(Received for publication, November 30, 1994; and in revised form, January 19, 1995)
From the
The promoter region of the rabbit serum amyloid A (SAA) gene
contains two adjacent C/EBP and one NF-B binding element.
Involvement of these elements in SAA gene induction, following
lipopolysaccharide (LPS) stimulation of the liver, has been studied by
investigating LPS-activated transcription factors and their interaction
with the promoter elements of the SAA gene. Appearance of complexes in
the electrophoretic mobility shift assay has indicated that DNA-binding
proteins that interact with the NF-
B element of the SAA promoter
are induced in the LPS-treated rabbit liver. Presence of RelA (p65
subunit of NF-
B) in these complexes was demonstrated by the
ability of RelA-specific antisera to supershift the DNA-protein
complexes. LPS also induced several members of the C/EBP family of
transcription factors, which interacted with the C/EBP motifs of the
SAA promoter. Activated C/EBP and RelA form a RelA
C/EBP
heteromeric complex that associates with varying affinity to NF-
B
and C/EBP elements of the SAA gene. Transfection assays using both
transcription factor genes have demonstrated that the heteromeric
complex of NF-
B and C/EBP is a much more potent transactivator of
SAA expression than each transcription factor alone. The heteromeric
complex efficiently promotes transcription from both NF-
B and
C/EBP sites.
Serum amyloid A is the precursor of amyloid A (AA) ()protein, one of the chief constituents of amyloid fibrils
found in secondary and experimental amyloidosis (Husebekk et
al., 1985). The structure of protein AA is identical to the N
terminus of SAA (Anders et al., 1977), and a precursor-product
relationship between SAA and AA has been documented (Husebekk et
al., 1985). SAA is also a member of a group of acute phase
proteins whose synthesis is highly induced under different inflammatory
conditions such as tissue injury or infection (Kushner, 1982).
Cytokines such as interleukin-1, interleukin-6, and tumor necrosis
factor-
increase the synthesis of SAA in cultured cells via
transcriptional induction (Ganapathi et al., 1991). Studies
have shown that this protein is coded by multiple genes in human,
mouse, rabbit, and rat. All three murine SAA genes are induced in the
liver, and each gene accounts for approximately one-third of the total
SAA mRNA (Lowell et al., 1986a). Analyses of the promoter
region of the human, rat, and mouse SAA gene have shown that two of the
protein-coding genes, termed SAA1 and SAA2, contain binding elements
for both C/EBP and NF-
B transcription factors (Edbrooke et
al., 1989; Li and Liao, 1991). The third murine gene, SAA3,
contains the binding site for C/EBP in the upstream regulatory region
(Huang and Liao, 1994; Lowell et al., 1986a). Studies on a
rabbit SAA gene indicated the presence of both C/EBP and NF-
B
elements in the 5`-proximal promoter region (Ray and Ray, 1991, 1993a,
1993b). Many acute-phase stimuli induce transcription of the SAA gene
in liver, but induction mechanisms do not always follow the same route.
Turpentine, an inducer of rabbit SAA gene expression, activates only
C/EBP transcription factors (Ray and Ray, 1993a), while LPS induces
both C/EBP and NF-
B-like factors (Alam et al., 1992; Ray
and Ray, 1993b, 1994b). Activation of the
and
isoforms of
C/EBP and their interaction with the two C/EBP binding sites are
essential for turpentine-mediated acute-phase induction of the rabbit
SAA gene (Ray and Ray, 1994a).
