Identification of adiponectin and its receptors in human osteoblast-like cells and association of T45G polymorphism in exon 2 of adiponectin gene with lumbar spine bone mineral density in Korean women

Abstract

Objective The role of adiponectin in bone metabolism has been recently reported in in vitro and in vivo studies. There has been no report on the association of adiponectin gene polymorphism and bone mineral density (BMD). Therefore, we investigated whether two single nucleotide polymorphisms (SNPs), T45G and G276T, in the adiponectin gene were related to BMD in Koreans. We also report on the identification of adiponectin and its receptors 1 and 2 in human osteoblast-like cell lines. Patients and measurements MG-63 cells were cultured and osteogenic and adipogenic differentiations from human mesenchymal stem cells (hMSCs) were performed. RNA was then extracted from the cultured cells and reverse transcriptase-polymerase chain reaction (RT-PCR) was performed using primers for adiponectin and for the adiponectin receptor genes. In 249 female and 80 male subjects, measurements were made of their lumbar spine and femoral neck BMDs, and biochemical markers of bone turnover. The genotyping of the T45G polymorphism in exon 2 and the G276T polymorphisms in intron 2 in the adiponectin gene was performed using an allelic discrimination assay with a TaqMan probe. Analyses were performed separately in each cohort. Results We found that the mRNAs for adiponectin and for adiponectin receptor 1 (AdipoR1) and 2 (AdipoR2) were expressed in the MG-63 cells. Sequencing of the PCR products revealed that they were identical to human adiponectin, AdipoR1 and AdipoR2, respectively. mRNAs for adiponectin, AdipoR1 and AdipoR2 were also expressed in the osteoblastic and adipogenic cell lines differentiated from hMSCs. For the polymorphism study, the frequencies of T45G and G276T in the adiponectin gene were in compliance with Hardy-Weinberg equilibrium and the two polymorphisms were in complete linkage disequilibrium (D' = -1.0, P < 0.001). In the female cohort, subjects with G alleles at the T45G locus had significantly lower lumbar spine BMD than those subjects with the TT genotype. Although BMD levels showed no association with the G276T locus, the GT genotype group showed significantly higher urine deoxypyridinoline levels than other genotype groups. In the male cohort, no association was observed between adiponectin genotypes and BMD levels. Conclusions We observed the expression of adiponectin, AdipoR1 and AdipoR2 in the MG-63 cell line and the osteoblastic cell line differentiated from hMSCs. T45G polymorphism in exon 2 of the adiponectin gene is associated with lumbar spine BMD and G276T polymorphism in intron 2 of the adiponectin gene is associated with the urine deoxypyridinoline level in Korean women. Additional studies are needed to elucidate the precise contribution of adiponectin to bone mineral metabolism.