Combined control of the fibroblast contractile program by YAP and TAZ

Abstract

Yes-associated protein (YAP) and transcriptional coactivator with PDZ-binding motif (TAZ) are transcription cofactors implicated in the contractile and profibrotic activation of fibroblasts. Fibroblast contractile function is important in alveologenesis and in lung wound healing and fibrosis. As paralogs, YAP and TAZ may have independent or redundant roles in regulating transcriptional programs and contractile function. Using IMR-90 lung fibroblasts, microarray analysis, and traction microscopy, we tested whether independent YAP or TAZ knockdown alone was sufficient to limit transcriptional activation and contraction in vitro. Our results demonstrate limited effects of knockdown of either YAP or TAZ alone, with more robust transcriptional and functional effects observed with combined knockdown, consistent with cooperation or redundancy of YAP and TAZ in transforming growth factor β1 (TGFβ1)-induced fibroblast activation and contractile force generation. The transcriptional responses to combined YAP/TAZ knockdown were focused on a relatively small subset of genes with prominent overrepresentation of genes implicated in contraction and migration. To explore potential disease relevance of our findings, we tested primary human lung fibroblasts isolated from patients with idiopathic pulmonary fibrosis and confirmed that YAP and TAZ combined knockdown reduced the expression of three cytoskeletal genes, ACTA2, CNN1, and TAGLN. We then compared the contribution of these genes, along with YAP and TAZ, to contractile function. Combined knockdown targeting YAP/TAZ was more effective than targeting any of the individual cytoskeletal genes in reducing contractile function. Together, our results demonstrate that YAP and TAZ combine to regulate a multigene program that is essential to fibroblast contractile function.

INTRODUCTION

The activation of tissue resident fibroblasts to highly contractile myofibroblasts is critically important for normal developmental processes such as lung alveologenesis as well as normal wound healing (1–3). If aberrantly and persistently activated, myofibroblasts can contribute to tissue architectural distortion and fibrosis (4). Although several signals and transcriptional events govern this critical cellular transition, chief among these being transforming growth factor β (TGFβ) and downstream SMAD signaling, recent attention has extended to the key roles of the Hippo pathway effectors yes-associated protein (YAP) and transcriptional coactivator with PDZ-binding motif (TAZ) as regulators of myofibroblast activation downstream of both mechanical and biochemical stimuli (1–3, 5–7). Recent work has highlighted potentially unique roles for YAP or TAZ in mesenchymal cell function and tissue remodeling (8–13), whereas other studies have emphasized redundant or overlapping roles of YAP and TAZ in these processes (1, 14, 15). Thus, the exact extent to which YAP and TAZ operate independently or redundantly in the conversion of fibroblasts to myofibroblasts and their roles in regulating contractile phenotype remain ambiguous.

To delineate the contributions and potential transcriptional targets of YAP and TAZ in regulation of fibroblast activation to a contractile state, we compared transcriptional profiles and contractile function of human lung fibroblasts stimulated with TGFβ1 in tandem with small interfering RNA (siRNA)-mediated knockdown of YAP alone, TAZ alone, or both in combination. We identified an integrated program of transcripts encoding cytoskeleton-related proteins that were selectively reduced by combined YAP/TAZ knockdown. Contributions of these genes to the contractile fibroblast phenotype were further validated by traction microscopy, and direct interactions with YAP were assessed by chromatin immunoprecipitation and polymerase chain reaction (ChIP-PCR). Together, our studies identify cooperative roles of YAP/TAZ in fibroblast contractile function and define multiple YAP/TAZ target genes as essential to the activation of a fibroblast contractile state.

MATERIALS AND METHODS

Cell Culture

IMR-90 cells (ATCC, RRID:CVCL_0347) and idiopathic pulmonary fibrosis (IPF) cells (patient-derived, gift from Y.S. Prakash, Mayo Clinic, and Peter Bitterman, University of Minnesota) or purchased primary human lung fibroblasts (Lonza, CC-7231) were maintained in Eagle’s minimum essential medium (EMEM) (ATCC) containing 10% fetal bovine serum (FBS), 10,000 U/mL penicillin, 10,000 μg/mL streptomycin, and 25 μg/mL amphotericin B in a humidified 37°C, 5% CO₂ incubator.

siRNA Transfection

IMR-90 cells were transfected with siRNA (ON-TARGETplus SMARTpool; Dharmacon) targeting YAP and TAZ alone or in combination, or scrambled control for 72 h, using Lipofectamine RNAiMAX (Invitrogen). IPF-derived fibroblasts were transfected with siRNA (ON-TARGETplus SMARTpool, Dharmacon) targeting scrambled control, YAP, TAZ, CNN1, TAGLN, or ACTA2 and incubated for 72 h.

