INTRODUCTION

Growth factors transmit an extended array of regulatory information to eukaryotic cells by binding to specific receptors at the cell surface. With the identification of a great variety of growth factors and receptors in the recent years, it has become apparent that some factors often exert pleiotropic or redundant effects on target cells. For example, identical immune responses can be induced by several different cytokines acting either alone or synergistically in combinations (1). In contrast, other growth factors, such as transforming growth factor  (TGF-)¹ and tumor necrosis factor  (TNF-) are often functionally antagonistic. For instance, TGF- and TNF- antagonize on the regulation of nitric oxide production in cardiac myocytes (2), the development of cytotoxic T cells (3), and in certain autoimmune diseases such as collagen-induced arthritis (4) and experimental allergic encephalomyelitis (5).

Recent studies have indicated that both factors bind distinct cellular receptors at the cell surface of numerous target cell types. TGF- receptor activation involves serine/threonine autophosphorylation of signaling receptor complexes (reviewed in Ref. 6). In contrast, TNF- receptors have no apparent enzymatic activity, yet they may associate with a set of recently identified putative signal-transducing molecules such as TNF receptor-associated factors (TRAFs) (reviewed in Ref. 7). Thus, the mutual antagonism of TGF- and TNF- is not exerted through competition for the same cell surface receptors but involves differential regulation of specific target intracellular mediators. However, no such mediators have been identified to date, and the molecular mechanisms underlying this mutual antagonism of TGF- and TNF- are still unknown.

Here, we have analyzed the molecular mechanisms involved in the regulation of the procollagen gene expression by TGF- and TNF-. Collagen genes are well-known targets of mutually antagonistic TGF- and TNF- regulation (8, 9, 10). For example, TGF- has been shown to activate 2(I) procollagen gene expression in mesenchymal cells by acting at the level of transcriptional initiation (8). This activation correlates with increased collagen production and secretion by fibroblasts during wound healing, a process that is potently promoted by TGF- (11, 12). The proinflammatory cytokine TNF- is also present in the healing wounds (13), and it has been shown to down-regulate expression of the collagen 2(I) gene promoter, thereby antagonizing TGF- action on collagen gene expression (9, 15). The mechanism of antagonistic 2(I) collagen promoter regulation by TGF- and TNF- is unclear at present. However, binding sites for the ubiquitous transcription factors CTF/NF-I and Sp1 have been proposed to mediate hormone action (8, 14, 15). This suggests that TGF- and TNF- might antagonistically regulate the activity and/or the expression of these transcription factors. Alternatively, hormone regulation might be effected on other proteins that interact with the transactivators, such as components of the basal transcriptional initiation machinery, including transcription factor IIB, the TATA-binding protein or transactivator-specific TATA-binding protein-associated factors.

Consistent with the proposed key role for CTF/NF-I in the TGF- induction of the 2(I) collagen promoter (8), we have recently shown that CTF-1, a member of the CTF/NF-I family of proline-rich transcription factors (16), mediates TGF--regulated transcriptional activation in NIH3T3 cells (17). TGF- does not induce CTF-1 through direct phosphorylation of its proline-rich domain, suggesting that TGF- induction is mediated by CTF-1-interacting proteins. One candidate target protein is histone H3, which interacts specifically with the TGF--regulated transactivation domain of CTF-1, and which interaction leads to the alteration of the nucleoprotein architecture of chromatin-reconstituted target promoters (17).

Here, we addressed the possibility that specific CTF/NF-I family members might mediate antagonistic TGF- and TNF- responses. Indeed, we demonstrate that CTF-1 mediates TNF--regulated transcriptional repression, and that TNF- antagonizes the TGF--dependent induction of CTF-1 transcriptional activity in mouse fibroblasts. These effects are mediated by the extreme C-terminal portion of the CTF-1 proline-rich transactivation domain, previously termed the TGF- responsive domain (TRD). Although TNF- repression is mimicked by expression of genes regulating protein phosphorylation, such as the proto-oncogenes ras and raf, our results indicate that the TRD is not directly phosphorylated in response to TNF-. Rather, our findings imply the interaction of the CTF-1 transactivation domain (TAD) with histone H3 in this regulatory process.