Recent studies on eukaryotic gene
regulation show that the transcriptional control region often contains
multiple binding sites for the same or several different transcription
factors and a combined effect of these factors is important for the
overall transcriptional activation. Other genes, such as those encoding
IL-6, IL-8, and angiotensinogen proteins, also have adjacent or
overlapping binding elements for NF-B and C/EBP. NF-
B and
C/EBP cooperate in the regulation of IL-6 and IL-8 (Kunsch et
al., 1994; Matsusaka et al., 1993; Stein and Baldwin,
1993) but are antagonistic in angiotensinogen gene regulation (Ron et al., 1990). NF-
B is a pleiotropic inducible
transcription factor initially identified as a nuclear factor that
binds to the
B enhancer motif of immunoglobulin
light chains
(Sen and Baltimore, 1986). The NF-
B family includes NFKB1 (p50),
NFKB2 (p52), RelA (p65), RelB, v-Rel, and c-Rel proteins. NF-
B
proteins regulate transcription of a wide variety of genes, including
those encoding cytokines, viral proteins and immunoglobulin, through
the
B binding element present at their promoter regions (Grilli et al., 1993; Grimm and Baeuerle, 1993). C/EBP is a family of
transcription factors termed bZIP proteins (Vinson et al.,
1989). They contain a leucine zipper domain linked to a DNA binding
basic region, both located in the C-terminal region. C/EBP-
(Landschulz et al., 1988) was originally identified and shown
to be involved in the transcriptional activation of adipose-specific
genes during differentiation of 3T3-L1 preadipocytes (Christy et
al., 1989; Friedman et al., 1989). C/EBP-
(Akira et al., 1990; Cao et al., 1991; Poli et al.,
1990) and C/EBP-
(Cao et al., 1991; Kinoshita et
al., 1992; William et al., 1991) are induced in response
to IL-6 and involved in IL-6-mediated signal transduction. Members of
the C/EBP family are capable of dimerization through the leucine zipper
domain, and both C/EBP-
(Nakajima et al., 1993; Wegner et al., 1992) and C/EBP-
(Ray and Ray, 1994a) are
activated by phosphorylation. Since LPS-mediated acute-phase
inflammation activates both C/EBP and NF-
B transcription factors
in the liver, it is likely that SAA gene transcription will be
influenced by the concerted action of these two factors. Analyses of
various deletion promoter constructs in transient transfection assays
indicated that a potential cooperative interaction between C/EBP and
NF-
B is involved in the regulation of expression of rat SAA1 and
human SAA2 genes (Betts et al., 1993; Li and Liao, 1991,
1992), although identity of the members of the C/EBP and
B/Rel
family was not well documented. Furthermore, activation of any
C/EBP-like factors was not seen in conditioned medium-stimulated Hep3B
cells (Li and Liao, 1991), and no cross-coupling or physical
interaction was demonstrated between the two factors. Thus, the nature
of cooperativity between these two elements of SAA gene in the hepatic
expression following acute phase induction remained unclear. In the
present study, we characterized members of the NF-
B and C/EBP
family that are activated in the liver following LPS-mediated
inflammatory condition and investigated their interaction with SAA
promoter for its transcriptional activation under acute phase
condition. We also showed, by in vitro DNA-protein binding
assays, evidence of heteromeric complex of C/EBP and NF-
B and its
interaction with both C/EBP and NF-
B elements. In vivo cotransfection assays provided evidence that the heteromeric
complex is a stronger activator of SAA gene transcription than
homomeric complexes of either C/EBP or NF-
B.
For self-annealing, the oligonucleotides were heated to 95 °C for 2 min in 50 mM Tris, pH 7.4, 60 mM NaCl, 1 mM EDTA and allowed to cool slowly to room temperature in 2-3 h.
Figure 1:
Induction kinetics of
nuclear factors in LPS-treated rabbit liver that interact with the
promoter elements of SAA gene. EMSAs were performed with P-labeled SAA promoter DNA fragments containing NF-
B
elements (-112 to -79) and C/EBP element (-193 to
-136) and shown in panels A and B,
respectively. Nuclear extracts (10 µg of protein) from the liver
tissue of uninduced and LPS-induced rabbits were incubated with the
P-labeled NF-
B probe (lanes 2-6) and
C/EBP probe (lanes 2`-6`). The resulting DNA-protein
complexes were fractionated in a 6% native polyacrylamide gel. Lanes 1 and 1` contained probe
only.
Figure 2:
Characterization of the activated
NF-B nuclear factors in LPS-treated rabbit liver. A,
identification of RelA and its interaction with NF-
B element of
SAA promoter. EMSAs were performed with
P-labeled SAA
promoter DNA (-112 to -79) and nuclear extract (10 µg
of protein) from the rabbit liver treated with LPS 3-h, which has the
highest level of factors capable of interacting with this DNA probe.
