Anna-Maria Frischauf

Activation of the BCL2 Promoter in Response to Hedgehog/GLI Signal Transduction Is Predominantly Mediated by GLI2

Aberrant activation of the Hedgehog (HH)/GLI signaling pathway has been implicated in the development of basal cell carcinoma (BCC). The zinc finger transcription factors GLI1 and GLI2 are considered mediators of the HH signal in epidermal cells, although their tumorigenic nature and their relative contribution to tumorigenesis are only poorly understood. To shed light on the respective role of these transcription factors in epidermal neoplasia, we screened for genes preferentially regulated either by GLI1 or GLI2 in human epidermal cells. We show here that expression of the key antiapoptotic factor BCL2 is predominantly activated by GLI2 compared with GLI1. Detailed promoter analysis and gel shift assays identified three GLI binding sites in the human BCL2 cis-regulatory region. We found that one of these binding sites is critical for conferring GLI2-specific activation of the human BCL2 promoter and that the selective induction of BCL2 expression depends on the zinc finger DNA binding domain of GLI2. In vivo, GLI2 and BCL2 were coexpressed in the outer root sheath of hair follicles and BCC and in plasma cells that infiltrated BCC tumor islands. On the basis of the latter observation, we analyzed plasma cell-derived tumors and found strong expression of GLI2 and BCL2 in neoplastic cells of plasmacytoma patients, implicating HH/GLI signaling in the development of plasma cell-derived malignancies. The results reveal a central role for GLI2 in activating the prosurvival factor BCL2, which may represent an important mechanism in the development or maintenance of cancers associated with inappropriate HH signaling.

Human GLI2 and GLI1 are part of a positive feedback mechanism in Basal Cell Carcinoma

Transgenic mouse models have provided evidence that activation of the zinc-finger transcription factor GLI1 by Hedgehog (Hh)-signalling is a key step in the initiation of the tumorigenic programme leading to Basal Cell Carcinoma (BCC). However, the downstream events underlying Hh/GLI-induced BCC development are still obscure. Using in vitro model systems to analyse the effect of Hh/GLI-signalling in human keratinocytes, we identified a positive feedback mechanism involving the zinc finger transcription factors GLI1 and GLI2. Expression of GLI1 in human keratinocytes induced the transcriptional activator isoforms GLI2α and GLI2β. Both isoforms were also shown to be expressed at elevated levels in 21 BCCs compared to normal skin. Detailed time course experiments monitoring the transcriptional response of keratinocytes either to GLI1 or to GLI2 suggest that GLI1 is a direct target of GLI2, while activation of GLI2 by GLI1 is likely to be indirect. Furthermore, expression of either GLI2 or GLI1 led to an increase in DNA-synthesis in confluent human keratinocytes. Taken together, these results suggest an important role of the positive GLI1-GLI2 feedback loop in Hh-mediated epidermal cell proliferation.

The zinc-finger transcription factor GLI2 antagonizes contact inhibition and differentiation of human epidermal cells

In stratified epidermis, activation of the Hh/Gli signal transduction pathway has been implicated in the control of cell proliferation and tumorigenesis. The zinc-finger transcription factor Gli2 has been identified as critical mediator of the Hh signal at the distal end of the pathway, but the molecular mechanisms by which Gli2 regulates cell proliferation or induces epidermal malignancies such as basal cell carcinoma are still unclear. Here, we provide evidence for a role of human GLI2 in antagonizing contact inhibition and epidermal differentiation. We show by gene expression profiling that activation of the GLI2 oncogene in human keratinocytes activates the transcription of a number of genes involved in cell cycle progression such as E2F1, CCND1, CDC2 and CDC45L, while it represses genes associated with epidermal differentiation. Analysis of the proliferative effect of GLI2 revealed that GLI2 is able to induce G1–Su2009phase progression in contact-inhibited keratinocytes. Detailed time-course experiments identified E2F1 as early transcriptional target of GLI2. Further, we show that GLI2 expression in human keratinocytes results in a marked downregulation of epidermal differentiation markers. The data suggest a role for GLI2 in Hh-induced epidermal neoplasia by opposing epithelial cell cycle arrest signals and epidermal differentiation.

