Age-dependent increase of heme oxygenase–1 gene expression in the liver mediated by NFκB

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Abstract

Heme, the iron-porphyrin coordination complex, released from the degradation of hemoproteins, is a strong prooxidant. It is enzymatically degraded by heme oxygenase to free iron, carbon monoxide and biliverdin. Biliverdin and its reduced metabolite bilirubin are two potent physiological antioxidants. Here we show a progressive increase of steady-state levels of the mRNA encoding the inducible isoform of this enzyme (heme oxygenase-1) in the rat liver during aging. We had previously reported that aging is associated with increased activation of the nuclear factor κB (NFκB). We now provide evidence to establish that overexpression of NFκB in transfected liver-derived HepG2 cells can cause a marked induction of the endogenous heme oxygenase-1 (HO-1) mRNA and activation of the cotransfected HO-1 gene promoter. Taken together, these results support the conclusion that enhanced oxidative stress during aging is accompanied by compensatory induction of the antioxidant enzyme HO-1 through activation of the NFκB pathway.

Introduction

Aging in eukaryotic organisms is associated with a progressive accumulation of oxidative damages to cellular macromolecules. Age-associated increase of oxidized proteins has been noted in tissues which are composed of cells that either undergo rapid cell renewal such as the epidermis, or are largely mitostatic such as the brain (Stadtman, 1992). These two observations provide a strong correlative evidence for the accumulation of oxidative damage with aging irrespective of cell types or their state of differentiation. Additionally, human fibroblasts derived from young and old subjects, when replicated in vitro, maintain their age-specific levels of oxidized proteins as determined by the carbonyl residues (Oliver et al., 1987). The latter observation may suggest an age-dependent program in the regulation of the redox status of the intracellular environment. The initial hypothesis that superoxide radicals are major causes of the age-dependent deterioration of biological functions (Harman, 1981) has received substantial experimental support, especially from the observation that simultaneous overexpression of superoxide dismutase and catalase in transgenic Drosophila can considerably (≈30%) lengthen their maximum life-span potential (Orr and Sohal, 1994). Additionally, the C. elegans strains harboring the mutant form of succinate dehydrogenase cytochrome b (a component of mitochondrial electron transport chain) show oxygen hypersensitivity and premature aging, possibly due to an increased rate of superoxide production (Ishii et al., 1998). Thus, it appears that a balance between the rate of superoxide generation and superoxide inactivation may play a critical role in aging.

Oxidative damage to cellular macromolecules is mediated by reactive oxygen species (ROS) of intracellular origin such as hydrogen peroxide, oxygen free radical, singlet oxygen, hypochlorite anion, nitric oxide and peroxynitrite anion. Although these ROS molecules arise from different enzymatic reactions, they can also undergo non enzymatic interconversions (Khan and Wilson, 1995). The ubiquitous electron carrier iron-porphyrin complex, heme, is a prooxidant which actively participates in the generation of ROS. It is enzymatically inactivated by heme oxygenase (HO) which degrades heme to free iron, carbon monoxide and biliverdin (Maines, 1997). HO not only removes the excess heme, a prooxidant, it also contributes to the antioxidant defense mechanisms of the cell (Barlow et al., 1999). Biliverdin and its reduced metabolite bilirubin are two highly potent physiological antioxidants (Stocker et al., 1987) and a rapid rise of heme oxygenase activity immediately after birth is thought to serve as a protective mechanism against high levels of ROS due to a sudden flux of oxygen in the new born. Two HO isozymes coded by separate genes have been identified and characterized in humans and rodents. Of these two isozymes, heme oxygenase-1 (HO-1) is ubiquitously expressed and is inducible, while the expression of heme oxygenase 2 (HO-2) is relatively tissue-restricted with notably high expression in the brain and testis, the two organs that may require a greater degree of antioxidant protection due to their critical regulatory roles in organ function and genetic propagation (Maines, 1997). HO-1 and 2 knockout mice show increased sensitivity to oxidative stress and more pronounced hepatotoxicity to endotoxins, providing additional evidence for important roles of these enzymes in antioxidant defense (Poss and Tonegawa, 1997, Dennery et al., 1998).

It is increasingly becoming apparent that much of the gene regulatory effects of oxidative stress is mediated by the redox-sensitive transcription factor, nuclear factor κB (NFκB) (Muller et al., 1997). NFκB is a family of transcription factors which generally function as heterodimers to regulate specific gene expression. The subunit components of NFκB are maintained in the cytoplasmic compartment by sequestration with the inhibitory protein IκB. Activation of an IκB-specific protein kinase pathway by ROS leads to phosphorylation and proteolysis of IκB resulting in release of the NFκB proteins and their translocation into the cell nucleus for transcriptional regulation of target genes (Verma and Stevenson, 1997). In an earlier study we have shown that aging is associated with a progressive increase of the nuclear level of activated NFκB in the rat liver (Supakar et al., 1995). This observation has been extended to show similar age-dependent rise of the NFκB activity in nuclear extracts derived from other tissues including heart, brain and kidney (Helenius et al., 1996, Toliver-Kinsky et al., 1997, Poynter and Daynes, 1998, Walter and Sierra, 1998). Since, both human and rodent HO-1 gene promoters contain NFκB binding sites (Shibahara et al., 1989, Lavrovsky et al., 1994, Kurata et al., 1996, Lavrovsky et al., 1996, Bergeron et al., 1998), we were interested to examine whether the presumed rise of oxidative stress during aging and more specifically, the increased NFκB activity, can influence the expression of the HO-1 gene. Results presented in this article show a progressive increase in the steady-state level of HO-1 mRNAs in the rat liver and a direct effect of NFκB on the upregulation of HO-1 gene expression in the human liver-derived HepG2 cells.

Section snippets

Animals

Fischer 344 rats of different ages were obtained from the colony of the national institute on aging, maintained at Charles River breeding laboratories (Wilmington, MA). All experiments were performed in strict accordance with the National Institutes of Health Guide for Care and Use of Laboratory Animals and animal protocols were approved by the University of Texas Health Science Center at San Antonio Animal Care Committee.

Plasmid construction

Construction of 5′-deletion mutants of the HO-1 promoter region has been

Results

Quantification of steady-state levels of both the ubiquitously expressed HO-1 mRNA and the relatively tissue-restricted HO-2 mRNA in livers of 1, 6, 13, 17 and 24-month-old rats show an isozyme-selective increase of HO-1 mRNAs with aging (Fig. 1). Unlike the levels of the HO-2 mRNA which remained almost age-invariant, a progressive increase of HO-1 mRNA from 1 to 24 months of age was observed. The total hepatic RNA derived from the 24 month-old rat contained an ≈15-fold higher level of HO-1

Discussion

Heme oxygenases are ubiquitous and essential enzymes for all eukaryotic organisms which depend on aerobic oxidation and electron transport via heme containing proteins. Both isoforms of this enzyme catalyze the same enzymatic reaction resulting in the degradation of heme to biliverdin, carbon monoxide and free iron. Because of the inducible nature of HO-1, it is presumed to make a greater contribution to the maintenance of oxidant/antioxidant homeostasis during changes in cellular environments.

Acknowledgements

This work was supported by the NIH grant R37-AG 10486. B.C. is a senior career scientist of the veterans affairs. A.K.R. is recipient of a MERIT award from the national institute on aging.

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