Oxidative Stress and Cytoskeletal Alterationsa G I O R G I O BELLOMOh A N D FRANCESCA MIRABELLI Dipartimento di Medicinu Intema e Terupia Medica Clinicu Medica I University of Pavia Pavia, Italy Increasing numbers of studies suggest the involvement of multiple processes in the They are linked to the depletion pathogenesis of cell injury during oxidative of critical intracellular coenzymes as well as the activation of cytotoxic mechanisms that disrupt the structural organization and the physiological activities in different intracellular compartments. The demonstration of the multifactoriality of oxidative cell injury led to the search for and characterization of intracellular targets of the different pathophysiological processes. Among them are breakage and fragmentation of DNA,3 peroxidation of membrane lipid^,^ oxidation and fragmentation of proteins,5 mitochondria1 damage,h impairment of cell energy s t a t ~ sand , ~ disruption of ion h o m c o s t a s i ~ . ~ One of the early events in cell injury caused by oxidative stress is represented by the appearance of multiple surface protrusions called blebs.YThe pathophysiology of bleb formation has not been fully elucidated, probably because several mechanisms participate independently, but it is generally accepted that disruption of the cytoskeleta1 organization and of the membrane-cytoskeleton interaction could play a relevant role. This assumption is supported by the ultrastructural evidence of a marked reorganization of scvcral cytoskeletal elements preceding and accompanying the appearance of plasma membrane blebs“) and by the demonstration that wellknown cytoskeletal toxins such as cytochalasins and phalloidin cause blebbing in a variety of different cell types.12 Historically, these findings suggested the possibility that the cytoskeleton could actually represent an important target in oxidative stress-induced cell injury and stimulated active research to identify the biochemical mechanisms involved. CYTOSKELETAL ALTERATIONS BY ATP DEPLETION Multiple physiological reactions involving cytoskeletal proteins require ATP. For example, polymerization of monomeric actin (G-actin) to the filamentous form (F-actin) is strictly dependent on ATP.I3 Thus, the decrease in intracellular ATP concentration should result in actin depolymerization, breakdown of the actomyosin network, and dramatic alterations in subcellular organization and cell morphology. ‘Most of the studies mentioned in this paper and performed in our laboratory have been supported by grants from CNR (Special project “Biologia e Patologia del calcio”) and from MURST (Project “Patologia da radicali liberi”) to Giorgio Bellomo and from IRCCS Policlinico S. Matteo to Francesca Mirabelli. bAddress for correspondence: Dr. Giorgio Bellomo, Clinica Medica 1, Universita di Pavia, Policlinico S. Matteo, P. le Golgi 2,27100 Pavia, Italy. 97



This is what has been suggested to occur following ATP depletion in cells exposed to “chemical hypoxia” obtained with iodoacetic acid (to inhibit glycolysis) and potas~~ it is still unclear sium cyanide (to block oxidative p h o ~ p h o r y l a t i o n ) .However, whether the cytoskeletal alterations are entirely and directly dependent on ATP depletion or are linked to the disruption of intracellular ion (Ca2+)homeostasis that has been observed in these conditions.Is ATP depletion has been reported to occur in cells challenged with oxidants. At least three different mechanisms have been claimed to be involved in causing ATP depletion: direct mitochondria1 damage,lh inhibition of the glycolytic flux,” and ATP consumption following NAD+ depletion caused by activation of poly(ADP-ribose) polymcrase.lX Specific investigations to ascertain the relative contribution of ATP depletion to the induction of cytoskeletal abnormalities during oxidative stress have not been done. However, in studies of isolated rat hepatocytes exposed to the oxidative stress generated during the metabolism of redox-cycling quinones, no correlation was found between the time course of ATP depletion and bleb formation, taken as a morphological signature of cytoskeletal damage.I9 This finding argues against a major role played by ATP depletion in causing cytoskeletal alterations during oxidative cell injury.

