©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Overlapping Pathways Mediate the Opposing Actions of Tumor Necrosis Factor- and Transforming Growth Factor- on 2(I) Collagen Gene Transcription (*)

(Received for publication, November 21, 1994)

Yutaka Inagaki (1) (2) Sharada Truter (1) Shizuko Tanaka (1) Maurizio Di Liberto (1) Francesco Ramirez (1)(§)

From the  (1)Brookdale Center for Molecular Biology, Mt. Sinai School of Medicine, New York, New York 10029 and the (2)First Department of Internal Medicine, Kanazawa University School of Medicine, Kanazawa, 920, Japan

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

Transforming growth factor-beta (TGF-beta) and tumor necrosis factor-alpha (TNF-alpha) are multifunctional peptides intimately involved in the process of extracellular matrix remodeling. We recently showed that TGF-beta stimulates the human alpha2(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-alpha. Nuclear proteins from cells treated with TNF-alpha bind to the TbRE sequence substantially more strongly than those from untreated cells. Additionally, TNF-alpha 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-alpha counteracts the TGF-beta-elicited stimulation of collagen gene expression through overlapping nuclear signaling pathways. One modifies the TGF-beta-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-beta and TNF-alpha pathways on the same sequence of the alpha2(I) collagen promoter is yet another example of combinatorial gene regulation achieved through composite response elements.


INTRODUCTION

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-beta (^1)is a key player in the physiopathology of tissue repair(3, 4) . TGF-beta 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-beta-dependent pathway(8) . Likewise, foci of activated fibroblasts in patients with idiopathic pulmonary fibrosis produce TGF-beta along with abnormally elevated levels of collagen(9) . TNF-alpha, on the other hand, is a cytokine released by activated macrophages whose matrix-remodeling function is opposite to that of TGF-beta(10) . TNF-alpha induces synovial cell proliferation and metalloprotease synthesis while concomitantly suppressing collagen production(11, 12, 13) . Several lines of evidence indicate that TNF-alpha 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-alpha(14) . Additionally, mice carrying a mutant TNF-alpha transgene have been shown to develop chronic inflammatory polyarthritis(15) .

TGF-beta and TNF-alpha 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-beta stimulates type I collagen synthesis by acting mostly at the transcriptional level(6, 16) . In contrast, inhibition of type I collagen transcription by TNF-alpha 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 alpha2(I) collagen gene or COL1A2(17, 18, 19) .

In the most recent of these reports, we located a strong TGF-beta-responsive element (TbRE) within the 3.5-kb upstream sequence of the human COL1A2 gene(19) . We also showed that TGF-beta 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-beta 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-beta is only observed when boxes 3A and B are together in the same DNA fragment. We interpreted this finding as suggesting that TGF-beta 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-beta. 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-alpha inhibition. The evidence strongly suggests that TNF-alpha activates an intracellular cascade that converges into the same final pathway as that stimulated by TGF-beta. As a result, TNF-alpha 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.


MATERIALS AND METHODS

Cell Transfection Experiments

Primary human fetal dermal fibroblasts (CF-37) were maintained in Dulbecco's modified Eagle's medium supplemented with 10% fetal calf serum (Hyclone, Logan, UT). 5 h after transfection, cells were treated with 15% glycerol for 105 s and then placed in medium containing 0.1% fetal calf serum. TGF-beta (Collaborative Biomedical Products, Bedford, MA) and TNF-alpha (Boehringer Mannheim) were added after 2 h at the concentration of 2 and 10 ng/ml, respectively. Chimeric constructs containing wild-type and mutated collagen sequences linked to a reporter gene (chloramphenicol acetyltransferase) (CAT) have been already described along with the conditions for DNA purification, cell transfection, and CAT assay(17, 18, 19) . Transcriptional activity of each construct was normalized against the co-transfected thymidine kinase (TK)-driven growth hormone gene construct, pTKGH(23) . Assessment of growth hormone concentration in tissue culture media was carried out according to the manufacturer's recommendation using a commercial kit (Nichols Institute, Los Angeles, CA). An additional plasmid used in these experiments was the TK promoter-driven CAT gene construct, pBLCAT2(24) . Determination of the statistical value of the functional data was based on the Mann-Whitney U test.

