Regulation of hypertrophic and apoptotic signaling pathways by reactive oxygen species in cardiac myocytes

Increasing evidence suggests that oxidative and nitrosative stress play an important role in regulation of cardiac myocyte growth and survival. The cardiovascular system is continuously exposed to both reactive oxygen species (ROS) and nitrogen species (RNS), collectively termed reactive inflammatory species (RIS), and imbalances between the enzymes that regulate their bioavailability are associated with cardiac hypertrophy and the pathogenesis of cardiomyopathies, myocardial infarction and heart failure. It is now clear that RIS act as critical regulators of cardiac myocyte hypertrophy and apoptosis through control of redox-sensitive signaling cascades, such as tyrosine kinases and phosphatases, protein kinase C, and mitogen-activated protein kinases. This review will focus on the mechanisms by which ROS/RNS modulate cardiac myocyte growth and apoptosis induced by neurohormones and cytokines, and will discuss evidence for a role in the pathophysiology of heart failure.     

Protein tyrosine phosphorylation and protein tyrosine nitration in redox signaling

Reversible phosphorylation of protein tyrosine residues by polypeptide growth factor-receptor protein tyrosine kinases is implicated in the control of fundamental cellular processes including the cell cycle, cell adhesion, and cell survival, as well as cell proliferation and differentiation. During the last decade, it has become apparent that receptor protein tyrosine kinases and the signaling pathways they activate belong to a large signaling network. Such a network can be regulated by various extracellular cues, which include cell adhesion, agonists of G protein-coupled receptors, and oxidants. It is well documented that signaling initiated by receptor protein tyrosine kinases is directly dependent on the intracellular production of oxidants, including reactive oxygen and nitrogen species. Accumulated evidence indicates that the intracellular redox environment plays a major role in the mechanisms underlying the actions of growth factors. Oxidation of cysteine thiols and nitration of tyrosine residues on signaling proteins are described as posttranslational modifications that regulate, positively or negatively, protein tyrosine phosphorylation (PTP). Early observations described the inhibition of PTP activities by oxidants, resulting in increased levels of proteins phosphorylated on tyrosine. Therefore, a redox circuitry involving the increasing production of intracellular oxidants associated with growth-factor stimulation/cell adhesion, oxidative reversible inhibition of protein tyrosine phosphatases, and the activation of protein tyrosine kinases can be delineated.     

Oxidative stress and growth-regulating intracellular signaling pathways in cardiac myocytes

The toxic effects of oxidative stress on cells (including cardiac myocytes, the contractile cells of the heart) are well known. However, an increasing body of evidence has suggested that increased production of reactive oxygen species (ROS) promotes cardiac myocyte growth. Thus, ROS may be 'second messenger' molecules in their own right, and growth-promoting neurohumoral agonists might exert their effects by stimulating production of ROS. The authors review the principal growth-promoting intracellular signaling pathways that are activated by ROS in cardiac myocytes, namely the mitogen-activated protein kinase cascades (extracellular signal-regulated kinases 1/2, c-Jun N-terminal kinases, and p38-mitogen-activated protein kinases) and the phosphoinositide 3-kinase/protein kinase B (Akt) pathway. Possible mechanisms are discussed by which these pathways are activated by ROS, including the oxidation of active site cysteinyl residues of protein and lipid phosphatases with their consequent inactivation, the potential involvement of protein kinase C or the apoptosis signal-regulating kinase 1, and the current models for the activation of the guanine nucleotide binding protein Ras.     

