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CCAAT/enhancer-binding protein delta (C/EBPdelta) maintains amelogenin expression in the absence of C/EBPalpha in vivo.

ABSTRACT: C/EBPalpha is implicated to regulate mouse amelogenin gene expression during tooth enamel formation in vitro. Because enamel formation occurs during postnatal development and C/EBPalpha-deficient mice die at birth, we used the Cre/loxP recombination system to characterize amelogenin expression in C/EBPalpha conditional knock-out mice. Mice carrying the Cre transgene under the control of the human keratin-14 promoter show robust Cre expression in the ameloblast cell lineage. Mating between mice bearing the floxed C/EBPalpha allele with keratin-14-Cre mice generate C/EBPalpha conditional knock-out mice. Real-time PCR analysis shows that removal of one C/EBPalpha allele from the molar enamel epithelial organ of 3-day postnatal mice results in dramatic decrease in endogenous C/EBPalpha mRNA levels and coordinately altered amelogenin mRNA abundance. Conditional deletion of both C/EBPalpha alleles further diminishes C/EBPalpha mRNA levels; however, rather than ablating amelogenin expression, we observe wild-type amelogenin mRNA abundance levels. We examined C/EBPbeta and nuclear factor YA expression, two transcription factors that had previously been shown to modestly participate in amelogenin expression, in vitro but found no significant changes in either of their mRNA abundance levels comparing conditional knock-out mice with wild-type counterparts. Although the abundance of C/EBPdelta is also unchanged in C/EBPalpha conditional knock-out mice, in vitro we find that C/EBPdelta activates the mouse amelogenin promoter and synergistically cooperates with nuclear factor Y, suggesting that C/EBPdelta can functionally substitute for C/EBPalpha to produce an enamel matrix competent to direct biomineralization.

SUBMITTER: Xu Y

PROVIDER: S-EPMC4445686 | biostudies-literature | 2007 Oct

REPOSITORIES: biostudies-literature

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CCAAT/enhancer-binding protein delta (C/EBPdelta) maintains amelogenin expression in the absence of C/EBPalpha in vivo.

Xu Yucheng Y Zhou Yan Larry YL Gonzalez Frank J FJ Snead Malcolm L ML

The Journal of biological chemistry 20070817 41

C/EBPalpha is implicated to regulate mouse amelogenin gene expression during tooth enamel formation in vitro. Because enamel formation occurs during postnatal development and C/EBPalpha-deficient mice die at birth, we used the Cre/loxP recombination system to characterize amelogenin expression in C/EBPalpha conditional knock-out mice. Mice carrying the Cre transgene under the control of the human keratin-14 promoter show robust Cre expression in the ameloblast cell lineage. Mating between mice b ...[more]

PMID: 17704518

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Project description:α-Synuclein is the main component of Lewy bodies, the intracellular protein aggregates representing the histological hallmark of Parkinson's disease. Elevated α-synuclein levels and mutations in SNCA gene are associated with increased risk for Parkinson's disease. Despite this, little is known about the molecular mechanisms regulating SNCA transcription. CCAAT/enhancer binding protein (C/EBP) β and δ are b-zip transcription factors that play distinct roles in neurons and glial cells. C/EBPβ overexpression increases SNCA expression in neuroblastoma cells and putative C/EBPβ and δ binding sites are present in the SNCA genomic region suggesting that these proteins could regulate SNCA transcription. Based on these premises, the goal of this study was to determine if C/EBPβ and δ regulate the expression of SNCA. We first observed that α-synuclein CNS expression was not affected by C/EBPβ deficiency but it was markedly increased in C/EBPδ-deficient mice. This prompted us to characterize further the role of C/EBPδ in SNCA transcription. C/EBPδ absence led to the in vivo increase of α-synuclein in all brain regions analyzed, both at mRNA and protein level, and in primary neuronal cultures. In agreement with this, CEBPD overexpression in neuroblastoma cells and in primary neuronal cultures markedly reduced SNCA expression. ChIP experiments demonstrated C/EBPδ binding to the SNCA genomic region of mice and humans and luciferase experiments showed decreased expression of a reporter gene attributable to C/EBPδ binding to the SNCA promoter. Finally, decreased CEBPD expression was observed in the substantia nigra and in iPSC-derived dopaminergic neurons from Parkinson patients resulting in a significant negative correlation between SNCA and CEBPD levels. This study points to C/EBPδ as an important repressor of SNCA transcription and suggests that reduced C/EBPδ neuronal levels could be a pathogenic factor in Parkinson's disease and other synucleinopathies and C/EBPδ activity a potential pharmacological target for these neurological disorders.

Project description:BACKGROUND: Microglia and astrocytes respond to homeostatic disturbances with profound changes of gene expression. This response, known as glial activation or neuroinflammation, can be detrimental to the surrounding tissue. The transcription factor CCAAT/enhancer binding protein ? (C/EBP?) is an important regulator of gene expression in inflammation but little is known about its involvement in glial activation. To explore the functional role of C/EBP? in glial activation we have analyzed pro-inflammatory gene expression and neurotoxicity in murine wild type and C/EBP?-null glial cultures. METHODS: Due to fertility and mortality problems associated with the C/EBP?-null genotype we developed a protocol to prepare mixed glial cultures from cerebral cortex of a single mouse embryo with high yield. Wild-type and C/EBP?-null glial cultures were compared in terms of total cell density by Hoechst-33258 staining; microglial content by CD11b immunocytochemistry; astroglial content by GFAP western blot; gene expression by quantitative real-time PCR, western blot, immunocytochemistry and Griess reaction; and microglial neurotoxicity by estimating MAP2 content in neuronal/microglial cocultures. C/EBP? DNA binding activity was evaluated by electrophoretic mobility shift assay and quantitative chromatin immunoprecipitation. RESULTS: C/EBP? mRNA and protein levels, as well as DNA binding, were increased in glial cultures by treatment with lipopolysaccharide (LPS) or LPS + interferon ? (IFN?). Quantitative chromatin immunoprecipitation showed binding of C/EBP? to pro-inflammatory gene promoters in glial activation in a stimulus- and gene-dependent manner. In agreement with these results, LPS and LPS+IFN? induced different transcriptional patterns between pro-inflammatory cytokines and NO synthase-2 genes. Furthermore, the expressions of IL-1? and NO synthase-2, and consequent NO production, were reduced in the absence of C/EBP?. In addition, neurotoxicity elicited by LPS+IFN?-treated microglia co-cultured with neurons was completely abolished by the absence of C/EBP? in microglia. CONCLUSIONS: These findings show involvement of C/EBP? in the regulation of pro-inflammatory gene expression in glial activation, and demonstrate for the first time a key role for C/EBP? in the induction of neurotoxic effects by activated microglia.

Dataset Information

CCAAT/enhancer-binding protein delta (C/EBPdelta) maintains amelogenin expression in the absence of C/EBPalpha in vivo.

Publications

CCAAT/enhancer-binding protein delta (C/EBPdelta) maintains amelogenin expression in the absence of C/EBPalpha in vivo.

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