Mechanosignaling through YAP and TAZ drives fibroblast activation and fibrosis

Abstract

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.

Keywords: Hippo; extracellular matrix; idiopathic pulmonary fibrosis; mechanotransduction; plasminogen activator inhibitor 1.

Fig. 1.

Yes-associated protein (YAP) (homolog of drosophila Yki) and transcriptional coactivator with PDZ-binding motif (TAZ) (also known as Wwtr1) expression and nuclear localization are enhanced in remodeled fibrotic lungs. A: immunohistochemical staining of TAZ (WWTR1) in representative low-power and high-power fields from normal (healthy) and idiopathic pulmonary fibrosis (IPF) lungs and quantification of nuclei staining positive for TAZ (WWTR1+/total nuclei) from 4 donors per group, 4 low-power images per donor, and 2–3 high-power sampling areas per image. B: immunohistochemical staining of YAP and quantification of cells (nuclei) staining positive for YAP from 4 donors per group, 4 low-power images per donor, and 2–3 high-power sampling areas per image. C: immunohistochemical staining of TAZ in IPF lung tissue and comparison with corresponding high-resolution stiffness maps measured in adjacent serial sections by atomic force microscopy (AFM). Scale bar in immunohistochemistry (IHC), 50 mm. Box in each IHC image indicates the approximate stiffness mapping area (80 × 80 μm). Color scale in stiffness maps indicates shear modulus in kPa. D: tissue stiffness (shear modulus) measured in several regions from snap-frozen unfixed normal and IPF lungs by AFM (n = 6 each of IPF and normal lungs).

Fig. 2.

YAP and TAZ respond to pathophysiological increases in matrix stiffness by relocalizing to fibroblast nuclei. A: despite immunohistochemical evidence for enhanced YAP/TAZ expression in IPF, IPF-derived fibroblasts do not overexpress YAP/TAZ in a cell-autonomous fashion. Cells from 7 different normal and IPF lungs were grown on tissue culture plastic, and YAP/TAZ protein levels were analyzed by Western blotting with mouse anti-YAP (63.7) antibody (Santa Cruz Biotechnology) and rabbit anti-YAP/TAZ (D24E4) antibody (Cell Signaling). Both antibodies cross react with YAP and TAZ, which appear at molecular weights of ∼70 kDa and ∼55 kDa, respectively. GAPDH was used as a loading control. Densitometry normalized to GAPDH demonstrates a modest decrease in YAP protein levels in IPF fibroblasts relative to normal lung fibroblasts. NL, normal cells. B: YAP and TAZ transcript levels vary little across matrix stiffness conditions spanning the pathophysiological range in IPF-derived, normal, and IMR-90 fibroblasts (n = 3 independent experiments). C: in contrast, YAP and TAZ nuclear localization is dramatically altered by matrix stiffness. Immunostaining with mouse anti-YAP (63.7) antibody (Santa Cruz Biotechnology, detects both YAP and TAZ) and Hoechst 33342 after culturing 48 h on 0.4- and 25.0-kPa shear moduli polyacrylamide hydrogels demonstrates pronounced nuclear localization on stiff matrices. D: cells with predominantly nuclear YAP/TAZ localization were counted in a blinded fashion on 0.4- and 25.0-kPa gels and expressed as a fraction of total cell number. N = total number of cells analyzed from 2 independent experiments from 3 normal (NL29, NL43, and NL45) and 3 IPF (IPF14, IPF26, and IPF32) lines.

Fig. 3.

YAP/TAZ siRNA (YAPi/TAZi) attenuates YAP/TAZ message (relative mRNA) and protein levels (Western blot) in IMR-90 fibroblasts. A: YAP and TAZ transcript levels measured by qPCR, normalized to GAPDH, and to 25-kPa control samples. B: Western blotting with mouse anti-YAP (63.7) antibody (Santa Cruz Biotechnology) detects YAP (65 kDa) and TAZ (45 kDa) bands in siCTRL but not YAPi/TAZi samples.

Fig. 4.

