Ducharme, Merrick T.

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 .

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.