CYTOSKELETAL ALTERATIONS BY PHOSPHORYLATION Protein phosphorylation is a posttranslational modification of proteins that integrates the biological responses to extra- and intracellular stimuli by modulating the expression of specific functions of various proteins and enzymes.2o These reactions are catalyzed by a variety of protein kinases, and the degree of protein phosphorylation is strictly controlled by the balance between the activity of the kinases and that of the counteracting phosphatases.2’ It is well known that abnormal protein phosphorylation caused by a variety of stimuli (xenobiotics, mutations, introduction of viral genomes) can be responsible for uncontrolled cell growth and eventually cancer.22In addition, abnormal phosphorylation reactions have also been detected in Alzheimer’s disease, in glutamate and aluminium toxicity of the nervous system, and in other neurodegenerative disorden2? Recent investigations report that activation of protein kinases occurs during oxidative stress. For instance, activation of protein kinasc C has been detected during oxidative stress caused by the metabolism of redox-cycling quinones in h e p a t ~ c y t e s and , ~ ~ Cerutti et a1.2shave demonstrated enhanced phosphorylation of the major ribosomal protein S6 by the Ca2+ and calmodulin-dependent protein kinase in JB6 cells exposed t o oxygen radicals. A common feature in most of the aforementioned conditions is the overphosphorylation of different cytoskeletal polypeptides,23and this led to the hypothesis that abnormal phosphorylation of cytoskeletal structures may represent an important step in the development of cell injury. Several cytoskeletal proteins are substrates for phosphorylation catalyzed by Ca2+/calmodulin-dependentprotein kinase,2“2x protein kinase C (PKC),29-31CAMPdependent protein kinase (PKA),32-36tyrosine- and serine-protein k i n a s e ~ , ~ and ’~’ cdc2 protein k i n a ~ eTABLE . ~ ~ ~ ~ some of the biologic events associated with the 1 lists phosphorylation of cytoskeletal proteins by these kinases. Interestingly, some of the reported effects, such as alterations in actin, tubulin, and intermediate filament protein polymerization, may obviously represent a toxic event. The concept that abnormal phosphorylation of cytoskeletal elements may result



in abnormal cytoskeletal function and ccll injury has cmcrged from recent invcstigations using specific toxins. Microcystin-LR, a cyclic peptide toxin isolated from certain species of cyanobacteria (blue-green algae), is reported to induce blebbing and a rapid reorganization of microfilaments in isolated rat hepatocytes.jh Thesc effects are apparcntly independent of any appreciable variations in intraccllular free Ca2+ concentration, ATP level, oxidation, or alkylation of macromolcculcs. Thc subsequent dcmonstration that microcystin-LR is a potent inhibitor of type 1 and type 2A protein pho~phatases~' Icd to the proposal that an altcrcd balance of protein kinase/phosphatase activity could induce abnormal phosphorylation of various cellular proteins (including cytoskeletal proteins), ultimately leading to cell injury and death. This conclusion seems t o be supported by the demonstration that

TABLE1. Examples of Kinase-Mediated Phosphorylation o f Cytoskclctal Proteins Proteins Microfilament proteins Actin Vinculin Fragmin Talin Caldesmon ABP Microtubule proteins MAP- 1A MAP-IB MAP-2 Tau Synapsin I Tubulin Intermediate filament proteins Vimentin Cytokeratins Lamins NF-L

Kinase( s) Involved

Biological Effects

PKA PKC Serine kinase cdc2 kinase Actin kinase

Modulation of actin polyrnerization Modulation of interaction between cytoskeleton and plasma membrane Inhibition of Ca'+-dependent proteolysis

Ca? +-,CaM kinase Serine kinase Tyrosine kinase cdc2 kinase Casein kinase I 1 PKA

Modulation of microtubule polymerization Modulation of organelle movement

PKA PKC Ca?+-.CaM kinase cdc2 kinase Serine kinase

IF depolymerization Breakdown of nuclear lamina Change in genome exposure

calyculin-A, another inhibitor of cellular phosphatases, caused enhanced phosphorylation of virnentin and 20-kD myosin light chain in 3T3 fibroblasts and promoted their detachment from thc growing substrate.4x CYTOSKELETAL ALTERATIONS BY CaZ+-DEPENDENTMECHANISMS