DNA binding assays were performed in the presence of antisera to RelA,
NFKB1, and v-Rel (1 µl of 1:10 dilution of each). For RelA and
NFKB1, two different antibody preparations, one raised against the
N-terminal end of the protein (lanes3 and 5) and the other raised against the C-terminal end of the proteins (lanes4 and 6) were used. Lane1 contained probe incubated in the absence of
any nuclear factors. Lane2 contained LPS 3-h nuclear
extract depicting the DNA-protein complexes A, B, and C. In addition to
nuclear extract, antiserum to either RelA (lanes3 and 4), NFKB1 (lanes5 and 6),
or v-Rel (lane7) was also included in some binding
reactions. Lane8 contains a nonspecific serum.
Supershifted complexes in lanes3 and 4 are
indicated by an arrow. B, binding of NFKB1 and v-Rel
to the SAA promoter. Radiolabeled DNA probe containing the NF-
B
element of rabbit SAA gene (-112 to -79) was incubated with
recombinant NFKB1 (lanes1 and 3) or v-Rel
protein (lanes2 and 4) in the absence (lanes1 and 2) or in the presence (lanes3 and 4) of a competitor
oligonucleotide containing NF-
B core binding element whose
sequence is described under ``Materials and Methods.'' Lane5 contained probe only. The complexes were
analyzed in a 6% native polyacrylamide gel. C, transactivation
of SAA-CAT reporter plasmids containing NF-
B element by the
NF-
B expression plasmids. Reporter plasmids SAA wtNF-
B-CAT
(designated by
,
, and
) and SAA mtNF-
B-CAT
(designated by
,
, and +) (10 µg of each) were
transfected into BNL CL.2 cells along with increasing concentrations of
pCMV-NFKB1 (
and
), pCMV-RelA (
and
), or
pCMV-v-Rel plasmids (
and +). As a control, SAA
wtNF-
B-CAT was cotransfected with pCMV4 vector plasmid (
).
CAT activity was measured as described under ``Materials and
Methods.'' The results represent an average of three independent
transfection assays.
To examine whether these
proteins can activate transcription from the SAA-NF-B element,
transient transfections were performed using BNL CL.2 liver cells. One
copy of the SAA promoter containing NF-
B element was ligated to
the pBLCAT2 reporter gene containing minimal tk promoter
(Luckow and Schutz, 1987) and cotransfected along with the expression
plasmids encoding NFKB1, RelA, and v-Rel (Fig. 2C).
These members of the NF-
B family activated the transcription of
the reporter gene carrying NF-
B motif of SAA promoter in a
dose-dependent manner.
Figure 3:
Identification of C/EBP isoforms induced
by LPS and their interaction with the C/EBP element of SAA promoter.
EMSAs were performed with P-labeled SAA promoter DNA
(-193 to -136) and nuclear extracts (10 µg of protein)
prepared from the liver tissues of uninduced (lanes
2-8), LPS 3-h (lanes 9-15), and LPS 24-h (lanes16-22) induced rabbits. DNA binding
assays were performed in the presence of two concentrations (0.5 and 1
µl of a 1:10 dilution) of antibody for three C/EBP isoforms. No
cross-reactivity between the antisera was noticed. Lane1 contained probe only, and lanes 2, 9, and 16 contained nuclear extract without any antisera. Migration
positions of DNA-protein complexes 1, 2, and 3a-3c are indicated. Arrowheads indicate the positions of some supershifted
complexes.
Figure 4:
Binding of NF-B and C/EBP to the SAA
promoter. Radiolabeled SAA probes (5 pmol/assay) containing
wtC/EBPwtNF-
B (panelA), mtC/EBPwtNF-
B (panelB), or wtC/EBPmtNF-
B (panelC) sequences were incubated with either NF-
B(RelA)
prepared from pCMV-RelA-transfected COS-7 cells (3 µg of protein
preparation) or C/EBP prepared from nuclear extract of
turpentine-treated rabbit liver (4 µg of protein preparation) or
both NF-
B and C/EBP as indicated. As competitors, oligonucleotides
(50 pmol/assay) containing binding elements for NF-
B and C/EBP
(sequences described under ``Materials and Methods'') were
used in some binding assays as indicated.
