MINIREVIEW Marine toxins and the cytoskeleton: okadaic acid and dinophysistoxins Carmen Vale and Luis M. Botana Departamento de Farmacologı ´ a, Facultad de Veterinaria, USC, Lugo, Spain Introduction The syndrome diarrheic shellfish poisoning (DSP) was first recognized in Japan 30 years ago. Although fatali- ties associated with DSP-contaminated shellfish have not been reported, this intoxication has become a seri- ous problem for public health and for the economy of aquaculture industries in several parts of the world. Symptoms of DSP poisoning are mainly gastrointesti- nal problems such as diarrhea, nausea, vomiting, and abdominal pain. The major toxin involved in DSP is a polyether derivative named dinophysistoxin-1 (DTX1). Another previously identified polyether fatty acid com- pound, named okadaic acid (OA), was found to be one of the toxic components of DSP [1]. OA was first isolated from the marine sponges Halichondria okadaii and Halichondria melanodocia, and it was subsequently shown to be produced by marine dinoflagellates of the genera Dinophysis and Prorocentrum [2,3]. DTX1 was confirmed to be 35S-methylokadaic acid [1]. The molecular structures of OA and its analogs are shown in Fig. 1. Molecular and cellular effects of diarrheic shellfish toxin exposure The Ser/Thr protein phosphatases Ser ⁄ Thr protein phosphatases represent a class of enzymes in eukaryotic cells that catalyze the dephos- Keywords actin; cytoskeleton; diarrheic shellfish poisoning; dinophysistoxins; DSP; methyl okadaate; microtubules; OA; okadaic acid; phycotoxin Correspondence C. Vale, Departamento de Farmacologı ´ a, Facultad de Veterinaria, Campus Universitario s/n 27002, USC, Lugo, Spain Fax ⁄ Tel: +34 982 252 242 E-mail: mdelcarmen.vale@usc.es (Received 4 July 2008, revised 15 September 2008, accepted 25 September 2008) doi:10.1111/j.1742-4658.2008.06711.x Okadaic acid (OA) and its analogs, the dinophysistoxins, are potent inhibi- tors of protein phosphatases 1 and 2A. This action is well known to cause diarrhea and gastrointestinal symptons when the toxins reach the digestive tract by ingestion of mollusks. A less well-known effect of these group of toxins is their effect in the cytoskeleton. OA has been shown to stimulate cell motility, loss of stabilization of focal adhesions and a consequent loss of cytoskeletal organization due to an alteration in the tyrosine-phosphory- lated state of the focal adhesion kinases and paxillin. OA causes cell round- ing and loss of barrier properties through mechanisms that probably involve disruption of filamentous actin (F-actin) and ⁄ or hyperphosphoryla- tion and activation of kinases that stimulate tight junction disassembly. Neither methyl okadaate (a weak phosphatase inhibitor) nor OA modify the total amount of F-actin, but both toxins cause similar changes in the F-actin cytoskeleton, with strong retraction and rounding, and in many cases cell detachment. OA and dinophysistoxin-1 (35S-methylokadaic acid) cause rapid changes in the structural organization of intermediate fila- ments, followed by a loss of microtubules, solubilization of intermediate filament proteins, and disruption of desmosomes. The detailed pathways that coordinate all these effects are not yet known. Abbreviations AD, Alzheimer’s disease; DSP, diarrheic shellfish poisoning; DTX1, dinophysistoxin-1; F-actin, filamentous actin; FAK, focal adhesion kinase; IF, intermediate filament; OA, okadaic acid; TPA, 12-O-tetradecanoylphorbol-13-acetate. 6060 FEBS Journal 275 (2008) 6060–6066 ª 2008 The Authors Journal compilation ª 2008 FEBS phorylation of phosphoserine or phosphothreonine residues. In mammalian cells, four major classes of Ser ⁄ Thr protein phosphatases, termed PP1, PP2A, PP2B (calcineurin) and PP2C, have been identified. The number of physiological processes in which the Ser ⁄ Thr protein phosphatases are involved is immense, including regulation of glycogen metabolism and coor- dination of the cell cycle and gene expression. The crit- ical importance of phosphatases in cell metabolism is underlined by the fact that they are targets of natural toxins such as OA. Protein phosphorylation and dephosphorylation events have been established as key factors in the regulation of cytoskeletal structure and function [4]. In this minireview, we focus on how diar- rheic shellfish toxins (OA, dinophysistoxins and ana- logs) affect Ser ⁄ Thr protein phosphatases and their effects on cell adhesion and cytoskeletal dynamics, the disruption of which is linked to loss of cell polarity, and increased cell motility and invasiveness. Molecular