Profibrotic responses to matrix stiffness are dramatically reduced by YAP/TAZ knockdown. A: IMR-90 fibroblasts were transfected with YAP/TAZ siRNA on 0.4- and 25.0-kPa shear moduli polyacrylamide hydrogels functionalized with collagen I for 72 h, fixed, and stained with mouse anti-vinculin antibody to visualize focal adhesions and phalloidin to visualize F-actin. Regions of interest indicated by white dashed boxes are shown at higher magnification in second row of images, with contrasting large and small vinculin-positive adhesions indicated by white arrows. B: increasing matrix stiffness enhances and YAP/TAZ knockdown attenuates focal adhesion (FA) length, F-actin assembly, and cell spreading. ***P < 0.0001 by 1-way ANOVA; FA length, n = number of cells analyzed from 2 independent experiments; F-actin, n = number of images analyzed from 3 independent experiments; cell area, n = number of cells analyzed from 3 independent experiments. C: immunostaining with mouse anti-procollagen I antibody and Hoechst 33342 to visualize cell nuclei. D: increased matrix stiffness enhances and YAP/TAZ knockdown attenuates procollagen I-positive cells (***P < 0.0001 by 1-way ANOVA, n = number of cells analyzed from 3 independent experiments) and soluble collagen production in cell culture medium, as measured with SirCol assay (**P < 0.05, by 1-way ANOVA from 2 independent experiments). E: increasing matrix stiffness promotes and YAP/TAZ knockdown attenuates cell accumulation over 72 h, with the converse trend observed for caspase 3/7 activity (*P < 0.05 by t-test from 2 independent experiments). F: increasing matrix stiffness enhances contractility, as measured by net contractile moment and peak tractions in traction force microscopy. YAP/TAZ siRNA knockdown selectively attenuates tractions on stiff matrices (***P < 0.0001 by 1-way ANOVA, n = number of cells analyzed from 2 independent experiments). Representative traction maps are shown from 0.4- and 6.4-kPa matrices, where color scale indicates traction magnitudes (kPa) and scale bar indicates 20 μm.

Fig. 5.

IPF fibroblast responses to matrix stiffness are ablated by YAP/TAZ knockdown. IPF fibroblasts were transfected with YAP/TAZ siRNA for 72 h on 0.4- and 25.0-kPa shear moduli polyacrylamide hydrogels functionalized with collagen I. A: equivalent knockdown of YAP and TAZ on soft and stiff matrices was observed (n = 3 independent experiments). B: increasing matrix stiffness enhances IPF fibroblast contractility, as measured by net contractile moment and peak tractions in traction force microscopy. YAP/TAZ siRNA knockdown selectively attenuates tractions on stiff matrices. ***P < 0.0001 by 1-way ANOVA, n = number of cells analyzed from 2 independent experiments. C: increasing matrix stiffness promotes cell accumulation over 72 h. YAP/TAZ siRNA knockdown selectively reduces cell accumulation on stiff matrix (n = 2 independent experiments). D: increasing matrix stiffness promotes IPF fibroblast gene expression of extracellular matrix transcripts COL1A1 and FN1. YAP/TAZ siRNA knockdown selectively attenuates matrix stiffness-promoted gene expression on stiff matrices (n = 2 independent experiments). Similarly, increasing matrix stiffness enhances expression of EDA fibronectin (FN) protein, as measured by Western blot from cell lysates, and YAP/TAZ siRNA knockdown reduces EDA FN expression on pathologically stiff but not physiologically compliant matrix (n = 3 independent experiments). E: YAP/TAZ siRNA knockdown evokes consistent effects on EDA FN protein expression across 3 independent IPF fibroblast lines. cFN, cellular fibronectin.

Fig. 6.

Matrix stiffness promotes profibrotic functions through the YAP/TAZ target plasminogen activator inhibitor (PAI)-1. A: increasing matrix stiffness promotes IMR-90 fibroblast PAI-1 (SERPINE1) gene expression and PAI-1 protein levels in conditioned media while decreasing plasmin activity (n = 3 independent experiments). YAP/TAZ siRNA knockdown selectively attenuates matrix stiffness-promoted PAI-1 expression on stiff matrices and correspondingly increases plasmin activity on stiff matrices. IPF fibroblasts exhibit similar stiffness- and YAP/TAZ-dependent PAI-1 (SERPINE1) expression (n = 2 independent experiments). B: exogenous plasmin (0.1 U/ml) attenuates matrix stiffness-promoted procollagen I and α-smooth muscle actin (SMA) expression on stiff matrices in a fashion similar to YAP/TAZ siRNA knockdown. Plasmin also significantly reduces the proportion of fibroblasts with predominant nuclear localization of YAP/TAZ on stiff matrices; n = number of cells analyzed per condition. C: inhibition of transforming growth factor (TGF)-β receptor with ALK5 inhibitor SB431542 (1 μM) or a pan anti-TGF-β antibody (10 μg/ml) has no effect on matrix stiffness-induced PAI-1 protein production or plasmin activity decrease in IMR-90-conditioned media (n = 2 independent experiments). D: exogenous TGF-β (2 μg/ml) potentiates matrix stiffness-promoted PAI-1 gene expression. YAP/TAZ siRNA double knockdown (YAPi/TAZi) ablates TGF-β effect on stiff matrices but has no effect on soft matrices (n = 2 independent experiments).