Figure 5:
Effect of increasing concentrations of
C/EBP and NF-B(RelA) on the binding of homo- and heteromeric
complexes of the two transcription factors. EMSAs were performed using
either mtC/EBPwtNF-
B (panelA) or
wtC/EBPmtNF-
B (panelB) probe. A, a
constant amount of RelA (3 µg of the protein preparation) was
incubated with increasing concentrations of C/EBP (0, 2, 4, and 6
µg of C/EBP preparation) (lanes 1-4), and the
resulting complexes were resolved in a 6% native polyacrylamide gel. B, a constant amount of C/EBP (5 µg of the protein
preparation) was incubated with increasing concentrations of RelA (0,
1, 2, and 3 µg of RelA preparation) (lanes5-8). The products were fractionated in a 6% native
polyacrylamide gel. Migration positions of NF-
B(RelA), C/EBP, and
C/EBP
NF-
B(RelA) are indicated.
Figure 6:
Relative affinity of C/EBPNF-
B
heteromers to interact with the NF-
B binding site. Panel
A, radiolabeled wtNF-
BmtC/EBP probe (5 pmol/assay) was
incubated with a mixture of NF-
B(RelA) (3 µg of protein) and
C/EBP (6 µg of protein) preparations in the absence (lanes
2, 9, and 10) or in the presence of increasing
concentrations of competitor NF-
B oligonucleotide (10, 25, and 50
pmol in lanes3, 4 ,and 5,
respectively) or C/EBP oligonucleotide (10, 25, and 50 pmol in lanes6, 7, and 8, respectively). Panel B, prior to the addition of the probe, protein
preparations were preincubated with antisera to C/EBP (lane9) or a nonspecific serum (lane10).
The complexes were resolved in a 6% native polyacrylamide
gel.
Although the results above (Fig. 4, lane8 in panelB and lane13 in panel C) showed that the mutated NF-B and C/EBP
sites used in these probes prevented binding of the corresponding
factors to the respective mutated sites, it can still be argued that
the slower migrating complex (the presumable heteromer seen in Fig. 4, lanes9 and 15) may arise due
to some cooperative binding of NF-
B to its mutated site when C/EBP
is present and vice versa. To rule out such a possibility, we
performed similar experiments, as those in Fig. 4(B and C), but using probes that contained only wtNF-
B
or wtC/EBP binding elements of SAA promoter. Appearance of identical
slower migrating complexes, such as those seen in Fig. 4(lanes9 and 15), when both
C/EBP and NF-
B were added (data not shown) asserted that indeed
these complexes are composed of a heteromer of NF-
B and C/EBP.
EMSAs were performed using SAA mtC/EBPwtNF-B probe and a
combination of constant amount of RelA and increasing amounts of C/EBP
factors for further characterization of the heteromer. Increasing
amounts of C/EBPs considerably enhanced the formation of
C/EBP
NF-
B heteromeric complex (Fig. 5A, lanes 1-4). In a reciprocal experiment, an increasing
dose of RelA (NF-
B) was seen to favor the formation of
C/EBP
NF-
B heteromer (Fig. 5B, lanes
5-8). It was further noticed that the intensity of the
C/EBP
NF-
B heteromeric complex was somewhat higher with the
NF-
B site than that with the C/EBP site (Fig. 5, compare
the level of C/EBP
NF-
B heteromeric complex between panelsA and B). This finding suggested that
C/EBP
NF-
B heteromer might have a higher affinity of binding
to the NF-
B site than to the C/EBP site.
To test this
possibility, EMSA was performed using SAA mtC/EBPwtNF-B DNA as
probe and molar excesses of NF-
B or C/EBP oligonucleotides as
competitors of DNA-protein complex formation (Fig. 6). The
heteromeric complex of NF-
B(RelA) and C/EBP was easily competed in
the presence of excess NF-
B oligonucleotides (lanes3-5) but less efficiently inhibited by the excess
C/EBP-specific oligonucleotide (lanes 6-8). If the
affinity of the C/EBP
NF-
B heteromeric complex for the C/EBP
or NF-
B element was similar, the level of competition by both
oligonucleotides would be comparable. Lack of efficient competition by
C/EBP oligonucleotide (lanes 6-8) indicated that the
NF-
B
C/EBP heteromer interacts more avidly with the NF-
B
site than with the C/EBP site of the SAA gene. Inclusion of C/EBP
antisera in EMSA supershifted only the slower migrating
C/EBP
NF-
B heteromer (lane9), whereas
nonspecific antiserum had no effect on it (lane10).
