matrix is both a cause and consequence of fibroblast activation. However, the molecular mechanisms underlying this process remain poorly understood. Here we identify yes-associated protein (YAP) (homolog of drosophila Yki) and transcriptional coactivator with PDZ-binding motif (TAZ) (also known as Wwtr1), transcriptional effectors of the Hippo pathway, as key matrix stiffness-regulated coordinators of fibroblast activation and matrix synthesis. YAP and TAZ are prominently expressed in fibrotic but not healthy lung tissue, with particularly pronounced nuclear expression of TAZ in spindle-shaped fibroblastic cells. In culture, both YAP and TAZ accumulate in the nuclei of fibroblasts grown on pathologically stiff matrices but not physiologically compliant matrices. Knockdown of YAP and TAZ together in vitro attenuates key fibroblast functions, including matrix synthesis, contraction, and proliferation, and does so exclusively on pathologically stiff matrices. Profibrotic effects of YAP and TAZ operate, in part, through their transcriptional target plasminogen activator inhibitor-1, which is regulated by matrix stiffness independent of transforming growth factor-β signaling. Immortalized fibroblasts conditionally expressing active YAP or TAZ mutant proteins overcome soft matrix limitations on growth and promote fibrosis when adoptively transferred to the murine lung, demonstrating the ability of fibroblast YAP/TAZ activation to drive a profibrotic response in vivo. Together, these results identify YAP and TAZ as mechanoactivated coordinators of the matrix-driven feedback loop that amplifies and sustains fibrosis.