MATERIALS AND METHODS

Human TGF-1 was purchased from Nacalai-Tesque (Kyoto, Japan). Mouse TNF- and human PDGF-BB were obtained from Boehringer Mannheim. Human TNF- was from Calbiochem. Dulbecco's modified Eagle's medium and fetal bovine serum were supplied by Life Technologies, Inc.. Arachidonic acid, indomethacin, and NDGA were from Sigma.

The mammalian expression vectors for the GAL4-CTF1 fusion proteins contain the DNA binding domain of the yeast transcription factor GAL4 (amino acids 1-147; Ref. 18) and appropriate portions of the human CTF-1 cDNA (amino acids 1-399; Ref. 16). They are termed GAL x-y, where x and y indicate CTF-1 insert end point amino acid positions. The construction of GAL 399-472, GAL 486-499, GAL 472-499, GAL 479-499 W.T., GAL 479-499 3xM, GAL 399-499 3xM, and GAL 399-499 Y491F and Y497F point mutants has been described in detail (17). GAL 399-499 S495A contains two point mutations at positions 1550 and 1552 of the wild-type human CTF-1 cDNA sequence changing serine 495 to alanine (TCC  GCA). GAL 399-499, GAL 1-399, GAL 399-438, and GAL 438-499 vectors have been described (19). GAL-AP2 and GAL-Sp1 contain amino acids 31-76 and 132-243 of the AP2 and Sp1 TADs, downstream of the GAL-DBD, respectively (17). The mammalian expression vectors pMLV-VP and pMLV-VP-H3 encode the VP16 transcriptional activation domain either unfused or fused to amino acids 10-136 of the coding sequence of human histone H3.3 (17). pSG5-PPAR (a kind gift of Prof. W. Wahli) is an SV40-driven mammalian expression vector encoding the  isoform of the Xenopus laevis peroxisome proliferator-activated receptor, a member of the superfamily of nuclear hormone receptors (20). The pCAT-55 and pCAT-87-3xAd (16), G5BCAT (21), and 5xTRE-TATA-CAT reporters (22) contain either zero and three high affinity CTF/NF-I binding sites or five GAL4 and five AP-1 binding sites in front of a TATA box and the cat gene, respectively, whereas the ACO-A.TK.CAT vector contains the upstream region of the PPAR-responsive rat acetyl-CoA oxidase gene (from positions 1273 to +20; Ref. 20) placed in front of a thymidine kinase basal promoter and the cat gene. pRCBX2 (23) and pRSV-Raf BXB (24), expressing oncogenically activated forms of the proto-oncogenes ras and raf downstream of the conalbumin and Rous sarcoma virus promoters, were generous gifts of Drs. B. Wasylyk and U. Rapp, respectively. The Ras empty plasmid containing the conalbumin promoter was prepared by excision of the large HindIII fragment encoding the ras sequences and vector religation.

Two clones of NIH3T3 cells were used in this study: clone 63 (which contains constitutive endogenous AP-1 activity; Figs. 1, 2, 3, 6, and 7); and clone 4 (in which AP-1 is inducible by Ras and Raf expression, used in Figs. 4 and 5). Both cell lines were grown at 37 °C in a humidified incubator containing 5% CO₂ in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum and antibiotics. Cells were transiently transfected by electroporation, essentially as described (17). Briefly, 4.5 × 10⁶ cells were mixed with 70 µg of total plasmid DNA and pulsed once at 960 microfarads and 250 V at room temperature, according to the instructions of the electroporator manufacturer (Bio-Rad). The contents of one pulsed cuvette were split in four, and cells were plated in DMEM plus 0.5% fetal bovine serum for 3-5 h. Cultures were then induced for a period of 18-40 h with either ethanol vehicle, 5 ng/ml human TGF-, or 50 ng/ml human TNF-, as appropriate. (The results presented in the figures have been obtained using human TNF-). Cells were lysed in 1 × Reporter Lysis Buffer (Promega Corp.), and CAT activities were determined using standard procedures (25) and normalized according to the activity of -galactosidase resulting from a cotransfected internal control plasmid (CMVgal; Clontech). The activity of the SV40 and CMV promoters was not significantly affected by any hormone or pharmacological agent used in this study.

Antagonistic regulation of a CTF/NF-I-responsive promoter by TGF- and TNF- in NIH3T3 cells.

