RESUMO
Although deregulated Hedgehog signalling and elevated Gli transcription factor expression are known to promote the development of basal cell carcinoma (BCC), little is known about molecular mechanisms driving the development of specific growth pattern subtypes. Using gene array analysis, we have previously observed that over-expression of GLI1 in human keratinocytes promotes increased expression of the neuronal differentiation markers ARC and ULK1. We asked whether neuronal differentiation is a characteristic of BCC and whether there is any correlation with BCC subtype. Using RT-PCR and immunohistochemistry, we confirmed that the neuronal markers ARC, beta-tubulin III, GAP-43 and Neurofilament are expressed in human BCC but not in normal epidermis. Moreover, we found that expression of these neuronal differentiation markers showed strong correlation to BCC subtype, with more aggressive infiltrative and morphoeic BCC showing low levels or lack of expression compared to nodular, superficial and micronodular subtypes. Primary human keratinocytes retrovirally expressing GLI1(-) and GLI2(-) showed elevated levels of beta-tubulin III and ARC but not Neurofilament or GAP-43, suggesting that beta-tubulin III and Arc may be early targets of aberrant Gli expression in BCC, whereas expression of Neurofilament and GAP-43 are either later, downstream targets or under control of alternative pathways. We propose that neuronal differentiation is a feature of BCC and that expression of these markers is in part due to aberrant Hedgehog signalling. Moreover, we suggest that correlation between loss of expression of neuronal markers in infiltrative and morphoeic BCC subtypes reflects dedifferentiation of more aggressive BCC subtypes.
Assuntos
Carcinoma Basocelular/patologia , Regulação Neoplásica da Expressão Gênica , Proteínas Hedgehog/genética , Neurônios/patologia , Análise de Variância , Biomarcadores/análise , Carcinoma Basocelular/genética , Carcinoma Basocelular/metabolismo , Estudos de Casos e Controles , Diferenciação Celular , Células Cultivadas , Proteínas do Citoesqueleto/genética , Proteína GAP-43/genética , Proteínas Hedgehog/metabolismo , Humanos , Interpretação de Imagem Assistida por Computador , Imuno-Histoquímica , Queratinócitos/metabolismo , Fatores de Transcrição Kruppel-Like/genética , Proteínas do Tecido Nervoso/genética , Proteínas de Neurofilamentos/genética , Plasticidade Neuronal , Proteínas Nucleares/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa/métodos , Transdução de Sinais/fisiologia , Fatores de Transcrição/genética , Transdução Genética , Tubulina (Proteína)/genética , Proteína GLI1 em Dedos de Zinco , Proteína Gli2 com Dedos de ZincoRESUMO
Hedgehog (HH)/GLI signaling plays a critical role in epidermal development and basal cell carcinoma. Here, we provide evidence that epidermal growth factor receptor (EGFR) signaling modulates the target gene expression profile of GLI transcription factors in epidermal cells. Using expression profiling and quantitative reverse transcriptase PCR, we identified a set of 19 genes whose transcription is synergistically induced by GLI1 and parallel EGF treatment. Promoter studies of a subset of GLI/EGF-regulated genes, including the genes encoding interleukin-1 antagonist IL1R2, Jagged 2, cyclin D1, S100A7, and S100A9, suggest convergence of EGFR and HH/GLI signaling at the level of promoters of selected direct GLI target genes. Inhibition of EGFR and MEK/ERK but not of phosphatidylinositol 3-kinase/AKT abrogated synergistic activation of GLI/EGF target genes, showing that EGFR can signal via RAF/MEK/ERK to cooperate with GLI proteins in selective target gene regulation. Coexpression of the GLI/EGF target IL1R2, EGFR, and activated ERK1/2 in human anagen hair follicles argues for a cooperative role of EGFR and HH/GLI signaling in specifying the fate of outer root sheath (ORS) cells. We also show that EGF treatment neutralizes GLI-mediated induction of epidermal stem cell marker expression and provide evidence that EGFR signaling is essential for GLI-induced cell cycle progression in epidermal cells. The results suggest that EGFR signaling modulates GLI target gene profiles which may play an important regulatory role in ORS specification, hair growth, and possibly cancer.
