(Received for publication, June 26, 1995; and in revised form, September 8, 1995)
From the
The widely used phosphatase 1 and 2A inhibitor okadaic acid is
one of the many stimuli activating transcription factor NF-B in
cultured cells. Phosphorylation of I
B-
, one of
NF-
B's inhibitory subunits, is a prerequisite for I
B
degradation and the subsequent liberation of transcriptionally active
NF-
B. This observation suggested that the phosphorylation status
of I
B is influenced by an okadaic acid-sensitive phosphatase. In
this study, we provide evidence that the effect of okadaic acid on
NF-
B activation is indirect and dependent on the production of
reactive oxygen intermediates rather than the inhibition of an
I
B-
phosphatase. Okadaic acid was found to be a strong
inducer of cellular H
O
and superoxide
production in two distinct cell lines. The structurally unrelated
phosphatase inhibitor calyculin A also induced oxidative stress. The
delayed onset of reactive oxygen production in response to okadaic acid
correlated with the delayed activation of NF-
B. Moreover,
NF-
B induction was optimal at the same okadaic acid concentration
that caused optimal H
O
production. Both
reactive oxygen intermediates production and NF-
B activation were
inhibited by the antioxidant pyrrolidine dithiocarbamate and
8-(diethylamino)octyl-3,4,5-trimethyoxybenzoate, a Ca
chelator. Future experiments using phosphatase inhibitors in
intact cells must consider that the compounds can act as strong
inducers of oxidative stress, which provides one explanation for their
tumor-promoting activity.
A hallmark of the higher eukaryotic transcription factor
NF-B is its rapid post-translational activation by a plethora of
distinct, mostly pathogenic stimuli, including viral and bacterial
infections, inflammatory cytokines, UV and
radiation, and
oxidants (reviewed in Liou and Baltimore(1993), Baeuerle and
Henkel(1994), and Siebenlist et al.(1994)). Many of these
stimuli cause oxidative stress in cells, i.e. there is an
increased production of ROIs, (
)such as superoxide, hydrogen
peroxide, hydroxyl radicals, and secondary reactive intermediates
(reviewed in Schreck and Baeuerle(1991) and Schreck et al. (1992a)). Four lines of evidence suggest that ROIs, in particular
H
O
, serve as common second messenger-like
molecules in the various pathways leading to NF-
B activation.
First, many structurally unrelated antioxidants suppress NF-
B
activation in response to most inducing conditions (reviewed in Schreck et al. (1992a) and Meyer et al. (1993a)).
Dithiocarbamates (Schreck et al., 1992b), NAC (Staal et
al., 1990; Schreck et al., 1991), and vitamin E
derivatives (Suzuki et al., 1993) are particularly well
studied in this respect. Second, in some cell lines NF-
B is
directly activated upon addition of micromolar amounts of
H
O
to the culture medium (Schreck et
al., 1991; Meyer et al., 1993b). Third, stable
overexpression of catalase impairs NF-
B activation while
overexpression of Cu/Zn superoxide dismutase superinduces NF-
B
activation in response to tumor necrosis factor
and OA (Schmidt et al., 1995). Last, re-exposure of hypoxic cells to dioxygen
is a very potent NF-
B-inducing condition. (
)In view of
this requirement for ROIs we wondered whether there are inducers that
bypass the ROI step and act more proximal on the NF-
B
I
B
system. A recent study by Packer and colleagues (Suzuki et
al., 1994) suggested that the phosphatase inhibitors OA and
calyculin A are such ROI-independent inducers.
In unstimulated
cells, NF-B is known to reside in a latent form in the cytoplasm
(Baeuerle and Baltimore, 1988a, 1988b). This form is stabilized by an
inhibitory subunit, called I
B, which is tightly bound to a dimer
frequently composed of the DNA-binding subunits p50 and p65 (RelA)
(reviewed in Beg et al.(1993)). Upon cell stimulation, I
B
is removed and the liberated NF-
B able to translocate into the
nucleus and activate target genes by binding to regulatory elements in
enhancers and promoters. The removal of I
B is controlled by
protein phosphorylation. A yet unidentified kinase rapidly
phosphorylates human I
B-
on serines 32 and 36 in response to
various inducers (Brown et al., 1995; Traenckner et
al., 1995). By phosphorylation, I
B-
which is still bound
to NF-
B has apparently turned into a high affinity substrate for
an ubiquitin-conjugating enzyme (Traenckner et al., 1994;
Traenckner and Baeuerle, 1995). Following this
phosphorylation-controlled ubiquitination, I
B-
is rapidly and
completely degraded by the 20 S or 26 S proteasome (Palombella et
al., 1994; Traenckner et al., 1994). Specific peptide
inhibitors of the proteasome which stabilize the phosphoform of I
B
have proven as potent inhibitors of NF-
B activation. Antioxidants
also prevent the decay of I
B, however, by inhibition of the
phosphorylation step and not the proteasome. Hence, antioxidants may
act upstream of the kinase/phosphatase system, suggesting that the
I
B phosphorylation is redox-controlled.
