Smad3-Smad4 and AP-1 Complexes Synergize in Transcriptional Activation of the c-Jun Promoter by Transforming Growth Factor β

Transforming growth factor β (TGF-β) is a multifunctional cytokine with a wide range of physiological as well as pathological effects (reviewed in references 31 and 42). Its physiological roles include inhibition of the proliferation of a variety of cell types, negative regulation of the immune system, and positive regulation of extracellular matrix deposition. Dysregulation of these processes can result in various fibrotic as well as malignant diseases. Indeed, many late stage cancers have lost expression of TGF-β receptors, which renders them resistant to TGF-β-mediated growth inhibition (19, 29, 36, 38, 50, 55); restoration of TGF-β pathways in these cells can often restore growth inhibition and decrease the malignant phenotype. TGF-β-mediated immune system suppression and stimulation of extracellular matrix (ECM) production may also contribute to tumor-promoting effects. Regulation of transcription of specific sets of genes by TGF-β mediates many of these physiological roles. Upregulation of two cyclin-dependent kinase inhibitor genes, p21 and p15, has been shown to mediate TGF-β-induced growth arrest in certain cell types (7, 12, 41), while upregulation of ECM genes, including plasminogen activator inhibitor 1 (PAI-1), fibronectin, and collagen genes, may mediate other effects of TGF-β. However, many of the genes regulated by TGF-β are also regulated by a variety of other signals, including some signals which appear to play very distinct roles at the physiological level. Of particular note is a subset of TGF-β immediate-response target promoters, including the TGF-β1 ligand gene and most of the TGF-β-responsive extracellular matrix genes, in which AP-1 binding sites have been found to be involved in mediating the TGF-β signal (4, 22, 51). The use of AP-1 sites in TGF-β-dependent transcription has been particularly puzzling, given the extensively described mitogenic signaling pathways which also activate transcription through AP-1; the mechanism by which TGF-β regulates these promoter sequences has not been clarified. An additional level of complexity is introduced by the regulation by TGF-β of the expression of AP-1 family members themselves. This suggests that there can be both primary and secondary effects on transcription through AP-1 by TGF-β. The regulation of AP-1 transcription factors by TGF-β varies with the specific family member and with cell type. The upregulation of c-Jun transcript occurs in a wide range of cell lines derived from both normal and transformed cells. This response to TGF-β is early and immediate, with mRNA induced within 15 to 30 min. While cycloheximide studies have been inconclusive, due to the inducing effects of the cycloheximide itself on c-jun transcription, the time course of induction strongly suggests that this gene could be a primary target of TGF-β (24, 26, 39), which is supported by the current study describing specific promoter elements capable of mediating TGF-β’s induction of c-Jun. The model for TGF-β activation of transcription continues to undergo rapid development. The Smads are a recently identified family of proteins which operate downstream of various members of the TGF-β superfamily (reviewed in references 13, 14, 23, 30, and 37). Smad2 and Smad3 are downstream effectors of the TGF-β signaling pathway. Upon ligand binding, they are phosphorylated by the TGF-β type I receptor kinase and translocate to the nucleus in a complex with Smad4 (28, 35, 59). Recent work has identified a potential consensus Smad3-Smad4 DNA binding site, GTCTAGAC (58), by random oligonucleotide screening, as well as similar sequences in the PAI-1 promoter (9), the engineered TGF-β-responsive reporter construct, p3TP-lux promoter (57), the JunB promoter (18), and the COL7A1 collagen promoter (54). It was found that four copies of the oligonucleotide consensus site or nine copies of the PAI-1 site could confer TGF-β responsiveness on a minimal promoter. In addition, mutation of all three putative Smad3-Smad4 binding sites in the PAI-1 promoter could eliminate TGF-β responsiveness of that promoter in HepG2 cells. Although these studies demonstrate the importance of Smad3-Smad4 binding sites in the mediation of TGF-β responsiveness, they do not fully address the issue of whether binding elements for other transcription factors are also required for TGF-β-mediated transcriptional activation of target promoters. Biochemical and overexpression studies have demonstrated that Smads are capable of functional interaction with Sp1 (33) and with AP-1; in fact, direct physical interaction between Smads and AP-1 family members has been demonstrated in model systems (27, 60). Cooperation between Smad2-Smad4 complexes and FAST-1 has been demonstrated at an activin responsive Xenopus promoter (2, 3). Finally, a very recent study reports that a binding site for the transcription factor muE3 (TFE3), as well as one for Smad3 and Smad4, is required for TGF-β-mediated transcription of a reporter controlled by a specific region of the PAI-1 promoter (16). While the TGF-β-responsive elements in the c-Jun promoter have not previously been characterized, extensive work has established the importance of two AP-1/CRE sequences in the c-Jun promoter in regulation by phorbol-12-myristate-13-acetate (TPA), serum, UV, E1A, and interleukin 1 (IL-1) (1, 15, 34, 43, 53). Furthermore, a reporter construct controlled by the −79 to +170 sequence of the c-Jun promoter, which contains only the more proximal AP-1/CRE site (−71 to −64), has proved sufficient for a maximal response to most of these signals. Interestingly, none of these stimuli appears to change the occupancy of any identified binding sites in the c-Jun promoter. Thus, the prevailing model of activation by these other signals is thought to be through modification of a constitutively promoter-bound complex, in most cases c-Jun-ATF-2. Here, we identify two DNA binding elements within this −79 to +170 region which are indispensable in TGF-β-mediated induction of c-Jun: the proximal AP-1/CRE site known to be important for the response to several other signals, and a novel Smad3/Smad4 binding site. Mutation of either site alone is found to abolish or severely reduce promoter upregulation by TGF-β, despite the presence of the remaining element. Our results suggest that the two complexes can cooperate synergistically in activating TGF-β-mediated transcription of this c-Jun promoter region.