Microarray

IMR-90 cells were stimulated with TGFβ1 (2 ng/mL) for 72 h in the presence of scrambled, YAP, TAZ, or both YAP and TAZ siRNA (n = 3 replicates per condition). Transcript levels were evaluated using the Affymetrix GeneChip PrimeView Human Gene Expression Array. Transcripts changed by more than twofold, with a P value < 0.05, were analyzed using Ingenuity Pathway Analysis (Qiagen, RRID:SCR_008653). Raw and analyzed microarray data from this study are available through the Gene Expression Omnibus (RRID:SCR_005012) under the GEO Accession No. GSE175853.

qPCR Analysis

RNA was isolated using the RNeasy Plus Mini Kit (Qiagen) according to the manufacturer’s instructions. Isolated RNA (500 ng) was then used to synthesize cDNA using SuperScript VILO (Invitrogen). Quantitative PCR was performed using FastStart Essential DNA Green Master (Roche) and analyzed using a LightCycler 96 (Roche). Data are expressed as a fold change by ΔΔCt relative to GAPDH. Primer sequences can be found in Supplemental Table S1 (see https://doi.org/10.6084/m9.figshare.16661377).

Traction Force Microscopy

Polyacrylamide substrates were prepared [Young’s modulus of 20 kPa (16)] or purchased (25 kPa, Matrigen, EasyCoat SoftWell). Fluorescent sulfate-modified latex microspheres (0.2 μm, 505/515 excitation/emission, FluoSpheres, Life Technologies) were conjugated to the gel surfaces. IMR-90 cells or primary fibroblasts were plated on gels overnight after prior treatment for traction force measurements. Images of gel surface-conjugated fluorescent beads were acquired for each cell before and after trypsinization using a Nikon ECLIPSE Ti microscope (×10 PLAN DL, NA 0.30 objective; Olympus Ph1) or a Cytation 5 (×10 UPLFLN, NA 0.30 objective; Olympus 10X2PH) Two-dimensional tractions were calculated by measuring bead displacement fields and computing corresponding traction fields using TractionsForAll (http://www.mayo.edu/research/labs/tissue-repair-mechanobiology/software).

Western Blotting

Fibroblasts in six-well plates were lysed in radioimmunoprecipitation assay (RIPA) buffer (Thermo Scientific, 89901) supplemented with Pierce protease and phosphatase inhibitor (Thermo, 78440). From the lysates, protein concentration was determined using the Pierce BCA Protein Assay Kit. Protein samples (30 μg/sample) were run on a 4%–15% gradient sodium dodecyl-sulfate polyacrylamide gel electrophoresis (SDS-PAGE) gel. Proteins were then transferred to immunoblot polyvinylidene difluoride membranes (Bio-Rad, Hercules, CA). Membranes were blocked for 1 h in blocking buffer (Bio-Rad; 5% milk, 0.1% Tween-20 in 1× TBS). Membranes were then incubated overnight at 4°C with the following primary rabbit monoclonal antibodies: anti-calponin (1:5000, Abcam ab46794, RRID:AB_2291941), anti-transgelin (1:1000, Abcam, ab170902), anti-α-smooth muscle actin (1:1000, Novus Biologics NB300-978, RRID:AB2273628), or anti-GAPDH (1:1000, Cell Signaling, 14C10, RRID:AB_561053). The next morning, blots were then incubated with a secondary anti-rabbit IgG horseradish peroxidase (HRP)-conjugated antibody for 1 h at room temperature (1:3000, Promega, W401B, RRID:AB_430833). Changes in expression levels of the target proteins normalized to GAPDH were determined from optical densitometry of immunoblots and shown as relative intensity using the Bio-Rad Gel Doc System (Bio-Rad Laboratories, Hercules, CA, RRID:SCR008426).