OA, a C fatty acid polyether compound, is a toxin which causes diarrhetic
shellfish poisoning in humans (reviewed in MacKintosh and
MacKintosh(1994) and Hardie et al. (1991)). It is produced by
dinoflagellates and accumulates in the food chain in fish via sponges.
OA and functionally related toxins, such as calyculin A, share two
biological effects. First, they are very specific and potent inhibitors
of protein phosphatases (PPs), which preferentially inhibit type 2A and
1 Ser/Thr-specific PPs. Second, OA and related toxins have nonphorbol
tumor promoting activity, meaning they do not act by direct binding to
PKC. In cell-free systems, nanomolar concentrations of OA can inhibit
PPs whereas close to micromolar concentrations are required in intact
cells (Hardie et al., 1991). This is because the concentration
of PPs in the cytoplasm is in the micromolar range. The tumor promoting
activity of OA and related toxins is not understood in detail.
OA
has been reported to be a potent activator of NF-B
(Thévenin et al., 1990), supporting a
role of protein phosphorylation in the process of NF-
B activation.
One study provided evidence that OA and calyculin A activate NF-
B
independently of ROIs (Suzuki et al., 1994), whereas another
study suggested that the OA effect is sensitive toward antioxidants and
that H
O
and L-buthionine-(S,R)-sulfoximine, a glutathione
inhibitor, have a costimulatory effect on OA (Menon et al.,
1993). Moreover, Menon et al.(1993) found that only tumor
cells, but not primary cell lines, strongly respond to OA by the
activation of NF-
B. In the present study, we have used a tumor
cell line (HeLa) and a primary fibroblast line (F26) to address these
contradictory results and investigate the possibility that OA relies on
ROI production for NF-
B activation. We report that OA and
calyculin A are very strong inducers of cellular H
O
and O
production in both a tumor
and primary fibroblast line. This was optimal at approximately 0.9
µM OA, coinciding with the optimal induction of NF-
B.
OA required a considerable lag time of 15-30 min to stimulate ROI
production which again correlated with the delayed activation of
NF-
B. The antioxidant PDTC prevented both the increased ROI
production and NF-
B activation by OA but did not detectably
influence the pattern of protein phosphorylation induced by the
PP1/PP2A inhibitor in intact cells. We suggest that OA activates
NF-
B indirectly via production of ROIs and that the tumor
promoting activity of OA and functionally related compounds is due to a
prooxidant activity, as suggested previously for phorbol esters
(Cerutti, 1985).
Figure 1:
The
effect of OA on NF-B activation and I
B-
stability in
intact cells. HeLa cells were treated for 1 h with the indicated
concentrations of OA. A, activation of NF-
B DNA binding
activity by OA. Total cell extracts were prepared from control (lanes 1, 3, and 5) and OA-treated cells (lanes
2, 4, and 6) and analyzed by EMSA using a
P-labeled oligonucleotide probe containing a high-affinity
B motif. Cell extracts were incubated with a control antibody
directed against human I
B-
(lanes 3 and 4)
or an antibody against the p65 NF-
B subunit (lanes 5 and 6). An OA-inducible protein-DNA complex which is abbrogated by
anti-p65 but not by anti-I
B-
is indicated by a filled
arrowhead. A faster-migrating nonspecific complex was unaffected.
