title: State of methylation of the human osteocalcin gene in bone-derived and other types of cells.
abstract: DNA methylation is a general mechanism of controlling tissue-specific gene expression. Osteocalcin is a bone matrix protein whose expression is limited almost entirely to osteoblasts. We were interested in determining whether the state of methylation of the osteocalcin gene plays a role in its expression by studying human bone-derived (MG-63, U2-Os, SaOs-2) and other types (normal lymphocytes, A-498, Hep G2) of cells. Reverse transcription-polymerase chain reaction (RT-PCR) analysis revealed that osteocalcin mRNA production is stimulated by 1,25(OH)2D3 in MG-63 and induced in SaOs-2 but not in U2-Os osteoblast-like osteosarcoma cells. Genomic analysis of the human osteocalcin gene showed that the local surroundings of this single-copy gene are identical in all cell lines studied. Using an isoschizomeric pair of restriction enzymes and Southern analysis, we found that the osteocalcin gene is identically methylated in all three osteosarcoma cell lines. The same sites are also methylated in human normal lymphocytes and A-498 kidney cells, whereas the degree of methylation is higher in Hep G2 human hepatocellular carcinoma cells. Furthermore, the osteocalcin gene was identically protected against enzymatic digestion at the chromatin level in normal lymphocytes and in all cell lines studied. Induction of hypomethylation of DNA by 5-azacytidine treatment did not cause an induction of osteocalcin synthesis in these cell lines. On the contrary, it attenuated the induction by 1,25(OH)2D3 in MG-63 cells. In gel mobility shift assays, human vitamin D receptor and the AP-1 transcription factor bound to an unmethylated response element oligonucleotide of the osteocalcin gene with greater affinity than to an in vitro methylated response element. These results indicate that the in vivo methylation state of the osteocalcin gene at sites determined in this study does not correlate with the inducibility of this gene. Nevertheless, the in vitro results clearly indicated that hypomethylation of critical regions of the osteocalcin gene promoter is a potential mechanism influencing effective binding of specific nuclear factors and, consequently, gene expression.
authors.affiliation: Department of Biochemistry and Biotechnology, University of Kuopio, Finland. sryhanen@messi.uku.fi
authors.forename: S
authors.initials: S
authors.lastname: Ryhänen
authors.forename: A
authors.initials: A
authors.lastname: Pirskanen
authors.forename: T
authors.initials: T
authors.lastname: Jääskeläinen
authors.forename: P H
authors.initials: PH
authors.lastname: Mäenpää
mesh_terms: D000964:Antimetabolites, Antineoplastic / Q000494:pharmacology; D001374:Azacitidine / Q000494:pharmacology; D001665:Binding Sites; D015139:Blotting, Southern; D001842:Bone and Bones / Q000166:cytology* / Q000378:metabolism; D002843:Chromatin / Q000235:genetics / Q000378:metabolism; D003596:Cytosine / Q000378:metabolism; D019175:DNA Methylation; D019003:Deoxyribonuclease HpaII / Q000378:metabolism; D005786:Gene Expression Regulation / Q000187:drug effects; D006801:Humans; D008214:Lymphocytes / Q000378:metabolism; D009928:Organ Specificity; D015675:Osteocalcin / Q000187:drug effects / Q000235:genetics* / Q000378:metabolism; D011506:Proteins / Q000378:metabolism; D012333:RNA, Messenger / Q000096:biosynthesis; D014807:Vitamin D / Q000494:pharmacology
chemical_list: D000964:Antimetabolites, Antineoplastic; D002843:Chromatin; D011506:Proteins; D012333:RNA, Messenger; D015675:Osteocalcin; D014807:Vitamin D; D003596:Cytosine; D019003:Deoxyribonuclease HpaII; D001374:Azacitidine