Fig. 7.

Overexpression of YAP5SA or TAZ4SA promotes fibroblast proliferation and matrix synthesis. YAP5SA-expressing NIH3T3 fibroblasts exhibit nuclear YAP localization on soft (0.4 kPa) matrices comparable to control NIH3T3 cells grown on stiff (25 kPa) matrices. In contrast, control NIH3T3 cells exhibit little nuclear localization on soft matrices. Immunostaining performed with mouse anti-YAP (63.7) antibody (Santa Cruz Biotechnology, detects both YAP and TAZ) and Hoechst 33342. All cells exposed to 100 ng/ml doxycycline (DOX) for 48 h. Dashed boxes in top row indicate areas detailed under higher power in bottom row. B: YAP5SA-expressing NIH3T3 fibroblasts exhibit a trend toward enhanced proliferative capacity on soft matrix (0.4 kPa) comparable to that of control cells on stiff matrix (25 kPa). Data shown are fold increases in cell number relative to seeding density, n = 2 independent experiments. C: YAP5SA- and TAZ4SA-expressing NIH3T3 fibroblasts exhibit increased PAI-1 (SERPINE1) transcript levels on soft matrix (0.4 kPa) comparable to control cells on stiff matrix (25 kPa), as measured by qPCR normalized to GAPDH and 0.4 kPa control (Ctrl) samples, n = 2 independent experiments. D: doxycycline-induced YAP5SA or TAZ4SA expression augments expression of COL1A1 transcripts in NIH3T3 cells cultured 48 h on 25-kPa hydrogels. *P < 0.05 compared with 0 ng/ml doxycycline, unpaired t-test. E: doxycycline-induced TAZ4SA expression increases extracellular matrix protein deposition on a per-cell basis by NIH3T3 cells on gelatin-coated coverslips. *P < 0.05 compared with NIH3T3 control cells (Ctrl) treated with same dose doxycycline. F: naïve NIH3T3 adhesion is enhanced on matrices derived from doxycycline-treated TAZ4SA cells relative to control cells (Ctrl). *P < 0.05 compared with NIH3T3 control cells treated with the same dose of doxycycline.

Fig. 8.

Conditional overexpression of YAP5SA or TAZ4SA confers fibrogenic potential to fibroblasts adoptively transferred to mouse lung. A: adoptive transfer of control 3T3 fibroblasts or 3T3 fibroblasts expressing doxycycline-inducible YAP5SA or TAZ4SA to immunocompromised mice results in cellular nodule formation in the lungs evident under low magnification in Masson's trichrome-stained tissue sections. Feeding with doxycycline dramatically increases nodular lung fraction in mice with YAP5SA- or TAZ4SA-expressing cells. B: higher magnification images from Masson's trichrome-stained sections demonstrate collagen deposition in nodules from doxycycline-fed mice with YAP5SA- or TAZ4SA-expressing cells. C: robust increases in lung hydroxyproline were observed in doxycycline-fed mice with YAP5SA- or TAZ4SA-expressing cells relative to no doxycycline feeding and relative to doxycycline-fed mice with control 3T3 cells, demonstrating the fibrogenic capacity of adoptively transferred cells expressing YAP5SA or TAZ4SA. *P < 0.05 relative to Untreated, #P < 0.05 between No Dox and Dox within same group. D: schematic illustrates key findings of this work: increasing matrix stiffness promotes nuclear YAP/TAZ localization, shifting fibroblasts from a relatively quiescent state to a more activated fibrogenic and matrix-remodeling state, supported in part by upregulation of PAI-1 and attenuation of pericellular plasmin activity.