ABSTRACT
Transforming growth factor beta (TGFß) is a key regulator of epithelial cell proliferation, immune function and angiogenesis. Because TGFß signaling maintains epithelial homeostasis, dysregulated TGFß signaling is common in many malignancies, including head and neck squamous cell carcinoma (HNSCC). Defective TGFß signaling in epithelial cells causes hyperproliferation, reduced apoptosis and increased genomic instability, and the compensatory increase in TGFß production by tumor epithelial cells with TGFß signaling defects further promotes tumor growth and metastases by increasing angiogenesis and inflammation in tumor stromal cells. Here, we review the mouse models that we used to study TGFß signaling in HNSCC.
Subject(s)
Carcinoma, Squamous Cell/metabolism , Head and Neck Neoplasms/metabolism , Signal Transduction/physiology , Transforming Growth Factor beta/metabolism , Animals , Disease Models, Animal , Humans , MiceABSTRACT
Glucocorticoid-dependent negative feedback of the hypothalamic-pituitary-adrenal axis is mediated in part through direct inhibition of hypothalamic CRH gene transcription. In the present study, we sought to further localize and characterize glucocorticoid receptor (GR) and AP-1 interactions at a functionally defined negative glucocorticoid response element (nGRE) of the CRH promoter. Transient transfection studies in mouse corticotroph AtT-20 cells demonstrated that internal deletion of the nGRE (-278 to -249 nucleotides) within the context of 1 kb of the intact CRH promoter resulted in decreased 8-BrcAMP stimulation and glucocorticoid-dependent repression of CRH promoter activity. The nGRE conferred transcriptional activation by both cAMP and overexpressed c-jun or c-fos AP-1 nucleoproteins as well as specific glucocorticoid-dependent repression to a heterologous promoter. A similar profile of regulation was observed for the composite GRE derived from mouse proliferin promoter. The CRH nGRE was clearly distinct from the consensus cAMP response element (CRE) at -224 nucleotides, which increased basal activity and cAMP responsiveness of a heterologous promoter but did not confer glucocorticoid-dependent repression. High-affinity binding sites for both GR and AP-1 nucleoproteins were identified at adjacent elements within the nGRE. Mutations that disrupted either GR or AP-1 binding activity were associated with loss of glucocorticoid-dependent repression. These results are consistent with a composite mechanism of glucocorticoid-dependent repression involving direct DNA binding of GR and AP-1 nucleoproteins at discrete adjacent sites within the CRH promoter.
Subject(s)
Corticotropin-Releasing Hormone/genetics , Glucocorticoids/metabolism , Response Elements/physiology , Animals , Base Sequence , Binding Sites , Cell Line , Conserved Sequence , Corticotropin-Releasing Hormone/metabolism , Cyclic AMP/metabolism , Cyclic AMP Response Element-Binding Protein/genetics , Cyclic AMP Response Element-Binding Protein/metabolism , Genes, fos , Genes, jun , Humans , Luciferases/genetics , Luciferases/metabolism , Mice , Mutation , Pituitary Gland, Anterior/cytology , Pituitary Gland, Anterior/metabolism , Promoter Regions, Genetic , Receptors, Glucocorticoid/genetics , Receptors, Glucocorticoid/metabolism , Recombinant Proteins/genetics , Recombinant Proteins/metabolism , Regulatory Sequences, Nucleic Acid , Sequence Deletion , Transcription Factor AP-1/metabolism , Transcription, GeneticABSTRACT
Corticotropin releasing hormone (CRH) plays a primary role in mediating suprapituitary activation of the hypothalamic-pituitary-adrenal axis and is an important physiologic target of negative feedback regulation by glucocorticoids. We sought to define cis-acting regions of the CRH promoter responsible for cAMP-dependent activation and glucocorticoid-dependent repression of CRH promoter activity. In transiently transfected AtT-20 cells, cAMP-dependent transcriptional activation was mediated largely through a classical, consensus, cAMP-response element (CRE) at - 224 bp. Dexamethasone (DEX) produced a specific 2-3-fold repression of cAMP-stimulated, but not basal, CRH promoter activity. Using a series of 5' nested deletions, dexamethasone-dependent repression of cAMP-stimulated CRH promoter activity was localized to promoter sequences between -278 and -249 bp. Specific, high-affinity binding of glucocorticoid receptor (GR) DNA-binding domain to this promoter region was observed using an eletrophoretic mobility shift assay (EMSA). We conclude that (i) cAMP dependent activation of the CRH promoter is mediated primarily by the CRE at -224 bp, (ii) glucocorticoid-dependent repression is specific for the CRH promoter, and not a generalized effect of glucocorticoid signaling or interference with the protein kinase A (PKA) signaling pathway, (iii) a highly conserved region between -278 and -249 bp is critical for glucocorticoid dependent repression, and (iv) GR is capable of interacting directly with this functionally defined negative glucocorticoid response element of the CRH promoter.
