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B p65 Mediates the Assembly and Activation of the TNF-Responsive Element of the Murine Monocyte Chemoattractant-1 Gene1
Department of Microbiology and Immunology, Emory University School of Medicine, Emory University, Atlanta, GA 30322
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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transcriptionally regulates murine monocyte chemoattractant
protein-1 (MCP-1) expression. Three approaches were used to determine
the mechanism by which TNF regulates MCP-1. Mutation analysis showed
that two distal 
B sites, a novel dimethylsulfate-hypersensitive
sequence, and a promoter proximal SP-1 site were required for TNF
induction. Although the B sites and the hypersensitive sequence
function as a NF-B-mediated enhancer, regulating induction by TNF,
stereospecific alignment of the B sites was not critical.
Trans-activation studies conducted by cotransfection of
p50 and/or p65 expression vectors with MCP-1 constructions showed that
TNF regulates MCP-1 through NF-B. Examination of MCP-1 induction in
NF-B-disrupted embryonic fibroblasts showed that p65 was necessary
for both the induction and the TNF-induced protein occupancy of the
enhancer in vivo. The action of the antioxidant inhibitor of NF-B
activation, pyrrolidine dithiocarbamate, in wild-type and NF-B
mutant cells was examined. The results suggested that TNF activates
NF-B through both pyrrolidine dithiocarbamate-sensitive and
-insensitive mechanisms. This study illustrates the crucial role for
NF-B p65 in the induction of the MCP-1 gene by TNF and in the
assembly of a NF-B dependent enhancer in vivo. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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TNF is a pleiotropic cytokine (19) functioning in diverse biological
processes such as apoptosis (20, 21, 22), inflammation (23), and
immunomodulation (24, 25). Induction of MCP-1 by TNF is important for
the initiation of inflammatory responses, but also contributes to
MCP-1-associated diseases (18, 23). In vivo genomic footprinting (IVGF)
of the upstream flanking DNA of the MCP-1 gene in both resting and
TNF-treated BALB/3T3 murine fibroblasts (4) showed that TNF induced
multiple DMS-resistant and DMS-hypersensitive bases in two regions: a
distal regulatory region and a proximal regulatory region. In vivo
occupancy in the proximal regulatory region identified an SP-1 binding
site and two novel sites (B-3 and site B). The distal regulatory
region, which contains two B binding sites, a novel site (A) and a
DMS-hypersensitive (HS) sequence located between the B sites, was
required for TNF-activated expression of wild-type MCP-1 reporter
constructions (4). The two B sites are conserved in the human MCP-1
gene and were also found to be critical for TNF induction of the human
gene (26). Recently, human MCP-1 reporter constructions were shown to
be activated by p65/p65 homodimers and p65/c-Rel heterodimers in
transient transfections (27). Together, these experiments suggest that
NF-B is involved in MCP-1 induction, but the exact composition and
mechanism of promoter occupancy by NF-B have not been determined.
Additionally, the role of each of the in vivo occupied sites has not
been examined.
Mammalian NF-B family proteins are composed of homo- and
heterodimers of five distinct DNA binding subunits: p50, p52, p65/RelA,
c-Rel, and RelB. NF-B isoforms containing p65 and p50 are important
for TNF-induced expression of a variety of genes (28). The inducibility
of TNF-activated genes such as IB and granulocyte-macrophage CSF
was impaired in embryonic fibroblasts containing a targeted disruption
of the NF-B p65 subunit (29). In the majority of cell types, NF-B
is sequestered in the cytosol by IB proteins. Upon TNF treatment,
IB is phosphorylated and degraded, resulting in the release and
translocation of NF-B to the nucleus (30). Inhibition of TNF-induced
degradation of IB by antioxidants such as pyrrolidine
dithiocarbamate (PDTC) prevents activation of NF-B and reduces or
eliminates TNF-induced gene expression in a variety of cell types (31, 32). However, in BALB/3T3 fibroblasts we observed that TNF was still
able to induce protein occupancy on the coding strand of the distal
B sites of the murine MCP-1 gene in the presence of PDTC (4),
suggesting the possibility that TNF may also activate NF-B by a
PDTC-insensitive pathway.