A small filled arrowhead indicates the position of an immune
complex. The open arrowhead marks the position of the unbound
DNA probe. A fluorogram of a native gel is shown. B, OA causes
a degradation of I
B-
. Total cell extracts of control (lane 1) and OA-treated HeLa cells (lane 2) were
subjected to reducing SDS-PAGE and proteins transferred on filters for
Western blotting. I
B-
was visualized by an affinity-purified
polyclonal antibody against recombinant human I
B-
and
enhanced chemiluminescent staining as described (Henkel et
al., 1993). A fluorogram of a section of the filter is shown. An arrowhead indicates the position of a 38-kDa
I
B-
-specific signal.
Figure 2:
The effect of the antioxidant PDTC on the
activation of NF-B by OA. A, PDTC prevents NF-
B
activation by OA. HeLa cells were pretreated for 1 h with PDTC (lanes 3 and 4) followed by addition of the indicated
amount of OA (lanes 2 and 3). Total cell extracts
were analyzed by EMSA for the DNA binding activity of NF-
B. For
illustration, see legend to Fig. 1. B, PDTC does not
detectably interfere with OA-induced protein phosphorylation. HeLa
cells were grown in phosphate-free medium followed by the addition of
carrier-free inorganic [
P]phosphate. Control
HeLa cells (lanes 1 and 2) or cells preincubated with
100 µM PDTC (lanes 3 and 4) were treated
with the indicated concentration of OA (lanes 2 and 4). Total cell extracts were analyzed by reducing 10%
SDS-PAGE. An autoradiogram is shown. The molecular mass standards in
kDa are indicated on the left (200, myosin heavy chain; 97,
phosphorylase b; 67, bovine serum albumin; 46, ovalbumin; 30,
carbonic anhydrase; 21, trypsin inhibitor; 14,
lysozyme).
Figure 3:
The effect of PDTC on OA-stimulated
cellular peroxide production. HeLa cells were treated as described
under ``Materials and Methods'' with the indicated compounds.
Thereafter, cells were resuspended in PBS, incubated with DCFH, and
analyzed by FACS. A, the effect of HO
and PDTC on the oxidation state of cellular DCFH. Results from
one representative experiment are shown. B, the effect of OA
and PDTC on the oxidation state of cellular DCFH. Results from three
independent experiments are shown. The bar reflects the
standard deviation from the mean.
Figure 4:
The effect of phosphatase inhibitors on
the cellular production of ROIs. A, OA-induced
HO
production in HeLa cells. B,
OA-induced H
O
production in F26 cells.
H
O
released from HeLa and F26 cells at the
indicated concentrations of OA was determined as described (Loschen et al., 1971). Approximately five times higher rates were
determined for superoxide release with an identical dose-response
behavior. C, calyculin A-induced H
O
and O
production in HeLa cells.
HeLa cells were treated with the indicated concentrations of calyculin
A and the release of H
O
(right) determined. The
relation to the release of O
(not
shwon) is indicated on the left. Several independent
experiments were performed from which typical results are
shown.
Figure 5:
Dose
dependence of NF-B activation by OA in HeLa tumor cells. A, EMSA analysis. HeLa cell cultures were treated with the
indicated concentrations of OA and cell extracts analyzed for
B-specific DNA binding activity using EMSA. For details see legend
to Fig. 1. A section of a fluorogram from a native gel is shown.
A filled arrowhead indicates the position of the OA-induced
NF-
B
DNA complex. A faster-migrating nonspecific complex was
unaffected. B, quantitation of NF-
B activity. The
P radioactivity in the NF-
B
DNA complexes was
quantitated by a
imager and is shown plotted against the OA
concentration. The maximal NF-
B activation seen at 0.9 µM OA was set to 100%.
Figure 6:
Dose dependence of NF-B activation by
OA in F26 primary fibroblasts. A, EMSA analysis. F26 cell
cultures were treated with the indicated concentrations of OA and cell
extracts analyzed for
B-specific DNA binding activity using EMSA.
For details, see the legend to Fig. 1. A section of a fluorogram
from a native gel is shown. A filled arrowhead indicates the
position of the OA-induced NF-
B
DNA complex. A
faster-migrating nonspecific complex was unaffected. B,
quantitation of NF-
B activity. The
P radioactivity in
the NF-
B
DNA complexes was quantitated by a
imager and
is shown plotted against the OA concentration. The maximal NF-
B
activation seen at 1 µM OA was set to
100%.
