Chronic ethanol feeding causes a significant increase of oxidative stress in rats liver cells. This is caused by the induction of CYP2E1 activity that metabolizes ethanol [Bardag-Gorce et al., 2006a
]. It is our hypothesis that proteasome inhibitor treatment is responsible for the up regulation of antioxidative stress response elements (ARE), which, in turn, is expected to counteract the ethanol-induced oxidative stress
Microarray analysis of liver of rats fed ethanol chronically did not show an increase of ARE mRNA levels, even if the levels of ROS were significantly increased by ethanol (data not shown). However, data mining of a microarray analysis performed on the liver of rats given the proteasome inhibitor PS-341 only, one time, IP, at 0.5 mg/kg body weight (bw), and sacrificed 24h later, showed a significant increase in the mRNA transcripts levels of several important antioxidative enzymes (), which play an important role in recycling glutathione (GSH) and in reducing the level of ROS (). This dosing protocol also caused a significant inhibition of proteasome activity ().
Data Mining of Microarray Analysis Comparing Antioxidative Enzymes mRNA Levels in the Livers of Rat Given PS-341 and its Control.
Figure 1 A. Transsulfuration pathway and the role of antioxidative enzymes in recycling glutathione (GSH). Note that GSR, GSS, GPX2, and SOD2 enzymes were up regulated by the proteasome inhibitor (↑ arrows). B. Proteasome chymotrypsin-like activity was (more ...)
qRT-PCR experiments were performed and results confirmed the data obtained in the microarray analysis: the gene expression of these antioxidative stress enzymes was significantly induced when proteasome was inhibited by PS-341 ().
Figure 2 A. qRT-PCR analysis of GSS, GSR, GPX2, and SOD2 enzymes gene expression showed a significant increase of these antioxidative enzymes. Mean ± SEM, n=3. B. Proteasome chymotrypsin-like activity. rats were treated with PS-341 at a lower dose (0.25mg/kg) (more ...)
To evaluate the cytoprotective effects of proteasome inhibitor treatment, we hypothesized that the administration of proteasome inhibitor to rats fed ethanol may enhance the antioxidant response and play a protective role against ethanol. Therefore, rats were fed ethanol for 1 month and injected with proteasome inhibitor PS-341 IP, once a week, 4 times, at 0.5 mg/kg body weight (bw). Proteasome activity was then measured and a significant inhibition of proteasome activity was found (). This marked inhibition was similar to the inhibition observed with PS-341 alone (), indicating that proteasome inhibition by PS-341 is different from proteasome dysfunction caused by ethanol feeding, which suggests that there is no accumulative inhibitory effects of PS-341 administration and ethanol feeding. In our previous study [Bousquet-Dubouch et al., 2009
], we demonstrated that chronic ethanol feeding causes a dysfunction of the ubiquitin proteasome pathway altering, not only the binding of the 20S proteasome to its regulatory complexes, but also the proteasome interacting proteins, which is totally different from the drug-induced proteasome inhibition. PS-341 is a reversible proteasome inhibitor and there is a stimulation of the proteasome activity 48h post injection (). These results indicate that proteasome inhibitor beneficial effects resides in this reversibility.
Figure 3 A. Proteasome chymotrypsin-like activity in the liver of rats fed ethanol and given PS-341. B. qRT-PCR analysis of GSS, GSR, GPX2, and SOD2 enzymes gene expression in the liver of rats fed ethanol and rats fed ethanol and treated with PS-341. Proteasome (more ...)
As expected, we found a significantly up regulation of the antioxidative enzymes mRNA in the liver of rats fed ethanol and treated with PS-341. On the other hand, ethanol feeding alone did not increase the gene expression of these enzymes (). These results thus confirm that ethanol-induced proteasome inhibition is different from that of PS-341 since ethanol alone has no effects in up regulating the antioxidative response. The mechanisms are yet to be determined.
Rats fed ethanol tolerated well the once a week PS-341 0.5 mg/kg treatment. ER stress proteins were analyzed to examine the potential side effects of this 4 times/PS-341 administration and sacrifice 4h post injection. We did not find any changes in ER stress markers Grp78 and PDI protein levels, indicating that this proteasome inhibitor treatment did not cause ER stress (data not shown). However, there was an increase in the polyubiquitinated proteins levels because of the proteasome activity inhibition (). The effect of proteasome inhibitor treatment in increasing apoptosis was evaluated using the TUNEL method. The results showed an increase in DNA fragmentation, indicating that the administration of proteasome inhibitor at 0.5 mg/kg caused an increase in apoptosis (). However, since PS-341 is a reversible proteasome inhibitor, and proteasome activity rebounds higher than the control 48h and 72h post injection (), it is expected the polyubiquitinated protein to be cleared up by the stimulated proteasome activity. We believe that this reversibility helped the rats fed ethanol for 4 weeks to tolerate the 0.5 mg/kg, once a week, 4 times dosing. PS-341 increased apoptosis was not greatly different from the ethanol increased apoptosis, which, we believe, indicates that a lower dose of PS-341 is certainly more beneficial.
Figure 4 A. Polyubiquitinated levels analysis in the liver homogenates of rats fed ethanol for 4 weeks and given proteasome inhibitor 4 times once a week. Note that polyubiquitinated proteins significantly accumulated with proteasome inhibitor treatment. B. Morphology (more ...)
To verify that proteasome inhibitor treatment was efficient in reducing the ethanol-induced oxidative stress at this dosing, we analyzed the protein levels of the antioxidative enzymes GPX2 and GCLC. show that a significant increase of the levels of these proteins occurred.
Figure 5 Western blot analysis of GPX2 (A) and GCLC (B) levels in the liver of rats fed ethanol and rats fed ethanol+PS-341. Proteasome inhibitor treatment increased the protein levels of these antioxidative enzymes. (C) Left: Immunodetection of oxidized protein (more ...)
Carbonyl proteins levels were also measured using Western blot analysis. The results show that proteasome inhibitor administration to rats fed ethanol prevented the protein carbonyl formation ().
Nrf2 stabilization in the cytoplasm was assessed by immunohistochemistry analysis to investigate the mechanism of proteasome inhibitor treatment in up regulating the antioxidative enzymes gene expression. Nrf2 is the transcription factor that plays a key role in the activation of cellular responses to oxidative stress [Kobayashi et al., 2002
]. shows that the gene expression of Nrf2 was surprisingly decreased by the combination of ethanol feeding and proteasome inhibitor treatment. The protein levels and the nuclear extract were also found down regulated (data not shown). It has been documented that when Nrf2 levels are high, the de novo
synthesized Nrf2 translocates to the nucleus, and not only activates the ARE gene expression, but also autoregulates its own gene expression [Kwak et al., 2002
Figure 6 A: (A) qRT-PCR analysis of Nrf2 gene expression, in the livers of rats fed ethanol and given PS-341. The combination of ethanol feeding and proteasome inhibitor treatment decreased the gene expression of Nrf2. Mean ±SE, n=3. B : qRT-PCR analysis (more ...)
Therefore, it was clear that different mechanism was involved in the beneficial effects of proteasome inhibitor treatment against the oxidative stress. ATF4 is a transcriptional factor with a well documented role in up regulating the antioxidative stress response. shows that ATF4 gene expression was significantly increased when the rats were fed ethanol and treated with PS-341. However, ethanol feeding alone did not significantly increase ATF4 gene expression, indicating that proteasome inhibitor treatment stabilized ATF4, which would stimulate the antioxidative stress response.