ChIP-PCR

IPF fibroblasts (range = 8–14 × 10⁶ cells per condition) were treated as indicated earlier with YAP/TAZ siRNA. Chromatin immunoprecipitation (ChIP) was performed using the Pierce Magnetic ChIP Kit (26157, Thermo Scientific) as per manufacturer’s directions. Briefly, cells were crosslinked with 1% formaldehyde in culture medium that was quenched with glycine. Cells were scraped off the plate surface and then pelleted. The cells were lysed with the membrane extraction buffer. After lysing, 1 µL of MNase per 2 × 10⁶ cells was added to digest the DNA before sonicating three times for 20 s each time. For each pull-down, we used 10 µL (10%) of the digested chromatin. Immunoprecipitation was performed using antibodies for YAP (Cell Signaling, D8H1X, RRID:AB2650491), anti-rabbit IgG (Cell Signaling, 2729, RRID:AB_103106), and TEA Domain Transcription Factor 4 (TEAD4, ab58310, Abcam, RRID:AB_945789), diluted 1:50, with overnight mixing at 4°C. The next morning, Protein A/G magnetic beads were incubated for 2 h at 4°C. Immunoprecipitated complexes were then eluted to isolate the DNA.

To identify putative YAP/TAZ/TEAD occupancy locations in the proximal promoters of TAGLN, CNN1, and ACTA2, we used the Eukaryotic Promoter Database to scan for TEAD motifs within 1 kb of the transcriptional start site (17). Primers amplifying these genomic regions are listed in Supplemental Table S2 (see https://doi.org/10.6084/m9.figshare.16661413). The amount of immunoprecipitated DNA in each sample was determined as the percent input $[10\text{ × }{2}^{\left(Ct\left(10\%input\right)-log2\left(10\right)\right)- Ct \left(ChIP\right)}]$.

Controls

ON-TARGETplus Non-Targeting Pool siRNA was used as controls for siRNA experiments. Dimethyl sulfoxide (DMSO) was used to solubilize TGFβ1 and as a vehicle control for TGFβ1 experiments. For ChIP experiments, anti-rabbit IgG was used as a negative antibody control and TEAD4 served as a positive antibody control.

Statistical Analysis

For statistical analysis, results were analyzed using unpaired t tests, one-way ANOVA with Tukey’s posttest, or nested one-way ANOVA on parametric data sets. We used GraphPad Prism 9 (RRID:SCR_002798) for statistical analysis, with statistical significance defined as P < 0.05. Only statistically significant comparisons are shown.

RESULTS

To first test the individual and combined functional roles of YAP and TAZ in controlling the contractile phenotype of fibroblasts, we performed traction force microscopy (TFM). Knockdown of YAP or TAZ alone had a modest but significant effect on tractions generated by IMR-90 fibroblasts (40% and 48% reduction, respectively; Fig. 1), but combined knockdown of both was more effective (73% reduction). These findings suggested cooperative and/or redundant roles for YAP and TAZ in regulating contractile function, with TAZ potentially playing a more prominent role. To similarly assess whether YAP and TAZ are essential to TGFβ1-mediated increases in tractions, we treated scrambled control and combined YAP/TAZ siRNA-treated cells for 72 h with 2 ng/mL TGFβ1. We previously observed that tractions are increased by TGFβ1 stimulation on matrices of elastic modulus >13 kPa (18), and verified that result here (Fig. 1, 28% increase). YAP and TAZ siRNA significantly reduced tractions (58% decrease) in the presence of TGFβ1 to levels below the baseline observed in unstimulated cells. Together these results emphasize the essential role that YAP and TAZ play in regulating both the baseline contractile state of fibroblasts and the amplified contractile state observed in TGFβ1-activated fibroblasts.

Figure 1.

Combined YAP and TAZ knockdown reduces baseline and TGFβ1-induced traction forces. IMR-90 cells treated for 72 h ± 2 ng/mL TGFβ1 in the presence of 5 µM siRNA (nontargeting, YAP alone, TAZ alone, or YAP and TAZ combined) were passaged onto microsphere and collagen-coated polyacrylamide substrates with an elastic modulus of 25 kPa overnight before traction force measurements. Points are individual cell measurements from 4 independent experiments. Statistical analysis was performed using a nested one-way ANOVA with Tukey’s multiple-comparisons test. Ctrl, control; RMS, root mean square; TAZ, transcriptional coactivator with PDZ-binding motif; TGFβ1, transforming growth factor β1; YAP, yes-associated protein.

