Tschumperlin, Daniel J.

Abstract Idiopathic pulmonary fibrosis (IPF) and rheumatoid arthritis‐associated interstitial lung disease (RA‐ILD) are two fibrotic interstitial lung diseases that share the usual interstitial pneumonia (UIP) injury pattern. Here, we report that RNA sequencing of lung biopsies from patients with RA‐ILD and IPF revealed shared and distinct disease‐causing pathways. Analysis of transcriptomic data identified a JAK2 related JAK/STAT signaling pathway gene signature that distinguishes RA‐UIP from idiopathic UIP. This was further confirmed by immunohistostaining, which identified JAK2 phosphorylation with two distinct forms of activation: a cytoplasmic form of JAK2 activation in most IPF cases (13/20) and a nuclear form of p‐JAK2 in RA‐UIP (5/5) and a minority of IPF (6/20) cases. Further immunohistostaining identified STAT5A&B as the downstream transcriptional activator for JAK2‐mediated canonical signal transduction and phosphorylation of Tyr41 on histone H3 (H3Y41ph) as the downstream epigenetic regulation site for JAK2‐mediated noncanonical signal transduction. Gene Set Enrichment Analysis (GSEA) of the RNA‐Seq data further supported this shared pathogenic mechanism for the two diseases with the enrichment of STAT5A&B target gene sets as well as the JAK2 regulated H3Y41ph target gene set. This regulatory role of JAK2 in the pathogenesis of pulmonary fibrosis was further demonstrated by the attenuation of bleomycin‐induced murine pulmonary fibrosis using a JAK2‐selective pharmacological inhibitor CEP33779. In vitro studies with normal and IPF derived lung fibroblasts revealed a central role for JAK2 as an essential intermediary molecule in TGF‐β‐mediated myofibroblast trans‐differentiation, proliferation, and extracellular matrix protein production. These observations support a crucial role for JAK2 as an intermediary molecule in fibrotic lung disease development.

Idiopathic pulmonary fibrosis (IPF) is an aging-associated disease characterized by myofibroblast accumulation and progressive lung scarring. To identify transcriptional gene programs driving persistent lung fibrosis in aging, we performed RNA-Seq on lung fibroblasts isolated from young and aged mice during the early resolution phase after bleomycin injury. We discovered that, relative to injured young fibroblasts, injured aged fibroblasts exhibited a profibrotic state characterized by elevated expression of genes implicated in inflammation, matrix remodeling, and cell survival. We identified the proviral integration site for Moloney murine leukemia virus 1 (PIM1) and its target nuclear factor of activated T cells-1 (NFATc1) as putative drivers of the sustained profibrotic gene signatures in injured aged fibroblasts. PIM1 and NFATc1 transcripts were enriched in a pathogenic fibroblast population recently discovered in IPF lungs, and their protein expression was abundant in fibroblastic foci. Overexpression of PIM1 in normal human lung fibroblasts potentiated their fibrogenic activation, and this effect was attenuated by NFATc1 inhibition. Pharmacological inhibition of PIM1 attenuated IPF fibroblast activation and sensitized them to apoptotic stimuli. Interruption of PIM1 signaling in IPF lung explants ex vivo inhibited prosurvival gene expression and collagen secretion, suggesting that targeting this pathway may represent a therapeutic strategy to block IPF progression.

The fibrogenic wound-healing response in liver increases stiffness. Here, macro and microheterogeneity of liver stiffness correlate with HSC heterogeneity in a hepatic fibrosis mouse model. Fibrogenic HSCs localized in stiff collagen-high areas upregulate the expression of focal adhesion molecule FHL2, which, in turn, promotes extracellular matrix protein expression. These results demonstrate that stiffness heterogeneity at the whole organ, lobular, and cellular level drives an amplification loop of fibrogenesis through specific focal adhesion molecular pathways.

