(Received for publication, September 29, 1994; and in revised form, November 17, 1994)
From the
Rel proteins are important intracellular mediators of
cytokine-induced signal transduction. To understand how cytokines
affect different cell populations in the brain, we have characterized
Rel activation in astrocytes. A RelA homodimer is uniquely activated in
cytokine-stimulated astrocytes. Cytokine-dependent phosphorylation of
the RelA inhibitor MAD-3 occurred on discrete peptides prior to its
dissociation from RelA. A transient hyperphosphorylation of RelA was
also induced. Antioxidant treatment inhibited both RelA activation and
phosphorylation of the RelAMAD-3 complex. These results
demonstrate that cytokine-dependent activation of the RelA homodimer
involves phosphorylation of both RelA and its associated inhibitor. The
sole activation of a RelA homodimer suggests that cytokines will
activate a unique set of Rel-regulated genes in astrocytes.
The Rel family is defined by a highly conserved 300-amino acid
domain, referred to as the Rel homology domain. Within the Rel homology
domain lie the amino acid residues necessary for sequence-specific DNA
binding, dimerization, and association with members of the IB
family of proteins (inhibitor of
B; (1) ). The I
B
proteins regulate the cellular location, sequence-specific DNA binding
activity, and transcriptional activation properties of Rel proteins.
Rel proteins modulate inducible expression of a variety of genes
through cis-acting Rel binding sites. Cytokines, including
tumor necrosis factor-
(TNF
) (
)and interleukin-1,
induce DNA binding and transcriptional activation by Rel
proteins(2) . Induction of Rel activity by these stimuli is a
post-translational event that targets an inactive cytosolic complex
containing Rel family members and an I
B protein. These stimuli
induce the phosphorylation and degradation of I
B followed by an
increase in nuclear Rel DNA binding activity(3, 4) .
Although in vitro phosphorylation of Rel proteins has been
correlated with enhanced DNA binding activity(5, 6) ,
the role of Rel phosphorylation in the activation of the cytosolic
Rel
I
B complex in mammalian cells is not known. The
demonstration that Toll-dependent phosphorylation of the dorsal morphogen from Drosophila correlates with increased
nuclear import of Dorsal suggests that signal-dependent phosphorylation
of Rel may also occur in mammalian cells(7) .
Rel proteins
are important mediators of cytokine-dependent signal transduction in
both lymphoid and non-lymphoid cells. As astrocytes are the primary
cytokine-responsive cell in the brain(8) , we have examined
cytokine-dependent activation of Rel proteins in astrocytes. We find
that treatment of both primary rat astrocytes or the astrocyte cell
line DITNC (9) with TNF results in the rapid activation of
DNA binding by a RelA homodimer. Induction of RelA DNA binding was
preceded by the TNF
-dependent phosphorylation of both RelA and the
RelA-specific I
B protein, MAD-3. The ability of an N-acetyl-L-cysteine (NAC) to inhibit signal-dependent
phosphorylation of RelA and MAD-3 implicates a role for reactive oxygen
intermediates upstream of this event. These results suggest that
TNF
-dependent phosphorylation of RelA and MAD-3 is a critical step
in the cytokine-dependent activation of the preexisting
Rel
I
B complex.
Figure 1:
A,
induction of RelA DNA binding activity. Nuclear fractions from
untreated (lanes 1-5) and LPS-treated (lanes
6-10) DITNC cells were analyzed by EMSA with an
oligonucleotide containing a palindromic B site. To identify the
Rel proteins present in the EMSA complex, antiserum raised against p50 (lanes3 and 8), p52 (lanes4 and 9), RelA (lanes6 and 10),
or normal rabbit serum (lanes2 and 7) were
added to the nuclear extract prior to the DNA binding reaction. The
protein
DNA complexes are denoted by the arrows to the left of the gel. B, immunoprecipitation of the
RelA
DNA adduct. Nuclear extracts from LPS-treated DITNC cells
were subjected to solution UV-cross-linking, followed by
immunoprecipitation with antisera raised against RelA (lane
2), p50 (lane 3), p52 (lane 4), or normal rabbit
serum (lane 1). The position of the full-length RelA
protein
DNA adduct is denoted by the arrow to the right of the gel. The position of molecular weight markers is
indicated to the right of the gel. C,
activation of
B-dependent gene expression. 5.0 µg of a plasmid
containing only TATA and initiator sites (TATA-Inr) driving expression
of the fire fly luciferase reporter gene or 5 µg of the same
plasmid in which the
B binding sites from the HIV-1 long terminal
repeat had been cloned in front of the TATA box were transfected into
DITNC cells. Media containing 10 ng/ml TNF
or media without
TNF
was added to the cells 30 h after transfection. Cell lysates
were collected and assayed for luciferase activity 7 h
post-transfection. Transfections were done in triplicate, and the
results shown are the average of three independent
experiments.