targets of OA and dinophysistoxin OA was first identified as a potent inhibitor of Ser ⁄ Thr protein phosphatases about 30 years ago. The toxic activity of OA is often attributed to inhibition of two major Ser ⁄ Thr protein phosphatases present in mamma- lian cells, PP1 and PP2A, with IC 50 values of 0.2 and 20 nm for PP2Ac and PP1c inhibition, respectively [5–7]. Being hydrophobic, OA can enter cells, and it has been shown that it blocks the dephosphorylation of pro- teins that are substrates for several protein kinases [8,9]. Recent advances in the analysis of the molecular interac- tions of OA with Ser ⁄ Thr protein phosphatases have contributed to our understanding of the role of these enzymes in cellular homeostais. Among the substrates already identified in different cell types, the cytoskeleton plays a pivotal role in the cellular response to OA. In addition, OA and DTX1 belong to the class of non-12- O-tetradecanoylphorbol-13-acetate (TPA)-type tumor promoters, which do not bind to the phorbol ester receptors in cell membranes or activate protein kinase C in vitro. They have potent tumor-promoting activities on mouse skin, as strong as those of TPA-type tumor promoters. It is well known that transformation of cells requires notable changes in their cytoskeletal organiza- tion and adhesive properties, and this fact has led to sev- eral studies exploring the mechanism of OA-induced cytoskeletal alterations. Not surprisingly, the decreased protein phosphatase activity observed in human carci- noma, metastatic and melanoma BL6 cells is associated with increased cell motility and invasiveness. OA-medi- ated PP2A inhibition also enhances cell motility. In these cells, altered PP2A activity induced by pharmaco- logical treatment with OA is accompanied by decreased cell adhesion and cytoskeletal reorganization [10]. Interaction of diarrheic shellfish toxins with cytoskeletal dynamics and organization Although there have been numerous studies employing DSPs, and mainly OA, to examine the role of protein phosphatases in cytoskeletal dynamics and cytoskeletal organization, most of the reports were focused on the role of phosphatases in the maintenance of cell cyto- skeleton integrity. With this purpose, DSP toxins have been widely employed, with OA being one of the most useful tools. As the only known targets for OA are Ser ⁄ Thr protein phosphatases, its toxic effects have been usually related to the inhibition of PP1 and PP2A, which are considered to account for most of the Ser ⁄ Thr protein phosphatase activity in mammalian cells. However, the fact that nonphosphatase targets are not known for OA does not mean that they do not exist. Below we review some of the findings related to the interaction of DSP toxins with cytoskeleton ele- ments, effects that are attributed almost exclusively to their action as phosphatase inhibitors. Toxin R 1 R 2 HHOAO DTX 1 OH CH 3 Methyl okadaate 3 H OH OH O OH O O O O OO O OH R 2 R 1 OH O O O O O OO O OH RR OCH Fig. 1. Molecular structures of OA and analogs. C. Vale and L. M. Botana DSPs and the cytoskeleton FEBS Journal 275 (2008) 6060–6066 ª 2008 The Authors Journal compilation ª 2008 FEBS 6061 Diarrheic shellfish toxins and cell adhesion The adherence of cells to each other and to the elabo- rate mesh that comprises the extracellular matrix is mediated by multiprotein complexes. Taking advantage of the effects of DSP toxins on protein phosphatase activity, different studies have employed these toxins (mainly OA) to examine cell–matrix interactions and cell–cell contacts, either directly by controlling struc- tural adhesion proteins, or indirectly by affecting pro- teins involved in the signaling pathways that regulate cell adhesion. Cell adhesion to the extracellular matrix leads to the formation and stabilization of focal adhesions, special- ized sites of convergence for the actin cytoskeleton, integrins and several interconnection protein com- plexes. Thus, focal adhesions provide sites of signal transduction that play a pivotal role in several cellular functions, including cytoskeletal tension. At the center of this transduction pathway is the focal adhesion kinase (FAK), which, through interactions with several other proteins, regulates cell motility. In endothelial cells, OA has been shown to stimulate cell motility [11]. Similarly, OA caused the loss of stabilization of focal adhesions and a consequent loss of cytoskeletal organization in keratinocytes, due to an alteration in the tyrosine-phosphorylated state of the focal adhesion proteins FAK and paxillin [12]. Cell–cell interactions