Assuntos
Fator de Crescimento Epidérmico/farmacologia , Regulação da Expressão Gênica/efeitos dos fármacos , Queratinócitos/efeitos dos fármacos , Transdução de Sinais/efeitos dos fármacos , Transativadores/metabolismo , Fatores de Transcrição/metabolismo , Sítios de Ligação/genética , Proliferação de Células , Células Cultivadas , Receptores ErbB/metabolismo , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Folículo Piloso/citologia , Proteínas Hedgehog , Humanos , Queratinócitos/citologia , Regiões Promotoras Genéticas/genética , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Receptores de Interleucina-1/metabolismo , Receptores Tipo II de Interleucina-1 , Células-Tronco/citologia , Fatores de Transcrição/genética , Proteína GLI1 em Dedos de ZincoRESUMO
Efficient manipulation of Hedgehog (HH)/GLI signaling activity is crucial to the analysis of molecular events underlying HH/GLI-regulated cell fate determination and tumor growth. In this article, we describe the use of retroviral expression systems as a valuable tool to activate or repress Hh-pathway activity in a broad spectrum of mammalian cells-including human cells-either by forced expression of the major Hedgehog-effectors GLI1 and GLI2 or by expression of the short-hairpin RNAs-targeting GLI mRNAs. We focus on two distinct retroviral systems that allow efficient and sustainable expression of GLI proteins in primary cells and cell lines of human origin: (i) a Moloney Murine Leukemia Virus-based and (ii) an HIV-derived lentivirus expression system, which allows transduction of both dividing and quiescent cells.
Assuntos
Proteínas Hedgehog/metabolismo , Proteínas Oncogênicas/metabolismo , Retroviridae/genética , Transdução de Sinais , Transativadores/metabolismo , Transdução Genética/métodos , Linhagem Celular , Proliferação de Células , Clonagem Molecular , Regulação da Expressão Gênica , Vetores Genéticos , Proteínas Hedgehog/genética , Humanos , Proteínas Oncogênicas/genética , Transativadores/genética , Transfecção , Transgenes , Montagem de Vírus , Proteína GLI1 em Dedos de ZincoRESUMO
The current concept of tumourigenesis holds that cancer results from the progressive acquisition of mutations that endow affected cells with selective growth advantages by activating multiple processes including intrinsic mitogenic and pro-survival pathways. Constitutive activation of the Hedgehog (HH)/GLI signalling cascade has recently been implicated in the growth of a number of human malignancies ranging from semi-malignant tumours of the skin to highly aggressive cancers of the brain, lung, pancreas and prostate. This review focuses on the role of the GLI zinc finger transcription factors, which mediate Hedgehog signalling at the distal end of the pathway. We summarise recent data on the mechanisms by which latent GLI proteins are activated in response to stimulation of Hedgehog signalling. Based on the identification of a growing number of direct GLI target genes, we propose that HH-driven tumourigenesis relies on multiple cellular processes such as promotion of G1/S phase progression, enhancement of cell survival by providing anti-apoptotic cues, increase in metastatic potential of Hedgehog responsive cells, and activation of potential tumour stem cells. In view of the critical role of GLI genes in Hedgehog-associated cancers, strategies that aim at interfering with GLI function are likely to represent efficient approaches in future targeted cancer therapy.
Assuntos
Proteínas de Transporte/genética , Glicoproteínas de Membrana/genética , Mutação/genética , Neoplasias/genética , Transdução de Sinais/genética , Fatores de Transcrição/genética , Humanos , Oncogenes/genética , Proteína GLI1 em Dedos de ZincoRESUMO
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.
Assuntos
Proteínas de Ligação a DNA/fisiologia , Proteínas Nucleares/fisiologia , Regiões Promotoras Genéticas , Proteínas Proto-Oncogênicas c-bcl-2/genética , Transdução de Sinais/fisiologia , Transativadores/fisiologia , Sequência de Bases , Sítios de Ligação , Células Cultivadas , Proteínas Hedgehog , Humanos , Fatores de Transcrição Kruppel-Like , Dados de Sequência Molecular , Proteínas Oncogênicas/fisiologia , Plasmócitos/metabolismo , Plasmocitoma/metabolismo , Fatores de Transcrição/fisiologia , Proteína GLI1 em Dedos de Zinco , Proteína Gli2 com Dedos de ZincoRESUMO
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 GLI2alpha and GLI2beta. 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.