Although the predominant NF-B isoform is a p50/p65 heterodimer,
multiple NF-B isoforms were detected in nuclear extracts of
TNF-treated cells (4, 27, 33). These isoforms may compensate for each
other and/or regulate TNF-induced gene expression in a
sequence-dependent manner (34, 35). Experiments involving
NF-B-mediated expression of several genes suggest that NF-B does
not function alone but, rather, forms cooperative interactions with
other factors (30, 33). One model of NF-B-mediated gene expression
involves the formation of a multiprotein complex, termed an
enhanceosome, containing architectural proteins such as high mobility
group-I(Y) (36, 37, 38). It is not known whether the distal
regulatory region of MCP-1 also functions using the structure of an
enhanceosome or whether TNF regulates MCP-1 induction by a novel
pathway.
In this study we examined the mechanism of TNF induction of MCP-1
through NF-B using three approaches: mutagenesis of potential MCP-1
regulatory sequences, trans-activation of MCP-1 expression
using p50 and p65 NF-B expression vectors, and analysis of NF-B
protein occupancy in vivo in B p50-, p65-, and p50/65-targeted
disrupted embryonic fibroblasts. The results showed that 1) the distal
B elements, the HS sequence, and the proximal SP-1 binding site are
required for induction of the MCP-1 gene; 2) TNF-induced DMS
hypersensitivity of the HS sequence is probably formed by the
interaction of a factor rather than solely through the interactions of
the neighboring B binding factors; 3) although p65 is required, TNF
induction of MCP-1 is most likely mediated through a p50/p65
heterodimer; 4) in vivo occupancy of the distal B sites requires
p65, but is less stable in the absence of p50; and 5) PDTC inhibition
of TNF-induced MCP-1 expression is effective only in cells containing
p50.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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NF-B p50-/-, p65-/-,
p50/p65-/-, and wild-type embryonic fibroblasts were
provided by Drs. A. Hoffmann and D. Baltimore (Massachusetts Institute
of Technology, Cambridge, MA). BALB/3T3 clone A31 and NIH-3T3
fibroblasts were purchased from the American Type Culture Collection
(Manassas, VA). Cells were grown in DMEM supplemented with 10% calf
serum (HyClone, Logan, UT), penicillin (50 U/ml), streptomycin (50
µg/ml), and L-glutamine (1 mM; Life Sciences, St.
Petersburg, FL). Human rTNF was purchased from Genzyme (Cambridge, MA)
and used at 500 U/ml. The cells were grown to about 90% confluence
before TNF treatment. PDTC was purchased from Sigma and was added 90
min before TNF treatment.
Plasmids
NF-B p50 and p65 mammalian expression vectors were obtained
from Dr. T. Collins (Brigham and Women’s Hospital, Boston,
MA). The pJECAT2.6 and pJECAT0.3 CAT reporter constructs
containing the region from -2642 to +81 and from -329 to +81 of the
murine MCP-1 promoter, respectively, were described previously (4).
Oligo-mediated PCR was used to create point mutations, base insertions,
and B site conversion constructs on the wild-type pJECAT2.6 plasmid.
Briefly, complementary mutagenizing oligos containing base changes at
the target site (Table I
) and two oligos
corresponding to the ends of the MCP-1 insertion (containing unique
restriction sites) were designed. The overlapping PCR products were
made using pJECAT2.6 as the template with the appropriate combination
of mutant and end primers. The PCR products were purified and annealed
together as the template for a second PCR reaction using only the end
primers. Both the final PCR product and pJECAT2.6 plasmid were cut with
the same restriction enzymes and ligated together to make the desired
construct. The changes in the distal regulatory region were made using
SalI and SpeI sites. The coding end oligo was
PCATM5 (5'-GACCATGATTACGCCAAGCTTGC), and the noncoding end oligo was
JEM3 (5'-TGATCTTGCTAGTCACTGTCCTCC). All mutations were verified by
DNA sequencing.
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.
Constructs juxtaposing the distal region 5' to the proximal regulatory
region were created by PCR using pJECAT2.6 as the template and primer
pairs of JEB5–9 (5'-GCTCTTGGGAGGCTGGGGATTGATGT) and JEB3–2
(5'-AGTTAGCACAGGAGGCAGCGCAA), and JEB5–4 (5'-CCCGAAGGGTCTGGGAA) and
JEB3–4 (5'-GGATAAGAGCGTGGAAATTCCC). The pJECAT0.3 was cut with
SalI and then filled in. Ligation of the linear blunt-end
pJECAT0.3 with the PCR products generated both the direct or forward
orientation (p0.3/D0.4F and p0.3/BF) and the reverse orientation
(p0.3/D0.4R and p0.3/BR) constructs.