Mechanisms of cell death in oxidative stress

Reactive oxygen or nitrogen species (ROS/RNS) generated endogenously or in response to environmental stress have long been implicated in tissue injury in the context of a variety of disease states. ROS/RNS can cause cell death by nonphysiological (necrotic) or regulated pathways (apoptotic). The mechanisms by which ROS/RNS cause or regulate apoptosis typically include receptor activation, caspase activation, Bcl-2 family proteins, and mitochondrial dysfunction. Various protein kinase activities, including mitogen-activated protein kinases, protein kinases-B/C, inhibitor-of-I-kappaB kinases, and their corresponding phosphatases modulate the apoptotic program depending on cellular context. Recently, lipid-derived mediators have emerged as potential intermediates in the apoptosis pathway triggered by oxidants. Cell death mechanisms have been studied across a broad spectrum of models of oxidative stress, including H2O2, nitric oxide and derivatives, endotoxin-induced inflammation, photodynamic therapy, ultraviolet-A and ionizing radiations, and cigarette smoke. Additionally ROS generated in the lung and other organs as the result of high oxygen therapy or ischemia/reperfusion can stimulate cell death pathways associated with tissue damage. Cells have evolved numerous survival pathways to counter proapoptotic stimuli, which include activation of stress-related protein responses. Among these, the heme oxygenase-1/carbon monoxide system has emerged as a major intracellular antiapoptotic mechanism.     

Redox-mediated gene therapies for environmental injury: approaches and concepts

Cellular redox state has been increasingly recognized as a critical component of stress-induced cellular responses and disease. Inherent in these responses are reactive oxygen species (ROS), which inflict direct cellular damage in addition to acting as intracellular second messengers modulating signal transduction pathways. These intracellular highways of communication are critical in determining cell fates and whole-organ responses following environmental injury. Although gene therapy for inherited and acquired disorders has exploded in the last decade, the application of gene therapeutic approaches for transient pathologic conditions resulting from environmental stress is just beginning to be recognized. This review will summarize the theoretical and practical applications of gene therapy for the treatment of environmental injury by modulating redox-activated cellular responses. Several approaches can be utilized to achieve this goal. These include the application of gene targeting to modulate the cellular redox state directly by expressing recombinant genes capable of degrading ROS at pathophysiologic important subcellular sites. The use of mitochondrial superoxide dismutase (MnSOD), which degrades superoxides arising from ischemia/reperfusion injury, is one example of this approach. MnSOD serves as a "garbage disposal" for potentially toxic ROS prior to cellular injury and the activation of signal transduction cascades important in whole-organ pathology and inflammation. In contrast, some ROS have been suggested to have beneficial effects on cellular responses following environmental injury. Hence, expressing the nitrogen oxygen synthetase gene (NOS) to enhance the levels of nitric oxide (NO.) and augment the beneficial effects of this compound has also been suggested as a useful redox-modulating gene therapy approach. Lastly, indirect intervention in signal transduction pathways following environmental stress by expressing dominant inhibitory proteins of redox-activated signal transduction cascades has also been useful in modulating cellular responses to redox stress. Two such examples have utilized dominant inhibitory forms of the retinoblastoma gene product (Rb) and IkappaBalpha which prevent activation of cyclin-dependent protein kinases and NF-kappaB, respectively. Ultimately, the most efficacious therapeutic approach or combination of approaches that alter the redox responsiveness of cells and organs to environmental injury will be determined through a comprehensive understanding of the relevant pathophysiologic processes.     

Reduced redox state allows prolonged survival of axotomized neonatal retinal ganglion cells

Axonal injury to CNS neurons results in apoptotic cell death. The processes by which axotomy signals apoptosis are diverse, and may include deprivation of target-derived factors, induction of injury factors, bursts of reactive oxygen species (ROS), and other mechanisms. Our previous studies demonstrated that death of a dissociated retinal ganglion cell, an identified CNS neuron, is ROS-dependent. To better define the mechanisms by which ROS induce retinal ganglion cell death after axotomy, we studied their effects in dissociated neonatal rat retinal cultures. Postnatal day 2-4 Long-Evans rat retinal ganglion cells were retrogradely labeled with the fluorescent tracer 1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine (DiI). Postnatal day 7-9 retinas were dissociated and cultured in the presence of specific ROS generating systems, scavengers, or redox modulators. Retinal ganglion cells were identified by DiI positivity and viability determined by metabolism of calcein-acetoxymethyl ester.We found that ROS scavengers protected against retinal ganglion cell death after acute dissociation, and the effects of ROS appeared to be due to shifts in the redox potential, as retinal ganglion cell survival was critically dependent on redox state, with greatest survival under mildly reducing conditions. Culture of retinal ganglion cell with the non-thiol-containing reducing agent tris(carboxyethyl)phosphine resulted in long-term survival equivalent to or better than with neurotrophic factors.Our data suggest that axotomy-associated neuronal death induced by acute dissociation may be partly dependent on ROS production, acting to shift the redox state and oxidize one or more key thiols. Understanding the mechanisms by which ROS signal neuronal death could result in strategies for increasing their long-term survival after axonal injury.     

