Research Article Induction of Sonic Hedgehog Mediators by Transforming Growth Factor-B: Smad3-Dependent Activation of Gli2 and Gli1 Expression In vitro and In vivo Sylviane Dennler,1 Jocelyne Andre´,1 Ismini Alexaki,1 Allen Li,2 Thierry Magnaldo,3 Peter ten Dijke,4 Xiao-Jing Wang,2 Franck Verrecchia,1 and Alain Mauviel1 1Institut National de la Sante et de la Recherche Medicale U697, Paris, France; 2Department ofOtolaryngology, Oregon Health and Science University, Portland, Oregon; 3Centre National de la Recherche Scientifique UPR 2169, Institut Gustave-Roussy, Villejuif, France; and 4Department ofMolecular Cell Biology, Leiden University Medical Center, Leiden, the Netherlands Abstract Gli1 expression via direct binding to its promoter region (6). Gli Hedgehog (Hh) and transforming growth factor-B (TGF-B) transcription factors regulate multiple cellular functions associated family members are involved in numerous overlapping with malignant transformation, such as cell cycle progression and processes during embryonic development, hair cycle, and apoptosis (4, 7). cancer. Herein, we show that TGF-B induces the expression of The importance ofthe Hh signaling pathway in tumorigenesis the Hh signaling molecules Gli1 and Gli2 in various human was established through the discovery ofinactivating mutations in cell types, including normal fibroblasts and keratinocytes, the Ptch gene in patients with familial (Gorlin’s syndrome) basal as well as various cancer cell lines. Gli2 induction by TGF-B is cell carcinomas (BCC) and sporadic BCC (8, 9). Other tumors exhibit inappropriate Hh pathway activation. For example, some rapid, independent from Hh receptor signaling, and requires a functional Smad pathway. Gli1 expression is subsequently esophageal squamous cell sarcomas and transitional cell carcino- activated in a Gli2-dependent manner. In transgenic mice mas of the bladder may carry loss-of-function mutations of the overexpressing TGF-B1 in the skin, Gli1 and Gli2 expression is Ptch gene (10), whereas gain-of-function mutations of Smo have also elevated and depends on Smad3. In pancreatic adeno- been identified in a subset of small cell lung carcinoma (11). carcinoma cell lines resistant to Hh inhibition, pharmacologic Additionally, overexpression ofthe main Hh member Sonic Hh blockade of TGF-B signaling leads to repression of cell pro- (Shh), leading to activation ofSmo, has been identifiedin some Gli2 gastrointestinal cancers (12) and pancreatic adenocarcinomas (13). liferation accompanied with a reduction in expression. h h We thus identify TGF-B as a potent transcriptional inducer Similar to Hh members, transforming growth factor- (TGF- ) of Gli transcription factors. Targeting the cooperation of Hh has emerged as a family of growth factors involved in various B essential physiologic processes that include embryonic develop- and TGF- signaling may provide new therapeutic opportuni- h ties for cancer treatment. [Cancer Res 2007;67(14):6981–6] ment, tissue repair, cell growth control, and differentiation. TGF- isoforms are expressed in a variety of tumor types and contribute to the aggressiveness and progression ofneoplasms (14, 15). Introduction TGF-h members signal via membrane-bound heteromeric serine- threonine kinase receptor complexes. In most cell types, TGF-h The hedgehog (Hh) signaling pathway is critical for stem cell binds to ThRII in combination with ThRI, also known as ALK5 (16). maintenance, embryonic patterning, and growth in both inverte- Receptor activation by TGF-h leads to phosphorylation ofcyto- brates and vertebrates (1). Deregulation ofthe Hh pathway is a plasmic proteins ofthe Smad family.Receptor-associated Smads, characteristic trait ofseveral pathologic states, including develop- Smad2 and Smad3, then heteromerize with Smad4, translocate mental syndromes with high proneness to cancer (1, 2). Cellular into the nucleus, and act as transcription factors to regulate target responses to the Hh signal are controlled by two transmembrane gene expression. Smad3 is thought to contribute most Smad- proteins, the tumor suppressor Patched-1 (Ptch) and the onco- dependent responses to TGF-h in the adult, whereas Smad2 is protein Smoothened (Smo; refs. 2, 3). The latter has homology to critical during embryogenesis (17, 18). G protein–coupled receptors and transduces the Hh signal. In In this study, we have examined the capacity ofTGF- h to the absence ofHh, Ptch maintains Smo in an inactive state, thus modulate the expression ofthe Hh signaling molecules Gli1 and silencing intracellular signaling. With the binding ofHh, Ptch Gli2. We provide definitive evidence, both in vitro and in vivo,for inhibition ofSmo is released and the signal is transduced. The Smad-dependent activation of Gli2 expression