(Received for publication, November 21, 1994)
From the
Transforming growth factor- (TGF-
) and tumor necrosis
factor-
(TNF-
) are multifunctional peptides intimately
involved in the process of extracellular matrix remodeling. We recently
showed that TGF-
stimulates the human
2(I) collagen gene by
increasing the affinity of an Sp1-containing transcriptional complex
bound to an upstream sequence termed the TbRE (Inagaki, Y., Truter, S.
and Ramirez, F.(1994) J. Biol. Chem. 269, 14828-14834).
Here, we report that the TbRE-bound complex also mediates the
inhibitory signal of TNF-
. Nuclear proteins from cells treated
with TNF-
bind to the TbRE sequence substantially more strongly
than those from untreated cells. Additionally, TNF-
increases
binding of a second protein complex that recognizes the negatively cis-acting element located immediately next to the TbRE. Thus,
we postulate that TNF-
counteracts the TGF-
-elicited
stimulation of collagen gene expression through overlapping nuclear
signaling pathways. One modifies the TGF-
-targeted transcriptional
complex, probably by reducing its stimulatory effect on collagen
transcription. The other acts on the binding of the adjacent factor,
presumably by increasing its effectiveness in repressing the activity
of the collagen promoter. The convergence of the TGF-
and
TNF-
pathways on the same sequence of the
2(I) collagen
promoter is yet another example of combinatorial gene regulation
achieved through composite response elements.
The balance between production and degradation of collagen plays a critical role in the development and maintenance of virtually every organ and tissue(1) . It also represents the most crucial element governing the process of tissue repair(2) . Following injury, inflammatory cells direct the activity of matrix-remodeling mesenchymal cells through a variety of cytokine-mediated stimuli(2) . Alteration of the dynamic equilibrium underlying the repair process leads to either excessive collagen deposition or inadequate tissue integrity(2) . The former is the histopathologic hallmark of several fibrotic diseases, such as liver cirrhosis, pulmonary fibrosis, and scleroderma(2) . In broad terms, fibrosis can therefore be viewed as a chronic and uncontrolled inflammatory/repair process that affects the normal interplay between cellular signals and matrix component-coding genes (2) .
An
increasing body of evidence indicates that TGF- (
)is a
key player in the physiopathology of tissue
repair(3, 4) . TGF-
stimulates fibroblast
proliferation, enhances collagen production, and inhibits collagenase
synthesis(5, 6, 7) . There is also some
evidence that intrinsic stimulation of collagen expression in cultured
sclerodermal cells utilizes a TGF-
-dependent pathway(8) .
Likewise, foci of activated fibroblasts in patients with idiopathic
pulmonary fibrosis produce TGF-
along with abnormally elevated
levels of collagen(9) . TNF-
, on the other hand, is a
cytokine released by activated macrophages whose matrix-remodeling
function is opposite to that of TGF-
(10) . TNF-
induces synovial cell proliferation and metalloprotease synthesis while
concomitantly suppressing collagen
production(11, 12, 13) . Several lines of
evidence indicate that TNF-
is intimately involved in the
processes of cartilage destruction and bone resorption(10) .
For example, synovial fluid of patients suffering from rheumatoid
arthritis has been found to contain elevated levels of
TNF-
(14) . Additionally, mice carrying a mutant TNF-
transgene have been shown to develop chronic inflammatory
polyarthritis(15) .
TGF- and TNF-
are therefore
invaluable experimental tools to study the mechanisms and factors that
orchestrate extracellular matrix remodeling in normal and diseased
conditions. Relatively little is known, however, about how these
cytokines influence the production of a variety of structural matrix
components, including collagen. Early work on cultured fibroblasts has
shown that TGF-
stimulates type I collagen synthesis by acting
mostly at the transcriptional level(6, 16) . In
contrast, inhibition of type I collagen transcription by TNF-
is a
relatively late effect that requires protein synthesis (13) .