How one TSH receptor antibody induces thyrocyte proliferation while another induces apoptosis

Thyroid stimulating hormone (TSH) activates two major G-protein arms, Gsα and Gq leading to initiation of down-stream signaling cascades for survival, proliferation and production of thyroid hormones. Antibodies to the TSH receptor (TSHR-Abs), found in patients with Graves' disease, may have stimulating, blocking, or neutral actions on the thyroid cell. We have shown previously that such TSHR-Abs are distinct signaling imprints after binding to the TSHR and that such events can have variable functional consequences for the cell. In particular, there is a great contrast between stimulating (S) TSHR-Abs, which induce thyroid hormone synthesis and secretion as well as thyroid cell proliferation, compared to so called “neutral” (N) TSHR-Abs which may induce thyroid cell apoptosis via reactive oxygen species (ROS) generation.In the present study, using a rat thyrocyte (FRTL-5) ex vivo model system, our hypothesis was that while N-TSHR-Abs can induce apoptosis via activation of mitochondrial ROS (mROS), the S-TSHR-Abs are able to stimulate cell survival and avoid apoptosis by actively suppressing mROS. Using fluorescent microscopy, fluorometry, live cell imaging, immunohistochemistry and immunoblot assays, we have observed that S-TSHR-Abs do indeed suppress mROS and cellular stress and this suppression is exerted via activation of the PKA/CREB and AKT/mTOR/S6K signaling cascades. Activation of these signaling cascades, with the suppression of mROS, initiated cell proliferation. In sharp contrast, a failure to activate these signaling cascades with increased activation of mROS induced by N-TSHR-Abs resulted in thyroid cell apoptosis.Our current findings indicated that signaling diversity induced by different TSHR-Abs regulated thyroid cell fate. While S-TSHR-Abs may rescue cells from apoptosis and induce thyrocyte proliferation, N-TSHR-Abs aggravate the local inflammatory infiltrate within the thyroid gland, or in the retro-orbit, by inducing cellular apoptosis; a phenomenon known to activate innate and by-stander immune-reactivity via DNA release from the apoptotic cells.     

Redox control of cell death

Cellular redox is controlled by the thioredoxin (Trx) and glutathione (GSH) systems that scavenge harmful intracellular reactive oxygen species (ROS). Oxidative stress also evokes many intracellular events including apoptosis. There are two major pathways through which apoptosis is induced; one involves death receptors and is exemplified by Fas-mediated caspase-8 activation, and another is the stress- or mitochondria-mediated caspase-9 activation pathway. Both pathways converge on caspase-3 activation, resulting in nuclear degradation and cellular morphological change. Oxidative stress induces cytochrome c release from mitochondria and activation of caspases, p53, and kinases, including apoptosis signal-regulating kinase 1 (ASK1), c-Jun N-terminal kinase, and p38 mitogen-activated protein kinase. Trx inhibits apoptosis signaling not only by scavenging intracellular ROS in cooperation with the GSH system, but also by inhibiting the activity of ASK1 and p38. Mitochondria-specific thioredoxin (Trx-2) and Trx peroxidases (peroxiredoxins) are suggested to regulate cytochrome c release from mitochondria, which is a critical early step in the apoptotis-signaling pathway. dATP/ATP and reducing factors including Trx determine the manifestation of cell death, apoptosis or necrosis, by regulating the activation process and the activity of redox-sensitive caspases. As mitochondria are the most redox-active organelle and indispensable for cells to initiate or inhibit the apoptosis process, the regulation of mitochondrial function is the central focus in the research field of apoptosis and redox.     

