scholarly journals CCAAT/enhancer binding protein β regulates expression of matrix metalloproteinase-3 in arthritis

2011 ◽  
Vol 71 (1) ◽  
pp. 99-107 ◽  
Author(s):  
Hidetoshi Tsushima ◽  
Ken Okazaki ◽  
Mitsumasa Hayashida ◽  
Takahiro Ushijima ◽  
Yukihide Iwamoto
Keyword(s):  
Matrix Metalloproteinase ◽  
Chromatin Immunoprecipitation ◽  
Binding Protein ◽  
Chromatin Immunoprecipitation Assay ◽  
Immunoprecipitation Assay ◽  
Matrix Metalloproteinase 3 ◽  
Enhancer Binding Protein ◽  
Ccaat Enhancer Binding Protein ◽  
And Cartilage ◽  
In Cells

ObjectivesTo investigate whether CCAAT/enhancer binding protein β (C/EBPβ) mediates the expression of matrix metalloproteinase-3 (MMP-3) and aggrecanases in arthritis.MethodsLocalisation of C/EBPβ and MMP-3 in synovium and cartilage from patients with rheumatoid arthritis and osteoarthritis was determined by immunohistochemistry. Cell lines SW982, C28/I2 and human fibroblast-like synoviocytes stimulated by interleukin 1β (IL-1β) were subjected to western blotting and quantitative PCR. Overexpression of C/EBPβ by adenovirus was performed in cells and organ culture of normal cartilage. Knockdown of C/EBPβ by small interference RNA was performed in cells. Activity of the human MMP-3 and aggrecanase-2 ADAMTS-5 (a disintegrin and metalloproteinase with thrombospondin motifs) promoters was analysed by a luciferase assay. To determine whether C/EBPβ directly binds to the MMP-3 or ADAMTS-5 promoter,a chromatin immunoprecipitation assay was performed.ResultsImmunohistochemistry showed that C/EBPβ and MMP-3 were co-localised in arthritic synovium and cartilage. Western blots revealed increased C/EBPβ expression in cells treated with IL-1β. Expression of MMP-3, MMP-13 and ADAMTS-5 mRNA was significantly increased by the overexpression of C/EBPβ. C/EBPβ stimulated MMP-3 expression and induced matrix degradation in cartilage explants. C/EBPβ knockdown reduced MMP-3 and ADAMTS-5 expression. C/EBPβ stimulated the 2011 bp MMP-3 promoter and the 1768 bp ADAMTS-5 promoter in a dose-dependent manner. Deletion and mutation analysis of the MMP-3 promoter showed that the C/EBPβ core responsive element was located between −108 bp and −100 bp. The chromatin immunoprecipitation assay showed that C/EBPβ was directly bound to MMP-3 and ADAMTS-5 promoters.ConclusionsThese data demonstrate that C/EBPβ is involved in expression of MMP-3 and ADAMTS-5 in arthritic synovium and cartilage.

scholarly journals Changes in Histone Modification and DNA Methylation of the StAR and Cyp19a1 Promoter Regions in Granulosa Cells Undergoing Luteinization during Ovulation In Rats

Endocrinology ◽  
2013 ◽  
Vol 154 (1) ◽  
pp. 458-470 ◽  
Author(s):  
Lifa Lee ◽  
Hiromi Asada ◽  
Fumie Kizuka ◽  
Isao Tamura ◽  
Ryo Maekawa ◽  
...  
Keyword(s):  
Gene Expression ◽  
Histone Modifications ◽  
Granulosa Cells ◽  
Chromatin Immunoprecipitation ◽  
Binding Protein ◽  
Mrna Levels ◽  
Chromatin Immunoprecipitation Assay ◽  
Immunoprecipitation Assay ◽  
Rapid Changes