Cleavage of pJECAT2.3 (4) with HindIII followed by
religation produced pJECAT0.5, a CAT construct carrying the region from
-526 to +81 of the murine MCP-1 5'-flanking sequence. To create
plasmids containing the distal B region 5' to the proximal
regulatory region in the forward orientation, the B region was
cloned by PCR using the primers BSTP5
(5'-TATCGGTAACCTCTCCCGAAGGGTCT) and BSUP3
(5'-CGTGGAATCCTCATCCTATTCTCGCA) in separate reactions with each of
the following templates, p2.6 mB1, p2.6 mB2, and p2.6 mHS. The
PCR products were cut with the restriction enzymes BstEII
and Bsu36I. Ligation of the cleaved PCR products with
BstEII/Bsu36I double-digested
pJECAT2.6, p2.6 mB1, and p2.6 mB2 plasmids generated
p2.6/B1, p2.6/B2, p2.6 mB1/B1, p2.6 mB2/B1, and p2.6
mB2/B2 constructs. Ligation of the cleaved PCR products with
BstEII/Bsu36I double-digested pJECAT0.5 produced
p0.5/B1, p0.5/B2, and p0.5/mHS constructs.
In vivo genomic footprinting
IVGF was conducted as previously described (4) with modifications in the preparation of genomic DNA. A 10-cm tissue culture plate was treated with DMS (1 µl/ml) for 2 min followed by three washes with cold HBSS. Thereafter, 4 ml of DNAzol (Molecular Research Center) was added to the plate. The lysate was collected into a tube, and genomic DNA was precipitated by addition of 2 ml of 100% ethanol. The DNA pellet was washed twice with 1 ml of a solution containing 70% DNAzol and 30% ethanol and twice with 1 ml of 95% ethanol. The DNA was dissolved in a solution of 10 mM Tris (pH 7.5) plus 1 mM EDTA and cleaved by piperidine. The IVGF primers were described previously (4).
Transfection assays
Transient transfections were conducted in NIH-3T3 cells as described previously (4) with addition of 1 µg of an alkaline phosphatase expression vector (pSV2AlkPhos) as an internal control of transfection efficiency (39). TNF (500 U/ml) was added 24 h post-transfection to one plate, and medium alone was added to the second plate. The cells were harvested 36 h post-transfection and assayed by ELISA as described by the manufacturer (Boehringer Mannheim, Indianapolis, IN). Alkaline phosphatase activity was assayed using a kit from Bio-Rad (Richmond, CA).
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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TNF induces murine MCP-1 expression at the transcriptional level
(40). IVGF of resting and TNF-treated BALB/3T3 cells revealed
TNF-induced protein occupancy at multiple sites within the MCP-1
5'-flanking DNA spanning 2.6 kb (4). These sites included two NF-B
binding sites (B-1 and B-2), an SP-1 binding site, and several
novel sites. To more precisely define the roles of these sites in
controlling MCP-1 expression, multiple base pair substitutions were
introduced into each element such that the site was randomized, and the
resulting constructions were analyzed for activity. The base vector for
these constructions was the wild-type pJECAT2.6 construct (Fig. 1), which contains the 2.6 kb of DNA 5'
to the start site of transcription and all sites identified by IVGF in
the previous study (4). Due to extremely poor transfection efficiency
of BALB/3T3 cells, the resulting mutant constructions were transiently
transfected into NIH-3T3 cells, a murine fibroblast cell line that is
highly transfectable. MCP-1 RNA blot hybridization showed that NIH-3T3
cells responded to TNF with similar kinetics as the BALB/3T3 cell line
(data not shown). Transfected cells were either treated with TNF or
left untreated, and the level of the CAT reporter protein was measured
by ELISA (Fig. 1A). In the distal regulatory region,
mutation of either B-1 or B-2 ablated the TNF inducibility,
suggesting that both sites are required for TNF induction of MCP-1. In
the proximal regulatory region, mutation of the SP-1 binding site
substantially decreased both basal and TNF-induced expression,
indicating that the SP-1 site is critical for MCP-1 expression.