Similar results were also obtained in a reciprocal experiment when SAA
wtC/EBPmtNF-
B element was used as probe (data not shown). These
results further verified that the slower migrating complex is indeed
composed of C/EBP and NF-
B proteins.
Figure 7:
Cotransfection analysis of the NF-B
and C/EBP expression plasmids on the SAA reporter genes. Three CAT
reporter plasmids, derivatives of pBLCAT2 and carrying SAA promoters
containing either wtC/EBPwtNF-
B, or mtC/EBPwtNF-
B or
wtC/EBPmtNF-
B elements, were cotransfected with plasmids
expressing either NF-
B(RelA), C/EBP (C/EBP-
), or both.
Reporter plasmids (10 µg of DNA) were transfected into BNL CL.2
cells alone (shadedbars) or cotransfected with 2
µg each of pCMV-RelA (solidbars),
pMSV-C/EBP
(stripedbars), or
pCMV-RelA+pMSV-C/EBP
(cross-hatched bars). CAT
activity in the transfected cells was measured as described under
``Materials and Methods.'' -Fold induction of the CAT
activity in the cotransfected cells relative to that of the reporter
plasmid alone was determined and plotted as relative CAT
activity.
Figure 8:
Cotransfection analysis of SAA promoter
activity. Panel A, stimulation of SAA NF-B promoter by
C/EBP-
. SAA-CAT reporter plasmid (10 µg of DNA) containing
mtC/EBPwtNF-
B element was used to transfect BNL CL.2 cells either
alone (solid bars) or with 2 µg of pCMV-RelA (shaded
bars) or 2 µg of pCMV-NFKB1 (stripedbars).
In addition, some transfection reactions also contained increasing
concentrations of C/EBP-
expression plasmid (2, 4, and 6 µg,
respectively). Panel B, stimulation of SAA C/EBP promoter by
NF-
B. SAA-CAT reporter plasmid (10 µg of DNA) containing
wtC/EBPmtNF-
B element was used to transfect BNL CL.2 cells either
alone (light shaded bars) or with 2 µg of pMSV-C/EBP-
(dark shaded bars). In some transfection assays, increasing
concentrations (2, 4, and 6 µg, respectively) of pCMV-NFKB1 or
pCMV-RelA were included. CAT activity in the transfected cells was
measured as described under ``Materials and Methods,'' and
the induction of CAT activity relative to that of the reporter plasmid
alone was presented.
Since the heteromeric complex of
NF-B and C/EBP can also interact with the C/EBP elements of SAA
promoter (seen in Fig. 4C and 5B), we studied
the transcriptional induction potential of SAA wtC/EBPmtNF-
B
promoter-containing CAT reporter gene in the presence of constant
amount of C/EBP-
and increasing amounts of NF-
B expression
plasmids. The results shown in Fig. 8B demonstrated
that the combination of C/EBP-
and NF-
B was a better
transcriptional activator than C/EBP-
alone. Western blot analysis
for C/EBP-
in the cotransfected cells (data not shown) indicated
that the expression of C/EBP-
was not altered by the increasing
presence of NFKB1 or RelA. Increased expression of the reporter gene
was due to simultaneous presence of the two families (NF-
B and
C/EBP) of transcription factors. However, the level of synergistic
transactivation was less than that obtained through the NF-
B
element. About 2-fold induction was detected at the C/EBP element due
to the presence of RelA (Fig. 8B), whereas more than
4-fold induction was detected at the NF-
B site (Fig. 8A). This was presumably due to the lower
affinity of the heteromeric NF-
B
C/EBP complex for the C/EBP
element seen earlier in the EMSAs ( Fig. 5and Fig. 6).
Figure 9:
Interaction of heteromeric
RelAC/EBP-
complex with the NF-
B element of SAA gene.
