Abstract
In diabetics, methylglyoxal (MG), a glucose-derived metabolite, plays a noxious role by inducing oxidative stress, which causes and exacerbates a series of complications including low-turnover osteoporosis. In the present study, while MG treatment of mouse bone marrow stroma-derived ST2 cells rapidly suppressed the expression of osteotrophic Wnt-targeted genes, including that of osteoprotegerin (OPG, a decoy receptor of the receptor activator of NF-kappaB ligand (RANKL)), it significantly enhanced that of secreted Frizzled-related protein 4 (sFRP-4, a soluble inhibitor of Wnts). On the assumption that upregulated sFRP-4 is a trigger that downregulates Wnt-related genes, we sought out the molecular mechanism whereby oxidative stress enhanced the sFRP-4 gene. Sodium bisulfite sequencing revealed that the sFRP-4 gene was highly methylated around the sFRP-4 gene basic promoter region, but was not altered by MG treatment. Electrophoretic gel motility shift assay showed that two continuous CpG loci located five bases upstream of the TATA-box were, when methylated, a target of methyl CpG binding protein 2 (MeCP2) that was sequestered upon induction of 8-hydroxy-2-deoxyguanosine, a biomarker of oxidative damage to DNA. These in vitro data suggest that MG-derived oxidative stress (not CpG demethylation) epigenetically and rapidly derepress sFRP-4 gene expression. We speculate that under persistent oxidative stress, as in diabetes and during aging, osteopenia and ultimately low-turnover osteoporosis become evident partly due to osteoblastic inactivation by suppressed Wnt signaling of mainly canonical pathways through the derepression of sFRP-4 gene expression.
Highlights
Many diabetic complications are induced by oxidative stress through advanced glycation end-products (AGEs)[1,2] derived from the accumulation of methylglyoxal (MG)[3], an intermediate metabolite of glucose that increases in the serum or the organs of diabetics [4,5,6]
To confirm the reproducibility of the effect of MG-derived oxidative stress on the altered gene expression as demonstrated by the comprehensive microarray analysis, we quantified the mRNA level of secreted Frizzled-related protein 4 (sFRP-4) and OPG and, at the same time, tested the expression level of RANKL, Thioredoxin 1 (Trx1, disulfide oxidoreductase), several osteoblastic or adipocytic differentiation markers, cell cycle protein (p16INK4a), and apoptosis executioner Caspase 3 (Casp-3) in the cultured ST2 cells
Differentiation of osteoblastic cells is regulated by bone morphogenetic protein (BMP)-Smad and Wnt associated pathways, followed by the induction of osteoblast-specific gene expression [19,20]
Summary
Many diabetic complications are induced by oxidative stress through advanced glycation end-products (AGEs)[1,2] derived from the accumulation of methylglyoxal (MG)[3], an intermediate metabolite of glucose that increases in the serum or the organs of diabetics [4,5,6]. It is well known that the diabetic condition evokes a state of low-turnover osteoporosis, characterized by a severe decrease in the rate of osteoblast/osteoid surface and bone mineral apposition and in reduced bone strength – diabetic osteopathy. Since bone is composed of two types of cells – boneforming osteoblasts and bone-resorbing osteoclasts – the net balance between these two cell types defines the rate of bone turnover and bone mass. Together with the fact that oxidative stress per se has little effect on the number and function of bone-resorbing osteoclasts either in vivo or in vitro [7,8], diabetic osteopenia can, by and large, be regarded as a condition induced by impaired anabolic functions of osteoblasts through oxidative stress [9]. In vitro oxidative stress (exemplified by H2O2) alters the function of cultured osteoblastic precursors by blocking the bone-anabolic function of canonical
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