Idiopathic pulmonary fibrosis (IPF) is a lethal lung disease targeting the alveolar gas exchange apparatus, leading to death by asphyxiation. IPF progresses on a tissue scale through aberrant matrix remodeling, enhanced cell contraction, and subsequent microenvironment densification. Although two pharmaceuticals modestly slow progression, IPF patient survival averages less than 5 years. A major impediment to therapeutic development is the lack of high-fidelity models that account for the fibrotic microenvironment. Our goal is to create a three-dimensional (3D) platform to enable lung fibrosis studies and recapitulate IPF tissue features. We demonstrate that normal lung fibroblasts encapsulated in collagen microspheres can be pushed toward an activated phenotype, treated with FDA-approved therapies, and their fibrotic function quantified using imaging assays (extracellular matrix deposition, contractile protein expression, and microenvironment compaction). Highlighting the system's utility, we further show that fibroblasts isolated from IPF patient lungs maintain fibrotic phenotypes and manifest reduced fibrotic function when treated with epigenetic modifiers. Our system enables enhanced screening due to improved predictability and fidelity compared to 2D systems combined with superior tractability and throughput compared to 3D systems.

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.

Epithelial-mesenchymal transition (EMT) creates an environment facilitating fibrosis following alveolar epithelial cell injury. IL-23 has important roles in chronic autoimmune conditions like rheumatoid arthritis (RA), but its role in the interstitial lung disease that affects patients with RA is unclear. This study aimed to determine the profibrogenic role of IL-23 on somatic alveolar type I (ATI) epithelial cells. Primary ATI cells were isolated from rats and cultured on plastic dishes for 1–3 wk. After prolonged culture (≥14 days) on rigid culture dishes, primary ATI cells gradually acquired a mesenchymal phenotype, identified by decreased expression of caveolin-1, and reorganization of F-actin cytoskeleton, indicating the initiation of EMT by matrix stiffness. To determine how IL-23 promotes EMT in vitro, transitioning ATI cells, cultured on a stiff substrate for ≥14 days were stimulated with IL-23. The EMT phenotype was significantly enhanced by IL-23, which upregulated α-smooth muscle actin (α-SMA), collagen I/III protein, and decreased caveolin-1. Furthermore, IL-23 significantly promoted cell invasion, as well as apoptotic resistance on transitioning ATI cells. Mechanistically, IL-23-induced EMT was mammalian target of rapamycin/ribosomal protein S6 (mTOR/S6) signaling dependent and reversible by rapamycin. Transcriptional sequencing analysis of human lung fibrosis biopsy tissue revealed key roles for IL-23 in rheumatoid arthritis-associated interstitial lung disease (RA-ILD). This result was further validated by significantly upregulated IL-23 expression at the mRNA level in RA-ILD lung sections. Notably, transitioning ATI epithelial cells were abundantly detected in RA-ILD tissue. Taken together, these data support a role for IL-23 in the pathogenesis of RA lung fibrosis by promoting EMT in alveolar epithelial cells through mTOR/S6 signaling.

Aging is accompanied by declining lung function and increasing susceptibility to lung diseases. The role of endothelial dysfunction and vascular remodeling in these changes is supported by growing evidence, but underlying mechanisms remain elusive. In this review we summarize functional, structural, and molecular changes in the aging pulmonary vasculature and explore how interacting aging and mechanobiological cues may drive progressive vascular remodeling in the lungs.