TNF- down-regulates the CTF-1 transcriptional activation domain in NIH3T3 cells.

The antagonistic regulation of CTF-1 transcriptional activity by TGF- and TNF- occurs through AA-independent pathways.

TNF- does not regulate CTF-1 through direct phosphorylation of the TGF- responsive domain.

Evidence for the down-regulation of the interaction of CTF-1 TAD with histone H3 by TNF-.

Lack of TNF--mediated repression of the CTF-1 TAD in NIH3T3 cells expressing oncogenic Ras.

Lack of TNF--mediated repression of the CTF-1 TAD in NIH3T3 cells expressing constitutively active Raf-1 kinase.

For gel-shift analysis of the endogenous CTF/NF-I or of the transiently expressed GAL4 fusion proteins, cells were lysed in extraction buffer (20 mM Tris, pH 7.5, 20% glycerol, 500 mM KCl, 1 mM dithiothreitol, and protease inhibitors) exactly as described in Ref. 19. Whole-cell lysates were normalized for protein concentration and incubated with end-labeled double-stranded DNA probes containing either the high-affinity CTF/NF-I binding site found within the first 50 base pairs of the adenovirus origin of replication (26) or the 17-base pair GAL4 binding site (27). Protein·DNA complexes were separated from free probe on native polyacrylamide gels and revealed by autoradiography.

RESULTS

CTF/NF-I Is Activated by TGF- and Repressed by TNF- in NIH3T3 Cells

Previous studies had indicated that TGF- activates the collagen 2(I) promoter in mouse NIH3T3 cells and identified binding sites for the transcription factor CTF/NF-I as necessary and sufficient mediators of TGF- induction (8). Conversely, in other experiments, TNF- was shown to suppress the basal activity of this promoter and to antagonize TGF- induction (9, 15). To address the possibility that CTF/NF-I might also mediate TNF--regulated transcriptional repression, a reporter promoter containing three high affinity CTF/NF-I binding sites in front of the -globin TATA box and the cat gene was transiently transfected in NIH3T3 fibroblasts. The CTF/NF-I binding sites conferred efficient TNF- transcriptional repression, because TNF- down-regulated both the basal as well as the TGF--stimulated CTF/NF-I transcriptional activity (Fig. 1A, pCAT-87-3xAd). In contrast, TNF- had no effect on the activity of a control promoter lacking CTF/NF-I binding sites, whereas it potently induced nuclear factor-B transcriptional activity under these conditions (Fig. 1A, pCAT-55 and data not shown). Gel mobility shift analysis of the endogenous CTF/NF-I proteins indicated that TNF- has no detectable effect on the binding activity and/or expression levels of CTF/NF-I species (Fig. 1B). Thus, these data together suggest that the endogenous mouse CTF/NF-I polypeptides specifically mediate antagonistic TGF- and TNF- transcriptional regulation.

The TGF--responsive Domain of CTF-1 Mediates Transcriptional Repression in Response to TNF-

To address the possibility that TNF- may regulate the transcriptional activity of specific CTF/NF-I variants such as CTF-1, we used a panel of expression vectors containing various portions of the CTF-1 cDNA fused to the sequences encoding the DNA binding and dimerization domains of the yeast GAL4 protein (amino acids 1-147, hereafter referred to as GAL-DBD). These GAL-CTF1 constructs were transfected in mouse NIH3T3 cells together with a reporter construct containing five GAL4 binding sites in front of the cat reporter gene (G5BCAT; Ref. 21). As shown previously (17), the prototype of the GAL4 fusion proteins, GAL 399-499, containing the entire proline-rich transactivation domain of CTF-1 (amino acids 399-499), activated transcription in NIH3T3 cells, and its activity was increased by TGF- (Fig. 2A). However, both the basal as well as the TGF--induced GAL 399-499 transcriptional activity were repressed by either human or mouse TNF- with identical potencies (Fig. 2A and data not shown). This effect was specific for TNF- because interleukin 1, serum, and PDGF-BB had no effect on GAL 399-499 transcriptional activity. Moreover, the TADs of other known transcription factors, such as that of the proline-rich transactivator AP2, the glutamine-rich TADs of Sp1 and Oct2, and the acidic GAL4 and VP16 TADs, all failed to mediate both TNF- repression and TGF- induction when fused to GAL-DBD (Fig. 2A and data not shown). GAL 1-399, a GAL4 fusion encoding a CTF-1 derivative without the proline-rich TAD, was not detectably repressed by TNF-, whereas a chimera containing the minimal GAL-DBD (amino acids 1-93) fused to the CTF-1 TAD did mediate TNF- repression. GAL4 fusions, containing either the proline-rich domain of CTF-2 or the entire CTF-2 coding sequence, were not detectably repressed by TNF- (data not shown), suggesting that TNF- repression is restricted to specific CTF/NF-I species. Moreover, gel mobility shift assays demonstrated that TNF- had no significant effect on the DNA binding affinity or the expression levels of the GAL4 fusion proteins when given either alone or in combination with TGF- (Fig. 2B; see below). Taken together, the above results demonstrate that the proline-rich domain of CTF-1 is sufficient to confer TNF- transcriptional repression when tethered to an heterologous DNA binding domain in NIH3T3 cells.

