<p> Uterine fibroids (UFs) are benign myometrial neoplasms that produce clinically-relevant symptoms in > 25% of all women and represent the leading cause of hysterectomy. Despite their importance, the mechanisms that promote UF growth are still not well-understood. Accumulating evidence indicates that the mechanical environment is altered and this may be a driver of fibroid pathobiology. There has been a proven regulatory mechanism for cellular proliferation with the Hippo pathway and the Yes-associated protein (YAP)/transcriptional coactivator with PDZ-binding domain (TAZ) in lung fibrosis which shares a response to gonadal steroids and serves as a model for this process. Here we assess the differences in YAP/TAZ nuclear localization and responsiveness to mechanical and hormonal cues in primary cells from UF compared to myometrium (Myo). Matched samples of UF and Myo were obtained from premenopausal women with IRB approval. Atomic force microscopy (AFM) was used to determine in-situ stiffness (N = 7). 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 sparsely on hydrogels of five defined stiffness or at confluence on tissue culture plastic (N = 4 for both). Immunostaining for YAP/TAZ, Fibronectin (FN), Collagen I (COLI), and Collagen III (COLIII) were performed (N = 4–5). To perform siRNA, cells were incubated with control or YAP1 (YAP)/WWTR1 (TAZ) siRNA for 72 hours prior to analysis. RNA expression for Endothelin 1 (ET-1) and Connective Tissue Growth Factor (CTGF) was determined. Paired t-test and Wilcoxon sign-rank test were used as appropriate. AFM-measured stiffness was significantly higher in UF compared to Myo (p = 0.03). 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). When seeded sparsely, decreasing substrate stiffness significantly (p = 0.05) reduced YAP/TAZ nuclear localization for both Myo and UF. Stimulation of confluent cells with 10nM estradiol and 100nM progesterone increased YAP/TAZ nuclear localization, but only in Myo (p = 0.01) as UF cells were constitutively active under these conditions. UFs also exhibited a trend toward increased FN, COLI, and COLIII deposition when compared to Myo. Following siRNA using YAP1 and WWTR1, ET-1 and CTGF were found to be statistically decreased which are known regulators of COLI and FN. These results demonstrate a possible feedback mechanism by which UFs progress by upregulation of matrix deposition, increase in tissue stiffness, and an increase in YAP/TAZ nuclear localization. </p><p>
| Identifer | oai:union.ndltd.org:PROQUEST/oai:pqdtoai.proquest.com:13806565 |
| Date | 06 April 2019 |
| Creators | Purdy, MacKenzie Phyllice |
| Publisher | College of Medicine - Mayo Clinic |
| Source Sets | ProQuest.com |
| Language | English |
| Detected Language | English |
| Type | thesis |
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