TRIM69 inhibits cataractogenesis by negatively regulating p53

Abstract

Ultraviolet B (UVB) irradiation can induce reactive oxygen species (ROS) production and apoptosis in human lens epithelial cells (HLECs), thus leading to the formation of cataracts. We studied the role of tripartite motif 69 (TRIM69) in cataract formation. The expression of TRIM69 protein was down-regulated in both human cataract capsule tissues and HLECs treated with UVB, whereas the expression of p53 protein exhibited an opposite trend. Ectopic expression of TRIM69 in HLECs significantly suppressed UVB-induced apoptosis and ROS production, whereas knockdown of TRIM69 promoted apoptosis and ROS production. TRIM69 can interact with p53 and induce its ubiquitination. The effects of TRIM69 overexpression in UVB-induced cell apoptosis and ROS production was clearly weakened by p53 overexpression, thus suggesting a role for p53 in TRIM69 functions. Furthermore, inhibition of ROS mitigated the effects of UVB irradiation on ROS production, cell apoptosis, forkhead box protein 3a (Foxo3a) phosphorylation, and TRIM69 expression. Additionally, Foxo3a overexpression significantly enhanced TRIM69 promoter activity, whereas Foxo3a knockdown had the opposite effect. In conclusion, we provide the first demonstration that Foxo3a is a potential transcription factor for TRIM69, and TRIM69 induces p53 ubiquitination. These results suggest that the Foxo3a/TRIM69/p53 regulatory network may be involved in cataract formation.

Keywords: Cataract; Foxo3a; TRIM69; UVB; p53.

Graphical abstract

Fig. 1

TRIM69 and p53 involved in cataract formation. (A) Western blot results of the relative levels of TRIM69 and p53 protein in cataracts (C1–C4) and normal (N1–N4) capsule tissues. The band intensity was determined by Image J software (http://rsb.info.nih.gov/ij/, Bethesda, MD, USA) (lower panel). ***P < 0.001. (B) Western blot results of the relative levels of TRIM69 and p53 protein in HLECs exposed to UVB (0.5, 1, 2, or 4 W/m²) for 10 min. HLECs without UVB irradiation served as the control.

Fig. 2

Overexpression of TRIM69 decreased cell apoptosis, p53 expression, and ROS production caused by UVB irradiation. (A) HLECs were transduced with TRIM69-overexpression virus (TRIM69 OE) or control vector virus (vector). Expression of TRIM69 protein was determined with western blot assays at 48 h of culture. HLECs were transduced with TRIM69 OE or vector for 24 h, then subjected to UVB irradiation (2 W/m²) for 10 min. (B) After 24 h of culture, Annexin V-FITC/PI staining was performed to analyze cell apoptosis. (C) Western blot assays were used to detect the expression of p53, Bax, and Bcl-2 protein. (D, E) DCFH-DA (D) and DHE (E) staining was performed to analyze ROS production. ***P < 0.001 vs. control; ###P < 0.001 vs. vector + UVB.

Fig. 3

Inhibition of TRIM69 expression induced apoptosis, p53 expression, and ROS production in HLECs. (A) HLECs were transduced with TRIM69 (TRIM69#1∼4) or control shRNA virus (shNC). Expression of TRIM69 protein was determined with western blot assays at 48 h of culture. HLECs were transduced with TRIM69#1, TRIM69#2, or shNC for 48 h. (B) Annexin V-FITC/PI staining was performed to analyze cell apoptosis. (C) Western blot assays were used to detect the expression of p53, Bax, and Bcl-2 protein. (D, E) DCFH-DA (D) and DHE (E) staining was performed to analyze ROS production. ns: not significant; ***P < 0.001 vs. control.

Fig. 4

TRIM69 interacted with p53 protein and induced ubiquitination. (A) HLEC lysates were immunoprecipitated (IP) with anti-TRIM69 or anti-p53, and western blot analysis was carried out as indicated. IgG was used as a negative control for IP. (B) HLECs were transduced with TRIM69 OE or vector for 48 h. HLEC lysates were subjected to IP with anti-p53, and western blot analysis was performed with anti-Ub. Half-open square bracket indicates the bands of ubiquitinated p53 (Ub-p53).

Fig. 5

p53 mediated the effects of TRIM69 in UVB-induced cell apoptosis and ROS production. HLECs were divided into the following five groups and treated as described in the Materials and Methods section before UVB irradiation: control; PFTα; vector; TRIM69 OE; and TRIM69 OE + p53 OE. (A) Cell apoptosis was detected with Annexin V-FITC/PI staining. (B, C) ROS production was assessed with DCFH-DA (B) and DHE (C). (D) Western blot assays were used to detect the expression of p53, Bax, and Bcl-2 protein. ***P < 0.001 vs. control, ###P < 0.001 vs. PFTα, &&&P < 0.001 vs. TRIM69 OE.

Fig. 6

Inhibition of ROS mitigated the effects of UVB irradiation on ROS production, cell apoptosis, and TRIM69 expression. HLECs were divided into the following three groups and treated as described in the Materials and Methods section: control; UVB; and UVB + NAC. (A, B) ROS production were assessed 24 h after UVB irradiation. (C) Cell apoptosis was assessed 24 h after UVB irradiation. (D) Protein expression was assessed 24 h after UVB irradiation. (E) HLECs were transduced with Fox3a overexpressing (Foxo3a OE)/vector or Fox3a shRNA (shFox3a#2)/control shRNA (shNC) and transfected with the TRIM69 promoter plasmid. The activity of the TRIM69 promoter was measured with dual luciferase reporter assays. ***P < 0.001 vs. control, ###P < 0.001 vs. UVB; &&&P < 0.001 vs. vector; $$$P < 0.001 vs. shNC.