Progressive deletion from the C-terminal end of the CTF-1 TAD eliminated both basal transcriptional activity and TGF- inducibility (Fig. 2A, GAL 399-472, GAL 399-438), whereas the N-terminal portion was dispensable for TGF- responsiveness (Fig. 2A, GAL 438-499, GAL 472-499). These results confirm our previous observations that both the basal transcriptional activity and the TGF- inducibility of individual GAL 399-499 deletion mutants requires the presence of the TGF--responsive domain at their extreme C termini (amino acids 479-499 of CTF-1; Ref. 17). Strikingly, TNF- repressed both basal and TGF--induced GAL 438-499 and GAL 472-499 transcriptional activities, whereas it did not detectably regulate the weak activators GAL 399-438 and GAL 399-472 (Fig. 2A). Gel-shift analysis confirmed that TNF- did not significantly affect the levels of these fusion proteins (Fig. 2B and data not shown). Thus, these results collectively demonstrate that TNF- represses the activity of the CTF-1 proline-rich domain, thereby antagonizing TGF- induction, and that the CTF-1 TRD mediates both responses in NIH3T3 cells.

Our subsequent experiments focused on the identification of signal transduction intermediates involved in the antagonistic regulation of CTF-1 transcriptional activity by TGF- and TNF-. Several intracellular mediators of TNF- action are known, including arachidonic acid (AA) and its metabolites prostaglandins and leukotrienes, reactive oxygen intermediates (ROIs), and protein kinases (reviewed in Refs. 7 and 28). Since it had been proposed that prostaglandins might at least partially mediate TNF- inhibition of collagen 1(I) transcription in fibroblasts (29), we initially assessed whether stimulation of mammalian cells with AA might affect GAL 399-499 transcriptional activity. However, exogenous AA did not regulate GAL 399-499, nor did it alter the TNF--mediated repression of basal or TGF--activated GAL 399-499 transcription (Fig. 3). In contrast, AA efficiently stimulated the transcriptional activity of the previously identified AA-responsive transcription factor PPAR (30) in fibroblastic cells (Fig. 3). Consistent with the lack of effect of exogenous AA on CTF-1 transcriptional activity, we found that overexpression of the 85-kDa cPLA₂ phospholipase, which generates intracellular AA, or specific inhibition of AA metabolism with NDGA or indomethacin, all failed to block or mimic TGF- and TNF- regulation of CTF-1 (data not shown). Taken together, these results thus indicate that AA and its metabolites are not involved in the signal transduction pathways that regulate CTF-1 activity in response TGF- and TNF-.

The failure of NDGA to block TNF- repression was particularly interesting because NDGA also possesses potent antioxidant activity, as it protects some cells from the cytotoxic effect of TNF- (28). The well-known compounds pyrrolidine dithiocarbamate and N-acetylcysteine are also potent antioxidants and efficient inhibitors of TNF--induced, ROI-mediated nuclear factor-B activation (31). However, treatment of NIH3T3 cells with these drugs or overexpression of the c-Myc oncoprotein, which potentiates TNF- cytotoxicity (32), did not prevent the regulation of GAL 399-499 by TGF- and TNF- in transient transfection assays (data not shown), implying that TGF- and TNF- signal on CTF-1 through ROI- and cellular growth-independent pathways.