are mediated by tight junc- tions, adherens junctions, desmosomes, and gap junc- tions. Again, several studies have explored the effects of DSP toxins on cell–cell interactions, taking advan- tage of the inhibitory effects of DSP toxins on phos- phatase activity. Among the structures implicated in cell–cell interactions, tight junctions are specialized contact sites between the cell membranes of adjacent cells in which the intercellular space is absent. High concentrations or prolonged incubations of epithelial cells with OA induce cell rounding and loss of barrier properties [13,14] through mechanisms that probably involve disruption of filamentous actin (F-actin) and ⁄ or hyperphosphorylation and activation of kinases that stimulate tight junction disassembly. In adherens junctions, E-cadherins connect to actin filaments by way of proteins called catenins. Treatment of keratino- cytes with OA decreases E-cadherin phosphorylation and causes adherens junction disruption [15] without affecting the expression levels of E-cadherin or its membrane distribution [10]. Similarly, OA has been shown to inhibit desmosome assembly in MDCK cells [16]. Desmosomes are intercellular junctions that pro- vide mechanical integrity to tissues by anchoring inter- mediate filaments (IFs) to sites of strong adhesion. The OA-induced desmosome dissasembly was presum- ably regulated by extracellular Ca 2+ via reversible pro- tein phosphorylation involving both protein kinases and protein phosphatases. Inhibition of endothelial cell PP2A by treatment with OA stimulated endothelial cell motility through mechanisms related to the focal adhe- sion proteins. Thus, it was found that OA inhibition of PP2A caused hyperphosphorylation of the paxillin Ser residues and dephosphorylation of its Tyr residues, causing dissolution of FAK–Src–paxillin that will eventually increase cell motility through increases in the activities of accessory protein complexes [11]. DSP toxins and cytoskeletal dynamics Cytoskeletal function and integrity rely on the inter- play of three filament systems, microtubules, microfila- ments, and IFs, which are integrated in a complex network regulated by associated proteins. Cytoskeletal structures play key roles in the maintenance of cell architecture, adhesion, migration, differentiation, divi- sion, and organelle transport. Cytoskeletal function is directly regulated by DSP toxins, presumably trough their interaction with Ser ⁄ Thr protein phosphatases, as assumed in numerous studies employing DSP toxins to examine cytoskeletal dynamics and integrity [17,18]. Diarrheic shellfish toxins and actin As Ser ⁄ Thr protein phosphatases are some of the main cytosolic enzymes involved in actin dynamics, numer- ous studies have examined the effect of OA on the actin cytoskeleton [9]. Thus, incubation of blood cells [19,20], hepatocytes [21], neuroblastoma cells [18,22,23] and other cell types with OA leads to F-actin dis- organization, cell rounding, and loss of cell polarity. OA-induced changes in F-actin have been extensively reported, and all of these reports demonstrate that OA-induced disruption of the F-actin cytoskeleton is a common event in a wide variety of tissues, thus con- firming the direct link between protein phosphatase inhibition and cytoskeletal changes. In fact, the well- documented effect of OA on the actin network even constitutes a diagnostic tool for the presence of DSP toxins in contaminated samples [24]. Recent studies in our laboratory have investigated the effect of OA and its methyl derivative, methyl okadaate, on the actin cytoskeleton in human neuroblastoma cells [18]. The results indicated that neither methyl okadaate nor OA modified the total amount of F-actin in neuroblastoma cells; however, both toxins caused similar changes to the F-actin cytoskeleton, with methyl okadaate being approximately 10-fold less potent than OA when DSPs and the cytoskeleton C. Vale and L. M. Botana 6062 FEBS Journal 275 (2008) 6060–6066 ª 2008 The Authors Journal compilation ª 2008 FEBS inducing morphological changes. Whereas control cells showed a flattened shape with multiple elongations around them, after treatment with 15 lm methyl okadaate or 1.5 lm OA for 4 h, cells showed strong retraction and rounding, and in many cases cell detach- ment was observed, with a subsequent reduction in cell number. This observation raised the question of whether OA-induced cytoskeletal changes can be exclu- sively attributed to its inhibition of protein phosphatase activity, as methyl okadaate has been reported not to inhibit PP1 and is a