Although an AP-1 binding site was not occupied in control or
TNF-treated BALB/3T3 cells in IVGF assays (4), mutation of this site
also reduced both basal and TNF-induced CAT expression (p2.6 mAP).
Thus, it is possible that the expression of the MCP-1 gene is regulated
through the AP-1 site. Mutation of the B-3 site, a site with
similarity to a NF-B binding site that was found to be protected by
IVGF (4), did not affect either basal or TNF-induced CAT expression,
suggesting that the site is not required for TNF induction.
Alternatively, this site may not be occupied in the NIH-3T3 cells.
Unfortunately, promoter sequence polymorphisms in an allele of the
MCP-1 gene in NIH-3T3 cells prevents IVGF analysis of MCP-1 in this
cell line.
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B-1
and B-2 sites became highly hypersensitive to DMS treatment after
TNF addition (Fig. 1B). Sequence analyses of this region,
termed HS, failed to identify a consensus protein binding site,
suggesting that the hypersensitivity could be due to either the binding
of a novel factor(s) to the HS sequence or to distortion of the DNA
caused by interactions between the factors bound to the adjacent B
sites. If the former is the case, sequence substitutions should have a
pronounced effect on TNF induction of the MCP-1 gene. Otherwise, the
sequence itself should not be critical for the induction. To
distinguish between the two possibilities, constructs containing point
mutations in the HS region were created, and TNF inducibility of the
constructs was assayed by transient transfection (Fig. 1). Random
mutagenesis of the entire HS sequence (10 bp) resulted in the loss of
TNF-induced expression (Fig. 1A, p2.6 mHS). Scanning
mutagenesis of the HS and the immediate flanking DNA in which 3 bp were
mutated in each construction revealed a critical sequence located in
the center of the HS that was required for TNF-induced expression (Fig. 1B, p2.6 mHS3). Mutations introduced surrounding these
central base pairs had little or no effect on induction by TNF (Fig. 1B, p2.6nHS1, p2.6 mHS2, and p2.6 mHS4). These results
suggest that the DMS hypersensitivity is due to the binding of a
specific protein(s) (the HS factor) to the middle of the sequence.
B-1, the HS sequence, and B-2 function together as an
enhancer
The above experiments suggest that TNF inducibility of the distal
regulatory region involves at least three TNF-responsive elements:
B-1, the HS sequence, and B-2. To determine the orientation and
position dependence of this region, a series of constructs was made and
tested in transient transfections (Fig. 2). The pJECAT0.3, a construct
carrying only the proximal region of the MCP-1 promoter, was not
inducible by TNF (4). When the distal region was juxtaposed 5' to the
proximal region of pJECAT0.3 in either orientation, TNF inducibility
was recovered, suggesting that the distal regulatory region could
function independent of the 2 kb of DNA separating the distal and the
proximal regulatory regions (p0.3/D0.4F and p0.3/D0.4R). TNF
inducibility was also restored when only the sequence from the B-1
to B-2 sites (B region) was fused 5' to the proximal regulatory
region, indicating that the B region can function independent of
site A (p0.3/BF and p0.3/BR). The inversion of the distal
regulatory region resulted in constructs that were expressed at higher
levels in both control and TNF-treated cells (p0.3/D0.4F and
p0.3/BR). This result may be due to a more efficient orientation of
the activating factors in relation to the downstream, proximal
regulatory elements or general transcription machinery. As described
above, mutation of B-1, B-2, or the HS sequence also ablated the
TNF inducibility (p0.5/B1, p0.5/B2, and p0.5/mHS). Additional
constructions in which the distal B region was placed 3' to the CAT
gene in either orientation were inducible by TNF (data not shown).
These results suggest that the B region constitutes a single
enhancer element mediating TNF induction of the MCP-1 gene.