Background Uterine leiomyomas, also known as fibroids (UF), are benign neoplasms of the uterus. UF consist of smooth muscle cells and fibroblasts, and display enriched extracellular matrix (ECM). Reports estimate a cumulative incidence of 70% in premenopausal women, with annual total health care costs attributed to UF exceeding 34 billion dollars in the United States alone. In spite of their prevalence, the underlying processes that drive UF development and growth are still poorly understood. Accumulating evidence implicates the canonical Hippo signaling pathway effectors Yes‐associated protein (YAP)/transcriptional coactivator with PDZ‐binding domain (TAZ) as key drivers of fibroblast biology. Thus, we seek to assess the differences in YAP/TAZ localization, ECM deposition, and gene expression between UF and normal myometrium (Myo). Methods Matched samples of UF and Myo were obtained from premenopausal women with IRB approval. Immunohistochemistry (N=5) was performed on OCT embedded tissue with YAP/TAZ antibody. For cell culture, UF and Myo were minced and digested with collagenase type 4. Cells (passages 1–4) were plated on plastic at confluence (N=4 for both). Immunostaining for YAP/TAZ, Fibronectin (FN), Collagen Type I (COL1), and Collagen Type III (COL3) were performed (N=4–5). Transcript levels were analyzed by treating cells with control or YAP/TAZ siRNA, and performing subsequent cDNA synthesis and qPCR reactions (N=4). Kruskall‐Wallis and Mann Whitney tests were used as appropriate. Results YAP/TAZ nuclear localization was upregulated in UF when compared to Myo tissue by immunofluorescence imaging. In freshly isolated cells cultured at confluence on tissue culture plastic, baseline YAP/TAZ nuclear localization was increased in UF when compared to Myo (p=0.03). UFs also exhibited a trend toward increased FN, COL1, and COL3 deposition when compared to Myo. UF displayed increased transcript levels for several matrix and pro‐fibrotic signaling genes (COL1A1, COL3A1, CTGF, EDN1, FN1) compared to the matched Myo. Following siRNA treatment for YAP/TAZ gene expression was reduced 40% and 50% for EDN1 (encoding endothelin‐1), as well as 70% and 65% for CTGF (encoding connective tissue growth factor), in UF and Myo respectively. Conclusions UF nuclear YAP/TAZ is increased at baseline in vivo as well as in vitro . UF also display upregulated deposition of the ECM proteins. Additionally, there are increased transcript levels of profibrotic genes in UF compared to Myo. Moreover, the observed decreases in gene expression following siRNA knockdown of YAP/TAZ suggests a role for the Hippo signaling pathway in regulation of certain profibrotic genes in UF. Together these results suggest that enhanced baseline YAP/TAZ localization in UF may drive upregulated expression of pro‐fibrotic genes promoting UF progression. Support or Funding Information Center for Biomedical Discovery‐Department of Obstetrics and Gynecology Team Science Pilot Award Program This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal .

The transcriptional co‐activator TAZ plays a critical role in lung alveolarization (Mitani et al. Am J Respir Crit Care Med. 2009). We recently observed enhanced TAZ expression in the nuclei of fibroblasts present in lung tissue from patients with idiopathic pulmonary fibrosis, suggesting that TAZ is reactivated during fibrosis. To test the role of TAZ in driving a fibroproliferative response, we developed scaffold‐free cultures in which fibroblasts were grown in 3D hanging drop spheroids. This spheroid model removes the influence of the artificial 2D cell culture environment on cell growth. NIH3T3 fibroblasts were stably transduced with doxycycline (dox)‐inducible constructs expressing nuclear‐localizing mutant TAZ4SA or empty vector (courtesy of X. Varelas, Boston University). Spheroids were initiated by seeding 500 cells in hanging drops, and were subsequently fed growth media supplemented with 0, 1, 10, or 100 ng/mL dox every other day up to 15 days. Spheroid growth was tracked by measuring 2D projected area under light microscopy. Control NIH3T3 fibroblast spheroids remained stable in culture, but neither increased nor decreased in size significantly. In contrast, TAZ4SA‐transduced spheroids grew over time, and by day 15 spheroids treated with 100 ng/ml dox had a 2D projected area ~2.2‐fold larger than those grown in the absence of dox (p=0.0054). Intermediate growth levels were observed in 1 and 10 ng/ml doxycycline treated spheroids. Our results demonstrate the utility of the spheroid culture model for studying fibroproliferation, and document a prominent role for TAZ in promoting fibroblast spheroid growth. *Supported by NIH grants RO1 HL092961 and NIH R21 HL113796.