Belka et al. (33) have recently shown that TNF- regulates a ROI-independent pathway leading to Raf-1 kinase phosphorylation and activation. Raf-1 operates downstream of Ras in the signaling pathway that links several activated growth factor receptors to downstream effector molecules (34). Interestingly, oncogenic Ras expression has been shown to down-regulate 1(I) and 2(I) collagen gene expression in Rat1A and NIH3T3 fibroblasts (35). We, therefore, examined whether Ras and Raf-1 might be involved in the regulation of CTF-1 transcriptional activity by TNF- and TGF-. Strikingly, expression of an oncogenically activated form of Ras (EJ-Ha-Ras; Ref. 23) significantly decreased both basal and TGF--induced GAL 399-499 transcriptional activities (Fig. 4, compare lane 1 with lane 5 and lane 2 with lane 6, respectively). Moreover, TNF- failed to further repress the basal and the TGF--induced GAL 399-499 activities in Ras-overexpressing cells, as compared to control cells (Fig. 4, compare lanes 5 and 7 with lanes 1 and 3 and lanes 6 and 8 with lanes 2 and 4). This effect of EJ-Ha-Ras was specific for GAL 399-499, because EJ-Ha-Ras did not significantly affect GAL-AP2 activity in the absence or presence of hormones (Fig. 4, lanes 9-16). TGF- was previously shown to potently induce endogenous AP-1 transcriptional activity in A-549 cells (36). However, little, if any, TGF- induction of AP-1 occurred in Mv1Lu cells (37), whereas the AP-1-responsive reporter promoter 5xTRE-TATA-CAT (22) was not regulated by TGF- in NIH3T3 cells (Fig. 4, lanes 17 and 18). Thus, the strong AP-1 induction by TGF- may be cell line- and/or construct-specific. In contrast, the endogenous AP-1 transcriptional activity was increased by treatment with TNF- (38) or, most efficiently, upon EJ-Ha-Ras expression in NIH3T3 cells (39) (Fig. 4, compare lanes 17, 19, and 21). We conclude that overexpression of oncogenic Ras fully replaces TNF- for the repression of CTF-1 transcriptional activity in NIH3T3 cells. Similarly, expression of a constitutively active mutant of Raf-1 kinase (Raf-BXB; Ref. 24) potently activated endogenous AP-1 in NIH3T3 cells (Fig. 5, lanes 13 and 16) (39), whereas it had no effect on the fold induction of GAL 399-499 by TGF- (Fig. 5, compare lanes 4 and 5 with 1 and 2). However, the ability of TNF- to repress TGF--induced GAL 399-499 activity was eliminated in the presence of Raf-BXB, and again Raf-BXB fully replaced TNF- for the repression effect (lanes 5 and 6 versus lanes 2 and 3). This was specific for Raf-1, because expression of several other kinases, including GSK3, JAK1, JAK2, src, abl, cdk2, cdk4, as well as the TNF--inducible JNK1 kinase (40), had no effect on TNF- repression under these conditions (results not shown). Thus, these data show that expression of oncogenic Ras or Raf mimics TNF- repression of CTF-1 transcriptional activity, which implies the Ras-Raf kinase signaling pathway in the TNF- repression of CTF-1.

TNF- Repression of CTF-1 Transcriptional Activity Is Not Mediated by Direct Phosphorylation of Its Proline-rich Domain