Prior studies have identified important functional roles for YAP/TAZ in TGFβ1 responses, with some studies documenting direct interactions between the canonical TGFβ1 response pathway and YAP/TAZ (7, 19). To determine how extensively YAP or TAZ contribute to TGFβ1-induced transcriptional programs, we performed a microarray analysis of transcripts expressed in IMR-90 cells under control conditions, after 72 h exposure to TGFβ1 alone, or exposure to TGFβ1 for 72 h in the presence of YAP, TAZ, or both YAP and TAZ siRNA. The microarray analysis identified over 18,000 unique genes, which we analyzed to identify differentially expressed genes (DEGs; summarized in Table 1; complete microarray in Gene Expression Omnibus GSE175853). Initially, we specifically focused on genes that were differentially expressed upon TGFβ1 stimulation compared with the control. We observed twofold (and P < 0.05) regulation of 299 unique transcripts by TGFβ1 (Fig. 2A, column 1). We then identified the subset of TGFβ1-regulated genes that were modulated through YAP, TAZ, or combined knockdown (Fig. 2A, columns 2–4). Based on prior literature demonstrating TGFβ-induced YAP/TAZ nuclear translocation (20–22), including in lung fibroblasts (23), we anticipated potentially broad influence of YAP and TAZ knockdown on transcriptional responses to TGFβ1 stimulation. Relative to TGFβ1 stimulation alone, transcripts for only five genes were significantly modulated by TAZ knockdown alone, zero by YAP knockdown alone, and 40 significantly altered by combined YAP/TAZ knockdown (Fig. 2, B and C). Once again, these results were consistent with largely cooperative or redundant roles of YAP and TAZ in controlling fibroblast transcriptional state. Notable among the cluster of YAP/TAZ regulated genes were several encoding cytoskeletal and actomyosin interacting proteins, including ACTA2, which encodes α-smooth muscle actin, and TAGLN, which encodes transgelin (also known as SM22α). These results suggested that YAP/TAZ knockdown exerts relatively selective effects on TGFβ1-dependent transcriptional programs, with a focused effect on cytoskeleton-related genes that may account for the attenuation of traction forces that we observed previously.

Table 1.

Summary of differentially expressed genes (P< 0.05, fold change = ±2) from each group

	Deg	Enhanced in Response to TGFβ1 Stimulation	Repressed in Response to TGFβ1 Stimulation	Genes Overlapping TGFβ1/Control Set
TGFβ1/control	299	167	132
TGFβ1 + TAZi/TGFβ1	21	12	9	5
TGFβ1 + YAPi/TGFβ1	4	3	1	0
TGFβ1 + YAPi + TAZi/TGFβ1	362	178	184	40

IMR-90 cells were stimulated with TGFβ1 and treated with YAP/TAZ siRNA for 72 h. TAZ, transcriptional coactivator with PDZ-binding motif; TGFβ1, transforming growth factor β1; YAP, yes-associated protein.

Figure 2.

Select TGFβ1-induced genes are modulated by YAP/TAZ combined knockdown. IMR-90 cells were incubated in the presence of TGFβ1 (2 ng/mL) and siRNA (5 µM; nontargeting, YAP alone, TAZ alone, or YAP and TAZ combined) for 72 h (n = 3 replicates per condition). A: transcript levels were evaluated using the Affymetrix GeneChip PrimeView Human Gene Expression Array. TGFβ1 significantly regulated 299 unique transcripts (P < 0.05 and fold change = >2). Each row represents a single transcript. B: TGFβ1 enhanced expression of 167 unique genes. Of those 167, YAP/TAZ knockdown significantly (P < 0.05 and fold change = >2) exaggerated transcription of 6 genes and reversed 28 genes. C: TGFβ1 repressed expression of 132 unique genes. Of those 132, YAP/TAZ knockdown significantly (P < 0.05 and fold change = >2) exaggerated 3 genes and reversed 3 genes. D: transcripts were compared with genes known to regulate cellular contraction from ingenuity pathway analysis. Of contraction-related genes, TGFβ1 significantly altered expression of 57 genes compared with the control (38 upregulated and 19 downregulated), whereas combined TGFβ1 stimulation with both YAP and TAZ knockdown altered 72 genes (35 upregulated and 37 downregulated) compared with TGFβ1 stimulation alone. E: focused heatmap of individual gene changes commonly reported to occur with both TGFβ1-induced fibroblast activation and cellular contraction. Raw and analyzed microarray data from this study are available through the Gene Expression Omnibus under the GEO Accession No. GSE175853. CTRL, control; siRNA, small-interfering RNA; TAZ, transcriptional coactivator with PDZ-binding motif; TGFβ1, transforming growth factor β1; YAP, yes-associated protein.