Assuntos
Carcinoma Basocelular/metabolismo , Retroalimentação Fisiológica/fisiologia , Proteínas Oncogênicas/fisiologia , Neoplasias Cutâneas/metabolismo , Fatores de Transcrição/fisiologia , Fosfatase Alcalina/metabolismo , Western Blotting , Bromodesoxiuridina , Carcinoma Basocelular/genética , Primers do DNA/química , DNA de Neoplasias/biossíntese , Ativação Enzimática , Citometria de Fluxo , Proteínas de Fluorescência Verde , Proteínas Hedgehog , Humanos , Queratinócitos/metabolismo , Fatores de Transcrição Kruppel-Like , Luciferases/metabolismo , Proteínas Luminescentes/metabolismo , Proteínas de Membrana/metabolismo , Proteínas Nucleares , Receptores Patched , Reação em Cadeia da Polimerase , RNA/metabolismo , Receptores de Superfície Celular , Retroviridae/genética , Transdução de Sinais , Neoplasias Cutâneas/genética , Transativadores/fisiologia , Transcrição Gênica , Transfecção , Proteína GLI1 em Dedos de Zinco , Proteína Gli2 com Dedos de Zinco , Dedos de ZincoRESUMO
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-S phase 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.
Assuntos
Ciclo Celular/fisiologia , Diferenciação Celular/fisiologia , Inibição de Contato/fisiologia , Queratinócitos/citologia , Fatores de Transcrição/genética , Sequência de Bases , Linhagem Celular , Transformação Celular Neoplásica , Primers do DNA , Células Epidérmicas , Epiderme/efeitos dos fármacos , Epiderme/fisiologia , Humanos , Queratinócitos/fisiologia , Fatores de Transcrição Kruppel-Like , Proteínas Nucleares , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Fatores de Transcrição/fisiologia , Proteína Gli2 com Dedos de Zinco , Dedos de Zinco/genética , Dedos de Zinco/fisiologiaRESUMO
Sonic hedgehog (Shh) binds to its receptor patched (PTCH), leading to the activation and repression of target genes via the GLI family of zinc-finger transcription factors. Deregulation of the Shh pathway is associated with basal cell carcinoma (BCC) due to upregulation of GLI1 and GLI2. We recently demonstrated a positive feedback loop between GLI1 and GLI2, which revealed that GLI1 may be a direct target of GLI2. Using band shift and luciferase reporter assays, we now show that GLI2 binds the GLI-binding consensus sequence in the GLI1 promoter. These data suggest that GLI2 directly activates GLI1 and that retrovirally expressed GLI2 induces expression of endogenous GLI1 in human primary keratinocytes. Finally, using in situ hybridization, we show that GLI2 is expressed in the interfollicular epidermis and the outer root sheath of hair follicles in normal skin as well as in BCC tumor islands. These results suggest an important role for GLI2 in regulating epidermal proliferation and skin tumorigenesis.
Assuntos
Carcinoma Basocelular/fisiopatologia , Epiderme/fisiologia , Proteínas Oncogênicas/genética , Neoplasias Cutâneas/fisiopatologia , Fatores de Transcrição/genética , Sequência de Bases , Carcinoma Basocelular/patologia , Linhagem Celular , Células Epidérmicas , Regulação Neoplásica da Expressão Gênica , Folículo Piloso/fisiologia , Humanos , Queratinócitos/citologia , Queratinócitos/fisiologia , Fatores de Transcrição Kruppel-Like , Dados de Sequência Molecular , Proteínas Nucleares , Proteínas Oncogênicas/metabolismo , Regiões Promotoras Genéticas/fisiologia , Neoplasias Cutâneas/patologia , Transativadores , Fatores de Transcrição/metabolismo , Transfecção , Proteína GLI1 em Dedos de Zinco , Proteína Gli2 com Dedos de ZincoRESUMO
Sonic hedgehog (Hh) signaling plays a key role in epidermal development and skin cancer. Mutational inactivation of the tumor suppressor gene patched (PTCH) leads to constitutive activation of the Hh signaling pathway, resulting in activation of target gene transcription by the zinc finger transcription factors GLI1 and GLI2. Recent experiments in mice point to GLI2 as the key mediator of Hh signaling in skin. We have concentrated on the identification of candidate mediators of GLI2 function in the human epidermis. We show here that the forkhead/winged-helix domain transcription factor FOXE1 is likely to be a direct GLI2 target gene. The kinetics of FOXE1 induction are similar to the known direct target PTCH, and a 2.5 kb upstream fragment containing five GLI-binding sites activates transcription in a reporter assay. We show by in situ hybridization that FOXE1 is expressed in the outer root sheath of the hair follicle, where murine Gli2 is also expressed. FOXE1 expression is also found in basal keratinocytes of the human epidermis and basal cell carcinoma (BCC). These data point to a putative role of FOXE1 in mediating Hh signaling in the human epidermis downstream of GLI2.
