Down-regulation and antiproliferative role of C/EBPalpha in lung cancer.
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The transcription factor, CCAAT/enhancer binding protein alpha (C/EBPalpha) is important in the terminal differentiation of granulocytes, hepatocytes, and adipocytes, and recurrent mutations of C/EBPalpha were described in acute myeloid leukemia. In the lung, C/EBPalpha is expressed in bronchial cells and type II pneumocytes. Abnormal proliferation of the latter cell type was reported in C/EBPalpha knockout mice. We determined the expression of C/EBPalpha by Northern blot analysis in 30 lung cancer cell lines and found significant down-regulation in 24 cell lines. Immunohistochemical study of primary tumor specimens showed undetectable or low expression of C/EBPalpha in 23 of 53 specimens. Its expression was more frequently down-regulated in adenocarcinoma and poorly differentiated cancer specimens than in squamous cell cancers. A higher frequency of reduced expression was found in more advanced stages. To investigate the consequences of C/EBPalpha expression in lung cancer cells, we stably transfected two cell lines that do not express the gene (Calu1 and H358) with a plasmid allowing for induction of C/EBPalpha protein expression. Induction of C/EBPalpha led to significant growth reduction attributable to proliferation arrest, morphological changes characteristic of differentiation, and apoptosis. These results suggest that C/EBPalpha is down-regulated in a large proportion of lung cancers and that it has growth-inhibitory properties in airway epithelial cells. Genetic analysis of the C/EBPalpha gene is in progress to fully evaluate its role as a novel tumor suppressor in lung cancer. (+info)
Interaction of C/EBPalpha and the glucocorticoid receptor in vivo and in nontransformed human cells.
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Belonging to the family of steroid hormones, glucocorticoids are essential for development and survival of vertebrates. The cellular response to glucocorticoids is attributed to the glucocorticoid receptor, which functions as a transcription factor. However, the majority of glucocorticoid-modulated genes lack a DNA binding site for the glucocorticoid receptor, raising the question of which mechanism mediates the responses to glucocorticoids. It has been suggested that besides direct DNA binding of the glucocorticoid receptor, interaction with members of other transcription factor families modulates the effect of the glucocorticoid receptor. However, the significance of such transcription factor interaction is not clear. In cultured human mesenchymal cells and peripheral blood leukocytes of human volunteers treated with glucocorticoids, we detected the formation of a complex between the GR and the CCAAT/enhancer binding protein alpha. In in vitro experiments, this interaction turned out to be responsible for the inhibitory action of glucocorticoids on lymphocytic and mesenchymal cell proliferation. Our results suggest that complex formation of the GR with C/EBPalpha accounts for a novel pathway of glucocorticoid action. (+info)
Protein kinase C-dependent, CCAAT/enhancer-binding protein beta-mediated expression of insulin-like growth factor I gene.
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The possible involvement of the protein kinase C (PKC) pathway in transcriptional regulation of the human insulin-like growth factor-I (IGF-I) gene has been suggested. In this study, we sought to determine whether a PKC-dependent pathway is implicated in the transcriptional control, and if it is, how this occurs. Treatment with 12-O-tetradecanoylphorbol 13-acetate (TPA) caused an increase in the activity of the human IGF-I gene major promoter in HepG2 cells. A CCAAT/enhancer-binding protein (C/EBP) binding site located at +22 to +30 was bound by C/EBP beta in a TPA-dependent manner and was solely responsible for the TPA responsiveness. This increase in C/EBP beta activity occurs through transcriptional and posttranslational regulation, and the latter is mediated by activation of p90 ribosomal S6 kinase (RSK): co-expression of dominant negative RSK abolished the TPA-responsive and C/EBP beta-dependent transactivation. Also, TPA-responsive activation of GAL4-C/EBP beta chimera required the Ser residue known as the RSK target. In SK-N-MC cells, which display constitutive, high expression of IGF-I on use of the major promoter, a large amount of C/EBP beta binding was observed with the C/EBP site in the basal state. Treatment with PKC inhibitors substantially reduced the promoter activity and mRNA amounts of IGF-I, with the binding of C/EBP beta to the C/EBP site also being reduced. When the C/EBP site was disrupted, the basal promoter activity was reduced, but the reduction by the PKC inhibitor was no longer observed. These observations suggest that the increase of C/EBP beta binding to the C/EBP site, which is in part mediated via activation of RSK, can primarily explain the TPA responsiveness of the IGF-I gene promoter. The intrinsic PKC activity in SK-N-MC cells should play a major role in the constitutive, high expression of IGF-I and may therefore contribute in part to the maintenance of the tumor phenotype of the cells. (+info)
Mutations in the gene encoding the transcription factor CCAAT/enhancer binding protein alpha in myelodysplastic syndromes and acute myeloid leukemias.