very poor inhibitor of PP2A in vitro [25]. However, both toxins showed similar levels of Ser ⁄ Thr phosphorylation on neuroblastoma cells [18]. This observation might support the idea that OA- and methyl okadaate-induced cytoskeletal changes could be due to their effect on phosphatases, although the effect of methyl okadaate on protein phosphatase inhibition might have been underestimated previously. In spite of the well-documented effect of OA on the actin cytoskeleton, the exact pathway leading to OA- induced cytoskeletal changes has not been elucidated. As rearrangement of the actin cytoskeleton can be induced by increases in the cytosolic Ca 2+ concentra- tion, the effects of OA and methyl okadaate on cyto- solic Ca 2+ have been also evaluated in neuroblastoma cells. None of the toxins modified the intracellular Ca 2+ concentration, indicating that Ca 2+ influx is not responsible for OA-induced F-actin reorganization [18]. In addition, neither OA nor methyl okadaate affected the cytosolic Ca 2+ concentration in primary cultures of cerebellar granule cells (Fig. 2), a fully characterized neuronal model that is widely used to study the effect of toxins on neuronal function. Similar alterations in the actin cytoskeleton were produced by OA and DTX1 in the human cell lines HEp-2 and Caco-2, derived from larynx and colon carcinomas respectively [26]. Although the relationship between cell viability and cytoskeletal alterations induced by DSP toxins had not been examined in detail, Oteri et al. [26] found that the DSP toxin-induced morpho- logical alterations could be detected earlier than the viability alterations. This observation was corrobo- rated by recent findings in primary cultures of neuro- nal cells, where we observed that 24 h of exposure of the neurons to different concentrations of OA caused a complete abolition of cell viability, whereas methyl okadaate at similar concentrations did not modify cellular viability (Fig. 3). However, exposure of the neurons to either 50 nm OA or the same amount of methyl okadaate for 1 h was enough to produce mor- phological changes in these neurons, with rounding of the cells and loss of neurites (data not shown). From these studies, it could be concluded that methyl okada- ate induces actin cytoskeleton rearrangement and mor- phological changes that are independent of cytosolic Ca 2+ but might be related to the increases in the levels of Ser ⁄ Thr phosphorylation of several cellular pro- teins. However, in view of the reported inhibition of PP2A and PP1 by methyl okadaate in vitro, it is possi- ble that more cellular targets for this OA derivative could exist. DSPs and microtubules In eukaryotic cells, microtubules form a well-organized network that is highly regulated both spatially and temporally. The microtubule is a dynamically regulated structure composed of a- and b-tubulins. Microtubules are stabilized by specific factors, including micro- tubule-associated proteins, such as tau, and post-trans- lational modifications (a-tubulin acetylation and detyrosination), and destabilized by dissociation of tau from microtubules or a-tubulin tyrosination. Accumu- Fig. 2. Time course of the effects of OA and methyl okadaate on the cytosolic Ca 2+ concentration in primary cultures of cerebellar granule cells. Intracellular Ca 2+ was monitored in neurons loaded with Fura-2. Mean ± SEM of three experiments. Fig. 3. Effects of different concentrations of OA and methyl okada- ate on cell viability in primary cultures of cerebellar granule cells. Cellular viability was assessed by the 3-(4,5-dimethylthiazole-2-yl)- 2,5-diphenyltetrazolium bromide assay after 24 h of exposure of the cells to different concentrations of the toxins. Values are means ± SEM of three independent experiments. C. Vale and L. M. Botana DSPs and the cytoskeleton FEBS Journal 275 (2008) 6060–6066 ª 2008 The Authors Journal compilation ª 2008 FEBS 6063 lating evidence indicates that Ser ⁄ Thr protein phospha- tases, such as PP1, PP2A, and PP2B, participate in the neurodegenerative process in Alzheimer’s disease (AD). OA, through its interaction with phosphatases, has emerged as an important research tool in the study of microtubule dynamics and microtubule-related dis- eases. In fact, OA is currently used in models of AD research to increase the degree of phosphorylation of various proteins, such as tau. One of the hallmarks of AD and tauopathies is the appearance of highly phos- phorylated tau isoforms in paired helical filaments. This might lead to dissociation of tau and microtu- bules, and subsequent cytoskeletal