In principle, the two cytokines could use either distinct or converging
signaling pathways to transduce their antagonistic cues on type I
collagen expression. The present study was undertaken to verify which
one of these two possibilities is correct; its premise was based on the
previous characterization of some regulatory sequences in one of the
human type I collagen genes, notably the
2(I) collagen gene or
COL1A2(17, 18, 19) .
In the most recent of
these reports, we located a strong TGF--responsive element (TbRE)
within the 3.5-kb upstream sequence of the human COL1A2
gene(19) . We also showed that TGF-
stimulation is
associated with increased affinity of an Sp1-containing nuclear complex
for its cognate binding site, the TbRE. Formation of the TbRE-bound
complex and response to TGF-
stimulation require the integrity of
two neighboring nuclear protein-bound sequences, termed boxes 3A and B.
Although Sp1 binds to box 3A in the absence of box B, enhanced binding
affinity in response to TGF-
is only observed when boxes 3A and B
are together in the same DNA fragment. We interpreted this finding as
suggesting that TGF-
may act by modifying specific Sp1 co-factors.
The nature of the postulated factor(s) interacting with box B and the
precise contribution of this DNA element to the augmented affinity of
the TbRE-bound complex remain obscure. During the course of that study,
we also mapped a functionally distinct cis-acting element, box
5A, upstream of and partially overlapping with box 3A(19) .
Deletion of box 5A enhances transcription of a chimeric construct
transfected into fibroblasts, suggesting that box 5A might represent a
negatively cis-acting element. The same functional test
excluded the participation of box 5A in mediating the transcriptional
induction by TGF-
. Interestingly, others have shown that the
region encompassing boxes 5A and 3A participates in the cell-specific
control of the mouse gene(20, 21, 22) .
Work presented here extends our previous study and demonstrates that
the same upstream region of the COL1A2 gene can also mediate TNF-
inhibition. The evidence strongly suggests that TNF-
activates an
intracellular cascade that converges into the same final pathway as
that stimulated by TGF-
. As a result, TNF-
inhibition is
transcriptionally elaborated by changes in the affinity of the box 5A
and TbRE-bound complexes. Altogether, the data indicate that tissue
specificity and cytokine responsiveness are inextricably connected
properties of a short upstream sequence of the COL1A2 gene.
Figure 1: Schematic representation of the human COL1A2 3.5-kb sequence. The composition of the upstream region encompassing the footprinted areas is shown beneath the map of the upstream COL1A2 sequence. Symbols identified the following elements: boxes 5A, 3A, and B (boxed), the Sp1 binding site of box 3A (stippledoval), and the putative AP-1 and NF-kB binding sites (dottedlines).
To this end, progressively shorter fragments
of the 3.5-kb sequence linked to the CAT gene were tested in cultured
fibroblasts subsequently grown for 48 h with or without TNF-. The
results showed that TNF-
treatment reduces the activity of the
-3500, -772, and -378COL1A2/CAT plasmids to about
half the level of untreated cells (Fig. 2A). In
contrast, TNF-
had little or no detectable effect on the
transcription of constructs containing sequence downstream of
nucleotide -235 (Fig. 2A). The data therefore
suggested that a relatively strong TNF-
-responsive element is
located between nucleotides -378 and -235, thus within the
upstream region that encompasses the TbRE. Consistent with this
finding, the -378 COL1A2/CAT construct exhibited nearly identical
CAT activity in untreated cells and in cells treated at the same time
with TGF-
and TNF-
(Fig. 2B).