DNA-Protein Binding Assays

Fibroblasts were plated at a density of 3 times 10^5 cells/100-mm culture dish and stimulated with 2 ng/ml TGF-beta for 3 h or with 10 ng/ml TNF-alpha for 24 h. Control dishes were left untreated for equivalent amounts of time. About 24 h prior to cytokine addition, cells were placed in medium containing 0.1% fetal calf serum. Nuclear extracts were prepared from cell cultures as previously described(19) . After oligonucleotide end-labeling, the binding reaction was carried out in a 25-µl reaction buffer containing 10 mM Tris, pH 7.5, 80 mM NaCl, 4% glycerol, 2 mM dithiothreitol, 1 mM EDTA, and 5 µg of poly(dI-dC). Following binding, DNA-protein complexes were separated from the unbound probe by electrophoresis in a 5% polyacrylamide gel; retarded complexes were visualized by autoradiography. In some experiments, nuclear extracts were pre-incubated with antisera against Jun and Fos (a kind gift of Dr. T. Curran, Roche Institute, Nutley, NJ) and NF-kB (Santa Cruz Biotechnology, Santa Cruz, CA) before incubating them with probes for box 5A and the TbRE or with a control probe (5`-GGTATGCAGACAACGAGTCAGAGTTTCCCCTTGAAAG-3`) that contains the consensus recognition sequences for both AP-1 and NF-kB(25) .


RESULTS

Functional Mapping of the TNF-alpha-responsive Element

Previous work in transiently transfected human skin fibroblasts and mouse NIH-3T3 cells has shown that TGF-beta and TNF-alpha modulate in opposite ways the transcription of a CAT construct, which contains 3.5 kb of COL1A2 upstream sequence(18) . Subsequent assays examined TGF-beta inducibility of chimeric plasmids carrying progressive 5` to 3` deletions of the 3.5-kb sequence(19) . This documented that the 196-base pair region residing between restriction sites BglII (nucleotide -378) and BstXI (nucleotide -183) is responsible for most of the TGF-beta induction (Fig. 1). In the present study, we used the same functional approach to ascertain whether this upstream region COL1A2 is also capable to mediate TNF-alpha inhibition.


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-alpha. The results showed that TNF-alpha 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-alpha 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-alpha-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-beta and TNF-alpha (Fig. 2B).


Figure 2: Mapping the TNF-alpha-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-alpha. The locations of box 5A (graybox) and the TbRE (whitebox) are shown. up triangle 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-alpha treatment. Ratio values (mean ± S.D.) represent the relative activities of TNF-alpha-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-beta (beta), TNF-alpha (alpha), and both (alpha, beta). 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-alpha. Values are expressed relatively to that of pBLCAT2 without TNF-alpha treatment (assumed as 100%); they represent the average of five independent tests ± S.D.



To obtain independent evidence for TNF-alpha 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-alpha 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-alpha-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-beta responsiveness (TbRE), whereas box 5A is a negatively cis-acting element that does not participate in mediating TGF-beta 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-alpha. This revealed that deletion of box 5A and nucleotide substitutions in the Sp1 recognition sequence of the TbRE substantially affect TNF-alpha responsiveness (Fig. 2A). We interpreted the data as suggesting that TNF-alpha-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-alpha.