We then loosened our selection criteria to fully consider genes significantly regulated by YAP/TAZ knockdown, independent of whether TGFβ1 alone altered their expression, based on the small number of genes differentially expressed in both the TGFβ1 and YAP/TAZ-knockdown groups, and because we also observed that YAP/TAZ knockdown reduces baseline tractions in the absence of TGFβ1 stimulation (Fig. 1). This analysis identified 362 DEGs (Table 2), with 178 unique genes exhibiting reduced transcript levels with combined YAP and TAZ siRNA, and 184 genes exhibiting increased transcript levels. We hypothesized that the genes that broadly contribute to fibroblast contractile function are encompassed within this larger list of genes regulated by YAP and TAZ knockdown. We used ingenuity pathway analysis (IPA) to cross-reference the differentially expressed genes with the term “contraction” that encompassed 750 genes. Among these 750, 57 were significantly altered (P < 0.05, fold change = ±2) in response to TGFβ1 stimulation, whereas 72 were significantly altered in response to YAP/TAZ knockdown. However, only seven genes overlapped between the two comparison groups (Fig. 2D), again emphasizing the limited overlap between TGFβ1 and YAP/TAZ target genes. Additional functional gene groupings overrepresented within the transcripts reduced with YAP and TAZ knockdown included “cell movement” as the top functional annotation (Table 2). Notable among the genes identified in this category were multiple genes implicated in fibrosis (IGF1, CAV1, GREM1, TGM2, and THBS1) (24–28) as well as genes associated with myofibroblast activation (ACTA2 and TRPC6) (29, 30) and additional actomyosin regulator (CNN1). The statistically significant overrepresentation of cytoskeletal/contractile and matrix synthetic-related genes influenced by YAP and TAZ knockdown is consistent with a focused effect of YAP and TAZ on fibroblast transcriptional programs that contributes to wound repair and fibrosis.

Table 2.

Ingenuity pathway analysis of the top biological functions regulated by TGFβ1 or combined inhibition of YAP and TAZ

Biological Functions	Control vs. TGFβ1[−log10(P Value)]	TGFβ1 vs. YAP/TAZ[−log10(P Value)]
Cell movement	24.7815	28.14746
Migration of cells	24.90333	27.43118
Development of vasculature	27.4003	23.92727
Angiogenesis	26.38825	23.45058
Vasculogenesis	26.94366	21.95053
Growth of epithelial tissue	21.91285	16.70681
Organismal death	24.26405	11.9369
Morphology of body cavity	19.33093	15.9761
Development of body trunk	16.16735	18.12851
Abnormal morphology of body cavity	19.37901	13.11802

IMR-90 cells were stimulated with TGFβ1 and treated with YAP/TAZ siRNA for 72 h. TAZ, transcriptional coactivator with PDZ-binding motif; TGFβ1, transforming growth factor β1; YAP, yes-associated protein.

Although ACTA2 is well described as a hallmark gene of activated fibroblasts and is implicated in contractile function of the fibroblast (4, 9, 29), we sought to understand whether additional candidate genes identified in our microarray analysis contribute to the contractile function of fibroblasts. We focused on TAGLN and CNN1 as likely mediators of fibroblast contractile function and first validated the microarray results (Fig. 2D) using qPCR analysis of the same samples submitted for microarray analysis. We confirmed potent TGFβ1-dependent increases in TAGLN and observed a significant increase in CNN1 by TGFβ1, an effect that was similar in magnitude but had failed to reach significance in our microarray analysis (Fig. 3, A–C). In agreement with our microarray results, transcripts for both genes were significantly reduced by combined YAP and TAZ siRNA. To extend these observations to protein levels, we repeated these analyses using Western blotting. We confirmed robust TGFβ1-mediated increases in expression of calponin I, transgelin, and α-smooth muscle actin proteins, as well as significant reductions in all three proteins when cells were subjected to YAP and TAZ siRNA (Fig. 3, D–I), demonstrating strong agreement between transcript and protein changes (Fig. 3).

Figure 3.

Combined YAP and TAZ knockdown reduces TGFβ1-stimulated ACTA2, CNN1, and TAGLN. IMR-90 cells were stimulated with TGFβ1 and treated with YAP/TAZ siRNA for 72 h. A–C: transcript levels of ACTA2, CNN1, and TAGLN were assessed by qPCR. D–F: representative Western blots of ACTA2, CNN1, and TAGLN measured in cellular lysates. G–I: quantification of relative expression of TAGLN, CNN1, and ACTA2 measured in cellular lysates, n = 3 replicates per condition. Statistical analyses were performed using one-way ANOVA with Tukey’s posttest. DMSO, dimethyl sulfoxide; qPCR, quantitative polymerase chain reaction; TAZ, transcriptional coactivator with PDZ-binding motif; TGFβ1, transforming growth factor β1; YAP, yes-associated protein.