Dose effect of oxidative stress on signal transduction in aging

Reactive oxygen species (ROS) during normal metabolism signal cells to stimulate proliferation or to cause cellular damages, depending on a specific concentration. Energy restriction (ER) increases life span in animals, which can explain an effective modulator for reducing oxidative stress. Oxidative stress can result from a decrease in the protection against ROS. The deleterious effects of oxidative stress generally occur after exposure to a relatively high concentration of ROS. Alternatively, it has been suggested that a low concentration of ROS can exert important physiological roles in cellular signaling and proliferation. Signal pathways are crucial for cell survival or death. It is generally acceptable that aged cells have less response to stresses such as ROS than young cells. Oxidative stresses induce JNK and p38 kinase pathways regulated by redox regulatory proteins: thioredoxin and glutathione s-transferase, respectively. Antioxidants such as selenium block apoptosis induced by ROS through blocking apoptotic signal ASK1 and stimulating survival signal Akt activity. Old hepatocytes are more susceptible to ROS-induced apoptosis than young hepatocytes, which is associated with low expression of ERK and Akt kinases. Pharmacological inhibition of ERK and Akt activation in the young cells markedly increase their sensitivity to H(2)O(2), and ER, by preventing loss of ERK and Akt activities, enhances survival of old hepatocytes to a level similar to those of young cells. Expressions of signal pathways such as survival and apoptotic signals can regulate cells' fate and aging process. Further studies on the interaction of signal pathways may change the scientific direction of the study of aging.     

Redox regulation of human thioredoxin network

Oxidative stresses are largely mediated by intracellular protein oxidations by reactive oxygen species (ROS). Host cells are equipped with antioxidants that scavenge ROS. The cellular reduction/oxidation (redox) balance is maintained by ROS and antioxidants. Accumulating evidence suggests that the redox balance plays an important role in cellular signaling through the redox modification of cysteine residues in various important components of the signal transduction pathway. Thioredoxin (TRX) is a small protein playing important roles in cellular responses, including cell growth, cell cycle, gene expression, and apoptosis, to maintain the redox circumstance. Moreover, many recent papers have shown that the redox regulation by TRX is deeply involved in the pathogenesis of various oxidative stress-associated disorders. This review focuses on TRX and its related molecules, and discusses the role of TRX-dependent redox regulation in oxidative stress-induced signal transduction.     

Activation of tyrosine kinases by reactive oxygen species in vascular smooth muscle cells: significance and involvement of EGF receptor transactivation by angiotensin II

Enhanced production of reactive oxygen species (ROS) such as H(2)O(2) and a failure in ROS removal by scavenging systems are hallmarks of several cardiovascular diseases such as atherosclerosis and hypertension. ROS act as second messengers that play a prominent role in intracellular signaling and cellular function. In vascular smooth muscle cells (VSMCs), a vascular pathogen, angiotensin II, appears to initiate growth-promoting signal transduction through ROS-sensitive tyrosine kinases. However, the precise mechanisms by which tyrosine kinases are activated by ROS remain unclear. In this review, the current knowledge that suggests how certain tyrosine kinases are activated by ROS, along with their functional significance in VSMCs, will be discussed. Recent findings suggest that transactivation of the epidermal growth factor receptor by ROS requires metalloprotease-dependent heparin-binding epidermal growth factor-like growth factor production, whereas other ROS-sensitive tyrosine kinases such as PYK2, JAK2, and platelet-derived growth factor receptor require activation of protein kinase C-delta. Each of these ROS-sensitive kinases could mediate specific signaling critical for pathophysiological responses. Detailed analysis of the mechanism of cross-talk and the downstream function of these various tyrosine kinases will yield new therapeutic interventions for cardiovascular disease.     