EMSAs were performed using
P-labeled SAA promoter DNA
(-112 to -79) and nuclear extract from the LPS 3-h treated
rabbit liver. Prior to the addition of the probe, nuclear extract was
incubated with anti-C/EBP-
(lane2),
anti-C/EBP-
(lane3), anti-C/EBP-
(lane4) antisera, or a nonspecific antiserum (lane5). In some reactions, antiserum to C/EBP-
was
blocked by adding either C/EBP-
-specific peptide (lane6) or a nonspecific peptide (lane 7) in the
preincubation reaction. The migration positions of the three complexes
A, B, and C are indicated. Supershifted complex in lanes4 and 7 is indicated by an arrow.
We have provided evidence of the concerted role of C/EBP and
NF-B in the regulation of SAA gene expression. The following novel
findings were obtained. (i) There is in vivo evidence of
RelA
C/EBP heteromer formation at the SAA promoter in the
LPS-induced rabbit liver; (ii) RelA and C/EBP-
cooperatively
transactivate the expression of SAA gene; (iii) the heteromeric complex
of NF-
B and C/EBP has a higher transactivation potential in
activating SAA expression than their homomeric counterparts; (iv) the
heteromeric complex of NF-
B and C/EBP can bind to both NF-
B
and C/EBP binding elements; (v) the NF-
B
C/EBP heteromer
interacts with the NF-
B element with a much higher affinity than
that with the C/EBP element of SAA promoter.
Several earlier studies
have shown that C/EBP or NF-B alone can increase the transcription
of SAA-CAT reporter gene quite effectively (Ray and Ray, 1993a, 1993b).
Under certain acute inflammatory conditions, when both of these
transcription factors are induced and activated, the expression of SAA
gene is likely to be regulated by the combinative effect of these two
factors. Although some previous studies suggested that both NF-
B
and C/EBP transcription factors cooperate in the inducible expression
of rat SAA gene (Li and Liao, 1991, 1992), no induction of any C/EBP
binding activity under inflammatory condition was shown. Thus, the role
of C/EBP was not established in the acute-phase induction of rat SAA1
gene expression. We have presented evidence indicating activation of
both NF-
B(RelA) and C/EBP-
in the nuclear extract of the
liver and their interaction with the SAA promoter using LPS-treated
rabbit liver that overexpresses SAA. We have also directly assessed the
ability of various members of C/EBP and
B/Rel family to interact
with SAA promoter and activate transcription of the SAA gene. Using
RelA protein from CMV-RelA-transfected COS-7 cells and C/EBP from
turpentine-induced liver, we have demonstrated the formation of homo-
and heteromeric complexes between these two transcription factors and
provided evidence that the heteromers of NF-
B and C/EBP are more
potent in transactivating the SAA-CAT reporter gene expression.
In vitro binding studies showed that both NF-B and
C/EBP are capable of interacting with the SAA promoter quite
efficiently and interactions of these factors to their cognate binding
sites were not dependent on the presence of the other ( Fig. 4and Fig. 5). However, presence of both NF-
B
and C/EBP factors resulted in the formation of slower migrating
complexes at both binding sites, indicating the interaction of these
factors as a heteromeric complex (Fig. 4). Using mutant
oligonucleotides, in which either the NF-
B element or the C/EBP
element was mutated by multiple sequence substitutions and probes
containing one factor binding element, we detected the formation of a
slower migrating complex that is presumably composed of both of these
factors. Such a heteromer could interact with either the C/EBP or the
NF-
B element of SAA gene (Fig. 5, A and B). Supporting our data, LeClair et al.(1992)
reported that p50 and C/EBP-
directly associate each other via the
bZIP domain and the Rel homology domain. Functional and physical
association between NF-
B and C/EBP family members have also been
reported by several other groups (Stein et al., 1993; Stein
and Baldwin, 1993; Matsusaka et al., 1993). However, in these
studies the formation of a heteromeric complex between C/EBP and
NF-
B protein, although suggested, could not be demonstrated under in vitro or in vivo condition (Stein et al.,
1993). We have presented evidence for the formation of heteromer of
C/EBP and NF-
B and demonstrated that such a heteromeric complex
interact with C/EBP or NF-
B element of SAA promoter under in
vitro and in vivo conditions (Fig. 4, Fig. 5, and Fig. 9).
