(Received for publication, April 24, 1995; and in revised form, June 26, 1995)
From the
Reactive free radicals have been implicated in mediating signal
transduction by a variety of stimuli. We have investigated the role of
p21 in mediating free radical signaling. Our
studies revealed that signaling by oxidative agents which modulate
cellular redox status, such as H
O
, hemin,
Hg
, and nitric oxide was prevented in cells in which
p21
activity was blocked either through
expression of a dominant negative mutant or by treating with a
farnesyltransferase inhibitor, as assessed by NF-
B binding
activity. Furthermore, the NF-
B response to these oxidative stress
stimuli was found to be enhanced when cells from the human T cell line,
Jurkat, were pretreated with L-buthionine-(S,R)-sulfoximine, an inhibitor
of glutathione synthesis. We directly assayed p21
and mitogen-activated protein kinase activities in Jurkat
cells and found both of these signaling molecules to be activated in
cells treated with the redox modulating agents. Blocking glutathione
synthesis made cells 10- to 100-fold more sensitive to these agents.
Finally, using recombinant p21
in
vitro, we found that redox modulators directly promoted guanine
nucleotide exchange on p21
. This study suggests
that direct activation of p21
may be a central
mechanism by which a variety of redox stress stimuli transmit their
signal to the nucleus.
Free radicals have been shown to play important roles in
carcinogenesis by directly damaging DNA and acting as tumor promoters (1, 2, 3, 4) . Free radicals and
redox stress are now thought to participate in cellular
signaling(5, 6, 7, 8, 9) ,
and, thus, additional targets may exist. The transcription factor
NF-B has been demonstrated to mediate signaling by reactive oxygen (5) and reactive nitrogen(10) . The exact target of
these species is unknown, although it has been postulated to be
upstream of p21
and involve tyrosine
phosphorylation(8, 9, 11) . We have
previously identified G proteins(12) , and particularly
p21
(13) , as central targets by which
nitric oxide transmits signals. Therefore, we explored whether
p21
is a more general target for reactive free
radicals and senses cellular redox status.
Figure 1:
Effect of
p21 inhibition on free radical signaling. A, wild type (wt) PC12 cells or cells harboring a
dominant negative mutation in p21
(mt) were treated with the indicated concentrations of
drugs for 4 h prior to isolation of nuclei. Cells treated with
H
O
were also co-treated with phorbol
12-myristate 13-acetate (PMA, 100 ng/ml). B, Jurkat T
cells were untreated (-FT inh) or treated with
-hydroxyfarnesylphosphonic acid (+FT inh, 10
µM) for 24 h prior to addition of the indicated drugs.
After a 4-h drug treatment, nuclei were isolated and assayed for
NF-
B binding activity. Bands were quantified via PhosphorImager
analysis, and their relative counts/min are indicated (Rel.
CPM). Arrow denotes migration of the NF-
B-DNA
protein complex.
Our previous work
implicated G proteins, and particularly p21, as
targets of the reactive nitrogen species, nitric
oxide(12, 13) . Therefore, we directly assessed the
activation state of p21
upon exposure of Jurkat
cells to free radicals and redox modulators. We found that treatment of
cells with H
O
, Hg
, or hemin
led to recovery of an activated form of p21
as
evidenced by an increase in GTP-bound p21
(Fig. 2). We then treated Jurkat cells for 24 h with L-buthionine-(S,R)-sulfoximine, a specific
inhibitor of
-glutamylcysteine synthetase(19) , the
rate-limiting enzyme in glutathione synthesis, and thus depleted cells
of this critical antioxidant. We found that depletion of intracellular
glutathione with L-buthionine-(S,R)-sulfoximine made cells
10- to 100-fold more sensitive to these agents (Fig. 2).