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B sites does not affect induction by TNF
Stereospecific alignment between adjacent cis elements
has been observed in several genes, including those containing B
motifs (36, 41). The finding that all three elements, B-1, the HS
sequence, and B-2, constitute an enhancer suggested that the factors
bound to these elements might have special constraints. To determine
whether the order of the elements or spacing between the elements was
critical for the inducibility, two mutants were made and assayed by
transient transfections. Insertion of 5 or 10 bp between B-1 and
B-2 did not significantly affect the induced CAT expression (Fig. 3, p2.6I5 and p2.6I10), suggesting that
the phasing and distance between the two B sites were not critical
for induction. The B1 and B2 sites are not identical in sequence,
suggesting that they may serve distinct functions. To determine whether
this was the case, constructions in which the sites were identical with
each other were made and tested. Conversion of the B-1 site to a
B-2 site or of the B-2 site to a B-1 site also had no effect
on induction (p2.6B11 and p2.6B22), indicating that TNF induction
of MCP-1 does not depend on the unique sequences of B-1 and B-2
but merely on the presence of two B sites.
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B elements are required,
it may be possible to separate the sites from each other. To determine
whether the B elements could function together when separated, a
series of constructions was made in which the B region was
duplicated between the distal and proximal regions of the wild-type and
B mutant plasmids. In these constructions, one B site of the
inserted B region was mutated to determine whether two wild-type
B sites were sufficient for TNF induction (Fig. 3). In comparison
with pJECAT2.6 control construction in which the distal region is
intact, no significant difference in reporter gene expression was
observed when an additional B site was inserted (p2.6/B1 and
p2.6/B2). Insertion of either a wild-type B-1 or B-2 between
the distal and proximal regulatory regions also did not restore TNF
inducibility of the B mutants (p2.6 mB1/B1, p2.6 mB2/B1,
and p2.6 mB2/B2). These results indicate that the two B sites
must function in close proximity and that there is considerable
flexibility in the orientation of the sites with respect to each other.
TNF induces MCP-1 through NF-B
Activation and translocation of NF-B from the cytoplasm to the
nucleus are involved in TNF induction of many genes (30). To determine
whether activation and translocation of NF-B by TNF are sufficient
to induce MCP-1 expression, transfection experiments were conducted in
which the pJECAT2.6 plasmid was either transfected into NIH-3T3 cells
alone or with NF-B p50 and/or p65 expression vectors (Fig. 4A). Basal expression of the
reporter gene was not affected by p50 cotransfection. However,
TNF-induced CAT expression was lower when the p50 expression vector was
cotransfected, suggesting that the p50/p50 homodimer not only does not
activate MCP-1 gene expression but may compete with active NF-B
isoforms to inhibit MCP-1 induction when overexpressed. The level of
reporter gene expression increased dramatically when p65 and p50/p65
expression vectors were cotransfected, indicating that both the p65/p65
homodimer and the p50/p65 heterodimer are capable of inducing MCP-1.
Interestingly, TNF was found to further increase expression of the
reporter in these trans-activation assays. This result may
be due to an increase in the amount of translocated p65/p50 or may
result from the activation or induction of another factor important for
the regulation of MCP-1.
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B, the cis elements that
are required for TNF induction should also be important for
NF-B-mediated expression. To test this hypothesis, the MCP-1
mutation constructs and the NF-B p50 and p65 expression vectors were
cotransfected into NIH-3T3 cells, and the level of CAT reporter was
assayed (Fig. 4B). Mutation of B-1, B-2, AP-1, or SP-1
resulted in a dramatic decrease in p50/p65-induced CAT expression,
indicating that these four sites are also required for NF-B
induction of MCP-1 (Fig. 4B). The pJECAT2.3, a construct
lacking the distal regulatory region, was not inducible by TNF (4) or
by p50/p65, indicating that NF-B acts through the distal B region
as anticipated. Mutation of the HS site did not display as robust an
effect on NF-B trans-activation (as that observed with
TNF treatment), suggesting that the role of the HS site may not be as
crucial when NF-B subunits are overexpressed. Alternately, this site
may only be responsive to TNF stimulation. Similar to the TNF-induced
expression in Fig. 1, mutations of the B-3 site did not affect
NF-B induction, implying that this site is not critical for TNF- or
NF-B-mediated expression of MCP-1.
