De-fense! De-fense! De-fense: Scavenging H₂O₂ While Making Cholesterol

It has been long appreciated that abnormal glucose, lipid, and bone metabolism, as well as atherosclerosis and neuronal degeneration, are common accompaniments of old age—often present as co-morbidities in the same individual. However, it is only recently that shared molecular pathogenetic mechanisms related to aging per se have been implicated in the development of these conditions. This forward stride is largely due to significant advances in our understanding of the biology of aging, genetic discoveries in animal models and humans, the demonstration of a similar function of the same genes in different organs, and better grasp of integrative physiology and pathophysiology.

To date the most durable and widely accepted mechanistic explanation of aging is oxidative stress. According to this theory, cellular stress caused by reactive oxygen species (ROS) is a major determinant of aging and life span (1). Approximately 90% of ROS arise in the mitochondria as byproducts of aerobic metabolism—the process that is fueled by nutrients such as glucose and is responsible for the formation of ATP. ROS are generated by the escape of somewhere between 0.1 and 2% of electrons passing through the electron transport chain. Escaped electrons are added to molecular oxygen to generate superoxide (O₂^·−), hydrogen peroxide (H₂O₂), and the hydroxyl radical (OH^·−). A series of important discoveries highlighted in recent review articles (2,3,4) indicate that H₂O₂ is a critical signal for the replicative capacity of regenerative cells, as well as for apoptosis, for changes in gene expression leading to aging and aging-related diseases. Compared with (O₂^·−) and (OH^·−), H₂O₂ has the highest oxidative activity, the highest stability, and the highest intracellular molar concentration. Importantly, proapoptotic signals including ROS release the adapter protein p66^shc from an inhibitory complex in the inner mitochondria membrane. Active p66^shc serves as a redox enzyme that catalyzes reduction of O₂ to H₂O₂ through electron transfer from cytochrome c. H₂O₂ in turn causes opening of the mitochondrial permeability transition pore, swelling, and apoptosis.

To defend against the adverse effects of oxidative stress, cells have developed several intricate antioxidant defense mechanisms that involve both enzymatic reactions and altered gene transcription. Antioxidant enzymes include various isoforms of superoxide dismutase, which catalyzes the conversion of O₂^·− to H₂O₂, which in turn is converted to water and oxygen by catalases. Alternative mechanisms against oxidative stress involve thiol-reducing buffers consisting of oligopeptides with redox-active sulfhydryl moieties, the most abundant of which are glutathione (GSH) and thioredoxin. GSH peroxidase converts peroxides to harmless alcohols in a reaction in which the enzyme oxidizes GSH to the disulfide GSSG; and GSH reductase converts it back to GSH (5). A similar defense mechanism is provided by the peroxiredoxin family of enzymes, which use thioredoxin as substrate. Some of these enzymes are localized inside the mitochondria, whereas others are located in the cytosol or the extracellular space.

In this issue of Endocrinology, Lu and co-workers (6) report an elegant series of studies demonstrating that 3β-hydroxysteroid-Δ24 reductase (DHCR24), also known as Diminuto/Seladin-1, is a H₂O₂ scavenger that protects cells from oxidative stress-induced apoptosis. Interestingly, whereas catalase is exclusively localized in the peroxisome and the major isoforms of GSH peroxidase and peroxiredoxin are mainly in the cytosol, DHCR24 is localized in the endoplasmic reticulum. Consistent with this localization, DHCR24 exerted cytoprotective effects in response to both H₂O₂ and endoplasmic reticulum stress-induced by tunicamycin. Based on this unique localization, the authors suggest that different antioxidant enzymes may play distinct roles in H₂O₂ elimination in an organelle-specific manner.

DHCR24 has another biologic role. It is an indispensable catalyst of the final step in cholesterol biosynthesis. Derangements of lipid metabolism including cholesterol accumulation and lipid oxidation in association with oxidative stress have been demonstrated in neuronal cell membranes in patients with Alzheimer’s disease. Specifically, 4-hydroxynonenal (4-HNE), a breakdown product of oxidized fatty acids is increased in brain tissues of Alzheimer’s disease patients (7). 4-HNE itself is highly reactive, and it induces oxidative stress via glutathione depletion. Moreover, DHCR24 is highly expressed in neuronal cells throughout mammalian brains, and its expression is decreased in neurons within selective vulnerable regions of Alzheimer’s disease (8). Hence, scavenging of H₂O₂ by an enzyme that also makes cholesterol makes sense in teleological terms.

Members of the FoxO transcription factors have recently emerged as another major cell defense mechanism against oxidative damage by virtue of their ability to up-regulate the transcription of free radical scavenging enzymes such as manganese superoxide dismutase, catalase, and DNA damage repair genes such as Gadd45 (3,4). FoxOs shuttle between the cytoplasm and the nucleus depending on the phosphorylation of specific sites by distinct sets of kinases. Phosphorylation of FoxOs in response to growth factors such as insulin and IGFs, via phosphatidylinositol 3-kinase and Akt kinases, favors their retention in the cytoplasm. On the other hand, phosphorylation of FoxOs (in different sites) in response to oxidative stress results in their translocation into the nucleus where they transactivate several cyclins, cyclin-dependent kinase inhibitors, DNA repair, and apoptosis control genes, as well as antioxidant enzymes. FoxOs not only promote mammalian cell survival by inducing cell cycle arrest and quiescence in response to oxidative stress, but they also regulate longevity in model organisms.