Growth factor-induced activation of the Ras-Raf pathway results in MAP/ERK-mediated phosphorylation and activation of transcription factors such as TCF/Elk-1 (reviewed in Ref. 34). Our data suggested that TNF- might repress CTF-1 transcriptional activity in a similar manner, by regulating a Ras/Raf-dependent direct phosphorylation of the CTF-1 TAD. Since TNF- repression is mediated by the TRD portion of CTF-1 (see above), which contains three potential phosphoacceptor sites, (Fig. 6), we mutated these residues to neutral amino acids and asked whether these mutations might block TNF- repression. However, GAL-CTF1 variants harboring a triple mutation of their TRD (where the tyrosines 491 and 497 are mutated to phenylalanines and serine 495 to alanine) were similarly repressed by TNF-, as compared to their parental constructs (Fig. 6, compare GAL 479-499 3xM and GAL 399-499 3xM with W.T. constructs). Moreover, other GAL 399-499 mutants containing a single point mutation of each phosphoacceptor (for example GAL 399-499 S495A, Fig. 6) were also regulated as wild-type GAL 399-499, thereby ruling out the possibility that the triple GAL 399-499 mutant might still mediate regulation because of simultaneous removal of positive and negative regulatory determinants. Consistently, "solid-phase" kinase assays (41) demonstrated that the phosphorylation of bacterially expressed GST-CTF1 fusion proteins by NIH3T3 cell extracts was not modified by prior treatment of the cells with TNF- and/or TGF- (data not shown). These results demonstrate that TNF- does not regulate CTF-1 through direct phosphorylation of the TRD. Thus, the TNF- regulatory effect might be exerted on another factor(s), which interacts and cooperates with the CTF-1 proline-rich domain in mediating TNF- transcriptional repression.

The Interaction of the CTF-1 Transactivation Domain with Histone H3 Is Down-regulated by TNF- in NIH3T3 Cells

We have recently identified histone H3 as such a CTF-1-interacting protein in the yeast two-hybrid system. In NIH3T3 cells, histone H3 interacts with the TRD portion of the CTF-1 TAD, and TGF- up-regulates this interaction (17). Since the TRD also mediates TNF- transcriptional repression, we asked whether the histone H3-CTF1 interaction might be a target of TNF- regulation. To address this question, we used the previously described two-hybrid assay in NIH3T3 cells (17) that detects protein-protein interactions between specific GAL4 fusions and a chimera consisting of the herpes simplex virus VP16 transactivation domain fused to the coding sequence of mouse histone H3.3 (hereafter referred to as VP-H3). In transiently transfected NIH3T3 cells, such an interaction between the two chimeric proteins recruits the potent VP16 TAD to a GAL4-responsive promoter, leading to higher levels of transcriptional activation (superactivation). Accordingly, VP-H3 efficiently superactivated GAL 399-499, the TRD-containing fusion GAL 486-499 and GAL-Sp1 in NIH3T3 cells (Fig. 7A). This effect was specific, because GAL-DBD, GAL-AP2, and several of other GAL4 fusion proteins did not mediate significant VP-H3 superactivation, whereas expression of either the VP or histone H3 moieties alone or of VP16 fusions to other histones had no effect on GAL 399-499 activity (Fig. 7A; Ref. 17). TGF- up-regulated the interaction of both GAL 399-499 and GAL 486-499 with VP-H3, because superactivation was increased in the presence of the factor, but it did not up-regulate the interaction of VP-H3 with GAL-Sp1 (Fig. 7A). These results are consistent with the previously proposed specific potentiation of the CTF-1-histone H3 interaction by TGF- in NIH3T3 cells (17). Strikingly, TNF- appeared to down-regulate the interaction of VP-H3 with GAL 399-499 and GAL 486-499, as superactivation was decreased when TNF- was present, either alone or in combination with TGF- (Fig. 7A). This effect was specific for GAL 399-499 and GAL 486-499, as TNF- did not decrease the superactivation of GAL-Sp1 by VP-H3. Control experiments demonstrated that the expression of the GAL4 derivatives was not affected by either TNF- or VP-H3 under these conditions (Fig. 7B and data not shown). Thus, these results imply that TNF- may down-regulate the interaction of CTF-1 TRD with histone H3, and they further suggest that histone H3 might be a direct molecular target of antagonistic TGF- and TNF- regulation.

DISCUSSION

CTF-1 is the prototype member of the CTF/NF-I family of proline-rich transcription factors that mediate regulation of a variety of viral and cellular genes, including several procollagen genes (8, 16, 42, 43). Collagen synthesis and secretion by mesenchymal cells is potently promoted by TGF- during the process of wound healing, whereas TNF- down-regulates collagen gene expression, thereby antagonizing TGF- action (8, 9, 14, 15). Previous studies of the human 2(I) collagen promoter had identified a promoter element termed TbRE (for TGF--responsive element, Ref. 14), which can confer antagonistic TGF- and TNF- regulation on heterologous promoters, and is sufficient to direct correct tissue-specific expression of a marker gene in transgenic mice (8, 9, 14, 15, 44). Putative CTF/NF-I- and Sp1-like binding sites were mapped on the TbRE, which led to the proposal that such transcription factors might mediate promoter regulation by TGF- and/or TNF- (8, 14, 15). However, no difference in the binding affinity of either CTF/NF-I or Sp1 in response to TGF- and TNF- could be demonstrated by these authors, and thus the molecular mechanisms underlying the functional antagonism of TGF- and TNF- on collagen gene expression remained obscure.