To determine if RelA
DNA binding activity correlated with activation of B-dependent
transcription, two copies of the
B binding site from the HIV-1
long terminal repeat were cloned into a reporter plasmid that contains
a canonical TATA and initiator sequences in front of the firefly
luciferase reporter gene. TNF
treatment of the astrocytes
transfected with the TATA-luciferase plasmid did not affect luciferase
activity from this plasmid (Fig. 1C). However,
treatment of astrocytes transfected with the
B-luciferase plasmid
resulted in a 4-fold activation of luciferase activity over the basal
luciferase activity in untreated cells expressing the
B-luciferase
plasmid (Fig. 1C). Thus, activation of RelA DNA binding
activity in astrocytes by cytokine stimulation correlates with
B-dependent transcriptional activation.
Figure 2:
A, induction of RelA DNA binding
by TNF. Whole cell lysates were prepared from DITNC cells treated
with 10 ng/ml TNF
for 0-60 min. Equivalent volumes of each
lysate were used for solution UV-cross-linking experiments. The
protein
DNA adducts were separated by SDS-polyacrylamide and
visualized by autoradiography. The position of the RelA
DNA adduct
is indicated by the arrow to the right of the gel,
and the position of molecular weight markers is indicated to the left of the gel. B and C, anti-RelA
immunoblot and anti-MAD-3 immunoblot of whole cell lysates prepared
from the TNF
-treated DITNC cells. Equivalent volumes of the whole
cell lysates prepared from untreated DITNC cells (lane1) or DITNC cells treated with TNF
for 2.5 min (lane2), 5 min (lane3), 10 min (lane4), 30 min (lane5), or 60
min (lane6) were electrophoresed through a 7.5%
SDS-polyacrylamide gel. Protein samples were subsequently transferred
to nitrocellulose and blotted with anti-RelA serum or anti-MAD-3
serum.
Although phosphorylation of MAD-3 prior to the activation
of Rel DNA binding in vivo has been demonstrated
previously(3, 4) , it is not clear if phosphorylation
occurs before or after dissociation of the RelAMAD-3 complex. To
determine if RelA-associated MAD-3 underwent phosphorylation after
TNF
treatment, DITNC cells were labeled with
[
P]orthophosphate or
[
S]methionine and stimulated with TNF
for 5
or 30 min. Whole cell lysates were subjected to immunoprecipitation
with either anti-MAD-3 serum or anti-RelA serum. The anti-MAD-3 serum
immunoprecipitated a 40-kDa protein from both
P- and
S-labeled astrocytes (Fig. 3A, lane1; top and middlepanels). A
protein of identical mobility co-immunoprecipitated with RelA (Fig. 3A, lane 5; top and middlepanels). Five minutes after treatment with TNF
,
P-labeled MAD-3 and RelA-associated MAD-3 proteins
possessing an altered mobility were detected, while at 30 min following
treatment, levels of
P-labeled MAD-3 was dramatically
reduced (Fig. 3A, lanes2 and 3 and lanes 6 and 7; middlepanels). The reduced level of
P-labeled
MAD-3 30 min following TNF
treatment is a reflection of MAD-3
proteolysis as demonstrated by the reduction in
S-labeled
MAD-3 following 30 min of TNF
treatment (Fig. 3A, lanes3 and 7; toppanel).