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The CCAAT/enhancer binding protein alpha (C/EBPalpha) protein is essential for proper lung and liver function and granulocytic and adipose tissue differentation. It was hypothesized that abnormalties in C/EBPalpha function contribute to the development of malignancies in a variety of tissues. To test this, genomic DNA from 408 patient samples and 5 cell lines representing 11 different cancers was screened for mutations in the C/EBPalpha gene. Two silent polymorphisms termed P1 and P2 were present at frequencies of 13.5% and 2.2%, respectively. Of the 12 mutations detected in 10 patients, silent changes were identified in one nonsmall cell lung cancer, one prostate cancer, and one acute myelogenous leukemia (AML) subtype M4. The 9 remaining mutations were detected in 1 of 92 (1.1%) myelodysplastic syndrome (MDS) samples and 6 of 78 (7.7%) AML (AML-M2 and AML-M4) samples. Some mutations truncated the predicted protein with loss of the DNA-binding (basic region) and dimerization (leucine zipper [ZIP]) domains by either deletions or nonsense codons. Also, inframe deletions or insertions in the fork region located between the leucine zipper and basic region, or within the leucine zipper, disrupted the alpha-helical phase of the bZIP domain. The inframe deletion and insertion mutations abrogated the transcriptional activation function of C/EBPalpha on the granulocyte colony-stimulating factor receptor promoter. These mutants localized properly to the nucleus, but were unable to bind to the C/EBP site in the promoter and did not possess dominant-negative activity. The mutations in the MDS patient and one AML-M2 patient were biallelic, indicating a loss of C/EBPalpha function. These results suggest that mutation of C/EBPalpha is involved in specific subtypes of AML and in MDS, but may occur rarely in other types of leukemias or nonhematologic malignancies. (+info)
Contrasting expression patterns of CCAAT/enhancer-binding protein transcription factors in the hair follicle and at different stages of the hair growth cycle.
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Hair follicles undergo repeated cycles of growth and regression, throughout the entire life of the organism. These dynamic changes require closely co-ordinated regulation of gene expression. The CCAAT/enhancer-binding proteins are a family of basic region/leucine zipper transcription factors that regulate gene transcription in various tissues. They have been implicated in epidermal differentiation and may therefore play an important role in the hair follicle. We have investigated the localization of four members of this family--CCAAT/enhancer-binding protein-alpha, -beta, and -delta, and Gadd153--in both human and murine hair follicles by immunohistochemistry. Furthermore, we examined CCAAT/enhancer-binding protein-alpha, -beta, and -delta immunoreactivity at different stages of the depilation-induced murine hair growth cycle. Distinct immunoreactivity patterns for CCAAT/enhancer-binding protein-alpha, -beta, and -delta, and Gadd153 were observed in the outer root sheath, sebaceous gland, dermal papilla, and connective tissue sheath of human anagen hair follicles. In murine follicles, CCAAT/enhancer-binding protein-alpha was expressed in the outer root sheath, sebaceous gland, and dermal papilla, whereas CCAAT/enhancer-binding protein-beta expression was confined to the matrix, sebaceous gland, and inner and outer root sheaths. Both CCAAT/enhancer-binding protein-alpha and -beta were upregulated during anagen, then downregulated in catagen follicles. In contrast, CCAAT/enhancer-binding protein-delta showed no hair cycle-dependent variation in immunoreactivity. These data suggests that the expression of CCAAT/enhancer-binding protein-alpha and -beta may, in turn, play a part in regulating hair cycle-dependent gene expression. Moreover, as CCAAT/enhancer-binding protein-alpha, -beta, and -delta are crucial in the regulation of adipocyte differentiation and lipid metabolism, their expression in sebocytes suggests they may also play a similar role in differentiation and lipid metabolism of the sebaceous gland. (+info)
C/EBPalpha triggers proteasome-dependent degradation of cdk4 during growth arrest.
