Cytoprotective effects of proteasome beta5 subunit overexpression in lens epithelial cells

Abstract

Purpose: To determine whether the overexpression of the proteasome catalytic beta5 subunit (PSMB5) can induce the expression of the catalytic subunits beta1 and beta2, enhance proteasome activity, and exert a cytoprotective effect in lens epithelial cells.

Methods: Cells from the human lens epithelial cell line SRA01/04 (LECs) were stably transfected either with a plasmid expressing the proteasome catalytic subunit beta5 or with an empty plasmid. beta-5-expressing LECs and controls were analyzed for the expression of beta1, beta2, beta5, and alpha6 proteasome subunits; chymotrypsin-like (CT-L) and peptidylglutamyl-peptide hydrolase (PGPH) catalytic activities; as well as for the accumulation of carbonylated proteins, rates of cell viability, and apoptosis after oxidative stress.

Results: Stable expression of the beta5 proteasome subunit resulted in increased expression of the catalytic subunits beta1 and beta2, increased CT-L and PGPH proteasome activities, and increased resistance to accumulation of carbonylated proteins and cell death after oxidative stress.

Conclusions: The proteasome activity can be genetically "upregulated" in lens cells by overexpression of the beta5 catalytic subunit. The resulting increase in proteasome activity leads to a decrease in the accumulation of oxidized proteins and enhanced cell survival following oxidative stress.

Figure 1

Western blot analysis confirming the overexpression of β5 subunit in LECs stably transfected with PSMB5 plasmid compared to control cells transfected with an empty plasmid. Cells stably transfected with the pcDNA3.1-PSMB5 plasmid also showed increased expression of the catalytic subunits β1 and β2, while expression of the constitutive subunit α6 subunit did not show any significant change. GAPDH was used as a control. This figure is representative of the three independent cell lines analyzed.

Figure 2

Effects of stable expression of the β5 proteasome subunit on chymotrypsin-like and peptidyl glutamyl-peptide hydrolase catalytic activities in LECs. Mean values of activity were calculated as percentage of activity compared to the control cells. Both proteasome chymotrypsin-like (CT-L) catalytic activity (157.34±6.03%) and proteasome peptidyl glutamyl-peptide hydrolase (PGPH) catalytic activity (132.00±17.11%) were significantly increased in PSMB5 transfected cells as compared with the control cells. Each column shows the average of three independent experiments (%). Similar results were obtained in the three independent cell lines analyzed.

Figure 3

Effects of stable expression of the β5 proteasome subunit on the accumulation of carbonylated proteins after H₂O₂ treatment in LECs. Basal level of carbonyl content measured prior to treatment with H₂O₂ showed no significant difference between LECs expressing β5 subunit and controls (0.41±0.10 nmol/mg and 0.45±0.12 nmol/mg, respectively, p>0.05). When LECs were exposed to 40 mM H₂O₂, carbonyl content increased both in LECs overexpressing β5 and control cells. The accumulation of carbonyl proteins was significantly lower in LECs expressing β5 (1.63±0.36 nmol/mg versus 2.65±0.44 nmol/mg, p<0.01). The result is representative of the three independent cell lines analyzed.

Figure 4

Effects of stable expression of the β5 proteasome subunit on the viability of LECs after H₂O₂ treatment. Cell viability was measured by the MTT method 4 h after addition of H₂O₂. Treatment of LECs with H₂O₂ resulted in a dose-dependent decrease in viability in both β5 expressing cells and nonexpressing controls. Stable expression of β5 resulted in a significant protective effect against H₂O₂ toxicity in LECs. Similar results were obtained in the three independent cell lines analyzed.

Figure 5

Effects of stable expression of the β5 proteasome subunit on the levels of apoptosis in LECs after H₂O₂ treatment. Frequency of apoptotic cells before H₂O₂ treatment was 7.1±1.1% for control LECs (A) and 5.9±0.8% for LECs expressing β5 (B). Twenty-four h after treatment with 40 mM H₂O₂, apoptotic cell frequency increased to 15.7±1.9% in control LECs (C) while LECs overexpressing the β5 proteasome subunit experienced a smaller rise in apoptosis (9.3±0.9%; D). The flow cytometric measurements are representative of three independent experiments.

Figure 6

Effects of stable expression of the β5 proteasome subunit on the nuclear morphology of LECs after H₂O₂ treatment. Staining with Hoechst 33342 revealed normal nuclear morphology in untreated LECs transfected with an empty plasmid (A) or with PSMB5 (B). After treatment with 40 mM H₂O₂, the nuclei of control LECs showed more shrinking and fragmentation (D) than those from PSMB5-transfected cells. Data is representative of three independent experiments.