Assuntos
Carcinoma Basocelular/fisiopatologia , Proteínas de Ligação a DNA/genética , Epiderme/fisiopatologia , Proteínas Repressoras/genética , Neoplasias Cutâneas/fisiopatologia , Fatores de Transcrição/metabolismo , Proteínas de Ligação a DNA/metabolismo , Células Epidérmicas , Fatores de Transcrição Forkhead , Regulação Neoplásica da Expressão Gênica , Proteínas Hedgehog , Humanos , Hibridização In Situ , Queratinócitos/fisiologia , Fatores de Transcrição Kruppel-Like , Proteínas Nucleares , Regiões Promotoras Genéticas , RNA Mensageiro/análise , Proteínas Repressoras/metabolismo , Transativadores/metabolismo , Transcrição Gênica/fisiologia , Proteína Gli2 com Dedos de ZincoRESUMO
We present a highly sensitive bioanalytical microarray assay that enables the analysis of small genomic sample material. By combining an optimized cDNA purification step with single molecule cDNA detection on the microarray, the platform has improved sensitivity compared to conventional systems, allowing amplification-free determination of expression profiles with as little as 600ng total RNA. Total RNA from cells was reverse transcribed into fluorescently labeled cDNA and purified employing a precipitation method that minimizes loss of cDNA material. The microarray was scanned on a fluorescence chip-reader with single molecule sensitivity. Using the newly developed platform we were able to analyze the RNA expression profile of a subpopulation of rare multiple myeloma CD138 negative progenitor (MM CD138(neg)) cells. The high-sensitivity microarray approach led to the identification of a set of 20 genes differentially expressed in MM CD138(neg) cells. Our work demonstrates the applicability of a straight-forward single-molecule DNA array technology to current topics of molecular and cellular cancer research, which are otherwise difficult to address due to the limited amount of sample material.
Assuntos
Perfilação da Expressão Gênica/métodos , Microscopia de Fluorescência/métodos , Mieloma Múltiplo/patologia , Células-Tronco Neoplásicas/metabolismo , Análise de Sequência com Séries de Oligonucleotídeos/métodos , Células-Tronco/metabolismo , Sindecana-1/metabolismo , Linhagem Celular Tumoral , Humanos , Mieloma Múltiplo/metabolismo , Células-Tronco Neoplásicas/química , Células-Tronco Neoplásicas/citologia , RNA Mensageiro/análise , RNA Mensageiro/genética , Células-Tronco/química , Células-Tronco/citologia , Sindecana-1/genéticaRESUMO
Persistent activation of the Hedgehog (HH)/GLI signaling pathway has been implicated in the development of a number of human cancers. The GLI zinc finger transcription factors act at the end of the HH signaling cascade to control gene expression, and recent studies have shown that the activity of GLI proteins can be additionally modified by integration of distinct signals, such as the MEK/extracellular signal-regulated kinase (ERK) and phosphinositide-3 kinase (PI3K)/AKT pathway. However, little is known about the identity of the upstream activators of these HH/GLI interacting signaling pathways in cancer. Here, we provide evidence that integration of the HH/GLI and epidermal growth factor receptor (EGFR) pathway synergistically induces oncogenic transformation, which depends on EGFR-mediated activation of the RAS/RAF/MEK/ERK but not of the PI3K/AKT pathway. EGFR/MEK/ERK signaling induces JUN/activator protein 1 activation, which is essential for oncogenic transformation, in combination with the GLI activator forms GLI1 and GLI2. Furthermore, pharmacologic inhibition of EGFR and HH/GLI efficiently reduces growth of basal cell carcinoma (BCC) cell lines derived from mice with activated HH/GLI signaling. The results identify the synergistic integration of GLI activator function and EGFR signaling as a critical step in oncogenic transformation and provide a molecular basis for therapeutic opportunities relying on combined inhibition of the HH/GLI and EGFR/MEK/ERK/JUN pathway in BCC.