NF-B p65 is required for TNF induction of MCP-1- and TNF-induced
occupancy of the distal B region
To further characterize the roles of p50 and p65 in TNF induction
of MCP-1, a matched series of wild-type, p50 and p65 knockout cell
lines was analyzed for TNF induction of MCP-1 and the ability of TNF to
induce the distal regulatory region. The level of MCP-1 mRNA in resting
and TNF-treated p50-/-, p65-/-,
p50/p65-/-, and wild-type murine embryonic fibroblasts
was analyzed by RNA hybridization (Fig. 5A). Basal MCP-1 expression
was not detected in any of the cell lines. In agreement with the
NF-B trans-activation transfection experiments, addition
of TNF rapidly induced high level expression of MCP-1 in the wild-type
and p50-/- cells but not in the p65-/- and
p50/p65-/- cells, indicating that p65 is required for TNF
induction of MCP-1 in vivo. It is important to note that prolonged
treatment (>4 h) of the p50-/p65- cells
resulted in apoptosis, highlighting the importance of NF-B in
protecting cells from TNF-mediated apoptosis.
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B region of MCP-1 (4). To
investigate whether activation and translocation of p50- and/or
p65-containing isoforms to the nucleus by TNF are required for changes
in site occupancy, IVGF of the distal regulatory region was conducted
in the wild-type and the B mutant cells (Fig. 5, B and
C). As previously observed, no differences between the in
vitro and in vivo methylation protection pattern were discerned in the
resting cells (lane V and 0 h). After 30 min of
TNF treatment, full occupancy of both the B-1 and B-2 sites was
observed in the wild-type and p50-/- cells, but not in
the p65-/- or p50/p65-/- cells, suggesting
that TNF-induced occupancy at both B sites requires p65. The effect
is more easily observed in the noncoding strand (Fig. 5C).
In BALB/3T3 cells (wild-type), the B sites remained occupied after
4 h of TNF treatment (4). Interestingly, the sites remained
partially occupied in the wild-type embryonic fibroblast cells, but
were faintly occupied in the p50-/- cells after 4 h
of TNF treatment, suggesting that the stability of the induced
protein-DNA complex may be NF-B isoform dependent. As observed in
BALB/3T3 cells, TNF treatment induced strong DMS-hypersensitive sites
in the HS sequence in the wild-type cells and p50-/-
cells (Fig. 5C). However, no DMS-hypersensitive site was
detected in the p65-/- cells and p50/p65-/-
cells. Thus, it appears that the TNF-induced hypersensitivity of the HS
sequence is related to the occupancy of the B sites and directed by
p65.
TNF-induced activation of NF-B p50-containing isoforms is
inhibited by PDTC
Antioxidants such as PDTC have been shown to inhibit TNF-induced
gene expression (32, 42). The inhibitory mechanism of PDTC is to block
TNF-induced phosphorylation and degradation of IB, preventing
NF-B translocation (43). However, we previously observed that in the
presence of PDTC, TNF could still induce partial occupancy on the
coding strand of the distal B sites and could partially induce MCP-1
expression in BALB/3T3 cells (4). Therefore, it is possible that some
NF-B isoforms are induced by TNF through PDTC-insensitive pathways.
Because expression of NF-B family members is not affected by
targeted disruption of either NF-B p50 or p65 subunits (29, 44), the
NF-B mutant cells provide an opportunity to investigate this issue
(Fig. 6). A slight induction by PDTC can
be observed in wild-type and p50-/- cells in this
overexposed gel. This is in contrast to the significant levels observed
previously in BALB/3T3 cells by PDTC (4). Such differences may reflect
subtle cell type differences, such as the differences in B isoforms.
After the cells were treated with PDTC for 90 min, TNF induction of the
MCP-1 was blocked in the wild-type cells but not in the
p50-/- cells. Notably, the low level of TNF-induced
expression of MCP-1 in the p65-/- cells was also totally
blocked by PDTC. These results suggest that PDTC strongly inhibits
TNF-induced activation of NF-B p50-containing isoforms. Western blot
analyses of IB in the control and PDTC-treated cells showed that
TNF-induced IB degradation was blocked by PDTC in the same manner
in both the wild-type and p50-/- cells (data not shown).
Although a preference for one NF-B factor (p50/65) over another may
exist in wild-type cells, it appears as though TNF can activate the
different NF-B isoforms through distinct and potentially independent
mechanisms.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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B from the cytosol to the nucleus (30, 45). While this process
is similar for most TNF-induced genes (30), TNF has been found to
function in a cell-type and time-dependent manner (28, 40), suggesting
that other factors and distinct mechanisms must be involved. The
analysis of the mechanism by which MCP-1 responds to TNF presented here
focused on the role of the upstream B motifs and suggests that
cell-type specific gene expression through NF-B may occur by several
mechanisms. These include the activation of multiple NF-B isoforms,
stimulation through PDTC-sensitive and -insensitive pathways, and the
coordinate assembly of gene-specific transcription factors throughout
the upstream region.
