title: Homocysteine suppresses the expression of the collagen cross-linker lysyl oxidase involving IL-6, Fli1, and epigenetic DNA methylation.
abstract: Elevated homocysteine (Hcys) serum levels represent a risk factor for several chronic pathologies, including cardiovascular disease, atherosclerosis, and chronic renal failure, and affect bone development, quality, and homeostasis. Hcys influences the formation of a stable bone matrix directly through the inhibition of the collagen cross-linking enzyme lysyl oxidase (Lox) and, as we have shown recently, by repressing its mRNA expression. The aim of this study was to investigate the mechanisms involved in this process. Through evaluation of gene arrays, quantitative RT-PCR, immunoblots, and ELISA, we identified a Hcys-dependent stimulation of interleukin 6 (IL-6) and genes involved in IL-6/Janus kinase 2 (JAK2)-dependent signal transduction pathways in pre-osteoblastic MC3T3-E1 cells. Moreover, up-regulation of genes essential for epigenetic DNA methylation (DNA (cytosine-5)-methyltransferases and helicase lymphoid-specific (Hells) was observed. Further investigations demonstrated that Hcys increased via IL-6/JAK2 the expression of Fli1 (Friend leukemia virus integration 1), a transcription factor, which we found essential for IL-6-dependent Dnmt1 stimulation. CpG methylation analysis of CpG-rich Lox proximal promoter revealed an increased CpG methylation status after treatment of the cells with Hcys indicating an epigenetic origin for Hcys-dependent Lox repression. Inhibition of the IL-6/JAK2 pathway or of CpG methylation reversed the repressive effect of Hcys on Lox expression. In conclusion, we demonstrate that Hcys stimulates IL-6 synthesis in osteoblasts, which is known to affect bone metabolism via osteoclasts. Furthermore, IL-6 stimulation results via JAK2, Fli1, and Dnmt1 in down-regulation of Lox expression by epigenetic CpG methylation revealing a new mechanism negatively affecting bone matrix formation.
authors.affiliation: 1st Medical Department, Hanusch Hospital, Ludwig Boltzmann Institute of Osteology, Hanusch Hospital, Wiener Gebietskrankenkasse and AUVA Trauma Center Meidling, 1140 Vienna, Austria.
authors.forename: Roman
authors.initials: R
authors.lastname: Thaler
authors.forename: Marlies
authors.initials: M
authors.lastname: Agsten
authors.forename: Silvia
authors.initials: S
authors.lastname: Spitzer
authors.forename: Eleftherios P
authors.initials: EP
authors.lastname: Paschalis
authors.forename: Heidrun
authors.initials: H
authors.lastname: Karlic
authors.forename: Klaus
authors.initials: K
authors.lastname: Klaushofer
authors.forename: Franz
authors.initials: F
authors.lastname: Varga
mesh_terms: D000818:Animals; D018899:CpG Islands / Q000235:genetics; D000076123:DNA (Cytosine-5-)-Methyltransferase 1; D004248:DNA (Cytosine-5-)-Methyltransferases / Q000235:genetics / Q000378:metabolism; D004265:DNA Helicases / Q000235:genetics / Q000378:metabolism; D019175:DNA Methylation / Q000187:drug effects* / Q000235:genetics; D044127:Epigenesis, Genetic / Q000187:drug effects* / Q000235:genetics; D016326:Extracellular Matrix Proteins / Q000096:biosynthesis* / Q000235:genetics; D006710:Homocysteine / Q000378:metabolism / Q000494:pharmacology*; D015850:Interleukin-6 / Q000096:biosynthesis* / Q000235:genetics; D053614:Janus Kinase 2 / Q000235:genetics / Q000378:metabolism; D051379:Mice; D010012:Osteogenesis / Q000187:drug effects / Q000235:genetics; D011401:Promoter Regions, Genetic / Q000235:genetics; D008249:Protein-Lysine 6-Oxidase / Q000096:biosynthesis* / Q000235:genetics; D051789:Proto-Oncogene Protein c-fli-1 / Q000096:biosynthesis* / Q000235:genetics
chemical_list: D016326:Extracellular Matrix Proteins; C497426:Fli1 protein, mouse; D015850:Interleukin-6; D051789:Proto-Oncogene Protein c-fli-1; D006710:Homocysteine; C077112:Lox protein, mouse; D008249:Protein-Lysine 6-Oxidase; D000076123:DNA (Cytosine-5-)-Methyltransferase 1; D004248:DNA (Cytosine-5-)-Methyltransferases; C000619381:Dnmt1 protein, mouse; C507926:Jak2 protein, mouse; D053614:Janus Kinase 2; D004265:DNA Helicases; C487055:Hells protein, mouse