Oncogene (2004) 23, 1263–1274 & 2004 Nature Publishing Group All rights reserved 0950-9232/04 $25.00 www.nature.com/onc The zinc-finger transcription factor GLI2 antagonizes contact inhibition and differentiation of human epidermal cells Gerhard Regl1, Maria Kasper1, Harald Schnidar1, Thomas Eichberger1, Graham W Neill2, Mohammed S Ikram2, Anthony G Quinn3, Mike P Philpott2, Anna-Maria Frischauf1 and Fritz Aberger*,1 1Institute of Genetics, University of Salzburg, Hellbrunner Strasse 34, A-5020 Salzburg, Austria; 2Center for Cutaneaous Research, Barts and The London Queen Mary’s School of Medicine & Dentistry, University of London, UK; 3Experimental Medicine, AstraZeneca R & D Charnwood, Leicestershire, UK In stratified epidermis, activation of the Hh/Gli signal portance of Hh signalling in vertebrate embryonic transduction pathwayhas been implicated in the control of development and in the control of cell proliferation is cell proliferation and tumorigenesis. The zinc-finger demonstrated by mutations in key components of the transcription factor Gli2 has been identified as critical pathway, which account for severe congenital malfor- mediator of the Hh signal at the distal end of the pathway, mations and tumour development in man (reviewed in but the molecular mechanisms bywhich Gli2 regulates Goodrich and Scott, 1998; Toftgard, 2000; Mullor et al., cell proliferation or induces epidermal malignancies such 2002; Wetmore, 2003). as basal cell carcinoma are still unclear. Here, we provide Basal cell carcinoma (BCC) of the skin represents one evidence for a role of human GLI2 in antagonizing contact of the most common malignancies in the Western world. inhibition and epidermal differentiation. We show bygene Ligand-independent activation of Hh signalling in expression profiling that activation of the GLI2 oncogene epidermis by mutational inactivation of the Hh-receptor in human keratinocytes activates the transcription of a PTCH, which in the absence of ligand represses the number of genes involved in cell cycle progression such as pathway, or by activating mutations in the Hh-signal E2F1, CCND1, CDC2 and CDC45L, while it represses transducer SMOH has been implicated as the critical genes associated with epidermal differentiation. Analysis eventin BCC development(Johnson et al., 1996; Stone of the proliferative effect of GLI2 revealed that GLI2 is et al., 1996; Xie et al., 1998). Further support for a able to induce G1–S phase progression in contact-inhibited causative role of inappropriate Hh signalling in BCC keratinocytes. Detailed time-course experiments identified has come from studies of transgenic mice expressing Shh E2F1 as earlytranscriptional target of GLI2. Further, we itself or a BCC-derived oncogenic form of SMOH in the show that GLI2 expression in human keratinocytes results basal layer of the epidermis. In either experiment, mice in a marked downregulation of epidermal differentiation developed BCC-like structures, showing that activation markers. The data suggest a role for GLI2 in Hh-induced of Hh-signal transduction in epidermal cells is sufficient epidermal neoplasia byopposing epithelial cell cyclearrest to induce skin tumorigenesis (Oro et al., 1997; Xie et al., signals and epidermal differentiation. 1998). Oncogene (2004) 23, 1263–1274. doi:10.1038/sj.onc.1207240 The zinc-finger transcription factors (TFs) GLI1 and Published online 22 December 2003 GLI2, which act at the very distal end of the Hh pathway, have been identified as putative mediators of Keywords: hedgehog signalling; GLI proteins; keratino- Hh-induced neoplasia, since overexpression of either TF cytes; basal cell carcinoma; epidermal differentiation; in the epidermis of transgenic mice induces various types cell cycle of tumours, some of which show BCC-like features (Grachtchouk et al., 2000; Nilsson et al., 2000; Sheng et al., 2002). Their relative contribution to Hh-induced tumorigenesis is, however, unclear at present. Introduction Although either TF is a potent oncogene in epidermal cells, evidence has accumulated suggesting that GLI2 The hedgehog (Hh)-signal transduction pathway, first rather than GLI1 may represent the primary mediator of identified by genetic analysis of Drosophila embryonic the Hh signal during embryogenesis and tumorigenesis: mutants, plays a critical role in a number of develop- firstly, Gli1 is dispensable for normal development and mental processes, including pattern formation, control for Shh-induced medulloblastoma formation (Park et al., of cell differentiation, proliferation and growth (re- 2000; Weiner et al., 2002), while loss of Gli2 function viewed in Ingham and McMahon, 2001). The im- results in severe developmental anomalies similar to those observed in Shh knockout mice (Ding et al., 1998; *Correspondence: F Aberger; E-mail: [email protected] Matise et al., 1998; Mill et al., 2003). Secondly, removal Received 22 September 2003; accepted 26 September 2003 of Gli2 but not of Gli1 can partially rescue the phenotype GLI2 expression in human epidermal cells G Regl et al 1264 of patched knockoutmice, which suffer from hyperacti- human epidermis. Differences between murine and vation of the Hh pathway (Bai et al., 2002). Further- human skin and the relatively high resistance of mice more, Gli2 has been shown to act upstream of Gli1, since to BCC development prompted us to use a purely removal of Gli2 function decreases levels of Gli1 mRNA human in vitro system to study the effect of GLI2 (Ding et al., 1998; Bai et al., 2002; Mill et al., 2003), and expression on epidermal cells in the absence of paracrine overexpression of GLI2 in epidermal cells results in signals derived from dermal cells. To identify GLI2- induction of GLI1 expression (Regl et al., 2002). regulated genes by DNA-array technology, we intro- While the genetic lesions involved in BCC development duced into the human keratinocyte line HaCaT (Bou- are well characterized, little is known about the down- kamp et al., 1988) a tetracycline-regulated GLI2 stream events leading to tumorigenic conversion of expression system (Figure 2d). This strategy ensures epidermal cells in response to inappropriate Hh signalling. highly reproducible and temporally controlled transgene Recentexperimentsaddressing the mechanism by which expression, which greatly facilitates data analysis of gene Hh signalling controls cell proliferation have shown that expression profiling experiments. The system also allows the pathway can interact with the cell cycle machinery at detailed time-course studies and conditional activation various points. In Drosophila, Hh regulates cell prolifera- of GLI2 in quiescentcells, which is very difficultto tion in the developing eye by activating cyclin D and achieve with retroviral expression systems. cyclin E expression. Analysis of the Drosophila cyclin E We have chosen the spontaneously immortalized, promoter showed that Cubitus interruptus – the Droso- nontumorigenic human keratinocyte line HaCaT as a phila homologue of vertebrate GLI proteins – directly model system, since, although aneuploid, it resembles stimulates cyclin E transcription (Duman-Scheel et al., primary human keratinocytes in that it has retained 2002). In vertebrates, Shh protein stimulates proliferation the capacity to differentiate and even to reconstitute of cerebellar neuronal precursor cells by regulating the stratified epidermis when grafted onto nude mice or used expression of D-type cyclins (Kenney and Rowitch, 2000), in organotypic cultures (Boukamp et al., 1988; Schoop and in human keratinocytes expression of SHH has been et al., 1999). Further, we have shown that with respect to shown to promote epidermal proliferation and oppose target gene expression, HaCaT and primary keratino- p21-induced epithelial cell cycle arrest (Fan and Khavari, cytes respond to GLI expression in a largely identical 1999). Further, loss of Shh or Gli2 function in mice results manner (Regl et al., 2002; data not shown). On the other in a significantdecrease of proliferating cells in the hair hand, the presence of cytogenetic aberrations and the follicle (Mill et al., 2003), and in vitro overexpression of immortalized phenotype of HaCaT cells (Boukamp GLI1 and GLI2 has been shown to stimulate S phase in et al., 1988; Lehman et al., 1993) may limitthe human keratinocytes (Regl et al., 2002). physiological relevance of data on cell cycle regulation. Although these data suggest that the proliferative We, therefore, validated results obtained with HaCaT effectof Shh and Gli proteinson neuronal and cells by using normal human keratinocytes expressing epidermal cells plays a critical role in brain and skin GLI2 via retroviral gene transfer. tumour development of vertebrates, the details of the To analyse the phenotypic changes of human molecular processes involved in Hh-mediated neoplasia keratinocytes in response to GLI2 expression on a remain to be established. molecular level, cDNA from confluent tetracycline- To elucidate the role of GLI2 in epidermal home- inducible GLI2-HaCaT cells (henceforth referred to as ostasis and disease, we analysed the effect of GLI2 tet-GLI2 HaCaT), either treated with tetracycline for expression on the molecular phenotype of human 96 h or untreated, was hybridized to high-density cDNA keratinocytes. Using DNA-array technology, we show arrays containing a set of 2135 sequence-verified EST that GLI2 induces the expression of key regulators of clones spotted in duplicate onto nylon membranes. cell cycle progression, while itrepresses genes associated Tetracycline-treated