Figure 3:
A, TNF-dependent
phosphorylation of MAD-3 and RelA-associated MAD-3. DITNC cells were
metabolically labeled with
S (top panel) or
P (middlepanel) and left untreated (lanes1, 4, 5, and 8) or
treated with 10 ng/ml TNF
for 5 min (lanes2 and 6), or 30 min (lanes3 and 7).
Whole cell lysates were immunoprecipitated with either anti-MAD-3 serum (lanes 1-4) or anti-RelA serum (lanes
5-8). Lanes 4 and 8 are
immunoprecipitations from
S-labeled cells performed in the
presence of excess peptide (lane 4, MAD-3 peptide; lane
8, RelA peptide). Gel slices containing MAD-3 and RelA-associated
MAD-3 isolated from
P-labeled cells were used for V8
protease mapping experiments (lower panel). MAD-3 and
RelA-associated MAD-3 were digested in situ with 50 ng of V8
protease. The arrows to the left of the gel indicate
the positions of TNF
-dependent phosphopeptides present in lanes2, 3, 6, and 7. B, TNF
-dependent phosphorylation of RelA. DITNC cells
were metabolically labeled with
S (toppanel) or
P (middlepanel)
and left untreated (lanes1 and 4) or
treated with 10 ng/ml TNF
for 5 min (lane2) or
30 min (lane3). Whole cell lysates were subjected to
immunoprecipitation with anti-RelA serum (lanes1-3) or anti-RelA serum plus excess peptide (lane4; peptide competition shown was performed on
lysates from
S-labeled cells). Gel slices containing
P-labeled RelA were digested in situ with 50 ng
of V8 protease (lowerpanel). Asterisks to
the left of the gel indicate the positions of phosphopeptides
that contained increased
P incorporation in the presence
of TNF
.
MAD-3 immunoprecipitated from P-labeled cells with
anti-MAD-3 serum or co-immunoprecipitated with anti-RelA serum was
subjected to one-dimensional V8 peptide mapping. Digestion of MAD-3 or
RelA-associated MAD-3 from unstimulated cells with 50 ng of V8 protease
resulted in five distinct phosphopeptides (Fig. 3A, lanes1 and 4; bottompanel). Digestion of MAD-3 or RelA-associated MAD-3 from
cells treated with TNF
for either 5 or 30 min with 50 ng of V8
protease resulted in the appearance of two new phosphopeptides (Fig. 3A, lanes2 and 3 and lanes 5 and 6; bottompanel). These
results demonstrate that MAD-3 undergoes TNF
-dependent
phosphorylation prior to dissociation from RelA.
Figure 4:
A, solution UV-cross-linking of primary
astrocytes and DITNC cells treated with TNF. Primary rat
astrocytes or DITNC cells were left untreated (lanes1 and 4) or treated with 10 ng/ml TNF
for 10 min (lanes2 and 5) or 30 min (lanes 3 and 6). Whole cell lysates were prepared and subjected to
solution UV-cross-linking experiments. The position of the
RelA
DNA adduct is indicated by the arrow to the right of the gel. The position of the molecular weight markers
is indicated to the left of the gel. B,
TNF
-dependent phosphorylation of RelA in primary astrocytes.
Primary astrocytes were metabolically labeled with
[
P]orthophosphate and left untreated (lane1) or treated with 10 ng/ml TNF
for 5 (lane2) or 10 min (lane3). Whole cell
lysates were prepared and subjected to immunoprecipitation with
anti-RelA serum. The position of RelA is indicated by the arrow to the right of the gel
slice.
Figure 5:
A,
NAC inhibits TNF-dependent activation of DNA binding by the RelA
homodimer. DITNC cells were left untreated (lane1),
treated with 10 ng/ml TNF
for 60 min (lane2),
or pretreated with 30 mM NAC for 1 h prior to addition of 10
ng/ml TNF
for 60 min (lane3). Whole cell
lysates from these three treatments were subjected to solution
UV-cross-linking experiments. The position of the RelA
DNA adduct
is indicated by the arrow to the right of the gel,
and the position of molecular weight markers is indicated to the left of the gel. B, NAC inhibits TNF
-dependent
phosphorylation of RelA and dissociation of RelA-associated MAD-3.