instability. Aberrant tau phosphorylation can be induced in several cellular models by treatment with OA [27]. The activities of protein phosphatases are compromised in the AD brain, and in metabolically active brain slices from adult rats, the inhibition of PP2A activity by OA pro- duces the abnormal hyperphosphorylation of tau that inhibits its binding and the promotion of microtubule assembly in vitro [28]. In primary cultures of cerebellar granule cells, treatment of the cells with 50 nm OA or 50 nm methyl okadaate for 1 h (Fig. 4) caused modifi- cations in the distribution of tau in these cells. The redistribution of tau inmunoreactivity after the treat- ment was accompanied by cell shrinkage and loss of neuronal prolongations after very short periods of time. These observations are in accordance with recent studies indicating an increase in tyrosinated tubulins in primary cortical neurons after treatment with OA [17]. As the regulation of microtubules by phosphatase activity also plays an important role during morpho- genesis and tumorigenesis, the effect of OA has also been investigated in human carcinoma cells. As PP2A is associated with microtubules, in these cells OA treat- ment results in increased cell motility and invasiveness [29]. DSP toxins and intermediate filaments IF proteins, a large family of tissue-specific proteins, undergo several post-translational modifications, with phosphorylation being the most studied of these. IFs maintain cell shape and the structural integrity of cell contents, and provide protection against various types of stress. The mechanism of action of OA as a potent tumor promoter and the biological significance of Ser ⁄ Thr and Tyr protein phosphatases have been extensively investigated in cancer-related research. The hyperphosphorylation of IFs is one of the early biochemical changes induced by OA-class tumor pro- moters. The hyperphosphorylation of keratins induced by OA treatment resulted in the reorganization of the keratin filament network, which collapsed into large perinuclear aggregates [30]. The effects of DSP toxins on IF integrity were revealed after treatment of BHK- 21 fibroblasts with DSP toxins. OA and DTX1 caused rapid changes in the structural organization of IFs, fol- lowed by a loss of microtubules [4]. In a similar way, incubation of human fibroblasts or rat brain tumor cells with OA [31,32] promotes the hyperphosphoryla- tion of major IF proteins, leading to the disassembly of IF networks, solubilization of IF proteins, and disruption of desmosomes. Conclusion and perspectives To date, a myriad of studies have exploited the inter- action of DSP toxins with phosphatases to examine the role of these proteins in cytoskeletal integrity; A B C Fig. 4. Short-term effects of methyl okadaate and OA on the microtubule-associated proteins in primary cultures of cerebellar granule cells. Control cells (A) and cells incubated for 1 h with 50 n M OA (B) or 50 nM methyl okadaate (C) were stained for the microtubule-associated protein tau. The results are representative of three experiments. DSPs and the cytoskeleton C. Vale and L. M. Botana 6064 FEBS Journal 275 (2008) 6060–6066 ª 2008 The Authors Journal compilation ª 2008 FEBS nevertheless, very few of these reports have focused on the detailed study of the intracellular pathways involved in the reorganization of cytoskeletal compo- nents caused by DSP toxins. To date, almost all of the effects of DSP toxins on cytoskeletal dynamics and integrity have been attributed to the well-documented interaction of DSP toxins with protein phosphatases. These effects were not related to changes in the amount of polymerized actin, cytosolic Ca 2+ concen- tration, or membrane potential. However, a recent study on the effect of methyl okadaate on the cyto- skeleton, and its reported IC 50 for Ser ⁄ Thr protein phosphatases in vitro, indicated that some other cellu- lar targets for this particular compound could exist. Taking advantage of new available markers to assess cytoskeletal dynamics in living cells, further detailed studies should be performed to investigate the effects of DSP toxins on the cytoskeleton as well as the intra- cellular mechanisms involved in the cytoskeletal dis- organization caused by DSP toxins. References 1 Vale P (2007) Chemistry of diarrhetic shellfish poison- ing toxins. 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Botana Departamento de. 2008) doi:10.1111/j.1742-4658.2008.06711.x Okadaic acid (OA) and its analogs, the dinophysistoxins, are potent inhibi- tors of protein phosphatases 1 and 2A. This action is