Proc. Natl. Acad. Sci. USA Vol. 96, pp. 4844–4849, April 1999 Biochemistry Smads bind directly to the Jun family of AP-1 transcription factors (Smad3ySmad4ycJunyJunBytransforming growth factor) NICOLE T. LIBERATI,MICHAEL B. DATTO,JOSHUA P. FREDERICK,XING SHEN,CAROLYN WONG, ELISSA M. ROUGIER-CHAPMAN, AND XIAO-FAN WANG* Department of Pharmacology and Cancer Biology, Box 3813, Duke University Medical Center, Durham, NC 27708 Communicated by Gordon G. Hammes, Duke University Medical Center, Durham, NC, September 28, 1998 (received for review July 21, 1998) ABSTRACT Smad3 and Smad4 are sequence-specific cyte chemoattractant protein-1 (JEyMCP-1), type I collagen, DNA-binding factors that bind to their consensus DNA- and TGFb1 itself depends on specific AP-1 DNA-binding sites binding sites in response to transforming growth factor b in the promoter regions of these genes (3, 5–10). Furthermore, (TGFb) and activate transcription. Recent evidence impli- TGFb-mediated transcriptional activation of several of these cates Smad3 and Smad4 in the transcriptional activation of genes requires AP-1 proteins (5, 8–10). Intriguingly, the consensus AP-1 DNA-binding sites that do not interact with expression of many AP-1 proteins themselves is induced as an Smads directly. Here, we report that Smad3 and Smad4 can early response to TGFb in a cell type-specific manner (11–14). physically interact with AP-1 family members. In vitro binding It has been demonstrated that this induced expression of studies demonstrate that both Smad3 and Smad4 bind all particular AP-1 family members is involved in TGFb-mediated three Jun family members: JunB, cJun, and JunD. The Smad regulation of subsequent target genes (10). In addition, genetic interacting region of JunB maps to a C-terminal 20-amino studies of TGFb signaling in Drosophila melanogaster reveal a acid sequence that is partially conserved in cJun and JunD. direct overlap between AP-1 and TGFb signaling and suggest We show that Smad3 and Smad4 also associate with an an evolutionarily conserved convergence of these pathways endogenous form of cJun that is rapidly phosphorylated in (15). Together, these studies demonstrate a link between response to TGFb. Providing evidence for the importance of TGFb signaling and AP-1 in the TGFb-regulated expression this interaction between Smad and Jun proteins, we demon- of various genes. The molecular mechanisms responsible for strate that Smad3 is required for the activation of concatamer- the TGFb-mediated transcriptional activation of these genes ized AP-1 sites in a reporter construct that has previously are just beginning to be elucidated. been characterized as unable to bind Smad proteins directly. Insight into the mechanism of TGFb-regulated gene expres- Together, these data suggest that TGFb-mediated transcrip- sion has come about with the discovery of the Smad family of tional activation through AP-1 sites may involve a regulated proteins. The Smads are phosphorylated by the activated type interaction between Smads and AP-1 transcription factors. I receptor in response to ligand (16). Specifically, Smad2 and Smad3 were shown to be inducibly phosphorylated in response Transforming growth factor b (TGFb) is a multipotent cyto- to TGFb (17–19). Smad phosphorylation results in hetero- kine that regulates a variety of cellular activities, such as cell merization of either Smad2 or Smad3 with Smad4 (20–23). proliferation, differentiation, and extracellular matrix (ECM) Smad4-containing heteromers then enter the nucleus where formation. The combined actions of these cellular responses they can activate transcription of specific genes (24, 25). are likely to mediate more global effects of TGFb including its Current research is focused on elucidating the role of Smads role in development, wound healing, immune responses, and in TGFb-induced transcriptional activation. the pathogenesis of cancer (1–3). The identification of genes Through attempts made at understanding the mechanism of transcriptionally regulated by TGFb and the elucidation of the Smad-mediated transcriptional activation, two distinct roles molecular mechanisms responsible for this transcriptional for Smads have emerged: Smads as DNA-binding factors and regulation will help define how TGFb exerts its cellular effects Smads as transcription factor-binding proteins. Several lines of and its role in resulting physiological processes. Although evidence suggest that Smads activate transcription by binding progress has been made in the identification of TGFb target directly to DNA. For instance, transcription of a reporter genes, including the cyclin-dependent kinase inhibitors p21 plasmid containing the concatamerized consensus Smad- and p15 (1, 2) and the ECM component plasminogen activator binding site is induced by TGFb in a Smad4-dependent inhibitor-1 (PAI-1) (3), which has subsequently contributed manner (26). Smad3 and Smad4 were recently shown to form toward our understanding of TGFb-mediated growth inhibi- a complex on similar DNA sequences derived from the