PGE2 induces interleukin-8 derepression in human astrocytoma through coordinated DNA demethylation and histone hyperacetylation.
We have recently reported that in astrocytoma cells the expression of interleukin-8 (IL-8) is upregulated by prostaglandin E2 (PGE2). Unfortunately, the exact mechanism by which this happens has not been clarified yet. Here, we have investigated whether IL-8 activation by PGE2 involves changes in DNA methylation and/or histone modifications in 46 astrocytoma specimens, two astrocytoma cell lines and normal astrocytic cells. The DNA methylation status of the IL-8 promoter was analyzed by bisulphite sequencing and by methylation DNA immunoprecipitation analysis. The involvement of DNA methyltransferases (DNMTs) and histone deacetylases (HDACs), as well as histone acetylation levels, was assayed by chromatin immunoprecipitation. IL-8 expression at promoter, mRNA and protein level was explored by transfection, real-time PCR and enzyme immunoassay experiments in cells untreated or treated with PGE2, 5-aza-2'-deoxycytidine (5-aza-dC) and HDAC inhibitors, alone or in combination. EMSA was performed with crude cell extracts or recombinant protein. We observed that PGE2 induced IL-8 activation through: (1) demethylation of the single CpG site 5 located at position -83 within the binding region for CEBP-β in the IL-8 promoter; (2) C/EBP-β and p300 co-activator recruitment; (3) H3 acetylation enhancement and (4) reduction in DNMT1, DNMT3a, HDAC2 and HDAC3 association to CpG site 5 region. Treatment with 5-aza-dC or HDAC inhibitors of class I HDACs strengthened either basal or PGE2-mediated IL-8 expression. These findings have elucidated an orchestrated mechanism triggered by PGE2 whereby concurrent association of site-specific demethylation and histone H3 hyperacetylation resulted in derepression of IL-8 gene expression in human astrocytoma.
Department of Surgical Specialities, University of Messina, Messina, Italy.
Isabella
I
Venza
Maria
M
Visalli
Cinzia
C
Fortunato
Manuela
M
Ruggeri
Simona
S
Ratone
Maria
M
Caffo
Gerardo
G
Caruso
Concetta
C
Alafaci
Francesco
F
Tomasello
Diana
D
Teti
Mario
M
Venza
D000107:Acetylation; D000293:Adolescent; D000328:Adult; D001483:Base Sequence; D001932:Brain Neoplasms / Q000235:genetics*; D045744:Cell Line, Tumor; D018899:CpG Islands; D000076123:DNA (Cytosine-5-)-Methyltransferase 1; D004248:DNA (Cytosine-5-)-Methyltransferases / Q000378:metabolism; D019175:DNA Methylation; D000090205:DNA Methyltransferase 3A; D004273:DNA, Neoplasm / Q000378:metabolism; D015232:Dinoprostone / Q000494:pharmacology*; D005260:Female; D015972:Gene Expression Regulation, Neoplastic; D005909:Glioblastoma / Q000235:genetics*; D056464:Histone Deacetylase 2 / Q000378:metabolism; D056572:Histone Deacetylase Inhibitors; D006655:Histone Deacetylases / Q000378:metabolism; D006657:Histones / Q000378:metabolism*; D006801:Humans; D016209:Interleukin-8 / Q000235:genetics* / Q000378:metabolism; D008297:Male; D008875:Middle Aged; D008969:Molecular Sequence Data; D011401:Promoter Regions, Genetic; D011499:Protein Processing, Post-Translational; D012333:RNA, Messenger / Q000096:biosynthesis; D017422:Sequence Analysis, DNA; D000099186:Histone Deacetylase 3
D000076123:DNA (Cytosine-5-)-Methyltransferase 1; D004248:DNA (Cytosine-5-)-Methyltransferases; D000090205:DNA Methyltransferase 3A; D004273:DNA, Neoplasm; D015232:Dinoprostone; D056464:Histone Deacetylase 2; D056572:Histone Deacetylase Inhibitors; D006655:Histone Deacetylases; D006657:Histones; D016209:Interleukin-8; D012333:RNA, Messenger; D000099186:Histone Deacetylase 3; C000717967:DNMT3A protein, human; C000619380:DNMT1 protein, human; C533423:HDAC2 protein, human