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
Trang 1Marine 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 activahyperphosphoryla-tion of kinases that stimulate tight junchyperphosphoryla-tion 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.
Trang 2phorylation 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 IC50 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
H H O A
DTX 1 OH CH 3
Methyl okadaate 3 H
OH
OH O
OH O
O
O O
O
R1
OH
O O
O
O O
O
OCH
Fig 1 Molecular structures of OA and
analogs.
Trang 3Diarrheic 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
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 Ca2+via reversible pro-tein phosphorylation involving both propro-tein 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
Trang 4inducing 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 Ca2+
concentra-tion, the effects of OA and methyl okadaate on
cyto-solic Ca2+have been also evaluated in neuroblastoma
cells None of the toxins modified the intracellular
Ca2+concentration, indicating that Ca2+influx is not
responsible for OA-induced F-actin reorganization
[18] In addition, neither OA nor methyl okadaate
affected the cytosolic Ca2+ 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
Ca2+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 Ca2+ 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.
Trang 5lating 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 tumorimorpho-genesis, 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;
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 n M methyl okadaate (C) were stained for the microtubule-associated protein tau The results are representative of three experiments.
Trang 6nevertheless, 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 Ca2+
concen-tration, or membrane potential However, a recent
study on the effect of methyl okadaate on the
cyto-skeleton, and its reported IC50 for Ser⁄ Thr protein
phosphatases in vitro, indicated that some other
cellu-lar targets for this particucellu-lar 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
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