USP13 regulates cell senescence through mediating MDM2 stability

Abstract

Aims: Lung aging results in altered lung function, reduced lung remodeling and regenerative capacity, and increased susceptibility to acute and chronic lung diseases. The molecular and physiological underlying mechanisms of lung aging remain unclear. Mounting evidence suggests that deubiquitinating enzymes (DUBs) play a critical role in tissue aging and diseases through regulation of cellular signaling pathways. Here we investigate the role of Ubiquitin-Specific Protease 13 (USP13) in cell senescence and lung aging and its underlying mechanisms.

Main methods: Protein levels of USP13 and MDM2 in lung tissues from aged and young mice were compared. Gene silencing and overexpression of USP13 in human cell lines were performed. MDM2 levels were examined by Quantitative Real-Time PCR and Western blotting analysis. The cell senescence levels of human cells were checked by the β-galactosidase staining.

Key findings: Lung tissues from aged mice showed higher levels of USP13 compared to younger mice. We found a negative correlation between USP13 and MDM2 expression in lung tissues of aged mice. The increased protein levels of MDM2 were detected in lung tissues of USP13 deficient mice. Furthermore, overexpression of USP13 promoted cell senescence. Knockdown of USP13 increased MDM2 levels in lung cells, while overexpression of USP13 reduced it. The degradation of MDM2 caused by USP13 was prevented by the proteasome inhibitor MG132. Furthermore, we showed that USP13 targeted and reduced K63-linked polyubiquitination of MDM2. These results demonstrate that USP13 is involved in the aging signaling pathway in lungs through regulation of MDM2.

Keywords: Cell senescence; Deubiquitination; Lung aging; MDM2; USP13.

Fig. 1.

USP13 protein levels are associated with lung aging and MDM2 protein expression. Protein levels of USP13, MDM2, and β-actin in both 8 weeks (n = 5) and 90 weeks (n = 5) wild type (WT) mouse lung tissue lysates containing a constant amount of proteins (20 μg) were determined by Western blotting with specific antibodies (A). Lung lysates from 12-week-old wild type (WT) mice (n = 4) and USP13 knockout (KO) mice (n = 4, 12-week-old) containing total 20 μg proteins were subjected to Western blotting and detected by anti-USP13, MDM2, and β-actin specific antibodies (C). Blotting of β-actin served as loading control. The Dot plots shown in (B) and (D) are quantifications of (A) and (C) respectively. Protein levels were normalized to β-actin. Data are present as the means ± SD,** p < 0.01, * p < 0.05. (E) and (F): The relative levels of IL-6 in bronchoalveolar lavage solution (BAL) of mice (n = 5/each group) were determined by the enzyme-linked immunosorbent (ELISA) assay. Data are present as the means ± SD,* p < 0.05. Two-way parametric test and Mann Whitney test (for n < 5) were used.

Fig. 2.

USP13 overexpression increases cell senescence. (A). HVSMC cells were transfected with Flag-tagged USP13 plasmid, and the positive control group cells were treated by Cobalt chloride (CoCl₂). The cells were immunostained with anti-Flag antibody followed by Alexa-546 conjugated anti-rabbit antibody. Senescence staining was performed using β-Galactosidase Staining Kit. The cell nucleus was stained with DAPI (blue). Red stands for Flag-USP13 and green stands for senescent cells. (B). The Dot plots shown here is quantifications of (A). Data are present as the means ±SEM,* p < 0.05. (C). The HVSMC cells were transfected with the V5-tagged USP13 plasmid. The cells were harvested, and protein levels of cell extract were detected by Western blotting with anti-V5, p27, p21, IL-6, and β-actin specific antibodies. (D). The Dot plots shown here is quantifications of (C). Red (▴) represents p21, blue (■) represents IL-6, and black (●) represents p27. The protein levels were normalized to β-actin. Data are present as the means ±SEM,* p < 0.05. Two-way parametric tests were used.

Fig. 3.

Overexpression of USP13 induces MDM2 degradation. The HVSMC cells were transfected with V5-tagged USP13 plasmid and the cells were collected (A). The A549 cells were co-transfected with the plasmids for expressing MDM2 (HA-MDM2, 1 μg) and USP13 (USP13-V5, 0.5, 2, and 4 μg) (C). The protein levels of cell extract were detected by Western blotting with anti-V5, MDM2, and β-actin specific antibodies. Western blot shown here is representative of 3 independent experiments. The blot of β-actin served as loading control. The Dot plots shown in (B) and (D) are quantifications of (A) and (C) respectively. The protein levels were normalized to β-actin. Data are present as the means ±SEM,** p < 0.01, * p < 0.05. (E). The HVSMC cells were transfected with V5 tagged USP13 plasmid (or control vector plasmid) and the cells were harvested after cycloheximide (CHX) treatment at indicated times. The protein levels of cell lysates were detected by Western blotting with anti-V5, MDM2, and β-actin specific antibodies. The shown in (F) and (G) are quantifications of (E). Data are present as the means ± SD,** p < 0.01. Two-way parametric tests were used. (H). The HVSMC cells were transfected with siRNA of USP13 (or control siRNA) and the cells were collected. The protein levels of cell extracts were identified by Western blotting with anti-USP13, p-MDM2, MDM2, and β-actin specific antibodies.