Fibroblasts derived from the lungs of subjects with IPF have been reported to exhibit important functional, transcriptional, and epigenetic differences compared with normal human lung fibroblasts or fibroblast model cell lines, with evidence of aberrant wound healing or constitutive profibrotic activation even in the absence of exogenous signals such as TGFβ1 (31–35). We, therefore, extended our efforts to investigate whether YAP and TAZ are critical to the regulation of ACTA2, CNN1, and TAGLN expression in a disease-relevant context by testing the ability of single or combined YAP and TAZ knockdown to reduce transcripts for each gene across a panel of independent lines of fibroblasts isolated from subjects with IPF (knockdown confirmed in Supplemental Fig. S1; see https://doi.org/10.6084/m9.figshare.16661290). Interestingly, IPF fibroblasts demonstrated mixed responses to YAP and TAZ siRNA, with ACTA2 transcripts reduced preferentially by YAP siRNA, TAGLN by TAZ siRNA, and CNN1 demonstrating little response to either individual siRNA. The contrasting effects of YAP and TAZ knockdown on ACTA2 differ from the IMR-90 cell responses in our microarray analysis but have been reported by others previously (9, 11, 19). Most importantly, the combined knockdown of YAP and TAZ did recapitulate our earlier findings, demonstrating that combined targeting of YAP and TAZ in IPF fibroblasts significantly and consistently reduced expression of ACTA2, TAGLN, and CNN1 across multiple patient-derived cells (Fig. 4, A–C). These results confirm the preserved responsiveness of IPF fibroblasts to YAP/TAZ interventions demonstrated previously (32) and highlight the key role that YAP/TAZ play in regulating cytoskeletal gene expression in disease-relevant IPF fibroblasts.

Figure 4.

In IPF fibroblasts, YAP and TAZ combined knockdown reduces transcripts for cytoskeletal genes. A: ACTA2, CNN (B), and TAGLN (C) mRNA transcript levels were significantly decreased by combined YAP and TAZ knockdown. Points are results from independent experiments. Statistical analysis was performed using a one-way ANOVA with Dunnett’s posttest with P values shown. D: YAP and TAZ combined knockdown reduced YAP binding in promoter region of ACTA2, CNN1, and TAGLN. Numbers below each gene name indicate the distance the respective TEAD-binding site is from the transcriptional start site of each gene, expressed in base pairs. Points are results from independent experiments. Statistical analyses were paired t tests for each promoter/enhancer region. Ctrl, control; IPF, idiopathic pulmonary fibrosis; TAZ, transcriptional coactivator with PDZ-binding motif; YAP, yes-associated protein.

To gain further insight into the direct regulatory role of YAP in cytoskeletal gene expression, we next inquired whether YAP interacts with the proximal promoter region of TAGLN, CNN1, and ACTA2 in IPF fibroblasts. We performed chromatin immunoprecipitation using antibodies against YAP in IPF-derived lung fibroblasts cultured in the presence of scrambled or combined YAP and TAZ siRNA. Using chromatin immunoprecipitation-quantitative polymerase chain reaction (ChIP-qPCR), we confirmed that YAP interacts with these regions of chromatin in IPF-derived fibroblasts, with reduced signal detected in the presence of combined silencing of YAP and TAZ (Fig. 4D). Interestingly, although combined YAP/TAZ knockdown resulted in decreased YAP pull-down of promoter regions, TEAD levels remained the same (Supplemental Fig. S2; https://doi.org/10.6084/m9.figshare.14712357). These ChIP-qPCR data confirm the engagement of YAP in the proximal regulatory regions of ACTA2, CNN1, and TAGLN.

Finally, having confirmed regulation of multiple cytoskeletal genes by YAP and TAZ, we sought to evaluate whether these cytoskeletal genes individually contributed to the contractile function of fibroblasts. We used siRNA to knock down expression of CNN1, TAGLN, and ACTA2, first validating siRNA conditions for effective knockdown of transcripts and protein for each (Fig. 5, A–C). We then used traction force microscopy to determine whether each siRNA was sufficient to reduce traction force generation across multiple IPF donor lines. We found consistent and modest reduction of traction forces by 32%, 31%, and 30%, for CNN1, TAGLN, and ACTA2 knockdown, respectively, all of which failed to reach statistical significance. In contrast, combined YAP and TAZ knockdown significantly decreased traction forces by 54% (Fig. 5D), exhibiting a greater effect than any of the individual cytoskeletal genes. Repeating this approach in TGFβ1-stimulated IMR-90 cells, we observed that both ACTA2 (30% decrease) and TAGLN (42% decrease), but not CNN1 knockdown, diminished contractile function (Supplemental Fig. S3; see https://doi.org/10.6084/m9.figshare.16661350) but again less effectively than in our initial combined YAP/TAZ knockdown studies (58% decrease, Fig. 1). Together, these results are consistent with cooperative roles of YAP and TAZ in mediating contractile force generation in fibroblasts via transcriptional effects on multiple cytoskeletal genes.