DITNC cells were left untreated (lane1), treated
with 10 ng/ml TNF
for 5 min (lane 2), or pretreated with
30 mM NAC for 1 h prior to addition of 10 ng/ml TNF
for 5
min (lane3). Whole cell lysates were collected and
subjected to immunoprecipitation with anti-RelA serum. The positions of
RelA and MAD-3 are indicated to the right of the gel, and the
positions of molecular weight markers are indicated to the left of the gel. C, NAC inhibits TNF
-dependent
phosphorylation of MAD-3. DITNC cells were treated as in B.
Whole cell lysates prepared from these cells were subjected to
immunoprecipitation with anti-MAD-3 serum. MAD-3 was isolated from a
one-dimensional preparative SDS-polyacrylamide gel and subjected to in situ digestion in a second SDS-polyacrylamide gel with 50
ng of V8 protease. The arrows to the right of the gel
indicate the positions of the TNF
-dependent phosphopeptides that
are present in lane2.
As TNF treatment causes increased phosphorylation of both RelA
and MAD-3 prior to activation of RelA DNA binding, we reasoned that the
TNF
-dependent phosphorylation of the RelA
MAD-3 complex might
also be inhibited by NAC. To determine if NAC inhibited
TNF
-dependent phosphorylation of RelA or MAD-3, DITNC cells were
labeled with [
P]orthophosphate and pretreated
with 30 mM NAC prior to treatment with TNF
. Whole cell
lysates prepared from these cells were subjected to immunoprecipitation
with anti-RelA serum. Pretreatment of astrocytes with NAC significantly
decreased the TNF
-dependent phosphorylation of RelA and inhibited
dissociation of MAD-3 (Fig. 5B, lane3; data not shown). To determine if NAC inhibited
TNF
-dependent phosphorylation of MAD-3, MAD-3 was
immunoprecipitated from cells labeled with
[
P]orthophosphate and subjected to
one-dimensional V8 peptide mapping. One-dimensional V8 peptide mapping
of MAD-3 from cells treated with TNF
resulted in the appearance of
two TNF
-dependent phosphopeptides (Fig. 5C, lane2). In cells pretreated with NAC, the
TNF
-dependent phosphopeptides were no longer apparent in
one-dimensional V8 peptide maps of MAD-3 (Fig. 5C, lane3). Thus, NAC inhibits TNF
-dependent
phosphorylation of both MAD-3 and RelA.
Induction of Rel activity by cytokines is a
post-translational event that targets an inactive, preexisting
cytosolic complex containing Rel family members and an IB
protein(3, 4) . The ability of several protein kinases
to phosphorylate I
B
and thereby relieve I
B
-mediated
inhibition of Rel DNA binding in vitro suggests that
phosphorylation of I
B
is a mechanism by which activation of
the preexisting Rel
I
B complex occurs in
vivo(18, 19, 20) . The demonstration of
signal-dependent phosphorylation of I
B prior to activation of the
Rel
I
B complex in vivo has provided additional
support for this model(3, 4) . The function of
signal-dependent phosphorylation of I
B
is not known, but it
is postulated to trigger dissociation of the Rel
I
B
complex or to act as a signal for the proteolytic degradation of
I
B
.
In this study, we find that treatment of rat
astrocytes with TNF results in the signal-dependent
phosphorylation of RelA-associated MAD-3. TNF
treatment resulted
in the appearance of two new MAD-3-derived phosphopeptides, as
determined by V8 protease mapping experiments. This result indicates
that sites of TNF
-dependent phosphorylation are distinct from the
sites of constitutive phosphorylation in MAD-3. Constitutive
phosphorylation of the avian I
B
homolog, p40, is localized to
a serine-rich C-terminal region between amino acids 282 and 301. (
)This serine-rich region is highly conserved between
p40 and MAD-3 and V8 protease digestion of
P-labeled MAD-3
and p40 isolated from untreated cells resulted in similar
one-dimensional peptide maps (data not shown). The detection of new
MAD-3-derived phosphopeptides upon TNF
treatment indicates that
signal-dependent phosphorylation of MAD-3 occurs N-terminal to serine
283 of MAD-3, possibly within the ankyrin domain. Signal-dependent
phosphorylation of MAD-3 within the ankyrin domain would be consistent
with a role for phosphorylation in the dissociation of the
Rel
I
B complex.