About 10 years ago we hypothesized that Ca2+could play a relevant role in bleb formation and cytoskclctal alterations during oxidative stress.4yThis assumption was supported by the dcmonstration that treatment o f cultured and freshly isolatcd cells



with the Ca2+ ionophore A23187 induced blebbing that was prevented by the omission of Ca2+from the incubation m e d i ~ m . 4Moreover, ~ marked alterations in intracellular Ca2+ homeostasis and a sustained increase in cytosolic-free Ca2+ concentration were demonstrated to precede bleb formation during cell injury caused by o ~ i d a n t s Furthermore, .~~~~~ the omission of Ca2+ from the incubation medium or loading of target cells with intracellular Ca2+ chelators prevented or delayed the appearance of blebbing.11Js~s2 Finally, an increased number of investigations clearly demonstrated the occurrence of several Ca2+-dependent events in the control of cytoskeletal organization and function.s3In particular, a key role for Ca2+ was detected in the sequence of events that control the appearance of plasma membrane protrusions in physiological conditions such as pseudopod formation following platelet activations4 and neurite outgrowth in mouse NB2a/dl neuroblastoma cells.s5

Ca2+-DependentModification of the Association between Actin and Actin-Binding Proteins during Oxidative Stress

Several among the different actin-binding proteins are Ca2+-dependent.Typical examples are caldesmon (that binds actin and prevents subsequent myosin binding),56 gelsolin (that severs actin micro filament^),^^ and villin (that severs actin microfilaments into short fragment^).^^ Other actin-binding proteins are directly involved in the association of microfilaments to the plasma membrane in a Ca2+dependent manner. One of them is alpha-actinin which, in addition, cross-links actin filaments into regular parallel arrays.s9 When the cytosolic-free Ca2+ concentration increases above the physiological levels, gelsolin and villin (in specialized cells) bind in a Ca2+-dependentmanner to actin and shorten actin filaments. The interaction between the actin filaments and alpha-actinin is strongly inhibited by Ca2+,and even a transient increase in cytosolicfree Ca2+ to micromolar levels promotes the dissociation of these two proteins.s9 Experimental work performed in these last few years has provided evidence for the occurrence of such a phenomenon in cells exposed to oxidative stress. The incubation of human platelets with the oxidant menadione, which promoted a marked increase in cytosolic-free Ca2+concentration, resulted in the dissociation of alphaactinin from the whole cytoskeleton.60 These changes were largely inhibited by conditions that prevented the increase in cytosolic-free Ca2+ and by intracellular Ca2+chelators. Furthermore, immunocytochemical investigations using anti-alphaactinin antibodies and NBD-phallacidin to stain actin revealed a dissociation of alpha-actinin from the actin filaments and suggested that this phenomenon could represent one of the causative factors responsible for bleb f0rmati0n.l~ Another example of Ca2+-dependentregulation of the actin-binding proteins was recently obtained by Harris and Morrow6' with fodrin. Fodrin is a ubiquitous cytoskeletal polypeptide able to link integral membrane proteins to cortical actin filaments and for this reason is involved in organizing receptor domains and in controlling vesicle traffic at the plasma membrane. As a result of an increase in cytosolic-free Ca2+,fodrin binds calmodulin. This process results in the loss of the ability of fodrin to bind actin and in the dissociation of microfilaments from the integral membrane proteins.


Activation of Ca”-Dependent Proteases Interest in calcium-dependent neutral proteases, also called calpains, has increased in recent years. Among their properties are an absolute dependence on Ca2+ for activity and an optimum p H between 7 and 8.62Among the various endogenous substrates of calpains are several cytoskeletal proteins including spectrin, fodrin, caldesmon, adducin, tubulin, microtubule-associated protein 2 (MAP-2), tau factor, vimentin, and c y t ~ k e r a t i n s . ~Two ~ - ~other ~ cytoskeletal proteins that are directly involved in the anchoring of microfilaments to the inner surface of plasma membrane, namely, vinculin and actin-binding protein (ABP, in platelets), have been described to represent preferential substrates for Ca2+-dependent pro tease^.^^ An increase in the cytosolic-free Ca2+ concentration to the micromolar level (high enough to activate the protease) would result in the proteolysis of these two polypeptides. This is what physiologically happens during platelet activations4 and toxicologically during oxidative stress caused by the metabolism of the redox-cycling compound menadione in CG5 cells” and platelets.60 Investigations by Steenbergen and coworkershSperformed on the canine heart during ischemia and reperfusion using anti-vinculin antibodies revealed a progressive loss of vinculin staining along the lateral margin of myocytes. A role for Caz+-activated protease has been proposed to explain the loss of vinculin because this protein is a substrate for protease and because cytosolic Ca2+ concentration during ischemia and reperfusion increases well above the critical level necessary for protease activation. The loss in vinculin staining was associated with the appearance of subsarcolemmal blebs and breaks in the plasma membrane. The notion that proteolysis of cytoskeletal proteins may be one of the pathophysiological factors in bleb formation during chemical injury is also supported by the demonstrations that indeed a Ca2+-dependentproteolysis of actin-binding proteins occurs in these conditions60 and that inhibitors of calpains efficiently protect against blebbing.66