Our results provide evidence regarding the potential mechanism of antagonistic regulation of the promoter by TGF- and TNF-. Using a reporter promoter containing high affinity CTF/NF-I binding sites, we demonstrate that CTF/NF-I binding sites specifically mediate opposing TGF- and TNF- transcriptional regulation. Both TGF- (8) and TNF- (Fig. 1B) have no apparent effect on the CTF/NF-I levels, suggesting that the hormones might antagonistically regulate the transcriptional activity of individual CTF/NF-I species. Indeed, we have shown recently that TGF- induces the proline-rich transactivation domain of CTF-1 in NIH3T3 fibroblasts (17). We now report that TNF- down-regulates the activity of the CTF-1 TAD, thereby antagonizing TGF- induction, and that the TGF--responsive domain of CTF-1 mediates both the transcriptional activation and repression effects. Importantly, the naturally occurring CTF-2 TAD, which lacks a TRD, as well as the TADs of other known transcription factors, does not mediate TNF- repression or TGF- induction in NIH3T3 cells. Moreover, growth factors such as PDGF-BB and interleukin 1 have no effect on CTF-1 transcriptional activity, consistent with their lack of effect on the 2(I) collagen promoter (8, 9, 10). In contrast, oncogenic Ras expression results in down-regulation of promoter activity in fibroblasts (35), and we find that it also represses CTF-1 transcriptional activity. Thus, our results provide a perfect correlation between the regulation of CTF-1 activity and the regulation of the collagen promoter by growth factors and oncogenes. CTF-1 may thus be a major regulator of 2(I) collagen and perhaps also of other genes subjected to antagonistic TGF- and TNF- regulation in vivo.

TGF- induces the growth of NIH3T3 cells and other embryonic fibroblasts through poorly understood mechanisms that may involve increased synthesis of PDGF, which would then act in an autocrine fashion (reviewed in Refs. 6 and 12). Conversely, long-term exposure of NIH3T3 cells to TNF- eventually leads to cell death (45). However, murine TNF- is more cytotoxic than human TNF- in inducing cell death, and this may result from the inability of the latter to bind to both the 55- and 70-kDa TNF- receptors at the cell surface, as does murine TNF- (32, 45). TNF- cytotoxicity may further be potentiated by expression of the c-Myc oncoprotein (32), and c-Myc overexpression had been associated with decreased CTF/NF-I transcriptional activity in 3T3-L1 cells (46). These findings raised the possibility that the antagonistic regulation of CTF-1 by TGF- and TNF- might be a consequence of their antagonistic effect on cell growth, and that c-Myc might perhaps mediate this antagonism. However, we have not observed any difference in the transcriptional activity and regulation of CTF-1 in either transiently or stably c-Myc-overexpressing NIH3T3 cells. Although this may simply reflect differential regulation of CTF-1 in 3T3-L1 compared to NIH3T3 cells, several independent lines of evidence further suggest that TGF- and TNF- initiate at least two distinct pathways in NIH3T3 cells, one leading to growth control and the second to CTF-1 regulation. (i) Serum or PDGF-BB treatment potently activates the growth of NIH3T3 cells, yet it does not increase GAL 399-499 transcriptional activity, and it does not prevent antagonistic CTF-1 regulation by TGF- and TNF-. (ii) TGF- induction and TNF- repression are similar in actively growing or quiescent NIH3T3 cells. (iii) NIH3T3 cells stably transfected with cdk4 plus cyclin D2, or cells transiently overexpressing the p27/Kip1 or p21/Cip1 cdk inhibitors, still mediate TGF- induction.² (iv) Pharmacological inhibitors of arachidonic acid and ROI generation such as NDGA and pyrrolidine dithiocarbamate, which have been proposed to interfere with TNF--induced cytotoxicity and nuclear factor-B activation (28, 47, 48), cannot block TNF- repression. (v) Murine TNF- and human TNF- were equally effective in mediating CTF-1 repression in NIH3T3 cells, yet mTNF- is far more cytotoxic (see above; Refs. 32 and 45). Thus, these data altogether strongly support the notion that the antagonistic regulation of CTF-1 activity by TGF- and TNF- in NIH3T3 fibroblasts occurs independently of the control of cell growth.