Figure 5.

YAP/TAZ knockdown decreases tractions in IPF-derived fibroblasts. A: qPCR validation of ACTA2, CNN1, and TAGLN knockdown. Statistical analysis was performed using one-way ANOVA with Dunnett’s posttest. B: representative Western blots of siRNA knockdown. C: siRNA knockdown confirmation for α-smooth muscle actin, calponin I, and transgelin protein levels relative to GAPDH protein levels. Statistical analysis was performed using a one-way ANOVA with Dunnett’s multiple-comparisons test. Points are results from independent experiments. D: traction force microscopy results from siRNA knockdown in IPF-derived fibroblasts showing combined YAP/TAZ knockdown reduces traction forces. Each point represents calculated root mean square (RMS) traction from an individual cell. Statistical analysis was performed using a nested one-way ANOVA with Tukey’s multiple-comparisons test. Ctrl, control; IPF, idiopathic pulmonary fibrosis; KD, knockdown; qPCR, quantitative polymerase chain reaction; siRNA, small-interfering RNA; TAZ, transcriptional coactivator with PDZ-binding motif; YAP, yes-associated protein.

DISCUSSION

YAP and TAZ have emerged as important regulators of mesenchymal cell function in development, repair, and fibrosis. Our results demonstrate that YAP and TAZ act cooperatively to control fibroblast transcriptional and functional activation in vitro. Individual knockdown of YAP or TAZ has little effect on baseline or TGFβ1-activated fibroblast contraction, and similarly, it has limited effect on TGFβ1-induced gene expression. However, combined knockdown of YAP and TAZ effectively reduced contractile function of IMR90 and IPF fibroblasts and reduced expression of a focused set of genes strongly linked to cellular contractility, including ACTA2, CNN1, and TAGLN. Our results are consistent with previous work showing that YAP and TAZ regulate genes involved in cell migration and extracellular matrix organization in IMR-90 fibroblasts (36) and similar ontologies in cancer-associated fibroblasts (37). Strikingly, we found that YAP and TAZ knockdown was more effective than individual knockdown of any of the individual cytoskeletal genes we studied. Moreover, CNN1 and TAGLN knockdown were similarly effective to ACTA2 knockdown in altering contractile function in IPF fibroblasts. Taken together, these data demonstrate that knockdown of both YAP and TAZ is needed to effectively reduce contractile gene expression and contractile force generating capacity, indicating cooperative or redundant roles of YAP and TAZ in these functions. Moreover, our results expand the list of YAP/TAZ target genes essential to IPF fibroblast contractile function to include CNN1 and TAGLN, indicating that multiple targets in the YAP/TAZ transcriptional program are important mediators of contractile function.

As paralogs, YAP and TAZ have similar structures and predicted effects. Although we show here cooperation or redundancy in controlling cellular traction and transcriptional control, other reports indicate unique roles of YAP and TAZ in other cellular functions or contexts. In the lung, TAZ is essential to normal lung morphogenesis, and loss of a single copy of Taz generates airspace enlargement but is protective against bleomycin-induced fibrosis (8). However, this study used global knockout, and TAZ is expressed in multiple cell types in the lung. Specific unique roles for YAP and TAZ in certain contexts may reflect cell- and tissue-specific preferential expression of one or the other or could relate to specific binding partners or unique upstream regulator pathways that confer differences in YAP and TAZ activation or function. For example, YAP and TAZ have some distinct transcriptional and functional roles in breast cancer epithelial cells (38), whereas YAP predominantly controls proliferation and TAZ migration in lung cancer epithelial cells (39). In corneal fibroblasts, YAP and TAZ have been shown to have both unique and redundant effects (11). In fibroblasts more broadly, overexpression of either TAZ (28, 32, 40, 41) or YAP (9, 32, 41) is sufficient to promote fibroblast activation, again largely consistent with overlapping roles. In total, our data did not identify unique functional roles for YAP and TAZ in IMR90 and primary IPF lung fibroblast contractile responses, and only very limited transcriptome-wide unique roles for YAP or TAZ, implying widespread cooperativity or functional redundancy between the two and the need for combined knockdown to elicit robust functional and transcriptional responses.

