Differential expression of stress-inducible proteins in chronic hepatic iron overload

Abstract

Introduction:

Oxidative stress can trigger a cellular stress response characterized by induction of antioxidants, acute phase reactants (APRs) and heat shock proteins (HSPs), which are presumed to play a role in limiting tissue damage. In rodents, hepatic iron overload causes oxidative stress that results in upregulation of antioxidant defenses with minimal progressive liver injury. The aim of this study was to determine whether iron overload modulates expression of other stress-responsive proteins such as APRs and HSPs that may confer protection against iron-induced damage in rodent liver.

Methods:

Male rats received repeated injections of iron dextran or dextran alone over a 6-month period. Hepatic transcript levels for a panel of APRs and HSPs were quantitated by real-time PCR and protein expression was evaluated by Western blot and immunohistochemistry.

Results:

Hepatic iron concentrations were increased > 50-fold in the iron-loaded rats compared to controls. Iron loading resulted in striking increases in mRNAs for Hsp32 (heme oxygenase-1; 12-fold increase vs. controls) and metallothionein-1 and -2 (both increased ∼ 6-fold). Transcripts for α1-acid glycoprotein, the major rat APR, were increased ∼ 3-fold, while expression of other classical APRs was unaltered. Surprisingly, although mRNA levels for the HSPs were not altered by iron, the abundance of Hsp25, Hsp70 and Hsp90 proteins was uniformly reduced in the iron-loaded livers, as were levels of NAD(P)H:quinone oxidoreductase 1, an Hsp70 client protein.

Conclusions:

Chronic iron administration elicits a unique pattern of stress protein expression. These alterations may modulate hepatic responses to iron overload, as well as other injury processes.

Introduction

Hepatic iron overload resulting from hemochromatosis or hematologic disorders such as thallasemia can cause cirrhosis, end-stage liver disease and hepatocellular carcinoma. Iron-catalyzed oxidative damage to cellular components is presumed to be the major causal factor in the development of hepatic fibrogenesis and carcinogenesis (Kowdley, 2004). In support of this concept, removal of excess iron by phlebotomy or chelation prevents the development of these complications. Interestingly, however, recent data indicate that hepatic complications of iron overload in individuals with hemochromatosis are much less common than previously assumed (Beutler et al., 2002). Furthermore, although significant hepatic iron burdens can be produced in experimental animals by a variety of methods, these models rarely result in significant liver fibrosis (Ramm, 2000), which is the precursor lesion of serious liver pathology in human iron overload. Taken together, these observations suggest that mammalian livers possess a substantial capacity to resist the toxicity of chronic iron exposure.

The mechanisms that confer resistance to iron-induced hepatotoxicity are incompletely understood. We and others have reported that iron administration is associated with enhanced glutathione synthetic machinery in rodent liver (Ogino et al., 1989, Brown et al., 1998, Brown et al., 2007). In addition, enzymes involved in glutathione-dependent detoxification of reactive intermediates are upregulated by iron loading (Kahn et al., 1995). Given that excess iron is presumed to increase oxidant production, upregulation of antioxidant defenses is likely an important factor contributing to resistance to iron-related toxicity. However, whether other types of protective mechanisms are induced in response to iron overload has not been investigated.

In other models of tissue injury such as those resulting from thermal injury, radiation or heavy metal administration, induction of antioxidant expression frequently occurs in parallel with the upregulation of other stress-inducible genes whose products are broadly regarded as playing a role in limiting tissue damage. These include the acute phase reactants (APRs) and the heat shock proteins (HSPs). The acute phase reaction is characterized by altered transcription (positive or negative) of numerous target genes, many of which are expressed exclusively or predominantly by the liver (Gabay and Kushner, 1999). The products of these genes (APRs) participate in diverse processes such as blood coagulation, innate immunity, sequestration of metals, antiproteolytic activities, etc. The acute phase reaction is typically triggered by localized or systemic inflammation, and cytokines such as IL-1 and IL-6 are important mediators of this response.

Although it is clear that inflammatory mediators are the primary stimulus for the acute phase reaction, oxidative stress appears to play a role in this response. For example, the acute phase reaction caused by localized inflammation is accompanied by evidence of hepatic oxidative stress (Proulx and du Souich, 1995, El-Kadi et al., 2000), while treatments that deplete hepatic glutathione stores increase the expression of some APRs (Tacchini et al., 2002). Notably, hepcidin and ferritin, two proteins that play key roles in the regulation of iron metabolism and whose expression is induced by iron, are also APRs (Nemeth et al., 2003, Pigeon et al., 2001). These observations suggest that iron-induced oxidative stress may stimulate expression of other APRs that contribute to protection from liver injury.

The HSPs are another important class of proteins whose expression is inducible by a variety of stressors. Collectively, the HSPs are believed to assist in the proper folding of newly synthesized proteins and/or in the refolding of denatured proteins. Destabilization and unfolding of proteins leading to aggregation have been proposed to be the common signal that elicits the heat shock response (Freeman et al., 1999). Although destabilization and unfolding occur in response to elevated temperature, oxidation of thiol or other residues can have similar effects on protein stability, thus accounting for the observation that treatments that cause oxidative stress frequently induce expression of heat shock proteins as well. In support of the relationship between oxidative stress and increased HSP expression, we have previously reported that Hsp32 (heme oxygenase-1, HO-1) levels are increased in iron-loaded livers (Brown et al., 2003). However, it is not known whether other HSPs are altered by iron loading. Therefore, the goal of these studies was to determine the effects of chronic iron overload on the expression of a variety of stress-inducible proteins including APRs and HSPs.