and, consequently, transcriptional response to Hh signaling is mediated by a family of that of Gli1, thereby identifying TGF-h as a cytokine ubiquitously zinc-finger transcription factors that comprises the Ci protein in capable ofactivating, enhancing, or prolonging Hh signals. In Drosophila and the three closely related Gli proteins in vertebrates, addition, we show that some cyclopamine-resistant pancreatic Gli1, Gli2, and Gli3 (4). Gli2 is thought to function upstream of adenocarcinoma cell lines are growth inhibited by a small-molecule Gli1 and to be the primary mediator ofHh signaling (5), inducing inhibitor ofTGF- h signaling. Requests for reprints: Alain Mauviel, Institut National de la Sante et de la Materials and Methods Recherche Medicale U697, Hoˆpital Saint-Louis, Pavillon Bazin, 1, Avenue Claude Cell cultures and reagents. Primary human dermal fibroblasts, WI-26 Vellefaux, 75010 Paris, France. Phone: 33-1-53-72-20-69; Fax: 33-1-53-72-20-51; E-mail: human transformed lung fibroblasts, HaCaT immortalized human kerati- [email protected]. I2007 American Association for Cancer Research. nocytes, MDA-MB-231 and MDA-MB-468 breast carcinoma cell lines, and doi:10.1158/0008-5472.CAN-07-0491 PANC-1 human pancreatic adenocarcinoma cells were all maintained in www.aacrjournals.org 6981 Cancer Res 2007; 67: (14). July 15, 2007 Downloaded from cancerres.aacrjournals.org on September 23, 2021. © 2007 American Association for Cancer Research. Cancer Research DMEM with 10% fetal bovine serum and antibiotics (Invitrogen). When Western blot analysis oftotal Gli2 production in dermal indicated, cells were serum starved for 16 h and treated with human fibroblasts showed undetectable expression levels in unstimulated h h recombinant TGF- 1 (5 ng/mL; referred to as TGF- ) and/or human cultures. Gli2 protein became detectable 6 h after addition of A recombinant NH2-terminal Shh peptide (1.5 g/mL), both purchased from TGF-h and accumulated over 48 h ofincubation (Fig. 1 D). Given R&D Systems. Cyclopamine, cycloheximide, and the kinase inhibitors the high levels ofGli2 protein detected at the 48-h time point, it is SB431542, SB203580, PD98059, and SP600125 were obtained from Euro- h medex. LY294002 was from Calbiochem-Merck. Small interfering RNAs possible that TGF- may stabilize GLI2 protein, in addition to (siRNA) were purchased from Ambion/Applied Biosystems and transfected increasing gene expression. into cells using the RNAiFect reagent (Qiagen). Transfection of MDA- Ofnote, Gli3 (see multiplex PCR panels), Ptch, and Smo (data not MB-468 cells with Smad3 (19) and Smad4 (20) expression vectors was shown) expression in response to TGF-h showed minimal done using an electroporation kit (Amaxa Biosystems). Cell growth was variation. estimated by 3-(4-5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4- Activation of Gli expression by TGF-B does not involve the sulfophenyl)-2H-tetrazolium salt (MTS) assay, using a specific kit (Promega) Ptch/Smo axis. To determine whether TGF-h–induced Gli1 and according to the manufacturer’s protocol. Gli2 expression was dependent on the Ptch/Smo axis, the effect of Multiplex PCR and real-time PCR. Total RNA was prepared using an exogenous Shh and TGF-h on Gli expression by human dermal RNeasy mini kit (Qiagen). Reverse transcription (Invitrogen) was done on fibroblasts was tested in the absence or presence of the Smo genomic DNA-free RNA using oligo(dT) as primer. cDNAs are then used in multiplex PCR according to Qiagen recommendations. Real-time PCR was inhibitor cyclopamine, known to prevent Gli activation by Shh (24). h done with either a Power SYBR Green Mix or Taqman probes (mouse As shown in Fig. 2A, both TGF- and Shh strongly elevated Gli1 experiments) on an AB7300 apparatus (Applied Biosystems). The absolute mRNA steady-state levels. As expected, cyclopamine abrogated copy number for each mRNA was normalized to the absolute cyclophilin A Shh-induced Gli expression (Fig. 2A, lane 4 versus lane 3) but had mRNA copy number. PCR primer sequences and conditions are available on no effect on TGF-h–mediated Gli1 activation (Fig. 2A, lane 6 versus request. lane 5). Furthermore, when added together to the culture medium, Western blot analyses. Cells were lysed with ice-cold lysis buffer [20 mmol/L HEPES (pH 7.9), 420 mmol/L NaCl, 0.5% NP40, 25% glycerol, 1.5 mmol/L MgCl2, 0.2 mmol/L EDTA], rapidly frozen and thawed, rotated 30 min at 4jC, and centrifuged for 30 min. Total protein (100 Ag) was separated by SDS-PAGE and blotted onto nitrocellulose membranes. After saturation with TBS+0.1% Tween 20+5% dry milk, membranes were incubated with either anti-Gli2 (Santa Cruz Biotechnology, Inc.) or anti- actin (Zymed) antibodies. Detection was done using horseradish peroxi- dase–conjugated