TGFβ1-induced contractile activation of fibroblasts has been well described in the literature through both canonical and noncanonical pathways (42, 43), but relatively little attention has focused on the transcriptional effects necessary to amplify contractile capacity. In agreement with the functional and transcriptional response we observed, TGFβ1 has previously been shown to increase contractile forces (44) and enhance expression of genes associated with cytoskeletal reorganization, including ACTA2, CNN1, and TAGLN (45). Interestingly, prior work in smooth muscle cells has implicated YAP/TAZ in the regulation of ACTA2, CNN1, and TAGLN (46), and studies of fibroblast activation have documented increased expression of ACTA2, CNN1, and TAGLN (47) during this transition. Human single-cell RNA-seq analyses of IPF lungs confirm increases in TAGLN, CNN1, and ACTA2 transcripts in fibroblasts and myofibroblasts, suggesting direct relevance of this program in the progression of IPF (Kaminski/Rosas data set, IPFCell atlas.com) (35). Our results expand on these findings by showing that neither knockdown of YAP nor TAZ alone has a dramatic effect on reversing expression of these TGFβ1-induced genes and fibroblast contractile function. Functionally, ACTA2 is known to regulate fibroblast contractile forces in addition to serving as a common indicator of fibroblast activation (29, 48). Both CNN1 and TAGLN are contractile proteins (49), inadequately described in myofibroblast activity, despite both being implicated as markers of TGFβ-induced myofibroblast activation (50). Calponin has been shown to increase forces generated by individual, stationary actin filaments (51). Both TAGLN and CNN1 have been described to stabilize F-actin (52, 53) by cross-linking actin bundles (54, 55), though potentially through different mechanisms (56). Our findings demonstrate for the first time, to our knowledge, that expression of TAGLN and CNN1, in addition to ACTA2, is essential to fibroblast contractile force generation in IPF fibroblasts. Importantly, we observed that knockdown of each of these cytoskeletal proteins individually reduced traction forces to a similar extent in IPF fibroblasts, but none was as effective as combined knockdown of YAP and TAZ. These results highlight YAP and TAZ as key transcriptional regulators of the contractile function of fibroblasts.

In conclusion, we have shown that YAP and TAZ combine to control a transcriptional program that is essential to the contractile function of fibroblasts. CNN1 and TAGLN emerge along with ACTA2 from our studies as YAP/TAZ transcriptional targets that are essential to IPF fibroblast contractile function. Together, these observations further our understanding of the gene programs that support fibroblast contractile function during wound repair and fibrosis. More broadly, our results highlight the importance of simultaneous targeting of YAP and TAZ in fibroblast populations to limit their contractile capacity.

SUPPLEMENTAL DATA

Supplemental Table S1: https://doi.org/10.6084/m9.figshare.16661377.

Supplemental Table S2: https://doi.org/10.6084/m9.figshare.16661413.

Supplemental Fig. S1: https://doi.org/10.6084/m9.figshare.16661290.

Supplemental Fig. S2: https://doi.org/10.6084/m9.figshare.14712357.

Supplemental Fig. S3: https://doi.org/10.6084/m9.figshare.16661350.

GRANTS

This work was supported by NIH Grant Nos. HL092961 and HL105355, Boehringer Ingelheim Discovery Award in Interstitial Lung Disease, American Lung Association Catalyst Award, and the Pulmonary Fibrosis Foundation Scholars Award.

DISCLOSURES

No conflicts of interest, financial or otherwise, are declared by the authors.

AUTHOR CONTRIBUTIONS

P.A.L., K.M.C., and D.J.T. conceived and designed research; P.A.L., K.M.C., A.M.D.E., D.L.J., A.Y.G., and A.J.H. performed experiments; P.A.L., K.M.C., A.M.D.E., D.L.J., A.Y.G., and A.J.H. analyzed data; P.A.L., K.M.C., A.M.D.E., D.L.J., A.Y.G., A.J.H., and D.J.T. interpreted results of experiments; P.A.L. and K.M.C. prepared figures; P.A.L. and K.M.C. drafted manuscript; P.A.L., K.M.C., A.M.D.E., D.L.J., A.Y.G., A.J.H., and D.J.T. edited and revised manuscript; P.A.L., K.M.C., A.M.D.E, D.L.J., A.Y.G., A.J.H., and D.J.T. approved final version of manuscript.