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CCAAT/enhancer binding protein alpha (C/EBPalpha) causes growth arrest via direct interaction with the cyclin-dependent kinases cdk2 and cdk4. In this paper, we present evidence showing that C/EBPalpha enhances a proteasome-dependent degradation of cdk4 during growth arrest in liver of newborn mice and in cultured cells. Overexpression of C/EBPalpha in several biological systems leads to a reduction of cdk4 protein levels, but not mRNA levels. Experiments with several tissue culture models reveal that C/EBPalpha enhances the formation of cdk4-ubiquitin conjugates and induces degradation of cdk4 through a proteasome-dependent pathway. As a result, the half-life of cdk4 is shorter and protein levels of cdk4 are reduced in cells expressing C/EBPalpha. Gel filtration analysis of cdk4 complexes shows that a chaperone complex cdk4-cdc37-Hsp90, which protects cdk4 from degradation, is abundant in proliferating livers that lack C/EBPalpha, but this complex is weak or undetectable in livers expressing C/EBPalpha. Our studies show that C/EBPalpha disrupts the cdk4-cdc37-Hsp90 complex via direct interaction with cdk4 and reduces protein levels of cdk4 by increasing proteasome-dependent degradation of cdk4. (+info)
Expression of C/EBPbeta from the C/ebpalpha gene locus is sufficient for normal hematopoiesis in vivo.
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CCAAT/enhancer-binding proteins (C/EBPs) are critical transcriptional regulators of differentiation of hematopoietic cells. Previous studies have shown that targeted disruption of the C/ebpalpha gene results in a lack of granulocytic differentiation with an arrest at the stage of immature myeloblasts. By using a gene replacement strategy in which C/EBPbeta was expressed from the C/ebpalpha gene locus of C/EBPalpha-null mice, we have evaluated the ability of C/EBPbeta to function for C/EBPalpha in directing differentiation along the granulocytic pathway. We show that the morphology and the differential cell counts of the bone marrow and peripheral blood cells from C/EBPbeta knockin mice are indistinguishable from those of their wild-type littermates, indicating that hematopoiesis occurs normally in these animals. Additionally, we analyzed expression of 21 myeloid-specific genes, including markers for distinct stages of granulocytic differentiation, and found no significant differences in their levels of expression in the bone marrow of C/EBPbeta knockin and wild-type mice. These results imply that C/EBPbeta can substitute for C/EBPalpha during hematopoiesis when expressed from the C/ebpalpha gene locus. (+info)
Mechanisms regulating adipocyte expression of resistin.
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Resistin, also known as Adipocyte Secreted Factor (ADSF) and Found in Inflammatory Zone 3 (FIZZ3), is a mouse protein with potential roles in insulin resistance and adipocyte differentiation. The resistin gene is expressed almost exclusively in adipocytes. Here we show that a proximal 264-base pair fragment of the mouse resistin promoter is sufficient for expression in adipocytes. Ectopic expression of the adipogenic transcription factor CCAAT/enhancer-binding protein (C/EBPalpha) was sufficient for expression in non-adipogenic cells. C/EBPalpha binds specifically to a site that is essential for expression of the resistin promoter. Chromatin immunoprecipitation studies of the endogenous gene demonstrated adipocyte-specific association of C/EBPalpha with the proximal resistin promoter in adipocytes but not preadipocytes. C/EBPalpha binding was associated with the recruitment of coactivators p300 and CREB-binding protein and a dramatic increase in histone acetylation in the vicinity of the resistin promoter. The antidiabetic thiazolidinedione (TZD) drug rosiglitazone reduced resistin expression with an ED(50) similar to its K(d) for binding to peroxisome proliferator activated receptor gamma (PPARgamma). Other TZD- and non-TZD PPARgamma ligands also down-regulated resistin expression. However, no functional PPARgamma binding site was found within 6.2 kb of the transcriptional start site, suggesting that if PPARgamma is involved, it is either acting at a long distance from the start site, in an intron, or indirectly. Nevertheless, rosiglitazone treatment selectively decreased histone acetylation at the resistin promoter without a change in occupation by C/EBPalpha, CREB-binding protein, or p300. Thus, adipocyte specificity of resistin gene expression is because of C/EBPalpha binding, leading to the recruitment of transcriptional coactivators and histone acetylation that is characteristic of an active chromatin environment. TZD reduces resistin gene expression at least in part by reducing histone acetylation associated with the binding of C/EBPalpha in mature adipocytes. (+info)