Fig. 4.

USP13 is associated with MDM2 in the nuclei. (A), (B). The cell lysates of A549 were immunoprecipitated (IP) with anti-MDM2 (or anti-USP13) antibody, and examined by Western blotting with anti-USP13 (or anti-MDM2) antibody. (C, D). The A549 cells were co-transfected with plasmids expressing HA-MDM2 or USP13-V5 as indicated. The cell lysates were immunoprecipitated with anti-V5 (or anti-HA) antibody, and the Co-immunoprecipitation (Co-IP) was examined by Western Blot with both anti-HA and anti-V5 antibodies. (E). Co-localization of USP13 and MDM2 was identified by Immunofluorescence Microscopy analysis. HVSMC cells were transfected with USP13 SiRNA (or control SiRNA) and the cells were immunostained with anti-USP13 and anti-MDM2 antibodies followed by Alexa-546 conjugated (red, USP13) and Alexa-488 conjugated (green, MDM2) antibodies. Scale bar is 10 μm.

Fig. 5.

USP13 reverses K63-linked polyubiquitination of MDM2. (A). The HVSMC cells were transfected with USP13-V5 plasmid (or control vector plasmid), and then cells were treated with Caspase 2 inhibitor (CSPS2-Inhi), ZVAD, or MG132. The cell lysates were subjected to use for Western blotting analysis with anti-V5, MDM2, USP13, and β-actin specific antibodies. (B). The Dot plots shown here is quantifications of (A). Red represents cells were treated with MG132, Green represents cells were treated with ZVAD, and blue represents cells were treated with CSPS2-Inhi. The levels of proteins were normalized by β-actin. Data are present as the means ± SD,** p < 0.01, *** p < 0.001. Two-way parametric tests were used. (C). HVSMC cells were transfected with siUSP13 (or control siRNA), and after 72 h incubation the cells were harvested in RIPA buffer and IP was performed with anti-MDM2 antibodies. The ubiquitination of MDM2 were detected by anti-ubiquitin and anti-MDM2 antibodies. Western blotting analysis of cell lysates with anti-USP13 antibody was performed to confirm the expression levels of USP13. Western blotting of anti-β-actin was performed as a loading control. (D). In vitro deubiquitination assay was performed using the purified human recombinant protein USP13 (rhUSP13) and the MDM2 autoubiquitination kit. The reaction mixture solution containing E3 active center of MDM2 (MDM2_E3), magnesium ions (Mg) and rhUSP13 was incubated at 37 °C for 1 h. Regarding the positive control, the reaction mixture did not contain magnesium ions (Mg), and as a negative control, BSA was used instead of rhUSP13. After the incubation, the reaction mixtures were analyzed by Western blotting with anti-ubiquitin and anti-MDM2 antibodies. (E). The HVSMC cells were transfected with siUSP13 (or control siRNA), and 72 h later the cells were harvested and IP was performed with anti-MDM2 antibody. The Western blotting analysis was performed with anti-K63 ubiquitin antibody. The normal IgG (CtrIgG) was serving as a control for IP.

Fig. 6.

Overexpression of MDM2 attenuates the effect of USP13 on cell senescence. (A). HVSMC cells were co-transfected with Flag tagged USP13 and HA-tagged MDM2 plasmid. The control group cells were transfected with Flag tagged USP13 plasmid only. The cells were then stained with β-Galactosidase Staining Kit and the cell nucleus was stained with DAPI (blue). The green stands for senescent cells. (B). The HVSMC cells were transfected with siMDM2 (or control siRNA), and 48 h later the cells were then stained with β-Galactosidase Staining Kit and cell nucleus was stained with DAPI (blue). The green indicates for senescent cells. (C). HVSMC cells were transfected with siMDM2 (or control siRNA), and 72 h later the cells were harvested and Western Blotting was performed with anti-MDM2, anti-USP13, anti-p21 and anti-β-actin antibodies. The β-actin was serving as a loading control. (D). The Dot plots shown here is quantifications of (C). The protein levels of p21 were normalized to β-actin. Data are present as the means ± SD, * p < 0.05. Two-way parametric tests were used.