TNF-alpha Action on the TbRE-bound Complex

To examine possible changes in the TbRE-bound complex, the radiolabeled -313 to -183 probe was incubated with equal amounts of nuclear proteins purified from untreated fibroblasts and from cells treated with TGF-beta for 3 h or with TNF-alpha for 24 h. As expected, TGF-beta treatment stimulated binding of the TbRE complex (Fig. 3). The slower migrating bands correspond to the Sp1 component of the TbRE-bound complex, whereas the nature of the faster complex (Cx in Fig. 3) is still unknown(19) . Like TGF-beta, a substantial increase in the affinity of the TbRE-bound complex was observed with nuclear proteins from fibroblasts grown for 24 h in the presence of TNF-alpha (Fig. 3). In contrast, nuclear proteins from fibroblasts treated with TNF-alpha for only 3 h exhibited the same binding affinity as those purified from untreated cells (data not shown). The different binding activity of the 3- and 24-h TNF-alpha-treated samples is consistent with the time of response of the endogenous COL1A2 gene to TNF-alpha inhibition(13) . Finally, there was a reproducible difference in the intensity, and perhaps the migration, of complex Cx in the TGF-beta versus the TNF-alpha-treated sample (Fig. 3).


Figure 3: Gel mobility shift assays of the TNF-alpha-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-beta (beta) or 10 ng/ml TNF-alpha (alpha). On the right, nuclear extracts from untreated(-) and TNF-alpha-treated (alpha) 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.



TNF-alpha Action on the Box 5A-bound Complex

In the next set of gel mobility shift assays, we examined if TNF-alpha treatment has any influence on the affinity of the box 5A-bound complex. Equal amounts of nuclear extracts purified from untreated cells and from cells treated with TNF-alpha for 24 h were incubated with the radiolabeled box 5A oligonucleotide (-330 to -297). In a parallel control sample, they were incubated with box 3A (-313 to -286), the probe that contains the Sp1 binding site of the TbRE.

Unlike TGF-beta, culturing fibroblasts in the presence of TNF-alpha 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-alpha 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-beta, TNF-alpha 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-alpha inhibition and TGF-beta 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-alpha responsiveness.

Exclusion of NF-kB and AP-1 Involvement in TNF-alpha Inhibition

It is well established that TNF-alpha stimulates expression of some genes by activating the nuclear internalization of transcription factor NF-kB and, to a lesser extent, by stimulating the production of the Fos-Jun (AP-1) protein complex(12, 26, 27, 28) . It could be argued that the same factors might also mediate TNF-alpha inhibition of some other genes in combination with other proteins or as a result of different post-translational modifications. Inspection of the protein-bound areas of the COL1A2 promoter identified three sequences resembling putative AP-1 (ATGAGTCAG) and NF-kB (GGGRNN(YYC)C) binding sites(25) . Potentially strong AP-1 and NF-kB binding sites (only one nucleotide mismatch) are in fact present in the coding and non-coding strands overlapping with and outside of box B, respectively (Fig. 1). There is also a possible NF-kB recognition sequence (two nucleotide mismatches) in the non-coding strand overlapping the 5` boundary of box 5A (Fig. 1).

To ascertain if these potential sites interact with the cognate nuclear factors, we performed an antibody interference experiment. To this end, TNF-alpha-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-alpha-treated cells. This point notwithstanding, the results of these experiments excluded participation of the NF-kB and AP-1 complexes in mediating the TNF-alpha 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-alpha-treated (+) nuclear extracts without pre-incubation with antisera (0) and with pre-incubation with NF-kB (alphaNk) or Fos-Jun (alphaFJ) 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.




DISCUSSION

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-beta and TNF-alpha 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-beta and TNF-alpha 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-beta 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-beta 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-beta and TNF-alpha could act through different cis-acting elements or overlapping nuclear signaling pathways. Results described in this report document the convergence of TNF-alpha-dependent signals upon the same sequence that mediates TGF-beta 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-alpha 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-alpha, 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 alpha-actin, another TGF-beta-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-beta and TNF-alpha 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-alpha compared with the TGF-beta-treated cells (see Fig. 3). In addition to counteracting TGF-beta stimulation by one of the aforementioned mechanisms, we postulate that TNF-alpha increases the negative effectiveness of the C1R complex. The dual action of TNF-alpha on two interacting complexes has the net effect of down-regulating COL1A2 gene transcription and counterbalancing TGF-beta stimulation.