CYTOSKELETAL ALTERATIONS BY THIOL OXIDATION The tripeptide glutathione (GSH) and a variety of GSH-dependent enzymes (peroxidases, reductases, transferases) represent important intracellular defense mechanisms against the toxicity of oxygen-reactive species.“’ As a result of peroxidase activity, GSSG is formed, and the intracellular GSH/GSSG balance is drastically shifted toward oxidation.68 Hinshaw and coworkers6Yrecently reported that increasing amounts of polymerized actin can be detected in P388D1 cells exposed to hydrogen peroxide o r to the oxidant diamide. Interestingly, this effect was separate from the ATP-dependent gross disruption of microfilaments and appeared to be strictly dependent on the oxidation of cellular glutathione and on the decrease of the GSH/GSSG ratio. In fact, conditions that maintained the glutathione couple in the reduced status also prevented actin polymerization. In conditions of “mild” oxidative stress, GSSG formed during the various peroxidase-catalyzed reactions is reduced to GSH by the activity of glutathione reductase at the expense of NADPH. However, when the generation of peroxides or other reactive species is particularly high, or when the availability of NADPH becomes rate-limiting, GSSG is formed in high amounts and subsequently extruded in the extracellular space.68 In these conditions of glutathione depletion, the cell


102 SH HS-Act-SH







Intramoleculardisulfide bond


FIGURE 1. Schematic representation of oxidative modifications of thiol groups in



SH I I HS-Act-S-S-Act-SH I


Intermoleculardisulfide bond Actin dimer

SH Intermoleculardisulfide bond Oxidative cross-linking

becomes more susceptible to the oxidative insult, and the thiol groups in cellular proteins represent preferential targets for oxidative modifications. In a series of studies performed in our laboratory we demonstrated that thiol groups in actin are oxidatively modified during the metabolism of peroxides and redox-cycling [email protected]~ As illustrated in FIGURE1, several mechanisms are involved in this process. The formation of an intramolecular disulfide bridge or a mixed disulfide with glutathione or other disulfides causes a modification of the physicochemical properties of the actin molecule, resulting in faster migration in an SDS-polyacrylamidegel (fast-migrating actin) .73 Furthermore, disulfide bridge formation between different actin molecules and with actin-binding proteins6()or other noncytoskeletal proteins73 has been found in several cells exposed to a variety of oxidizing conditions. This oxidative cross-linking caused the formation of high molecular weight protein aggregates that could easily be detectable when the cytoskeletal fraction isolated from oxidant-challenged cells was analyzed by means of polyacrylamide gel electrophore~is.~~ These aggregates and patches of actin bundles could also be observed morphologically by immunocytochemistry in treated cells.74 The formation of protein aggregates was clearly dependent on intermolecular disulfide bonds, because thiol reductants such as dithiothreitol led to the disgregation of these complexes and to the release of actin, actin-binding proteins, and other noncytoskeletal proteins.60 Thiol oxidation by oxygen-reactive species has also been detected in microtubular and intermediate-sized filament proteins.74As for microtubules, it is conceivable to hypothesize that oxidation of thiol groups in tubulin and in microtubuleassociated proteins (MAPS) could induce marked alterations in microtubule organization. Immunocytochemical studies performed in cultured mammalian cells have, in fact, demonstrated complete depolymerization of microtubules after exposure to oxidative stress.74Previous studies performed in several laboratories revealed that the oxidation of some of the 22-SH residues in tubulin, in addition to causing microtubule depolymerization, also affected the biochemical process of microtubule formation from cytosolic precursors induced by GTP.75 Complete inhibition of microtubule formation triggered by GTP has been observed when cytosolic precursors from oxidative stress-exposed cells were employed. Interestingly, this inhibition was completely reversed when dithiothreitol was included in the reaction medium.74