Four cis-acting elements were required for TNF induction of
murine MCP-1: the proximal SP-1 site, two distal B sites, and the
distal HS sequence. Although the SP-1 and the B sites have
previously been implicated in the activated expression of MCP-1 (4, 26, 27, 46), the requirement of the HS sequence in TNF induction of the
MCP-1 gene has not been reported. At present, we have not been able to
detect the binding of a HS factor(s) to the HS sequence in EMSAs.
Nevertheless, several lines of evidence suggest that a HS factor(s)
exists. First, mutation of the HS sequence ablated TNF inducibility.
Second, although TNF-induced DMS hypersensitivity of HS requires the
binding of NF-B to the adjacent B sites, the hypersensitivity
persisted even after NF-B pattern dissipated (4). Third, although
the human MCP-1 gene contains an additional CTC located in the HS
sequence, which appears to be a direct duplication of the preceding
basepairs, the central 7 bp of the HS sequence are completely conserved
between the mouse and human (Fig. 7).
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B sites and the HS sequence formed a complete TNF
responsive element (TNFRE). However, the mechanism involved in the
formation of this complex appears to be distinct from that described
for other genes. Considering that NF-B-dependent induction of genes
such as IFN-ß (36, 37) and HIV (47) involves specific spatial
arrangement of the adjacent cis elements and cooperative
interactions between NF-B and other transcription factors,
experiments were conducted to investigate whether the B sites and
the HS sequence function together in a stereospecific manner. The
current study showed that insertion of a half or full helical turn
between the B sites or site reversion between the B-1 and B-2
sites did not affect TNF inducibility. Therefore, formation of the
MCP-1 TNFRE complex probably does not involve a strict phasing between
the NF-B proteins bound to the two B sites or between NF-B
bound to the B-2 site and the HS factor. HMG-I(Y), an important
structural protein, is required for NF-B-dependent induction of many
genes (48, 49, 50). In the distal regulatory region of MCP-1, there are two
potential HMG-I(Y) binding sites (Fig. 7B) (51). However,
mutations of either of these sites did not affect the TNF inducibility
(data not shown), suggesting that HMG-I(Y) may not play a role in the
MCP-1 TNFRE. These results, therefore, distinguish the MCP-1 TNFRE from
others in which an enhanceosome is formed.
Mammalian cells contain five NF-B family members: p50, p52, p65,
c-Rel, and RelB. Although the p50/p65 heterodimer is the primary
isoform regulating gene expression, other isoforms may be important for
cell type and B sequence-specific gene expression (34, 52, 53, 54, 55). By
studying TNF induction of the MCP-1 gene in NF-B knockout embryonic
fibroblasts, we showed that MCP-1 was induced in the wild-type and
p50-/-, but not in the p65-/- and
p50/p65-/-, cells. Because NF-B p50, p52, c-Rel, and
RelB subunits are constitutively expressed in the p65-/-
embryonic fibroblasts (29), this result indicates that the MCP-1 distal
B sequences are specific for p65-containing isoforms, such as the
p50/p65 heterodimer in wild-type cells or a p65/p65 homodimer in
p50-/- cells. The observation that MCP-1 induction was
inhibited by PDTC in wild-type cells but not in p50-/-
cells implies that the mechanisms involved in the activation of NF-B
p50-containing isoforms are sensitive to PDTC inhibition. These data
also suggest that the p65/65 isoform does not form or is rare in the
wild-type embryonic fibroblast control cell line, as the PDTC block in
control cells is almost complete. However, not all wild-type cells
respond to PDTC similarly. We previously observed that PDTC could
stimulate the basal expression of MCP-1 in BALB/3T3 cells and that
subsequent treatment with TNF in these cells resulted in an increase in
MCP-1 mRNA and a partially occupied footprint (4). While the analysis
of the knockout cell lines is inconsistent with the previous data, one
explanation for the differences could be that the BALB/3T3 cells
contain a higher ratio of p65 homodimers to p50/65 heterodimers,
allowing an escape from a PDTC-sensitive pathway that affects
p50-containing isoforms. Thus, it is likely that TNF can induce the
activation of NF-B through both PDTC-sensitive and -insensitive
mechanisms, and changes in p50/65 or p65/65 formation may explain why
PDTC has different effects on induced activation of NF-B in
different cell types (56, 57). One implication of these observations is
that under certain conditions the activation of NF-B may proceed
without the generation of oxygen intermediates. If this is the case,
then these data describe a new pathway for TNF activation of gene
expression that may involve distinct combinations of IB inhibitory
factors and NF-B trans-activating transcription factors.
