Uterine fibroid cell cytoskeletal organization is affected by altered GPER1/PI3K signaling

Introduction:: Uterine fibroids are the most common reason for hysterectomy in the United States, with 30% of women seeking treatment in their lifetime. These benign tumors reside in the myometrium, or muscular layer of the uterus where they can cause dysmenorrhea, menorrhagia, and infertility. The tumors are exposed to cyclic forces due to uterine contractions, especially during menstruation when contractions are similar to those during childbirth. We hypothesize that these forces cause an increase in stress on the cells on the fibroid’s outer layer, resulting in fibroid growth due to increased ECM production.

Materials and Methods:: Patient-matched myometrial and fibroid cells from 3 patients were used in all studies. These cells were isolated from women undergoing either myomectomy or hysterectomy for the treatment of uterine fibroids after the subjects provided informed consent. Cells were exposed to cyclic strain mimicking the periovulatory phases (1%, 0.5 Hz), menses (5%, 0.1 Hz), or dysmenorrhea conditions (12%, 0.1 Hz). Static controls plated on the same uniaxial strain plates were used for comparison. Under menses mimetic conditions, cells were cultured with inhibitors to GPER1, PI3K, and ERα or vehicle control. Cell alignment was determined by image analysis using CurveAlign. Collagen content and cell number were determined by image analysis using FIJI. A two-way or three-way ANOVA with post-hoc Student’s t-test was performed depending on the number of dependent variables. A 95% confidence interval was used for assessing statistical significance.

Results, Conclusions, and Discussions:: Results and Discussion: Fibroid cells aligned more quickly to cyclic strain than myometrial cells, and the menses mimetic conditions led to the quickest alignment (p< 0.0001). Collagen production was increased in this condition with a 90% confidence interval (p=0.09). In the presence of GPER1 and PI3K inhibitors, cell alignment was significantly altered in fibroid, but not myometrial cells (p=0.046). Cells exposed to PI3K inhibitors visually appeared similar to static cultures. Inhibition of ERα led to a reduction of collagen (p=0.03); the cell proliferation was significantly higher in static cultures than in strained cultures that were exposed to ERα inhibitors (p< 0.0001).

Conclusions: Fibroid cells have dysregulated mechanotransduction signaling affecting the cytoskeletal organization when exposed to cyclic strain. This is regulated, at least in part, by the GPER1/PI3K pathway. Both the strain amplitude and frequency appear to affect cell alignment and should be further studied. Changes in collagen content were not highly significant, but given that cells do not produce much extracellular matrix in monolayer cultures, the results are interesting and should be considered in three-dimensional cultures.


Acknowledgements (Optional): : Thank you to Riccardo Barrile, PhD. for microscopy help and Paul J. Lee, M.D., Ph.D. for assistance with getting tissues through pathology.

References (Optional): :