Cell-type and Donor-specific Transcriptional Responses to Interferon-α USE OF CUSTOMIZED GENE ARRAYS

A sensitive, specific, reproducible, robust, and cost-effective customized cDNA array system based on established nylon membrane technology has been developed for convenient multisample expression profiling for several hundred genes of choice. The genes represented are easily adjusted (depending on the availability of corresponding cDNAs) and the method is accordingly readily applicable to a wide variety of systems. Here we have focused on the expression profiles for interferon-α2a, the most widely used interferon for the treatment of viral hepatitis and malignancies, in primary cells (peripheral blood mononuclear cells, T cells, and dendritic cells) and cell lines (Kit255, HT1080, HepG2, and HuH7). Of 150 genes studied, only six were consistently induced in all cell types and donors, whereas 74 genes were induced in at least one cell type. IRF-7 was identified as the only gene exclusively induced in the hematopoietic cells. No gene was exclusively induced in the nonhematopoietic cell lines. In T cells 12, and in dendritic cells, 25 genes were induced in all donors whereas 45 and 42 genes, respectively, were induced in at least one donor. The data suggest that signaling through IFN-α2 can be substantially modulated to yield significant cell-type and donor-specific qualitative and quantitative differences in gene expression in response to this cytokine under highly standardized conditions.

IL-4 and IL-13 Induce SOCS-1 Gene Expression in A549 Cells by Three Functional STAT6-Binding Motifs Located Upstream of the Transcription Initiation Site

Proteins of the suppressors of cytokine signaling (SOCS) family have important functions as negative regulators of cytokine signaling. We show here that SOCS-1 expression can be induced in the human epithelial lung cell line A549 by IL-4 and IL-13. Analysis of reporter gene constructs under control of the SOCS-1 promoter provides evidence that IL-4- and IL-13-induced up-regulation is dependent on three IFN-γ-activated sequence motifs of the sequence TTC(N)4GAA, which is known for binding STAT6. The three motifs are situated close to each other ∼600 bp upstream of the transcriptional initiation site. When mutations were inserted into all three IFN-γ-activated sequence motifs at the same time, IL-4-IL-13-induced luciferase activity was abrogated. With single and double mutants, promoter activity was diminished in comparison with the wild-type promoter. STAT6 is therefore required for IL-4-IL-13-dependent SOCS-1 expression in A549 cells, and the three identified binding motifs cooperate to induce maximal transcription. EMSAs conducted with nuclear extracts of IL-4- and IL-13-stimulated A549 cells showed that STAT6 was able to bind to each of the three binding motifs. Finally, cotransfection of a SOCS-1 expression vector inhibited activation of SOCS-1 promoter luciferase constructs. Thus, SOCS-1 is able to autoregulate its expression via a negative feedback loop.

Cooperation between GLI and JUN enhances transcription of JUN and selected GLI target genes

Sustained Hedgehog (HH) signaling is implicated in basal cell carcinoma of the skin and other types of cancer. Here we show that GLI1 and GLI2, the main transcriptional activators of the HH pathway, directly regulate expression of the activator protein 1 (AP-1) family member JUN, a transcription factor controlling keratinocyte proliferation and skin homeostasis. Activation of the JUN promoter by GLI is dependent on a GLI-binding site and the AP-1 sites known to be involved in self-activation of JUN. Transcription of JUN is greatly enhanced in the presence of GLI and requires activated JUN protein. GLI2act is a more potent activator than GLI1 in these experiments and physical interaction with phosphorylated JUN was only detected for GLI2act. The synergistic effect of GLI and JUN extends to the activation of further GLI target genes as shown by shRNA-mediated knockdown of JUN in human keratinocytes. Some of these cooperatively activated genes are involved in cell-cycle progression, which is consistent with a significant reduction of the proliferative potential of GLI in the absence of JUN. These results suggest a novel connection between HH/GLI pathway activity and JUN, which may contribute to the oncogenic activity of HH/GLI signaling in skin.

Non-consensus GLI binding sites in Hedgehog target gene regulation

BackgroundThe GLI transcription factors, mediators of the hedgehog signal bind with high affinity to the consensus sequence GACCACCCA. The affinity of variant single substitutions in GLI binding sites has been measured systematically, but the affinities of the variant binding sites appears low compared to the frequency of occurrence of variant sites in known GLI target gene promoters.ResultsWe quantified transcriptional activation by GLI using PTCH1 promoter based luciferase reporters containing all single substitutions of the GLI consensus binding site. As expected variants with very low affinity did not activate the reporter. Many lower affinity binding sequences are, however, functional in the presence of moderate GLI concentration. Using two natural non-consensus GLI site promoters we showed that substitution of the variant sequences by consensus leads to comparable activity.ConclusionsVariant GLI binding sites with relatively low affinity can within natural promoters lead to strong transcriptional activation. This may facilitate the identification of additional direct GLI target genes.