Under LPS-mediated inflammatory
conditions, mainly isoform of C/EBP and RelA (p65) are induced (Fig. 1Fig. 2Fig. 3). The appearance of NF-
B
is rapid and detectable within 1 h after LPS treatment. The inducible
isoform of C/EBP was detected well after 1 h of the onset of
inflammation. The induction of C/EBP-
is quite predominant, and
this activity declines within 12 h of the onset of LPS induction.
Cumulative accumulation of C/EBP isoforms and RelA thus represents a
key event in LPS-mediated induction of SAA gene. Earlier Lowell et
al. (1986b) had shown that SAA gene transcription reaches its
maximum at about 3 h following LPS induction. Our findings of the
appearance of the two families of transcription factors, which also
accumulate at a high concentration at 3 h following LPS induction,
suggest that these factors are likely to play a decisive role in SAA
gene expression. Betts et al.(1993) recently showed the role
of NF-
B and NF-IL6 (a human homolog of C/EBP
) in cytokine
induction of the human SAA2 gene. We have demonstrated that C/EBP-
and not C/EBP
is a major C/EBP isoform that is activated in LPS
induction of rabbit SAA gene, and further studies on transactivation
assays (Fig. 8) indicated that C/EBP-
is capable of
promoting transcription. This difference between the two findings may
have been related to use of different inducers or cell types. Previous
studies have shown that all
B-binding sites do not interact
equally well with each member of
B/Rel family (Kunsch et
al., 1992). In vitro DNA binding studies with purified
NFKB1 (p50) and v-Rel showed that these factors can interact
efficiently with the SAA promoter. Consistent with this observation, we
found that RelA (p65), NFKB1 (p50), or v-Rel expression plasmids can
transactivate the SAA NF-
B CAT reporter gene expression in a
dose-dependent manner (Fig. 2C). v-Rel has been shown
to be a weak transcriptional activator (Kamens et al., 1990)
in certain cells and may even inhibit transcription from the
B
sites (Inoue et al., 1991). It is quite possible that the
differential transactivator effect of v-Rel may be dependent upon the
cell types used in the transfection assay since we observed a positive
transactivating ability of v-Rel in inducing SAA gene expression.
Synergistic transactivation of SAA gene by C/EBP and NF-B
transcription factors was confirmed by cotransfection experiments (Fig. 8), which showed synergistic stimulation of SAA promoter
through both C/EBP and NF-
B elements. The synergy between RelA and
C/EBP-
was more prominent through the NF-
B element. This is
consistent with the result obtained from EMSAs ( Fig. 5and Fig. 6), which indicated that NF-
B
C/EBP heteromer
binds more efficiently with the NF-
B element of SAA gene. This
observation is different from some previous reports. Using multimerized
c-Fos response element and human immunodeficiency virus-1
B
enhancer motif linked to the ``TATA box,'' Stein et
al.(1993) showed that cross-coupling of C/EBP and NF-
B
results in the inhibition of promoter containing the NF-
B element
but synergistically stimulates the promoters containing the C/EBP
binding element. However, both binding sites are required for the
synergistic stimulation of expression of IL-6 and IL-8 genes (Matsusaka et al., 1993). These differences in findings may occur due to
the use of artificial promoters, reporter genes with different spatial
arrangement of the two factor binding elements, or different cell types
used in the transfection assays. We also noticed that RelA
C/EBP
has a higher transactivation potential than the NFKB1
C/EBP (Fig. 8). This could be due to a higher binding affinity of the
RelA
C/EBP to the SAA NF-
B site compared to that of
NFKB1
C/EBP. A similar effect of differential binding affinity has
been shown recently to be involved in Ig
chain expression during B
cell differentiation, where p50-Rel was found to have a higher binding
affinity to the Ig
promoter than the p50-p65 (Miyamoto et
al., 1994). In addition to RelA and NFKB1, the contribution of
c-Rel and RelB in SAA gene expression remains to be determined. In
summary, we have shown that cooperative interaction between two
transcription factors, C/EBP and NF-
B, is involved in the
regulation of rabbit SAA gene expression under LPS-mediated acute
inflammation. Accumulating evidence indicates that a gene is regulated
by the combined actions of a group of transcription factors and
interaction between them is a critical regulatory component of gene
expression. It will be of interest to find out if other factors also
participate in SAA gene regulation.