Figure 2:
Mapping the TNF--responsive region in
the 3.5-kb sequence. PanelA, percentage CAT
conversion of plasmids containing various amounts of the COL1A2
promoter transfected into cells grown without (whitehistogram) and with (hatchedhistogram)
TNF-
. The locations of box 5A (graybox) and the
TbRE (whitebox) are shown.
signifies deletion
of the box 5A, whereas the dottedline indicates the
relative position of the mutated Sp1 binding site in the TbRE. The
activity of each construct is expressed relatively to that of the
-3500 COL1A2/CAT without TNF-
treatment. Ratio values (mean
± S.D.) represent the relative activities of TNF-
-treated versus untreated fibroblasts with the number of independent
tests indicated in parentheses. The ratio values of the
constructs highlighted by oneasterisk are
statistically lower than those of the plasmids highlighted by twoasterisks (Mann-Whitney U test, p <
0.01). PanelB, representative CAT assays
illustrating the activity of the -378COL1A2/CAT construct
transfected into fibroblasts untreated(-) and treated with
TGF-
(
), TNF-
(
), and both (
,
). PanelC, percentage
CAT conversion of TK-driven plasmid pBLCAT2 (24) without (TK)
and with the insertion of the Bg1II/BstXI segment
(COL1A2/TK) transfected into cells grown without (whitehistogram) and with (hatchedhistogram)
TNF-
. Values are expressed relatively to that of pBLCAT2 without
TNF-
treatment (assumed as 100%); they represent the average of
five independent tests ± S.D.
To obtain
independent evidence for TNF- responsiveness, the BglII-BstXI segment was tested within the
heterologous context of the TK promoter. As we and others have noted
before(19, 20) , inclusion of the collagen sequence
nearly doubled the basal activity of the pBLCAT2 plasmid; more
important, it conferred TNF-
responsiveness to the otherwise
unresponsive TK promoter (Fig. 2C). Thus, the
heterologous promoter test confirmed the homologous promoter results,
suggesting the presence of a TNF-
-responsive element (TaRE) within
the -378 to -235 sequence.
Previous footprinting
experiments identified within the -378 to -235 segment two
protein-bound areas (boxes A and B) (Fig. 1)(19) . Gel
mobility shift assays divided box A into two distinct nuclear protein
binding sites (boxes 5A and 3A) (Fig. 1). Together, boxes B and
3A constitute the element responsible for TGF- responsiveness
(TbRE), whereas box 5A is a negatively cis-acting element that
does not participate in mediating TGF-
stimulation(19) .
To evaluate the individual contribution of each element, the activity
of mutant plasmids was assessed in transiently transfected fibroblasts
grown with or without TNF-
. This revealed that deletion of box 5A
and nucleotide substitutions in the Sp1 recognition sequence of the
TbRE substantially affect TNF-
responsiveness (Fig. 2A). We interpreted the data as suggesting that
TNF-
-elicited inhibition of COL1A2 transcription requires the
concerted contribution of both the box 5A and the TbRE-bound complexes.
In the next set of experiments, the gel mobility shift assay was used
to examine if the binding pattern of the two complexes is modified by
growing fibroblasts in the presence of TNF-
.
Figure 3:
Gel mobility shift assays of the
TNF--responsive element-bound protein complexes. On the left, the TbRE probe (Tb) was incubated with nuclear
extracts purified from untreated cells(-) and from fibroblasts
grown in the presence of 2 ng/ml TGF-
(
) or 10 ng/ml
TNF-
(
). On the right, nuclear extracts
from untreated(-) and TNF-
-treated (
) cells
were incubated with the box 5A (5A) or the box 3A (3A) probe. Equal amounts of proteins were used in each set of
experiments; identities of the complexes are indicated on the sides of the autoradiograms.
Unlike TGF-, culturing fibroblasts in
the presence of TNF-
for 24 h caused a substantial increase in the
affinity of the box 5A-bound complex (Fig. 3). Like the TbRE
probe experiment, the same binding increase was not observed with
nuclear proteins derived from cells that were treated with TNF-
for only 3 h (data not shown). We provisionally named the box 5A-bound
complex C1R, for collagen I repressor, because deletion of this
sequence has been shown to elevate transcription of the COL1A2
promoter(19) . Like TGF-
, TNF-
treatment had no
influence on the binding of Sp1 to box 3A in the absence of the
downstream box B element (Fig. 3). Thus, Sp1 participation in
mediating TNF-
inhibition and TGF-
stimulation apparently
requires additional DNA element(s) and interacting factor(s).