Based on the previous observation that oncogenic Ras expression leads to decreased procollagen transcription in fibroblasts (35), we examined the potential role of Ras and Raf in the antagonistic regulation of CTF-1 transcriptional activity by TGF- and TNF-. Expression of activated forms of either oncogene had no significant effect on the induction of GAL 399-499 by TGF-, suggesting that their expression does not interfere with TGF- regulation in NIH3T3 cells. However, we found that expression of activated Ras and Raf variants is sufficient to specifically mediate CTF-1 repression in NIH3T3 cells, thereby acting indistinguishably from TNF-. In contrast, expression of the JNK/SAPK kinase, which mediates TNF- induction of the c-Jun TAD (49, 50), and which is apparently induced by TGF- in MC3T3-E1 cells (51), did not affect CTF-1 regulation. Thus, our results imply Ras and Raf-1 in the TNF- signal transduction to CTF-1, and they further argue that the pathways leading to CTF-1 and c-Jun regulation are distinct. Nevertheless, further experiments are required to determine whether Ras and Raf are part of the TNF- signal transduction pathway to CTF-1.

Growth factor-regulated activation of the Ras-Raf pathway results in ERK-mediated phosphorylation of target transcription factors, which is often associated with changes in their transactivation potential (34). We, therefore, tested whether TNF- might similarly repress CTF-1 through direct phosphorylation of its proline-rich domain. Our analysis demonstrated that TNF- targets the same regulatory domain of CTF-1 previously shown to confer TGF- responsiveness, the TRD (17). However, the TRD is not the direct target of regulatory phosphorylation by either growth factor (Ref. 17 and Fig. 6), suggesting that TGF- and TNF- antagonistically regulate the binding of accessory proteins on the TRD. We have previously identified histone H3 as such a TRD-interacting protein in the yeast two-hybrid system and have shown that TGF- up-regulates this interaction in NIH3T3 cells (17). We, therefore, tested whether TNF- down-regulates the interaction of GAL 399-499 with histone H3. Indeed, we find that two TRD-containing GAL-CTF1 variants are able to mediate TNF--responsive histone H3 interaction, in contrast to GAL-Sp1, the interaction of which with histone H3 is constitutive. Thus, although a role for other CTF-1-interacting proteins cannot be excluded, our results are consistent with the notion that histone H3 might be implicated in the antagonistic regulation of CTF-1 transcriptional activity. Our data further argue that the CTF-1-histone H3 interaction might be down-regulated, directly or indirectly, in response to a Ras-Raf signaling cascade engaged by the TNF- receptors. Interestingly, other growth factors known to activate Ras, such as epidermal growth factor, have been shown to induce histone H3 phosphorylation, and this modification correlates with increased gene expression (52).

The proline-rich domain of CTF-1 mediates TNF- transcriptional repression (this study), and it is capable of binding to histone octamers, leading to reconfiguration of nucleosomal architecture in vitro (17). This suggests that TNF- might regulate chromatin remodeling through CTF-1 in vivo. Indeed, in the case of the human immunodeficiency virus 1 promoter (HIV-1 LTR), TNF- treatment induces disruption of a target nucleosome that is translationally positioned (53) close to CTF/NF-I binding sites (54). Thus, disruption of the HIV LTR nucleosome may occur through a TNF--regulated interaction of CTF-1 with histone H3.

In summary, the results presented in this study identify CTF-1 as a novel molecular target of TNF- and show that CTF-1 mediates antagonistic transcriptional regulation in response to TGF- and TNF-. Given the wide range of opposing TGF- and TNF- actions and the broad expression of the various CTF/NF-I proteins, these findings uncover novel mechanisms that may regulate gene expression in response to the two growth factors, and they illustrate how multiple signaling pathways are integrated by converging to common target regulatory proteins.