PAI-1 tion and ECM deposition, the mechanisms by which TGFb promoter (27). Mutation of these sequences in the PAI-1 controls gene expression remain largely unknown. promoter reduced TGFb responsiveness. Furthermore, Gal4 Numerous studies have characterized the differential ex- fusions with the C-terminal domains of Smad1 and Smad4 pression of specific genes in response to TGFb, revealing a activate transcription from concatamerized Gal4 DNA- common link in the ability of TGFb to regulate many of these binding sites (28). genes through the functions of the AP-1 family of transcription Other evidence suggests that Smads can activate transcrip- factors. This protein family, which includes the Fos and Jun tion by binding to other transcription factors. For example, the proteins, binds a specific DNA sequence and facilitates tran- interaction between Smad2ySmad4 heteromers and the tran- scriptional regulation (4). The ability of TGFb to induce the scription factor FAST-1 is critical for the formation of the expression of several genes, including PAI-1, clusterin, mono- Abbreviations: TGFb, transforming growth factor b; HaCaT, human The publication costs of this article were defrayed in part by page charge keratinocyte cells; JNK, cJun N-terminal kinase; CMV, cytomegalo- virus; GST, glutathione S-transferase; TNT, transcription and trans- payment. This article must therefore be hereby marked ‘‘advertisement’’ in lation. accordance with 18 U.S.C. §1734 solely to indicate this fact. *To whom reprint requests should be addressed. e-mail: PNAS is available online at www.pnas.org. [email protected]. 4844 Downloaded by guest on October 1, 2021 Biochemistry: Liberati et al. Proc. Natl. Acad. Sci. USA 96 (1999) 4845 activin responsive factor (ARF), an activin-inducible DNA- tained 5 mM 3-aminotriazole. Transformants (242) were pos- binding complex in Xenopus (24, 29). Overexpression of the itive for b-galactosidase activity, which was measured by the Smad-binding domain of FAST-1 blocked ARF formation and appearance of blue color on colony filter lifts incubated in the transcriptional induction of an activin-inducible early response presence of 5-bromo-4-chloro-3-indolyl-b-D-galactosidase (X- gene. Together, these data indicate that although Smads bind Gal). Bait dependence for each positive transformant was DNA directly, association with other transcription factors may established similarly. play a crucial role in Smad-mediated transcriptional activation. Binding Studies. Full length JunB in pGEM4 was digested In an attempt to identify transcription factors involved in with BspHI, BssHII, or DraI (NEB). The full length construct Smad-mediated transcriptional activation, we performed a and the digested DNAs were used as templates for in vitro yeast two-hybrid screen using Smad3 as a bait. Two interacting transcription and translation (TNT) with [35S]methionine in cDNAs encoding two different clones of the AP-1 family rabbit reticulocyte lysates (Promega). The TNT–JunB lysates member, JunB, were isolated, indicating that Smads may bind were incubated with an equal amount of bacterially purified to AP-1 members directly. Supporting a direct interaction glutathione S-transferase (GST), or GST-Smad3 or GST- between Smads and AP-1, we show that Smad3 and Smad4 Smad4 (30) in ByP (150 mM NaCly50 mM Tris, pH 7.5y0.1% bind all known members of the Jun family of proteins in vitro. Tweeny1 mM DTT) for 2.5 hours at 4°C. The GST reactions Furthermore, we demonstrate that Smad3 is critical for the were washed three times in TBS (500 mM NaCly25 mM Tris, ability of TGFb to activate AP-1 sites independent of Smad pH 7.5y0.1% Tween-20y1 mM DTT). Samples were resolved DNA binding. These data, therefore, provide insight into a by SDSyPAGE. The gels were treated with 10% sodium possible mechanism by which TGFb activates AP-1-mediated salicylate, dried, and exposed to film. Whole-cell COS lysates transcription through the induction of SmadyAP-1 complex overexpressing each AP-1 member were lysed as described formation. (30) and incubated with the GST fusions as described above. The binding reactions were washed three times with ByP and y MATERIALS AND METHODS separated by SDS PAGE. For endogenous protein interactions, HaCaT cells were Materials. TGFb1 was a generous gift of Amgen Biologicals. treated with 100 pM TGFb1 in DMEMy10% FBS for 15, 30, Human keratinocyte cells (HaCaT) were the generous gift of or 60 min. Cells were then lysed and either whole-cell or P. Baukamp and N. Fusenig. A HaCaT cDNA library in the nuclear extracts were prepared (30, 31). Four hundred fifty mg pACT2 expression vector was the generous gift of Y. Xiong. of each whole-cell lysate was incubated with an equal amount The full length cDNAs for murine Jun family members, FosB, of bacterially purified GST-Smad3 or GST-Smad4 normalized cFos, Fra2, and human Fra1, were the generous gifts of R. for protein by Coomassie blue and for volume of glutathione- Wisdom. Smad3 polyclonal antibody was generated against Sepharose added to each binding reaction.