Altogether, the DNA binding assays provided independent support to the
results of the transfection experiments that correlated the -378
to -235 sequence with TNF-
responsiveness.
To ascertain if these potential
sites interact with the cognate nuclear factors, we performed an
antibody interference experiment. To this end, TNF--treated
nuclear extracts were incubated with NF-kB or Fos-Jun antisera prior to
the addition of probes for box 5A and the TbRE. As a control, we used a
probe that contains high affinity AP-1 and NF-kB binding sites.
Pre-incubation of nuclear extracts with NF-kB or Fos-Jun antisera left
virtually unchanged protein binding to box 5A and the TbRE (Fig. 4). On the contrary, the same antibodies reduced protein
binding to the control probe (Fig. 4). It should be noted that
the two antisera affected to different degrees the intensity of the
band obtained with the control probe, conceivably as a reflection of
the relative amounts of induction of AP-1 and NF-kB complexes in
TNF-
-treated cells. This point notwithstanding, the results of
these experiments excluded participation of the NF-kB and AP-1
complexes in mediating the TNF-
signal. The conclusion is also
consistent with the three putative binding sites being partly or
entirely outside of the footprinted areas.
Figure 4:
Antibody interference assays. The three
autoradiograms show untreated(-) and TNF--treated (+)
nuclear extracts without pre-incubation with antisera (0) and
with pre-incubation with NF-kB (
Nk) or Fos-Jun (
FJ) antisera using as probes box 5A (5A), the
TbRE (Tb), and an oligonucleotide (C) that contains
the AP-1 and NF-kB consensus recognition sequences(25) . Equal
amounts of proteins were used in each sample; the identities of the
nuclear protein complexes are indicated on the rightside of the autoradiogram.
Excessive collagen accumulation is the histopathologic
hallmark of fibrotic diseases, which impair the function of several
organs such as lungs, kidneys, liver, and skin(2) . Central to
the development and progression of fibrosis are cytokines that are
normally involved in matrix remodeling(2) . Among them,
TGF- and TNF-
have opposite effects on type I collagen
production. Elucidation of how these cytokines modulate collagen gene
expression may therefore provide new insights into the complex
physiology and physiopathology of tissue repair. With this idea in
mind, a few years ago we began studying the regulation of the human
type I collagen genes(17, 18, 19) . First, we
confirmed previous mouse data (20) by showing that a
phylogenetically conserved region lying between nucleotides -378
and -183 is responsible for high and tissue-specific COL1A2 gene
expression(17) . Next, we documented that the 3.5-kb upstream
sequence of COL1A2 could replicate the transcriptional responses of the
endogenous gene to TGF-
and TNF-
treatment of cultured
fibroblasts(18) . Finally, we demonstrated that the major TbRE
within the 3.5-kb sequence co-localizes with the aforementioned
tissue-specific element(19) .
The last line of investigation
resulted in the characterization of the mechanism implicated in the
stimulation of COL1A2 transcription(19) . Extensive DNA binding
assays revealed that the TbRE consists of two distinct binding sites:
one of them (box 3A) is occupied by Sp1 and the other (box B) by
unknown factor(s). They also documented that TGF- treatment of
cultured dermal fibroblasts elevates the affinity of the TbRE-bound
complex, probably by modifying Sp1-specific co-factors. The study
identified another cis-acting element (box 5A) immediately
adjacent to but functionally distinct from the TbRE. Such a distinction
was based on transfection data that excluded box 5A participation in
TGF-
stimulation while implicating it in the negative control of
COL1A2 transcription(19) . As already mentioned, work in the
mouse gene has shown that the region comprising boxes 5A and 3A is
sufficient to confer tissue specificity to chimeric constructs
expressed in transgenic mice and cultured
cells(20, 21, 22) . Thus, different
combinations of motifs within the same footprinted region of the COL1A2
promoter are apparently responsible for distinct transcriptional
properties of the gene. This conclusion raised the question of whether
TGF-
and TNF-
could act through different cis-acting
elements or overlapping nuclear signaling pathways. Results described
in this report document the convergence of TNF-
-dependent signals
upon the same sequence that mediates TGF-
responsiveness and cell
specificity.