Some NF-B isoforms, including the p50/p50 homodimer and the p50/RelB
heterodimer, are implicated in the inhibition of NF-B-activated gene
expression. For example, LPS-induced chemokine expression was more
potent and long lasting in RelB-/- fibroblasts than in
wild-type cells (52). As observed by others, we also found that
cotransfection of a p50 expression vector down-regulated TNF induction
of the wild-type MCP-1 constructions. A mechanism involving competitive
binding of inhibitory NF-B isoforms (e.g., p50/50) to the target
B sites has been supported by studying both NF-B-dependent
transcription in vitro and NF-B-mediated chromatin reconfiguration
of the HIV enhancer (55, 58). However, IVGF of the NF-B mutant cells
showed that only p65-containing isoforms could induce occupancy of the
distal B sites in vivo. Therefore, under physiological
concentrations of B factors, it appears unlikely that inhibition of
p65-induced activation by non-p65 inhibitory isoforms involves direct,
stable binding of these isoforms to the target B sites.
IVGF also revealed that p65 is required for the formation of the
hypersensitive site at the HS sequence. This result suggests either
that the binding of the HS factor to the HS sequence is directed by the
binding of NF-B p65-containing isoforms to the adjacent B sites
or that p65 is required for the expression of the HS factor. Because we
detected the DMS hypersensitivity after 15 min of TNF treatment (data
not shown), p65 is unlikely to be involved in the expression of the HS
factor. The binding of one transcription factor stabilizing the binding
of another factor to an adjacent site has been demonstrated in several
other genes, including the MHC class II promoters (59, 60, 61, 62). In MHC
class II promoters, two transcription factors, RFX and X2BP,
cooperatively bind and occupy their respective sites in wild-type
cells, but neither of the sites is occupied in RFX-deficient cells. If
a similar situation exists for NF-B, it is possible that NF-B
could differentially regulate cell type-specific gene expression
through recruitment of other transcription factors to the adjacent
sites. Alternatively, the binding of NF-B p65-containing isoforms to
the B sites may be required to open the chromatin structure to allow
the binding of the HS factor or other factors to their respective
sites. By either mechanism, these studies show that NF-B p65 is
required for the assembly of the MCP-1 TNF response enhancer and
perhaps other NF-B-dependent enhancers as well.
|
Acknowledgments |
|---|
B-disrupted cell lines and Dr. T. Collins for p65
and p50 expression vectors. We also thank Drs. M. Brown,
A. Neish, and S. W. Caughman for their discussions about
this study. |
Footnotes |
|---|
2 Address correspondence and reprint requests to Dr. Jeremy M. Boss, Department of Microbiology and Immunology, Emory University, Rollins Research Center, Room 3120, 1510 Clifton Rd., Atlanta, GA 30322. E-mail address:
3 Abbreviations used in this paper: MCP-1, monocyte chemoattractant protein-1; IVGF, in vivo genomic footprinting; DMS, dimethylsulfate; HS, hypersensitive; PDTC, pyrrolidine dithiocarbamate; CAT, chloramphenicol acetyltransferase; AP-1, activating protein-1; TNFRE, TNF-responsive element.
Received for publication April 13, 1998. Accepted for publication September 30, 1998.
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D. Ping, G. Boekhoudt, and J. M. Boss trans-Retinoic Acid Blocks Platelet-derived Growth Factor-BB-induced Expression of the Murine Monocyte Chemoattractant-1 Gene by Blocking the Assembly of a Promoter Proximal Sp1 Binding Site J. Biol. Chem., November 5, 1999; 274(45): 31909 - 31916. [Abstract] [Full Text] [PDF]
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