Mitochondrion-associated protein peroxiredoxin 3 promotes benign prostatic hyperplasia through autophagy suppression and pyroptosis activation

Abstract

Benign prostatic hyperplasia (BPH) is one of the most common diseases in the senior men and age plays an important role in the initiation and development of BPH. Mammalian cells primarily use the autophagy-lysosome system to degrade misfolded/aggregated proteins and dysfunctional organelles such as mitochondria and suppress pyroptosis, a type of cell death that stimulates inflammatory responses and growth of other cells around. Peroxiredoxin 3 (PRDX3) is the only mitochondrion-associated member of peroxiredoxin family enzymes that exert their protective antioxidant role in cells through their peroxidase activity. We hypothesized that PRDX3 may inhibit autophagy to activate pyroptosis to induce growth of prostatic epithelial cells. Here we show that PRDX3 maintained the integrity of mitochondria and its depletion led to an enhancement of oxidative stresses. PRDX3-associated and PRDX3-free mitochondria co-existed in the same cells. PRDX3 expressed at higher levels in prostatic epithelial cells in prostate tissues from BPH patients and BPH-representative cell line than in prostate tissues from healthy donors and a cell line representing normal epithelial cells. PRDX3 suppressed autophagy flux and activated pyroptosis to induce inflammatory responses and stimulate the over-growth of prostate tissues. Therefore, higher levels of PDRX3 in prostatic epithelial cells may promote the initiation and development of BPH through autophagy inhibition and pyroptosis activation.

Keywords: BPH; LC3; autophagy; benign prostatic hyperplasia; caspase 1.

Figure 1. The expression of PRDX3 protein in human prostatic tissues

(A) Representative images showing the immunostaining patterns of PRDX3 in prostatic tissues from healthy donors or BPH patients. The insets in black boxes are enlarged images. Bar = 100 μm. (B, C) Comparison of the levels of PRDX3 in total prostate tissues (B) or epithelial cells (C) from normal healthy donors and BPH patients. Bars represent the relative score of PRDX3 intensity (Intens), frequency (Freq) and level. **P ≤ 0.01; and ****P ≤ 0.0001.

Figure 2. PRDX3 expression and its association with autophagy flux in cultured prostate cells

(A) Representative immunoblot showing the levels of PRDX3, TOM20 and LC3-II in in lysates prepared from three different cultures of BPH-1 and RWPE-1 cells in the absence (Ctrl) or presence of bafilomycin A1 (BAF). (B–D) The quantification of the relative levels of PRDX3 (B), TOM20 (C) and LC3-II (D) to β-Actin as shown in (A). Data are mean and standard deviation of three repeats and differences are tested with Student's T-test. *P ≤ 0.05; **P ≤ 0.01.

Figure 3. Mitochondrial association and impacts on oxidative stress of PRDX3

(A) Representative images showing the colocalization of PRDX3 (green) with TOM20 (red) in BPH-1 cells. Bar = 10 μm. (B) A image showing a part of the merge showing in (A). (C) A representative immunoblot showing the levels of PRDX3 in BPH-1 cells treated with random (MOCK) or PRDX3-specific siRNA (PRDX3). (D, E) Representative images (D) and quantification (E) oxidative stress as indicated by the intensities of red fluorescence after staining with dihydroethidine hydrochloride. Bar = 200 μm. Data are mean and standard deviation of three repeats and differences are tested with Student's T-test. ***P ≤ 0.001.

Figure 4. Impacts of PRDX3 protein on autophagy flux

(A–D) Representative immunoblot (A, C) and quantification (B, D) showing the levels of LC3-II in BPH-1 cells treated with random (MOCK) or PRDX3-specific siRNA (PRDX3) (A, B) or RWPE-1 cells transiently expressing different amount of PRDX3 (C, D) in the absence (Ctrl) or presence of bafilomycin A1 (BAF). Data are mean and standard deviation of three repeats and differences are tested with Student's T-test. *P ≤ 0.05; **P ≤ 0.01; ***P ≤ 0.001. (E–G) Representative immunoblot (E) and quantification (F, G) showing the levels of Beclin 1 (F) and PI3KCIII (G) in BPH-1 cells treated with random (MOCK) or PRDX3-specific siRNA (PRDX3). Ns, not significant; *P ≤ 0.05.

Figure 5. Impacts of PRDX3 on pyroptosis

(A–C) Representative immunoblot (A) and quantification (B–C) showing the levels of caspase 1 (P45) (B) and caspase 1 (P20) (C) in BPH-1 cells treated with random (−) or PRDX3-specific siRNA (+). (D) Plots of lactate dehydrogenase (LDH) activity released in medium from cultured BPH-1 cells treated with random (−) or PRDX3-specific siRNA (+). Data are mean and standard deviation of three repeats and differences are tested with Student's T-test. *P ≤ 0.05; **P ≤ 0.01.