β-Catenin, a critical component of the canonical Wnt signaling pathway, may be a pivotal molecule in defense against oxidative stress by serving as a cofactor of FoxOs. Like oxidative stress, the Wnt/β-catenin signaling pathway affects several biological processes ranging from embryonic development, patterning, and postembryonic stem cell fate, to bone formation and insulin secretion in adulthood. β-Catenin mediates canonical Wnt signaling by binding to and activating members of the T-cell factor (TCF) transcription factor family. Several lines of recent evidence have strongly suggested a linkage among the age-associated oxidative stress, FoxOs, Wnt/β-catenin signaling, osteoblastogenesis, adipogenesis, osteoporosis, and several features of the metabolic syndrome and atherosclerosis.

Oxidative stress (corresponding with decreased GSH reductase activity) is a pivotal pathogenetic factor of age-related bone loss and strength in mice, leading to, among other changes, an increase in osteoblast and osteocyte apoptosis and a decrease in osteoblast number and the rate of bone formation. Estrogens or androgens decrease oxidative stress in bone cells and the loss of these sex steroids accelerates the involution of the skeleton by age-associated oxidative stress (9). Moreover, skeletal involution with advancing age in mice is also associated with the diversion of a limited pool of β-catenin from TCF- to FoxO-mediated transcription in osteoblastic cells (10). Consistent with these findings, attenuation of Wnt-mediated transcription, resulting from autosomal-dominant missense mutations in LRP6 or LRP5, coreceptors for the Wnt-signaling pathway, has been linked genetically to osteoporosis. These mutations have also been linked to coronary artery disease in the same large kindreds as well as several features of the metabolic syndrome including hyperlipidemia, hypertension, and diabetes (11,12). Based on all these lines of evidence, we have proposed that antagonism of Wnt signaling by oxidative stress with increasing age may be a common molecular mechanism contributing to the development not only of involutional osteoporosis, but several pathologies such as hyperlipidemia, atherosclerosis, and insulin resistance, all of which become more prevalent with advancing age (Ref. 13 and Fig. 1). The same mechanism may be operational in neurodegenerative disorders like Alzheimer’s (14).

Figure 1.

Increased oxidative stress with advancing age antagonizes the beneficial effects of Wnt/β-catenin signaling on bone, glucose, and lipid metabolism, by diverting β-catenin from TCF- to FoxO-mediated transcription. LEF, Lymphoid enhancer-binding factor 1.

Closer to the subject of lipid oxidation and the causation of disease, a vast literature dating back to 1979 suggests a critical role for lipid oxidation in the development of atherogenesis (15). Furthermore, extensive epidemiological evidence, including an association between coronary artery disease and low bone mineral density has more recently indicated that atherosclerosis and osteoporosis may be mechanistically linked (16,17,18). Indeed, the levels of Alox15—a lipoxygenase that generates oxidized lipids—are inversely related to bone mass in mice (19,20). Furthermore, activation of peroxisome proliferator-activating receptor-γ by oxidized lipids (or the synthetic drug ligand rosiglitazone) promotes the development of adipocytes at the expense of osteoblasts in vitro (21). In addition, rosiglitazone induces bone loss in mice and humans, and this effect is associated with increased marrow adiposity, decreased osteoblast number and bone formation rate, and increased osteoblast and osteocyte apoptosis (22,23,24). Alox15 adds oxygen to polyunsaturated fatty acids and in the process the catalytic iron is reduced to ferrous. This hydroperoxide product decomposes into a hydroxy derivative to generate a stable oxidation product, which can then bind to peroxisome proliferator-activating receptor-γ to exert the skeletal effects. In the process of this decomposition, a hydroxy radical is released. This has two important consequences. First, the catalytic iron motif is oxidized to reactivate alox15. Second, it adds to the redox burden of the cell, which can generate by nonenzymatic means newly oxidized fatty acids. During aging, alox15 activity increases as does lipid oxidation; and this probably contributes to the increased osteoblast apoptosis and other cellular changes leading to osteoporosis. Furthermore, 4-HNE is as potent as H₂O₂ in inducing p66^shc phosphorylation and osteoblastic cell apoptosis (25).

The discovery in the article by Lu et al. (6) that DHCR24, an essential enzyme in cholesterol biosynthesis, is also an H₂O₂ scavenging enzyme adds a further element to the complex array of cellular mechanisms involved in defense against oxidative stress. At this stage, it is unknown whether DHCR24 deficiency plays a role in the pathogenesis of atherosclerosis, the metabolic syndrome, or osteoporosis, but these possibilities certainly need to be explored. Defense, defense, and more defense clearly seem to be part of Nature’s game plan in dealing with oxidative stress. Defects in any one of the components involved could have far-reaching consequences for aging and age-related degenerative diseases.