Treatment of Jurkat cells with L-buthionine-(S,R)-sulfoximine (100
µg/ml) yields cells with less than 20% of their original
intracellular glutathione levels(8) . We next examined whether
a known downstream effector of p21
was also
activated by redox stress. We found that MAP kinase immunoprecipitated
from cells treated with hemin or H
O
for 30 min
had an enhanced ability to phosphorylate myelin basic protein (Fig. 3). Furthermore, pretreatment of cells with L-buthionine-(S,R)-sulfoximine greatly
enhanced their MAP kinase activity (Fig. 3). The close
correlation between p21
and MAP kinase
activation in response to oxidative stimuli suggest that this pathway
is indeed important in transmitting redox stress signals. Furthermore,
downstream signaling in response to Hg
and sodium
nitroprusside, a nitric oxide-generating compound, was profoundly
enhanced by L-buthionine-(S,R)-sulfoximine
treatment, as assessed by the NF-
B binding assay (Fig. 4).
Therefore, depletion of intracellular glutathione resulted in enhanced
free radical signaling through p21
, MAP kinase,
and NF-
B. The disparity in the dose-response relationship between
the ability of Hg
and hemin to activate
p21
and NF-
B may lie in the chemical nature
of these reagents. Hemin is a known free radical generator which can
catalytically generate oxygen-free radicals, whereas
Hg
, a thiol-specific reagent, will stoichiometrically
bind thiols. Therefore, differences seen in dose-response relationships
between cell types may likely reflect their differential intracellular
thiol content. These data suggest that the p21
pathway acts to transmit cellular redox stress signals to
the nucleus.
Figure 2:
Effect of cellular redox stress on
endogenous p21 activity. Jurkat T cells, labeled
with
PO
in phosphate-free
media, were treated with the indicated concentrations of drugs for 10
min prior to lysis, immunoprecipitation of p21
,
and analysis of bound guanine nucleotides. Some cells were treated with L-buthionine-(S,R)-sulfoximine (BSO) 24 h prior to addition of drugs. Data represent the
average and standard deviation of 3 experiments, each assayed in
duplicate.
Figure 3: Effect of free radicals on MAP kinase activity. Jurkat T cells were either untreated or treated with L-buthionine-(S,R)-sulfoximine (BSO) 24 h prior to addition of drugs. Cells were treated with drugs for 30 min, and then their cytosol was analyzed for MAP kinase activity in an in vitro kinase assay. Data represent the average and standard deviation of 3 experiments.
Figure 4:
Effect of glutathione depletion on free
radical signaling. Jurkat T cells were either untreated or treated with L-buthionine-(S,R)-sulfoximine (BSO) 24 h prior to addition of drugs. Cells were treated with
drugs for 4 h, and then their nuclei were analyzed for NF-B
binding activity. SNP, sodium nitroprusside (a nitric
oxide-generating compound). Arrow denotes migration of the
NF-
B-DNA protein complex.
Figure 5:
Effect of free radicals on
p21in vitro. p21
(1 µM) was preloaded with GDP and then mixed
with [
-
P]GTP and the indicated
concentration of drug for 10 min. Hydrolyzed
PO
was quantified as
described under ``Experimental Procedures.'' Control GTPase
rates were 27.3 ± 2.1 fmol/min/mg. Data represent the average
and standard deviation of 3 experiments, each assayed in
duplicate.
We
have previously reported the mitogenic properties of iron compounds
such as hemin on lymphocytes and implicated free radical generation as
a mechanism of activation(20, 21) . HgCl,
a thiol-binding agent, was also shown to be mitogenic to
lymphocytes(22, 23) . The exact target of free
radicals responsible for initiating the mitogenic event was unknown.
Furthermore, the oncogenicity of free radicals is known to be mediated
through both physical damage to DNA and their properties as tumor
promoters(1, 2, 3, 4) . The recent
identification of free radicals as second messengers (5, 6, 7, 8, 9) suggests
that some of the pathophysiological consequences of free radical
generation may be due to effects on the enzymes controlling signaling
pathways. Our data suggest that free radicals can activate
p21
and lead to a nuclear signal. Mutated and
activated forms of p21
have been found in many
human cancers (24) and, thus, it is possible that the apparent
sensitivity of p21
to free radicals and redox
status, which we have identified here, provide an additional mechanism
by which free radicals promote oncogenesis.
A common mechanism by
which cells respond to redox signals and initiate gene expression has
been postulated(9, 11, 15) , although the
precise target remains unknown. Our data indicate that, under normal
conditions, p21 may represent the cellular
sensor for redox stress. Studies are currently underway to identify the
exact molecular alteration on p21
generated by
reactive free radicals.