YAP1 Recognizes Inflammatory and Mechanical Cues to Exacerbate Benign Prostatic Hyperplasia via Promoting Cell Survival and Fibrosis

Abstract Chronic prostatic inflammation promotes cell survival and fibrosis, leading to benign prostatic hyperplasia (BPH) with aggravated urinary symptoms. It is investigated whether yes‐associated protein 1 (YAP1), an organ size controller and mechanical transductor, is implicated in inflammation‐induced BPH. The correlation between YAP1 expression and fibrosis in human and rat BPH specimens is analyzed. Furthermore, the effects of YAP1 activation on prostatic cell survival and fibrosis, as well as the underlying mechanism, are also studied. As a result, total and nuclear YAP1 expression, along with downstream genes are significantly upregulated in inflammation‐associated human and rat specimens. There is a significant positive correlation between YAP1 expression and the severity of fibrosis or clinical performance. YAP1 silencing suppresses cell survival by decreasing cell proliferation and increasing apoptosis, and alleviates fibrosis by reversing epithelial‐mesenchymal transition (EMT) and extracellular matrix (ECM) deposition in prostatic BPH‐1 and WPMY‐1 cells. Mechanistically, inflammatory stimulus and rigid matrix stiffness synergistically activate the RhoA/ROCK1 pathway to provoke cytoskeleton remodeling, thereby promoting YAP1 activation to exacerbate BPH development. Overall, inflammation‐triggered mechanical stiffness reinforcement activates the RhoA/ROCK1/F‐actin/YAP1 axis, thereby promoting prostatic cell survival and fibrosis to accelerate BPH progression.


Figure S1 .
Figure S1.LPS reduces the inactivating phosphorylation of YAP1 at Ser-127 and promoted YAP1 nuclear localization.A, B) Immunoblotting analysis to investigate the impact of LPS on the expression of total-YAP1 (t-YAP1), phospho-YAP1-Ser127 (p-YAP1), CTGF in the whole cell lysate, as well as the expression of nuclear-YAP1 (n-YAP1) in the nuclear lysate.Data were presented as mean ± SD of at least 5 independent experiments.One-way ANOVA followed by Dunnett's post-hoc test was used for A, B) to measure statistical significance in comparison to control group.*p < 0.05, ** p < 0.01, *** p < 0.001.

Figure S2 .
Figure S2.LPS promotes prostatic cell proliferation in a concentration-dependent manner.EdU assay was applied to identify EdU-positive cells.The proliferating cell ratio was calculated through dividing the number of EdU-positive cells by the number of Hoechst-positive total cells.Data were presented as mean ± SD of at least 3 independent experiments.One-way ANOVA followed by Dunnett's post-hoc test was used to measure statistical significance in comparison to control group.** p < 0.01, *** p < 0.001.

Figure S3 .
Figure S3.LPS dose-dependently promotes apoptosis resistance in prostatic cells.A, B) Immunoblotting analysis revealed that LPS decreased the expression of the proapoptotic protein Bax but increased the expression of the anti-apoptotic protein Bcl-2, leading to a reduced Bax/Bcl-2 ratio in both BPH-1 and WPMY-1 cells.Data were presented as mean ± SD of at least 5 independent experiments.One-way ANOVA followed by Dunnett's post-hoc test was used to measure statistical significance in comparison to control group.** p < 0.01, *** p < 0.001.

Figure S4 .
Figure S4.YAP1 silencing participates in the occurrence of apoptosis in prostatic cells.A, B) Immunoblotting analysis to explore the influence of YAP1 knockdown on the expression of apoptosis-regulated proteins Bax and Bcl-2 in the presence of LPS stimulation.Data were presented as mean ± SD of at least 5 independent experiments.Two-tailed Student's t-test was used for A, B). *p < 0.05, ** p < 0.01, *** p < 0.001.

Figure S5 .
Figure S5.LPS dose-dependently promotes EMT switch of BPH-1 cell and ECM production of WPMY-1 cell.A) Immunoblotting analysis revealed that application of LPS triggered EMT process manifested as decreased E-cadherin expression but increased Vimentin expression.B) Immunoblotting analysis revealed that application of LPS accelerated ECM production characterized by enhanced α-SMA and Collagen I expression.Data were presented as mean ± SD of at least 5 independent experiments.One-way ANOVA followed by Dunnett's post-hoc test was used to measure statistical significance in comparison to control group.* p < 0.05, ** p < 0.01, *** p < 0.001.

Figure S6 .
Figure S6.Positive correlation between YAP1 and RhoA or ROCK1 expression in the normal prostate samples.A, B) Correlation analysis of YAP1 and RhoA in the normal prostate samples from TCGA database and GTEx database.C, D) Correlation analysis of YAP1 and ROCK1 in the normal prostate samples from TCGA database and GTEx database.Spearmen correlation analysis was applied to A-D).

Figure S7 .
Figure S7.Involvement of ROCK1/YAP1 pathway in regulating cell apoptosis process.A, B) Immunoblotting analysis was performed to investigate the occurrence of apoptosis following treatment with the ROCK1 inhibitor (Y27632), YAP1overexpression vector (YAP1), and control vector (vector) in BPH-1 and WPMY-1 cells.Data were presented as mean ± SD of at least 5 independent experiments.Twotailed Student's t-test was used for A, B). *p < 0.05, ** p < 0.01.

Figure S8 .
Figure S8.Blockade of YAP1-TEAD1 interaction triggers cell apoptosis in prostatic cells.A, B) Immunoblotting analysis to examine the curative effect of YAP1-TEAD1 binding inhibitor VP on apoptosis promotion.Data were presented as mean ± SD of at least 4 independent experiments.Two-tailed Student's t-test was used for A, B). *p < 0.05, ** p < 0.01, *** p < 0.001.

Table S1 .
Evaluating histopathological feature of rat prostate using histoscore protocol.

Table S2 .
The information of primary antibodies used in this study.

Table S3 .
The sequences of RT-PCR primers used in this study.