In conclusion, this is the first report documenting a change in DNA-protein interactions associated with TNF-alpha 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.


FOOTNOTES

*
This work was supported by National Institutes of Health Grant AR-38648 and by an award from the Mochida Memorial Foundation for Medical and Pharmaceutical Research. This is article 153 from the Brookdale Center for Molecular Biology. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom reprint requests should be addressed.

(^1)
The abbreviations used are: TGF-beta, transforming growth factor-beta; AP-1, activating protein-1; CAT, chloramphenicol acetyltransferase; COL1A2, the gene coding for the alpha2(I) collagen chain; NF-kB, nuclear factor-kB; Sp1, specificity protein 1; TaRE, TNF-alpha-responsive element; TbRE, TGF-beta-responsive element; TK, thymidine kinase; TNF-alpha, tumor necrosis factor-alpha; kb, kilobase(s).


ACKNOWLEDGEMENTS

We are very thankful to Dr. G. Karsenty for many helpful discussions and suggestions, to Dr. T. Curran for the generous gift of the Fos-Jun antisera, to H. Zhou and W. Hu for excellent technical assistance, and to M. Sozomenu for typing the manuscript.


REFERENCES

  1. Hay, E. D. (1991) in Cell Biology of Extracellular Matrix (Hay, E. D., ed) 2nd Ed., pp. 419-462, Plenum Publishing Corp., New York
  2. Diegelmann, R. F., Lindblad W. J., and Cohen, I. K. (1988) in Collagen: Biochemistry and Biomechanics (Nimni, M. E., ed) Vol. II, pp. 113-131, CRC Press, Boca Raton, FL
  3. Sporn, M. B., and Roberts, A. B. (1992) J. Cell Biol. 119, 1017-1021 [Medline] [Order article via Infotrieve]
  4. Border, W. A., and Ruoslahati, E. (1992) J. Clin. Invest. 90, 1-7 [Medline] [Order article via Infotrieve]
  5. Hill, D. J., Strain, A. J., Elstow, S. F., Swenne, I., and Miller, R. D. G. (1986) J. Cell. Physiol. 128, 322-328 [Medline] [Order article via Infotrieve]
  6. Ignotz, R. A., Endo, T., and Massagué, J. (1987) J. Biol. Chem. 262, 6443-6446 [Abstract/Free Full Text]
  7. Edwards, D. R., Murphy, G., Reynolds, J. J., Whitham, S. E., Docherty, A. J. P., Angel, P., and Heath, J. K. (1987) EMBO J. 7, 2977-2981 [Abstract]
  8. Kikuchi, K., Hartl, C. W., Smith, E. A., LeRoy, E. C., and Trojanowska, M. (1992) Biochem. Biophys. Res. Commun. 187, 45-50 [Medline] [Order article via Infotrieve]
  9. Broekelmann, T. J., Limper, A. H., Colby, T. V., and McDonald, J. A. (1991) Proc. Natl. Acad. Sci. U. S. A. 88, 6642-6646 [Abstract]
  10. Beutler, B., and Cerami, A. (1988) Annu. Rev. Biochem. 57, 505-518 [CrossRef][Medline] [Order article via Infotrieve]
  11. Butler, D. M., Piccoli, D. S., Hart, P. H., and Hamilton, J. A. (1988) J. Rheumatol. 15, 1463-1470 [Medline] [Order article via Infotrieve]
  12. Brenner, D. A., O'Hara, J. A., Angel, P., Chojkier, M., and Karin, M. (1989) Nature 337, 661-663 [CrossRef][Medline] [Order article via Infotrieve]
