[CANCER RESEARCH 63, 1371–1376, March 15, 2003] Synthetic Triterpenoids Enhance Transforming Growth Factor /Smad Signaling 1 Nanjoo Suh, Anita B. Roberts, Stephanie Birkey Reffey, Kohei Miyazono, Susumu Itoh, Peter ten Dijke, Elke H. Heiss, Andrew E. Place, Renee Risingsong, Charlotte R. Williams, Tadashi Honda, Gordon W. Gribble, and Michael B. Sporn 2 Dartmouth Medical School and Dartmouth College, Hanover, New Hampshire 03755 [N. S., E. H. H., A. E. P., R. R., C. R. W., T. H., G. W. G., M. B. S.]; National Cancer Institute, NIH, Bethesda, Maryland 20892 [A. B. R., S. B. R.]; Cancer Institute, Tokyo 170-8455, Japan [K. M.]; and The Netherlands Cancer Institute, 1066 CX Amsterdam, the Netherlands [S. I., P. t. D.] ABSTRACT We have studied the effects of two new synthetic triterpenoids, 2-cyano- 3,12-dioxooleana-1,9-dien-28-oic acid (CDDO) and its derivative, 1-(2- cyano-3,12-dioxooleana-1,9-dien-28-oyl) imidazole (CDDO-Im), on trans- forming growth factor (TGF)-/Smad signaling. These agents, at nanomolar concentrations, increase the expression of TGF--dependent genes, such as those for plasminogen activator inhibitor 1 and the type II TGF- receptor, and they synergize with TGF- in this regard. They prolong the activation of Smad2 induced by TGF- and markedly en- hance the ability of Smad3 to activate a Smad binding element, CAGA- luciferase. In transfection assays, they reverse the inhibitory effects of Smad7. CDDO and CDDO-Im also enhance Smad signaling in the path- ways of two other members of the TGF- superfamily, namely, activin and bone morphogenetic protein. Finally, these triterpenoids induce ex- pression of the transcriptional coactivator p300-CBP-associated factor and synergize with TGF- in this regard. These are the first studies to report enhancement of Smad signaling by synthetic triterpenoids and should further their optimal use for applications in prevention or treat- ment of diseases in which there is aberrant function of TGF-. INTRODUCTION Aberrant function of the TGF- 3 /Smad signaling pathway is a frequent occurrence in human disease (1– 4). In this context, it is important to develop new agents to modulate this pathway and to restore normal function (4, 5). At present, there is a paucity of molecules for this purpose. We report here, for the first time, the potent ability of some new synthetic triterpenoids to enhance TGF- /Smad signaling and to increase the effects of TGF- in cells of both epithelial and hematopoietic origin. The TGF- superfamily consists of more than 40 members, includ- ing TGF-s, activins, and BMPs; the molecules in this set are multi- functional cytokines that affect inflammatory and immune responses, cell growth and differentiation, apoptosis, and morphogenesis (6). They all signal through heteromeric complexes of type II and type I transmembrane Ser/Thr kinase receptors, which then initiate phospho- rylation cascades involving receptor-regulated Smads, a co-Smad, and inhibitory Smads (6 – 8). In the present studies, we have used synthetic triterpenoids, whose actions often mimic those of TGF- (9), to modulate the TGF-/Smad signaling pathway. We have recently developed many new synthetic triterpenoids to be more potent than the naturally occurring parent structure, oleanolic acid, as anti-inflammatory, antiproliferative, and cell-differentiating agents (9 –13). These molecules inhibit the expres- sion of genes for inflammatory mediators, such as iNOS and inducible cyclooxygenase (cyclooxygenase 2), inhibit proliferation of many cancer cells, and induce monocytic differentiation of leukemia cells and adipogenic differentiation of fibroblasts (9, 14). It is well known that TGF- also has potent effects on all of the above functions (6). Therefore, we have investigated interactions of triterpenoids with TGF- in the Smad pathway. In the present study, we have used a new synthetic