Initial identification of the TaRE was mostly based on
transfection data obtained with different COL1A2 sequences and then
confirmed using the heterologous TK promoter. Subsequent support was
generated by gel mobility shift assays, which correlated TNF-
treatment with increased affinity of the nuclear proteins bound to box
5A and the TbRE. These cis-acting elements are known to
contribute differently to COL1A2
transcription(19, 20) . Transfection data discussed in
this report implicated both of them in mediating the full response to
TNF-
, thus implying that the cognate nuclear factors are probably
interacting components of a larger complex. Taken together, the data
seem to indicate that the TaRE region contains at least three distinct
regulatory circuits. They include the one (box A) implicated in
restricting gene expression to a specific group of tissues and those
(boxes A and B, and boxes 3A and 3B) responsible for responding to two
cellular antagonists. In this respect, COL1A2 belongs to the increasing
list of genes whose diversified transcriptional properties are mediated
by combinatorial interactions of multifunctional
complexes(29) . One of such examples is skeletal
-actin,
another TGF-
-inducible gene that owes its tissue specificity and
cytokine responsiveness to this kind of regulatory
mechanism(30) . Transcription of this cardiac gene requires in
fact synergy between two functionally distinct cis-acting
elements that contain binding sites for serum-responsive factor, the
bifunctional YY1 protein, Sp1, and the SV40 enhancer binding factor
TEF-1(30) .
One of the components of the TbRE-bound complex is the ubiquitous activator Sp1, whose binding affinity and transcriptional properties are differentially modulated in response to cytokines, apparently through the indirect action of co-factors. The opposite activities of the Sp1-containing complex are supported by recent data that correlated binding of a specific co-factor with inhibition of Sp1-mediated transcription in vivo(31) . It is also possible that repression of Sp1-mediated activation might be attained by inducing binding of Sp3, the recently recognized inhibitory member of the Sp family(32) . We currently favor the first scenario because the increase in binding affinities was only observed when the Sp1 recognition sequence of the TbRE is coupled to box B. Supporting our model are several other examples of transcription factors with diversified properties(33) . The best known of them is probably MCM1, the yeast homolog of mammalian serum response factor. Alone, this nuclear protein is an ubiquitous activator; in combination with different co-factors, it becomes a cell-specific activator or a cell-specific repressor (34) .
By integrating
our evidence with the available data, we propose a model whereby the
cellular signals elicited by TGF- and TNF-
are elaborated
transcriptionally by changing the modification and/or the composition
of the TbRE-bound complex. Along these lines, there might be a
difference in the binding pattern of complex Cx in the TNF-
compared with the TGF-
-treated cells (see Fig. 3). In
addition to counteracting TGF-
stimulation by one of the
aforementioned mechanisms, we postulate that TNF-
increases the
negative effectiveness of the C1R complex. The dual action of TNF-
on two interacting complexes has the net effect of down-regulating
COL1A2 gene transcription and counterbalancing TGF-
stimulation.
In conclusion, this is the first report documenting a change in
DNA-protein interactions associated with TNF- inhibition of
collagen gene expression. It is also the first to establish the
convergence of antagonistic cellular signals on a final common pathway.
Altogether, the data indicate that a cluster of binding sites in the
upstream sequence is apparently responsible for the combinatorial
regulation of the COL1A2 gene. Work in progress is elucidating the
nature of the other components of this transcriptional complex as well
as the relevance of our regulatory model to fibrotic diseases.