Figure 7:
Time
dependence of HO
production and NF-
B
activation in response to OA in HeLa tumor cells. A,
H
O
production. The H
O
released by HeLa cells in response to a treatment with 0.7
µM OA was determined and is shown plotted against time. B, NF-
B activity. Total cell extracts from HeLa cells
treated for the indicated periods of time with 0.7 µM OA
were prepared and analyzed by EMSA as described in the legend to Fig. 1. A section of a fluorogram is shown. A filled
arrowhead indicates the position of the OA-inducible
NF-
B
DNA complex.
Figure 8:
Time dependence of HO
production and NF-
B activation in response to OA in F26
fibroblasts. A, H
O
production. The
H
O
released by F26 cells in response to a
treatment with 0.7 µM OA was determined and is shown
plotted against time. B, NF-
B activity. Total cell
extracts from F26 cells treated for the indicated periods of time with
0.7 µM OA were prepared and analyzed by EMSA as described
in the legend to Fig. 1. A section of a fluorogram is shown. A filled arrowhead indicates the position of the OA-inducible
NF-
B
DNA complex.
Figure 9:
The effect of TMB-8 on the production of
HO
and the activation of NF-
B in response
to OA. HeLa cells were pretreated for 1 h with the indicated
concentrations of TMB-8 followed by stimulation with 0.4 µM OA. A, H
O
release.
H
O
production in response to 0.4 µM OA is shown as % of maximal production in the absence of TMB-8. B, NF-
B activity. Total cell extracts from HeLa cells
were analyzed for
B-specific DNA binding activity by EMSA as
described in the legend to Fig. 1. A section of a fluorogram
from a native gel is shown. The filled arrowhead indicates the
position of the OA-induced NF-
B
DNA complex, the open
arrowhead indicates the position of nonspecific
complexes.
The mechanism by which the PP inhibitor OA activates
NF-B is a matter of debate. Packer and colleagues (Suzuki et
al., 1994) reported that the activation of NF-
B by OA or
calyculin A cannot be prevented by the antioxidants NAC (20
mM) or dihydrolipoate (1 mM), suggesting that OA does
not require a ROI step for signaling. However, the antioxidant rotenone
(5 µM), which inhibits tumor necrosis factor
signaling (Schulze-Osthoff et al., 1993), prevented OA-induced
NF-
B activation. It was speculated that the inhibitory effect of
rotenone on OA was not caused by its antioxidative potential but the
reduction of cellular ATP levels by approximately 50% (Suzuki et
al., 1994). However, it is questionable whether this degree of ATP
depletion was sufficient to inhibit protein kinases in the cell, most
of which have K
values for ATP in the micromolar
range. Here we show that the antioxidant PDTC is a potent inhibitor of
OA activity and ROI production without having an apparent influence on
overall protein phosphorylation. PDTC was also shown to prevent
H
O
production and NF-
B activation in
response to Fc
2a receptor stimulation in J774 cells (Muroi et
al., 1994). It seems that antioxidants profoundly differ with
respect to their effect on OA-induced NF-
B activation. These
differences may come from a distinct uptake, subcellular distribution,
or metabolism of the antioxidants in the cell. Non-thiol antioxidants,
such as PDTC and rotenone, are presumably less rapidly metabolized or
taken up better than the more physiological compounds NAC and lipoic
acid. Menon et al.(1993) have observed that 30 mML-cysteine prevents both OA- and tumor necrosis factor
-induced NF-
B activation in primary and transformed MRC-5
cells. It was also observed by these authors that the NF-
B
activation by OA was enhanced in the presence of either micromolar
concentrations of H
O
or 5 mM of the
glutathione synthesis inhibitor L-buthionine-(S,R)-sulfoximine in several cell lines.
We have observed that 30 mM NAC suppresses NF-
B
inhibition in HeLa cells (data not shown). These data would also be
consistent with a requirement of a pro-oxidant condition for OA
signaling.
All published studies relied on a pharmacological
approach to test for an involvement of ROIs in the signaling of OA. The
conflicting results prompted us to directly investigate whether OA
leads to an enhanced cellular production of ROIs. We found that OA is a
strong inducer of cellular ROI production by two distinct approaches.