Assuntos
Transformação Celular Neoplásica/patologia , Receptores ErbB/fisiologia , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , MAP Quinase Quinase Quinases/metabolismo , Neoplasias/patologia , Proteínas Proto-Oncogênicas c-jun/metabolismo , Fatores de Transcrição/fisiologia , Substituição de Aminoácidos , Animais , Divisão Celular/genética , Receptores ErbB/deficiência , Receptores ErbB/genética , Fibroblastos/fisiologia , Proteínas Hedgehog/fisiologia , Humanos , Queratinócitos/citologia , Fatores de Transcrição Kruppel-Like/genética , Camundongos , Camundongos Knockout , Neoplasias/enzimologia , Neoplasias/fisiopatologia , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transdução de Sinais , Fatores de Transcrição/genética , Proteína GLI1 em Dedos de ZincoRESUMO
We developed a microarray platform for PCR amplification-independent expression profiling of minute samples. A novel scanning system combined with specialized biochips enables detection down to individual fluorescent oligonucleotide molecules specifically hybridized to their complementary sequence over the entire biochip surface of cm2 size. A detection limit of 1.3 fM target oligonucleotide concentration--corresponding to only 39,000 molecules in the sample solution--and a dynamic range of 4.7 orders of magnitude have been achieved. The applicability of the system to PCR amplification-independent gene-expression profiling of minute samples was demonstrated by complex hybridization of cDNA derived from the equivalent of only 10(4) cells, which matches results obtained in ensemble studies on large samples. By counting each hybridized molecule on the microarray, the method is insusceptible to gene-specific variations of the labeling, thereby representing a principle advance to conventional ensemble-based microarray analysis.
Assuntos
Perfilação da Expressão Gênica , Linhagem Celular , Sondas de DNA , DNA Complementar , Hibridização de Ácido Nucleico , Análise de Sequência com Séries de Oligonucleotídeos , Reação em Cadeia da PolimeraseRESUMO
The GLI transcription factors mediate the hedgehog signal in development and carcinogenesis. Basal cell carcinoma can be caused by overexpression of either GLI1 or GLI2. Though GLI1 and GLI2 have identical or very similar DNA binding specificities, some of their activities are overlapping, some are clearly distinct. We analyzed target gene specificities of GLI1 and constitutively active GLI2 (GLI2DeltaN) by global expression profiling in an inducible, well-characterized HaCaT keratinocyte expression system. Four hundred fifty-six genes up- or downregulated at least twofold were identified. GLI target gene profiles correlated well with the biological activities of these transcription factors in hair follicles and basal cell carcinoma. Upregulation of largely overlapping sets of target genes was effected by both factors, repression occurred predominantly in response to GLI2. Also, significant quantitative differences in response to GLI1 and GLI2DeltaN were found for a small number of activated genes. Since we have not detected a putative processed GLI2 repressor, these results point to specific but indirect target gene repression by GLI2DeltaN via preferential activation of one or more negative regulators.
Assuntos
Regulação da Expressão Gênica , Fatores de Transcrição Kruppel-Like/genética , Proteínas Nucleares/genética , Fatores de Transcrição/genética , Animais , Células Cultivadas , Perfilação da Expressão Gênica , Proteínas Hedgehog/genética , Queratinócitos , Análise de Sequência com Séries de Oligonucleotídeos , Reação em Cadeia da Polimerase/métodos , Isoformas de Proteínas/genética , Proteínas Recombinantes/genética , Proteínas Repressoras/genética , Transdução de Sinais/genética , Fatores de Transcrição/metabolismo , Proteína GLI1 em Dedos de Zinco , Proteína Gli2 com Dedos de ZincoRESUMO
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-gamma-activated sequence motifs of the sequence TTC(N)(4)GAA, which is known for binding STAT6. The three motifs are situated close to each other approximately 600 bp upstream of the transcriptional initiation site. When mutations were inserted into all three IFN-gamma-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.