PA-FABP, a novel marker of human epidermal transit amplifying cells revealed by 2D protein gel electrophoresis and cDNA array hybridisation

Human epidermal stem cells express higher levels of β1 integrins than their more differentiated daughters, transit amplifying cells. In a search for additional stem and transit cell markers we used proteomics and differential cDNA hybridisation to compare keratinocytes fractionated on the basis of β1 integrin expression. There were remarkably few differences between the two populations and none of the RNAs differed in abundance by more than 2‐fold. Nevertheless, proteomics revealed upregulated expression of epidermal fatty acid binding protein (PA‐FABP, also known as E‐FABP), Annexin II and two keratin related proteins in the transit population. An unknown high molecular mass protein was upregulated in the stem cell population. The upregulation of PA‐FABP was confirmed by Northern blotting and conventional and whole mount labelling of human epidermis. We conclude that PA‐FABP is a novel marker of epidermal transit amplifying cells.

Imiquimod directly inhibits Hedgehog signalling by stimulating adenosine receptor/protein kinase A-mediated GLI phosphorylation

Imiquimod (IMQ), a nucleoside analogue of the imidazoquinoline family, is used in the topical treatment of basal cell carcinoma (BCC) and other skin diseases. It is reported to be a TLR7 and TLR8 agonist and, as such, initiates a Th1 immune response by activating sentinel cells in the vicinity of the tumour. BCC is a hedgehog (HH)-driven malignancy with oncogenic glioma-associated oncogene (GLI) signalling activated in a ligand-independent manner. Here we show that IMQ can also directly repress HH signalling by negatively modulating GLI activity in BCC and medulloblastoma cells. Further, we provide evidence that the repressive effect of IMQ on HH signalling is not dependent on TLR/MYD88 signalling. Our results suggest a mechanism for IMQ engaging adenosine receptors (ADORAs) to control GLI signalling. Pharmacological activation of ADORA with either an ADORA agonist or IMQ resulted in a protein kinase A (PKA)-mediated GLI phosphorylation and reduction in GLI activator levels. The activation of PKA and HH pathway target gene downregulation in response to IMQ were abrogated by ADORA inhibition. Furthermore, activated Smoothened signalling, which positively signals to GLI transcription factors, could be effectively counteracted by IMQ. These results reveal a previously unknown mode of action of IMQ in the treatment of BCC and also suggest a role for ADORAs in the regulation of oncogenic HH signalling.

Expression profiling of vitamin D treated primary human keratinocytes

Vitamin D has attracted much attention by its ability to stop cell proliferation and induce differentiation, which became of particular interest for the treatment of cancer and psoriasis. We performed an expression profile of 12 hours and 24 hours 1α,25‐dihydroxyvitamin D3 (1α,25(OH)2D3) treated primary human keratinocytes, to determine the changes in gene expression induced by the steroid in order to improve our understanding of the biological activity of 1α,25(OH)2D3. This we expect to be useful for establishing a test system for vitamin D analogs or might open new therapeutic targets or uses for the hormone. For the filter array experiments a non‐redundant set of 2135 sequence verified EST clones was used. The normalized raw data of 2 filters per time point were combined and subjected to SAM analysis to further increase the statistical significance. 86 positive and 50 negative genes were identified after 12 h. The numbers went down to 43 positive and 1 negative gene after 24 h of treatment. Fifteen genes are up‐regulated over a longer period of time (12 h and 24 h). Results were verified by real‐time PCR and/or Northern blots. Targets identified are involved in intracellular signaling, transcription, cell cycle, metabolism, cellular growth, constitution of the extracellular matrix or the cytoskeleton and apoptosis, immune responses, and DNA repair, respectively. Expression profiles showed an initial stop of proliferation and induction of differentiation, and resumed proliferation after prolonged incubation, most likely due to degradation of the hormone. J. Cell. Biochem. 100: 574–592, 2007.