The ovulatory LH surge induces rapid up-regulation of steroidogenic acute regulatory (StAR) protein and rapid down-regulation of aromatase (Cyp19a1) in granulosa cells (GCs) undergoing luteinization during ovulation. This study investigated in vivo whether epigenetic mechanisms including histone modifications are involved in the rapid changes of StAR and Cyp19a1 gene expression. GCs were obtained from rats treated with equine chorionic gonadotropin (CG) before (0 h) and after human (h)CG injection. StAR mRNA levels rapidly increased after hCG injection, reached a peak at 4 h, and then remained higher compared with 0 h until 12 h. Cyp19a1 mRNA levels gradually decreased after hCG injection and reached their lowest level at 12 h. A chromatin immunoprecipitation assay revealed that levels of histone-H4 acetylation (Ac-H4) and trimethylation of histone-H3 lysine-4 (H3K4me3) increased whereas H3K9me3 and H3K27me3 decreased in the StAR promoter after hCG injection. On the other hand, the levels of Ac-H3 and -H4 and H3K4me3 decreased, and H3K27me3 increased in the Cyp19a1 promoter after hCG injection. Chromatin condensation, which was analyzed using deoxyribonuclease I, decreased in the StAR promoter and increased in the Cyp19a1 promoter after hCG injection. A chromatin immunoprecipitation assay also showed that binding activities of CAATT/enhancer-binding protein β to the StAR promoter increased and binding activities of phosphorylated-cAMP response element binding protein to the Cyp19a1 promoter decreased after hCG injection. These results provide in vivo evidence that histone modifications are involved in the rapid changes of StAR and Cyp19a1 gene expression by altering chromatin structure of the promoters in GCs undergoing luteinization during ovulation.

scholarly journals CCAAT/Enhancer Binding Protein β, But Not Steroidogenic Factor-1, Modulates the Phthalate-Induced Dysregulation of Rat Fetal Testicular Steroidogenesis

Endocrinology ◽  
2007 ◽  
Vol 148 (12) ◽  
pp. 5851-5864 ◽  
Author(s):  
Adam J. Kuhl ◽  
Susan M. Ross ◽  
Kevin W. Gaido
Keyword(s):  
Cytochrome P450 ◽  
Protein Interactions ◽  
Chromatin Immunoprecipitation ◽  
Binding Protein ◽  
In Utero ◽  
Steroidogenic Factor 1 ◽  
Enhancer Binding Protein ◽  
Ccaat Enhancer Binding Protein ◽  
Steroidogenic Genes ◽  
Dna Protein Interactions

Prolonged in utero exposure of fetal male rats to dibutyl phthalate (DBP) can result in a feminized phenotype characterized by malformed epididymides, hypospadias, cryptorchidism, and retained thoracic nipples, among others. These symptoms likely result, in part, from decreased expression of steroidogenic enzymes and, therefore, reduced testosterone biosynthesis. However, the molecular mechanisms involved in these changes in gene expression profiles are unknown. To understand these mechanisms in rats, in vivo DNase footprinting was adapted to provide a semiquantitative map of changes in DNA-protein interactions in the promoter region of steroidogenic genes, including steroidogenic acute regulatory, scavenger receptor B-1, cytochrome P450 side chain cleavage, and cytochrome P450 17A1, that are down-regulated after an in utero DBP exposure. Regions with altered DNase protection were coordinated with a specific DNA binding protein event by EMSA, and binding activity confirmed with chromatin immunoprecipitation. Results demonstrated altered DNase protection at regions mapping to CCAAT/enhancer binding protein β (c/ebp β) and steroidogenic factor-1 (SF-1). Chromatin immunoprecipitation confirmed declines in DNA-protein interactions of c/ebp β in DBP treated animals, whereas SF-1 was reduced in both diethyl phthalate (nontoxic) and DBP (toxic) treatments. These results suggest that inhibition of c/ebp β, and not SF-1, is critical in DBP induced inhibition of steroidogenic genes. In addition, these observations suggest a pathway redundancy in the regulation of steroidogenesis in fetal testis. In conclusion, this study presents a snapshot of changes in the structure of transcriptional machinery and proposes a mechanism of action resulting from DBP exposure.

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