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Heat shock proteins are present in mallory bodies (cytokeratin aggresomes) in human liver biopsy specimens

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Abstract

Mallory bodies (MBs) are aggresomes, composed of cytokeratin and various other proteins, which form in diseased liver because of disruption in the ubiquitin-proteasome protein degradation pathway. Heat shock proteins (hsp’s) are thought to be involved in this process because it was discovered that MB formation is induced by heat shock in drug-primed mice. It has been reported that ubiquitin and a mutant form of ubiquitin (UBB+1) are found in aggresomes formed in the neurons in Alzheimer’s disease and in the liver MBs in various liver diseases. In addition, hsp 70 has been found in aggresomes in Alzheimer’s and in MBs in drug-primed mice. Therefore, we hypothesized that hsp’s might be involved in MB formation in human liver diseases. Liver biopsy sections were double-stained using ubiquitin and hsp 70 or 90b antibodies. Both hsps 70 and 90b were found in MBs in all liver diseases investigated including primary billiary cirrhosis, nonalcoholic steatohepatitis, hepatitis B and C, idiopathic cirrhosis, alcoholic hepatitis, and hepatocellular carcinoma. Ubiquitin and the hsp’s colocalized in all MBs in the diseased liver sections. These results indicate that hsp involvement in MB formation is similar to that seen in aggresome formation in other conformational diseases.

Introduction

Heat shock protein (hsp) 70 was first detected in liver tissue from patients with alcoholic hepatitis (Omar et al., 1990). Heat shock treatment of drug-primed mice caused increased expression of hsp 70 and development of Mallory bodies (MBs) in the livers (Yuan et al., 1995). In addition, Stumptner et al., 2001 found hsp 70 in MBs in a mouse model of hepatocellular injury. Mayer et al. (1991) noted that hsp 70 was located in MBs using immunogold electron microscopy. Even though hsp 90 is thought to play a role similar to hsp 70 in protein folding and degradation, hsp 90 has not been noted in MBs to date.

MBs are cytokeratin aggresomes that are induced in liver cells in response to a variety of cellular injuries acting through different mechanisms Yuan et al 1995, Yuan et al 1998, Yuan et al 2000, Zhang-Gouillon et al 1998, French et al 2001, which culminate in the failure of the proteasome to turnover cytokeratin proteins (Bardag-Gorce et al., 2002). Because hsp’s have been associated with protein folding and degradation pathways, it was postulated that hsps were involved in the process of MB formation (Yuan et al., 1995). In this study, human liver sections from patients with various liver diseases were double-stained with antibodies to ubiquitin and hsp 70 or 90 to establish if hsp’s are involved in MB formation in these diseases. To extend previous studies concerning hsp and MB formation in mice, we stained liver sections from a mouse model of hepatocellular injury to determine if hsp 70 and 90 are involved in MB formation in this model. These results have been reported in part in an abstract (Riley et al., 2001).

Section snippets

Materials and methods

Four-micron sections were cut from archived formalin-fixed, paraffin-embedded human liver biopsy specimens and mounted on poly-l-lysine–coated slides. Twenty patient liver biopsy specimens, which contained MBs, and nine patient liver biopsy specimens, which did not contain MBs, were examined. The sections were deparafinized and subject to citric acid–microwave treatment for antigen retrieval (Biogenex, San Ramon, CA). Then, the sections were double-stained using ubiquitin (DAKO, Carpinteria,

Results

Both hsps 70 and 90b were found in MBs in all liver diseases investigated including primary billiary cirrhosis, nonalcoholic steatohepatitis, idiopathic cirrhosis, hepatitis B and C, alcoholic hepatitis, and hepatocellular carcinoma. A representative section from a patient with idiopathic cirrhosis, stained for hsp 90 and ubiquitin, is shown in Figs. 1A and 1B. The photomicrographs show that the staining for ubiquitin and hsp 90 colocalized in the MB. The same result was obtained when an

Discussion

Cellular chaperones such as hsp 70 and 90 provide a quality control mechanism to distinguish normal proteins from misfolded proteins destined for degradation by the ubiquitin-proteasome pathway. Molecular chaperones, including hsps 70 and 90, are thought to recognize misfolded proteins and either convert the proteins back to their normal conformations or target them for degradation through the ubiquitin-proteasome pathway (Connell et al., 2001). The discriminating function of hsp 90 is thought

Acknowledgements

The authors thank Emmanuel J. Gorce for his expert help in designing Fig. 3, which was also used in a poster presentation at the December 2001 American Society of Cell Biology meeting (Riley et al., 2001). This research was supported by an Alcoholism Research Center grant for liver and pancreatic disease including the morphology and animal cores National Institutes of Health/National Institutes on Alcohol Abuse and Alcoholism (NIH/NIAAA) 011999.

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  • Bardag-Gorce, F., vanLeeuwen, F.W., Li, J., Riley, N.E., Nguyen, V., French, B.A., McPhaul, L.W., Lue, Y.H., French,...
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