Proteasome inhibition induces cytokeratin accumulation in vivo

https://doi.org/10.1016/j.yexmp.2003.11.004Get rights and content

Abstract

Chronic ethanol ingestion leads to inhibition of proteasomal activity. As a consequence, proteins accumulate in liver cells. Cytokeratin accumulation as seen in alcoholic hepatitis could lead to the formation of Mallory bodies. In order to study the phenomenon of cytokeratin accumulation in liver cells, rats were fed ethanol or dextrose for 1 month and some were given the proteasome inhibitor, PS-341, to augment the inhibitory effect of ethanol feeding. This was done to study the involvement of proteasome inhibition in the process of cytokeratin accumulation. There was a marked increase in the accumulation of polyubiquitinated proteins, and heat shock proteins (hsp) 25 and 70 in the liver of rats treated with PS-341. Similarly, cytokeratin-8 (CK-8) levels were markedly increased in the liver homogenates of rats fed ethanol when given PS-341. When normal mouse cultured hepatocytes were transfected with cytokeratin-18 (CK-18) tagged with red fluorescent protein (RFP), CK-18 aggresomes formed because proteasome was overloaded. These data provide new evidence that proteasome inhibition is involved in cytokeratin accumulation, when aggresomes are formed in tissue culture. Accumulation of cytokeratin in this way may ultimately lead to Mallory body formation as seen in alcoholic hepatitis.

Introduction

Proteasome inhibition or failure of the ubiquitin proteasome system in the cell alters cell functions. The critical role of the ubiquitin proteasome system failure in the neurodegenerative disease such as Parkinson disease has been documented (McNaught et al., 2002). Other studies have reported that elevated oxidative stress inhibited proteasome activity in neurodegenerative disorders such as Alzheimer's disease Keller et al., 2000, Lopez Salon et al., 2000. These results suggest that proteasome inhibition may play a role in neurotoxicity associated with oxidative stress. However, the direct mechanism of proteasome inhibition has not been determined.

Ding and Keller (2001) have proposed that heat shock protein (hsp) expression plays as a protective role against oxidative stress because it prevents proteasome inhibition, either by recruitment of substrates for proteasome digestion or through hsp–proteasome interaction. Proteasome inhibition has been shown to increase cellular hsp levels (Stangl et al., 2002).

Alcohol fed to rats causes inhibition of the proteasome in the liver Bardag-Gorce et al., 2000, Donohue et al., 1998, Fataccioli et al., 1999 and increases liver cell protein concentration due to the loss of proteasome catalytic activity. Proteasome inhibitor PS-341 causes inhibition of the catalytic activity of the proteasome in vivo and causes ubiquitin–cytokeratin-8 (CK-8) aggresome formation resembling Mallory bodies seen in alcoholic hepatitis (Bardag-Gorce et al., in press). Since alcohol and PS-341 both inhibit the proteasome, we studied the effect of proteasome inhibition on cytokeratin-8 and hsp levels in rats fed alcohol and given PS-341.

Section snippets

Animals

Male Wistar rats from Harleco (Hollister, CA), weighing between 250 and 300 g, were used. They were maintained according to the Guidelines of Animal Care, as described by the National Academy of Sciences and published by the National Institute of Health (1996).

Ethanol and PS-341 treatment

The rats (n = 12) were fed a liquid diet intragastrically containing ethanol (13 g/kg/day) for 30 days. Controls (n = 12) were pair-fed isocaloric dextrose. Four rats fed ethanol for 1 month and their pair-fed control (dextrose) were

Results

Rats were fed ethanol chronically for 1 month to induce proteasome inhibition. Another group of rats fed ethanol was treated with PS-341 four times once a week and sacrificed 4 h after the last injection of PS-341. The combination of PS-341 treatment and ethanol feeding accordingly was tolerated very well by the animals. Ethanol feeding and PS-341 treatment were combined to examine the effects of proteasome inhibition in the liver cells of rat fed ethanol. The inhibition of proteasomes was

Discussion

During alcoholic hepatitis and cirrhosis stages, alcoholic liver disease is characterized with Mallory body formation. They are composed of ubiquitin, proteasome, and cytokeratin proteins (Yuan et al., 2000). It was also reported that alcohol feeding induced MB formation in drug primed mice livers (Gouillon et al., 1999). The mechanism of aggresome formation has been shown to be a result of proteasome inhibition in the cells (Johnston et al., 1998). CK-8 turnover is controlled by the proteasome

Acknowledgements

This study was supported by NIH/NIAAA Grants AA08116 and P50 011999 (Alcohol Research Center Grant: Liver and Pancreas including the morphology core).

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