triterpenoid, CDDO-Im, as well as its parent molecule, CDDO, to study effects on TGF-/Smad signaling. We have first studied the effects of CDDO and CDDO-Im on expression of genes responsive to TGF-, such as those for PAI-1 and the TGF- type II receptor (15, 16), and then evaluated transcriptional effects on a Smad binding element, using the TGF-/Smad-dependent (CAGA)12-Luc reporter (17). Furthermore, we have explored the effects of triterpenoids on signaling by other members of the TGF- superfamily, such as activin and BMP2, because these cytokines share common intracellular/ nuclear proteins and similar mechanisms of regulation and degrada- tion, although the specific Smads involved may be different (8, 18). A final possible overlap between triterpenoids and TGF- is their use of common nuclear coactivators, such as the histone acetyl trans- ferases, p/CAF, and p300/cAMP-responsive element-binding protein (CREB)-binding protein, which are known to be involved in transac- tivation of TGF--dependent genes (19 –22). Therefore, we have also studied whether CDDO or CDDO-Im could modulate the synthesis of p/CAF. Our findings in all of the above studies show for the first time that synthetic oleanane triterpenoids such as CDDO-Im and CDDO have profound effects on TGF-/Smad signaling. Because of these effects, triterpenoids may provide a therapeutically useful way to modulate the TGF- pathway. MATERIALS AND METHODS Reagents. Details of the synthesis of CDDO and CDDO-Im have been published (11, 12). Sources of other materials were as follows: recombinant mouse IFN- (lipopolysaccharide content, 10 pg/ml), TNF-, and TGF- were from R&D Systems (Minneapolis, MN); polyclonal iNOS IgG and peroxidase-conjugated secondary antibody were from Santa Cruz Biotechnol- ogy (Santa Cruz, CA); antibody to phospho-Smad2 was from Upstate Bio- technology (Lake Placid, NY); antibody to Smad2 was from Zymed Labora- tories (South San Francisco, CA); FuGene 6 was from Roche Diagnostics (Indianapolis, IN); and all other chemicals were purchased from Sigma (St. Louis, MO) unless otherwise noted. BMP2 was a gift from Tony Celeste (Wyeth Genetics Institute, Cambridge, MA). Triterpenoids were dissolved in DMSO before addition to cell cultures or enzyme assays; final concentrations of DMSO were 0.1% or less. Controls with DMSO alone were run in all cases. Cell Culture, Nitric Oxide Production in Primary Mouse Macrophages, SDS-PAGE and Western Blot Analyses, and Northern Blot Analyses. These procedures have been described previously (9, 10). Plasmids and Transfection Assays. Plasmids have all been reported pre- viously as follows: FLAG-tagged Smad2, Smad3, and Smad4, ALK5, and Received 9/24/02; accepted 1/16/03. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. 1 Supported in part by NIH Grant R01 CA78814, the National Foundation for Cancer Research, the Oliver and Jennie Donaldson Trust, and two Department of Defense/AMRD Awards, DAMD17-98-1-8604 and 17-99-1-9168. M. B. S. is Oscar M. Cohn Professor. P. t. D. and S. I. were supported by the Dutch Cancer Society. 2 To whom requests for reprints should be addressed, at Department of Pharmacology, Dartmouth Medical School, Hanover, NH 03755. Phone: (603) 650-6557; Fax: (603) 650- 1129; E-mail: michael.sporn@dartmouth.edu. 3 The abbreviations used are: TGF, transforming growth factor; ALK, activin receptor- like kinase; BMP, bone morphogenetic protein; CDDO, 2-cyano-3,12-dioxooleana-1,9- dien-28-oic acid; CDDO-Im, 1-(2-cyano-3,12-dioxooleana-1,9-dien-28-oyl) imidazole; iNOS, inducible nitric oxide synthase; co-Smad, common partner Smad; PAI, plasmino- gen activator inhibitor; TNF, tumor necrosis factor; Luc, luciferase; -gal, -galactosid- ase; TPA, 12-O-tetradecanoylphorbol-13-acetate. 1371 Research. on November 22, 2015. © 2003 American Association for Cancer cancerres.aacrjournals.org Downloaded from