It was recently shown that, in addition to direct oxidation, thiol groups in tubulin can also be alkylated by a series of 4-hydroxy-alkenals produced during peroxidation of membrane lipids.7o It thus appears that oxidative stress can affect cytoskeletal protein thiols by either direct or indirect reactions.

DIFFERENT SUSCEPTIBILITY OF SULFHYDRYL GROUPS IN NUCLEAR MATRIX PROTEIN TO OXIDATIVE MODIFICATIONS In addition to microfilaments, microtubules, and intermediate filaments, another class of structural elements exists within the cell, the nuclear matrix scaffold,77which is bounded by an outer nuclear lamina connected to the cytoskeletal framework and is composed of a mesh network of inner filaments. The filaments range in diameter from 3-22 nm and are organized in an anastomosing network in which nucleoli are embedded. The nuclear lamina is apparently composed of intermediate-type filaments and lines the nucleoplasmic surface of the nuclear envelope.7xA physiological key role has been attributed to the nuclear matrix because it appears involved in the control of fundamental processes such as the regulation of DNA superhelicity, DNA replication, transcription, and R N A processing and transport.7y We have employed a procedure, adapted from one originally developed by Staufenbiel and Depper,x‘l to isolate nuclear matrix proteins from cultured cells. These procedures involved subsequent “dissection” of the entire cytoskeletal fraction isolated from starting cells, which ultimately led to the biochemical differentiation between an intermediate-filament fraction and a nuclear matrix fraction, on the basis of different solubility of the proteins in a urea-free medium.x1As reported in TABLE 2, it is clear that the amount of sulfhydryl groups present in nuclear matrix proteins is significantly higher than that in all other protein fractions. This unexpected finding was confirmed by a more sophisticated analysis of the thiol groups in structural proteins by digitized video microscopic imaging of cells labeled with the fluorescent indicator monobromobimane (FIG.2 ) . Interestingly, the -SH rcsidues of nuclear matrix proteins were extremely resistant to oxidative depletion induced by the oxygen-free radical-generating compound menadione (TABLE2). It is unlikely that the different susceptibility of this subset of cellular protein thiols to oxidation could be linked exclusively to inaccessibility of these proteins to

TABLE2. Depletion of Sulfhydryl Groups in Different Cytoskeletal Structures Isolated from Rat Hepatocytes Exposed to 2-Methyl-l,4-naphtoquinone (Menadione)” Protein Sulfhydryl Groups (nmolimg protein) Fractions Total cellular proteins Whole cytoskeleton KCI-extracted cytoskeleton Intermediate filaments Nuclear matrix proteins

Control Cells 982 842 97rt 81 rt 1142

II 10

11 10 18

MenadioneTreated Cells 54 rt 9 39 2 9 50 9 41 2 7 101 ’’ 10


“Isolated rat hepatocytes were incubated for 60 minutes without or with 0.2 mM menadione and then processed for the sequential extraction of the fractions indicated in the table. For experimental details, see Vairetti and coworkers.x1

with an extraction buffer containing 1% Triton X-100 to solubilize noncytoskeletal proteinss1 and then fixed in ethanol/acetic acid (3:l). The fixed cells were treated with 0.2 mM monobromobimane that gives a strong fluorescence upon reaction with -SH groups (excitation: 393 nm; DM 455 dichroic mirror, 470 nm barrier filter). The obtained fluorescent images (A) were taken using an MTI SIT66videocamera, processed using an IT1 150 imaging system, and depicted using a pseudocolor scale. The same cells were subsequently treated with 0.05 mM fluorescein isothiocyanate to label proteins, and images were acquired (B) (excitation: 490 nm; DM 510 dichroic mirror, 52C560 nm barrier filter). The image presented in C was obtained by dividing the image in A by the image in B followed by result enhancement. It represents the distribution of thiol groups in the cytoskeletal structures expressed per protein unit.