Role of reactive oxygen species and protein kinase C in ischemic tolerance in the brain

It is now understood that the mechanisms leading to neuronal cell death after cerebral ischemia are highly complex. A well established fact in this field is that neurons continue to die over days and months after ischemia, and that reperfusion following cerebral ischemia contributes substantially to ischemic injury. It is now well accepted that central to ischemic/reperfusion-induced injury is what occurs to mitochondria hours to days following the ischemic insult. For many years, it has been established that reactive oxygen species (ROS) and reactive nitrogen species (RNS) promote lipid, protein, and DNA oxidation that affects normal cell physiology and eventually leads to neuronal demise. In addition to oxidation of neuronal molecules by ROS and RNS, a novel pathway for molecular modifications has risen from the concept that ROS can activate specific signal transduction pathways that, depending on the insult degree, can lead to either normal plasticity or pathology. Two examples of these pathways could explain why lethal ischemic insults lead to the translocation of protein kinase Cdelta (deltaPKC), which plays a role in apoptosis after cerebral ischemia, or why sublethal ischemic insults, such as in ischemic preconditioning, lead to the translocation of epsilonPKC, which plays a pivotal role in neuroprotection. A better understanding of the mechanisms by which ROS and/or RNS modulate key protein kinases that are involved in signaling pathways that lead to cell death and survival after cerebral ischemia will help devise novel therapeutic strategies.     

Significance of ROS in oxygen sensing in cell systems with sensitivity to physiological hypoxia

Reactive oxygen species (ROS) are oxygen-containing molecular entities which are more potent and effective oxidizing agents than is molecular oxygen itself. With the exception of phagocytic cells, where ROS play an important physiological role in defense reactions, ROS have classically been considered undesirable byproducts of cell metabolism, existing several cellular mechanisms aimed to dispose them. Recently, however, ROS have been considered important intracellular signaling molecules, which may act as mediators or second messengers in many cell functions. This is the proposed role for ROS in oxygen sensing in systems, such as carotid body chemoreceptor cells, pulmonary artery smooth muscle cells, and erythropoietin-producing cells. These unique cells comprise essential parts of homeostatic loops directed to maintain oxygen levels in multicellular organisms in situations of hypoxia. The present article examines the possible significance of ROS in these three cell systems, and proposes a set of criteria that ROS should satisfy for their consideration as mediators in hypoxic transduction cascades. In none of the three cell types do ROS satisfy these criteria, and thus it appears that alternative mechanisms are responsible for the transduction cascades linking hypoxia to the release of neurotransmitters in chemoreceptor cells, contraction in pulmonary artery smooth muscle cells and erythropoietin secretion in erythropoietin producing cells.     

Mechanisms of cell signaling by nitric oxide and peroxynitrite: from mitochondria to MAP kinases

Many of the biological and pathological effects of nitric oxide (NO) are mediated through cell signaling pathways that are initiated by NO reacting with metalloproteins. More recently, it has been recognized that the reaction of NO with free radicals such as superoxide and the lipid peroxyl radical also has the potential to modulate redox signaling. Although it is clear that NO can exert both cytotoxic and cytoprotective actions, the focus of this overview are those reactions that could lead to protection of the cell against oxidative stress in the vasculature. This will include the induction of antioxidant defenses such as glutathione, activation of mitogen-activated protein kinases in response to blood flow, and modulation of mitochondrial function and its impact on apoptosis. Models are presented that show the increased synthesis of glutathione in response to shear stress and inhibition of cytochrome c release from mitochondria. It appears that in the vasculature NO-dependent signaling pathways are of three types: (i) those involving NO itself, leading to modulation of mitochondrial respiration and soluble guanylate cyclase; (ii) those that involve S-nitrosation, including inhibition of caspases; and (iii) autocrine signaling that involves the intracellular formation of peroxynitrite and the activation of the mitogen-activated protein kinases. Taken together, NO plays a major role in the modulation of redox cell signaling through a number of distinct pathways in a cellular setting.