Influence of an altered methylation potential on mRNA methylation and gene expression in HepG2 cells.
S-adenosylhomocysteine (AdoHcy), a by-product and inhibitor of S-adenosylmethionine (AdoMet)-dependent methylation reactions, is removed by AdoHcy hydrolase. The ratio of AdoMet and AdoHcy, also termed methylation potential (MP), is a metabolic indicator for cellular methylation status. In the present study, we have investigated the influence of hypoxia and inhibition of AdoHcy hydrolase on MP in HepG2 cells. Furthermore, we studied the impact of deviations in MP on mRNA and DNA methylation and the expression of selected genes: erythropoietin, VEGF-A, AdoHcy hydrolase, cyclophilin, and HIF-1alpha. Under hypoxic conditions, the MP raised from 53.4 +/- 3.3 to 239.4 +/- 24.8, which is the result of increased AdoMet and decreased AdoHcy levels. Inhibition of AdoHcy hydrolase by adenosine-2',3'-dialdehyde leads to a 40-fold reduction of the MP under both normoxic and hypoxic conditions. Hypoxia increases erythropoietin (2.7-fold) and VEGF-A (5-fold) mRNA expression. During a reduced MP erythropoietin mRNA expression is lowered under normoxia and hypoxia by 70%, whereas VEGF-A mRNA expression is only reduced under hypoxic conditions by 60%. The mRNA expression of AdoHcy hydrolase, HIF-1alpha, and cyclophilin is insensitive to an altered MP. Furthermore, decreased MP leads to a highly significant decrease in overall mRNA methylation. Our results show that the mRNA levels of the studied genes respond differentially to changes in MP. This implies that genes with a slower transcription rate and mRNAs with a slower turnover are insensitive to short-term changes in MP.
Hermes
Marina
M
Department of Pharmacology and Toxicology, Faculty of Medicine, University of Tübingen, D-72074 Tübingen, Germany.
Osswald
Hartmut
H
Mattar
Julia
J
Kloor
Doris
D
D000241:Adenosine / Q000031:analogs & derivatives* / Q000494:pharmacology; D043383:Adenosylhomocysteinase / Q000037:antagonists & inhibitors / Q000378:metabolism*; D045744:Cell Line, Tumor; D021983:Cyclophilins / Q000096:biosynthesis / Q000235:genetics; D019175:DNA Methylation; D015536:Down-Regulation / Q000187:drug effects / Q000235:genetics; D004921:Erythropoietin / Q000096:biosynthesis / Q000235:genetics; D005786:Gene Expression Regulation / Q000235:genetics*; D006801:Humans; D000860:Hypoxia / Q000378:metabolism*; D051795:Hypoxia-Inducible Factor 1, alpha Subunit; D008745:Methylation; D012323:RNA Processing, Post-Transcriptional / Q000187:drug effects / Q000502:physiology; D012333:RNA, Messenger / Q000378:metabolism*; D012435:S-Adenosylhomocysteine / Q000378:metabolism*; D012436:S-Adenosylmethionine / Q000378:metabolism*; D014157:Transcription Factors / Q000096:biosynthesis / Q000235:genetics; D015854:Up-Regulation / Q000187:drug effects / Q000235:genetics; D042461:Vascular Endothelial Growth Factor A / Q000096:biosynthesis / Q000235:genetics
C497442:HIF1A protein, human; D051795:Hypoxia-Inducible Factor 1, alpha Subunit; D012333:RNA, Messenger; D014157:Transcription Factors; D042461:Vascular Endothelial Growth Factor A; D004921:Erythropoietin; C027579:periodate-oxidized adenosine; D012436:S-Adenosylmethionine; D012435:S-Adenosylhomocysteine; D043383:Adenosylhomocysteinase; D021983:Cyclophilins; D000241:Adenosine