First, OA was shown to increase the cellular oxidation of the dye DCFH;
second, an increased HO
and
O
production was directly measured in
culture supernatants of OA-stimulated HeLa tumor cells and F26 primary
fibroblasts. ROI production and the activation of NF-
B correlated
both with respect to kinetics and dose dependence. We could not observe
that only the tumor cell line would respond to OA treatment; F26
primary fibroblasts showed a similar responsiveness as HeLa tumor
cells. NF-
B activation by OA was also reported in human B
lymphocytes, another type of primary cells (Rieckmann et al.,
1992). Hence, the finding that primary cells are less or unresponsive
toward OA cannot be generalized.
The slow kinetic of ROI production
in response to OA suggests that a number of reactions have to occur
before NF-B is activated. Alternatively, it may reflect a very
slow uptake of the PP inhibitor into the cell. Inhibition of PP1 and
PP2A may allow enhanced phosphorylation of a substrate X.
Phosphorylated X may then, for instance, cause directly or indirectly a
rise in intracellular calcium which is where TMB-8 might interfere in
the signaling pathway. Intracellular calcium is also required for
activation of NF-
B by endoplasmic reticulum stress-inducing
conditions and two drugs inhibiting the Ca
-ATPase in
the endoplasmic reticulum membrane turned out to be potent NF-
B
inducers (Pahl and Baeuerle, 1995). (
)There is one report
that calyculin A induces an increase in intracellular calcium (Ishihara et al., 1989). Whether PKC is activated and required in a
subsequent step needs future studies. Thévenin et al.(1989) provided evidence that PKC is not involved in OA
signaling. Very clearly, ROIs are produced in response to OA
stimulation of cells. It is likely that the ROI scavenger PDTC as well
as the costimuli H
O
and L-buthionine-(S,R)-sulfoximine act at this late stage
of OA signaling. This is also where signals from other NF-
B
inducers, such as tumor necrosis factor
, interleukin-1, and UV
light may converge into the pathway. As described in the Introduction,
an as yet unidentified kinase/phosphatase system appears to be
controlled by ROIs and ultimately activates NF-
B through a
phosphorylation-controlled proteolytic degradation of I
B-
.
The very slow action of OA and the apparent involvement of ROIs make it
very unlikely that an I
B-
phosphatase is a direct target for
OA inhibition. In conclusion, this study cannot support the previous
notion that OA activates NF-
B independently of ROIs. On the
contrary, OA was found to be a strong inducer of oxidative stress which
may provide a further explanation for its broad biological effects.
Our observation that both OA and calyculin A are potent inducers of
oxidative stress may well explain their tumor promoting potential.
Tumor promotion by OA inhibitors could not be correlated to events
controlling the cell cycle. On the contrary, in myeloid leukemic cells
OA induced cell cycle arrest and apoptosis (Ishida et al.,
1992) and in raf and ret-II-transformed fibroblasts the drug reversed
the transformed phenotype to that of normal contact inhibited cells
(Sakai et al., 1989; reviewed in MacKintosh and MacKintosh
(1994)). Based on our present finding that both OA and calyculin A
potently induce oxidative stress we would like to propose that PP1 and
PP2A inhibition has a tumor promoting effect by virtue of deregulating
a phosphorylation-controlled ROI-producing cellular process. A similar
mechanism was proposed for the tumor-promoting activity of phorbol
esters (Cerutti, 1985), which are activators of several PKC isozymes. A
number of observations are consistent with this hypothesis. OA has been
reported to cause mutagenesis of eukaryotic but not prokaryotic DNA
(Aonuma et al., 1991). DNA damage is a well documented effect
of oxidative stress (reviewed in Halliwell and Gutteridge (1989)). Upon
long-term treatment, OA can cause programmed cell death (Ishida et
al., 1992), a phenomena known to be associated with oxidative
stress (reviewed in Buttke and Sandström(1994)).
Although OA and calyculin A are tumor promoters which unlike PKC do not
directly bind phorbol esters, they nevertheless cause an activation of
the kinase in NRK cells (Gopalakrishna et al., 1992).
Intriguingly, the induction of PKC by OA and calyculin A showed a lag
phase of 15-30 min, as was observed in our study for the
production of ROIs and the activation of NF-B. This raises the
possibility that PKC is involved in tumor promotion, ROI production,
and NF-
B activation by the non-phorbol tumor promoter OA. An
involvement of PKC would also be consistent with the inhibitory effect
of the Ca
chelator TMB-8. Ca
is
required for activation of PKC and TMB-8 has been shown to inhibit PKC
activation (Christiansen et al., 1986).