We have found that RelA is also subject to
TNF-dependent phosphorylation prior to activation of RelA DNA
binding. A 2-3-fold increase in RelA phosphorylation was detected
after 5 min of TNF
treatment. This is the first demonstration that
signal-dependent phosphorylation of RelA occurs prior to activation of
RelA DNA binding. The fact that TNF
-dependent phosphorylation of
RelA precedes the induction of RelA DNA binding suggests that
TNF
-dependent phosphorylation is important for the activation of
RelA DNA binding. This conclusion is supported by the concurrent
inhibition of both TNF
-dependent phosphorylation of RelA and
induction of RelA DNA binding by NAC. At least two roles can be
envisioned for the TNF
-dependent phosphorylation of RelA. First,
phosphorylation may occur while RelA is still associated with MAD-3 and
thus be required for dissociation of the RelA
MAD-3 complex. In
favor of this hypothesis, TNF
-dependent phosphorylation of RelA
occurs in parallel with TNF
-dependent phosphorylation of MAD-3.
Alternatively, TNF
-dependent phosphorylation of RelA may occur
immediately after dissociation of the RelA
MAD-3 complex and thus
play a functional role in the nuclear import of RelA. In favor of this
latter possibility, Toll-dependent phosphorylation of Dorsal correlates
with its increased nuclear import during Drosophila embryogenesis (7) . Furthermore, a serine to glutamic acid
mutation within the conserved phosphorylation site for cyclic
AMP-dependent protein kinase of avian c-Rel allowed increased nuclear
import of avian c-Rel(21) . A direct correlation between
phosphorylation of this cyclic AMP-dependent protein kinase site,
I
B association, and nuclear transport remains to be established.
Antioxidants such as NAC inhibit both the activation of Rel DNA
binding and the proteolytic degradation of
IB(4, 17) , although the precise mechanisms by
which they function is not known. Our results demonstrate that NAC
inhibits the TNF
-dependent phosphorylation of both RelA and MAD-3.
Several mechanisms by which NAC inhibits TNF
-dependent
phosphorylation of RelA and MAD-3 can be proposed. First, antioxidants
may function early in the signal transduction pathway and thereby
prevent activation of the protein kinase(s) that targets RelA and
MAD-3. Second, RelA and MAD-3 might be substrates for one or more
cytokine-inducible protein kinases that are directly inhibited by NAC.
The recent identification of a Rel-associated kinase that demonstrates
substrate specificity for Rel proteins suggests that multiple protein
kinases may target the RelA
MAD-3 complex(5) . The role of
this putative Rel kinase in the cytokine-dependent activation of Rel
DNA binding remains to be established. Identification of the protein
kinase(s) that phosphorylate RelA and MAD-3 in vivo will be
critical to fully elucidate the regulatory steps that ultimately lead
to RelA activation.
The Rel protein family currently comprises seven
distinct polypeptides that are capable of forming a variety of homo-
and heterodimeric complexes with distinct functional properties
(reviewed in (1) ). Most cell types that have been examined
contain multiple Rel DNA binding complexes(4) . For example,
neurons contain both constitutive and inducible forms of the canonical
NF-B heterodimer(22) . In contrast, we have found that
astrocytes contain a single, cytokine-inducible Rel DNA binding complex
that comprises a RelA homodimer. RelA homodimers have previously been
detected in lymphoid cells, but as only one of multiple Rel DNA binding
complexes(4) . As RelA homodimers have distinct DNA binding
specificities (23) and transcriptional activation properties (24) relative to other homodimeric and heterodimeric
combinations of Rel proteins, activation of a RelA homodimer will
likely result in the expression of a distinct set of cytokine-inducible
genes in astrocytes.