  13. Solis-Herruzo, J. A., Brenner, D. A., and Chojkier, M. (1988) J. Biol. Chem. 263, 5841-5845 [Abstract/Free Full Text]
  14. Saxne, T., Palladino, M. A., Jr., Heinegard, D., Talal, N., and Wollheim, F. A. (1988) Arthritis Rheum. 31, 1041-1045 [Medline] [Order article via Infotrieve]
  15. Keffer, J., Probert, L., Cazlaris, H., Georgopoulos, S., Kaslaris, E., Kioussis, D., and Kollias, G. (1991) EMBO J. 10, 4025-4031 [Abstract]
  16. Penttinen, R. P., Kobayashi, S., and Bornstein, P. (1988) Proc. Natl. Acad. Sci. U. S. A. 88, 1105-1108 [Abstract]
  17. Boast, S., Su, M. W., Ramirez, F., Sanchez, M., and Avvedimento, E. V. (1990) J. Biol. Chem. 265, 13351-13356 [Abstract/Free Full Text]
  18. Kähäri, V. M., Chen, Y. Q., Su, M. W., Ramirez, F., and Uitto, J. (1990) J. Clin. Invest. 86, 1485-1495
  19. Inagaki, Y., Truter, S., and Ramirez, F. (1994) J. Biol. Chem. 269, 14828-14834 [Abstract/Free Full Text]
  20. Karsenty, G., Golumbek, P., and de Crombrugghe, B. (1988) J. Biol. Chem. 263, 13909-13915 [Abstract/Free Full Text]
  21. Goldberg, H., Helaakoski, R., Garrett, L. A., Karsenty, G., Pellegrino, A., Lozano, G., Maity, S., and de Crombrugghe, B. (1992) J. Biol. Chem. 267, 19622-19630 [Abstract/Free Full Text]
  22. Niederreither, K., D'Souza, R. D., and de Crombrugghe, B. (1992) J. Cell Biol. 119, 1361-1370 [Abstract]
  23. Selden, R. F., Burke-Howie, K., Row, M. E., Goodman, H. M., and Moore, D. D. (1986) Mol. Cell. Biol. 6, 3173-3179 [Medline] [Order article via Infotrieve]
  24. Luckow, B., and Schütz, G. (1987) Nucleic Acids Res. 15, 5490-5494 [Medline] [Order article via Infotrieve]
  25. Faisst, S., and Meyer, S. (1992) Nucleic Acids Res. 20, 3-26 [Medline] [Order article via Infotrieve]
  26. Lowenthal, J. W., Ballard, D. W., Bohnlein, E., and Greene, W. C. (1989) Proc. Natl. Acad. Sci. U. S. A. 86, 2331-2335 [Abstract]
  27. Osborn, L., Kunkel, S., and Nabel, G. J. (1989) Proc. Natl. Acad. Sci. U. S. A. 86, 2336-2340 [Abstract]
  28. Heller, R. A., and Kronke, M. (1994) J. Cell Biol. 126, 5-9 [Medline] [Order article via Infotrieve]
  29. Yamamoto, K. R., Pearce, D., Thomas, J., and Miner, J. N. (1992) in Transcriptional Regulation (McKnight, S. L., and Yamamoto, K. R., eds) pp. 1169-1192, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY
  30. MacLellan, W. R., Lee, T. C., Schwartz, R. J., and Schneider, M. D. (1994) J. Biol. Chem. 269, 16754-16760 [Abstract/Free Full Text]
  31. Murata, Y., Kim, H. G., Rogers, K. T., Udvadia, A. J., and Horowitz, J. M. (1994) J. Biol. Chem. 269, 20674-20681 [Abstract/Free Full Text]
  32. Hagen, G., Müller, S., Beato M., and Suske, G. (1994) EMBO J. 13, 3843-3851 [Abstract]
  33. Johnson, A. (1992) in Transcriptional Regulation (McKnight, S. L., and Yamamoto, K., eds) pp. 975-1006, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY
  34. Jarvis, E. E., Clark, K. L., and Sprague, G. F., Jr. (1989) Genes & Dev. 3, 936-945

©1995 by The American Society for Biochemistry and Molecular Biology, Inc.