FIGURE 2. Spatial distribution of cytoskeletal protein thiols in BT-20 (human adenocarcinoma) cells. BT-20 cells grown on coverslips were treated











A T P d r



p l clncreaae l u m coInjlclntratlon cytosollc

Activation 01 calclumdapendent protein kinase

ctln polymarlzatlon xldative croaa-llmkin 01 actin and ABPs


Microtubule epolymerlzatlo

l l

lrom ABPs Proteolysla 01 ABPs

Braakdown 01

xldatlve alterations 01 fllamenta

FIGURE 4. General scheme of oxidative stress-induced cytoskeletal alteration.

the oxygen-reactive species. In fact, despite their extremely short life, oxygen radicals easily reach the nucleus where they induce a wide variety of DNA breakages. It is more conceivable to hypothesize that a well-organized defense system may exist within the nucleus to prevent protein thiol oxidation or to “buffer” the oxidizing potential of oxygen-reactive species. It is well known, for instance, that a group of glutathione transferases and glutathione peroxidases is specifically located in the n~cleus.~~~~~ Recent work from our laboratory, using digitized video microscopic imaging of cells loaded with fluorescent glutathione indicator^?^ has provided experimental evidence of a high intranuclear concentration of glutathione, approximately three times higher than that in the cytoplasm (FIG.3). Intranuclear glutathione appears more resistant than cytoplasmic glutathione to the depletion caused by alkylating and oxidizing corn pound^.^^ Taken together, these findings suggest that nuclear matrix proteins are less sensitive to oxidative modifications of their sulfhydryl groups, because they are compartmentalized in a region of the cell where reduced glutathione and glutathionedependent defense-directed enzymes are particularly abundant. TABLE 3. Major Consequences of Oxidative Stress-Induced Cytoskeletal Alterations Alterations in transcription and gene expression Alterations in ribosome-mRNA interaction Alterations in membrane receptor turnover Alterations in membrane protein expression and distribution Organelle relocation Appearance of cell surface protrusions (blebs)


CONCLUSIONS A general scheme of pathophysiological reactions occurring during oxidative cell 4. As discussed in injury and affecting cytoskeletal proteins is presented in FIGURE the previous sections, at least four different mechanisms are involved, namely, ATP depletion, altered phosphorylation, Ca*+-dependent dissociative and degradative reactions, and thiol oxidation. Some consequences of the illustrated alterations in cytoskcletal structure and function are reported in TABLE 3. Because of the strict interconnection between cytoskeletal microfilaments and plasma membrane, the expression, distribution, and turnover of membrane receptors and proteins will take place. An altered distribution of intramembrane particles has, in fact, been, demonstrated by freeze-fracture techniques in cultured mammalian cells exposed to redox-cycling quinones. The physiological interaction bctween mRNA, ribosomes, and initiation factors will conceivably be perturbed and the translational flux will be disrupted. The cytoskeleton has strictly been involved in the intracellular trafficking of all the components of the translational machinery.84 One of the most evident (and best investigated) consequences of cytoskeletal alterations during oxidative stress is the formation of plasma membrane protrusions (blebs) whose rupture appears critical in precipitating cell death.y In these conditions, the formation of patches of bundled actin and the dissociation of the microfilament mesh network from the inner surface of the plasma membrane would generate sites of weakness where blebs would protrude and progress. This is a dramatic event that occurs during acute and marked oxidative injury. However, the molecular and biochemical processes that follow cytoskeletal alterations during oxidative stress that is not immediately associated with cell death are still largely unknown and poorly investigated.


We want to thank S. Orrenius, P. Nicotera, H. Thor, E. Albano, A. Benedetti, R. Fulceri, 0. Cantoni, W. Malorni, F. Iosi, M. Perotti, F. Taddei, M. Vairetti, and P. Richelmi for the work done in collaboration and for helpful discussions. REFERENCES

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Oxidative stress and cytoskeletal alterations.

Oxidative Stress and Cytoskeletal Alterationsa G I O R G I O BELLOMOh A N D FRANCESCA MIRABELLI Dipartimento di Medicinu Intema e Terupia Medica Clini...
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