The Role of Annexin A6 in Osteoporosis

Abstract

Osteoporosis is a common calcium and metabolic skeletal disease which is characterized by decreased bone mass, microarchitectural deterioration of bone tissue and impaired bone strength, thereby leading to enhanced risk of bone fragility.

In this study, we aimed to discover a novel herbal therapeutic drug for effective osteoporosis treatment and to further clarify its molecular mechanism of action. At first, ethanol or methanol extracts of 68 edible Korean native plants were screened and Poncirus trifoliata (PT) was selected as a final candidate because of its high inhibitory activity on glucocorticoid (GC)-induced apoptosis plus its novelty. The hexane extract of PT (PT-H) inhibited apoptotic cell death of osteoblastic cell lines, C3H10T1/2 and MC3T3-E1 induced by synthetic GC, dexamethasone (Dex). In vivo mouse results indicated that PT-H not only had an inhibitory effect on the bone loss caused by GC, but also promoted bone formation.

We further clarified the molecular mechanisms behind the effect of PT-H on GC-induced osteoporosis (GIO) by screening of differentially-expressed genes (DEGs) between Dex-induced osteoblastic cells with or without PT-H treatment. Finally, we found that the expression level of AnxA6 in Dex-induced osteoblastic cells and prednisolone-treated GIO-model mice was significantly decreased by PT-H treatment. These findings suggest that PT-H has a strong in vitro and in vivo inhibitory effect on GIO, and decreased expression of AnxA6 may play a key role in this inhibition. As the next step, we aimed to determine whether ANXA6 gene was associated with the susceptibility to osteoporosis and further identify novel genes for susceptibility to osteoporosis. At first, we performed a whole-genome comparative expression analysis. Ten genes were identified as the DEGs in the Dex-treated MC3T3-E1 cells. To validate and evaluate the identified DEGs, we performed quantitative real-time RT-PCR using the gene-specific primers and compared the expression levels of these genes between the Dex-treated and untreated MC3T3-E1 cells, and between the ovariectomized (OVX) and sham mice and found that the expression levels of 7 genes, Anxa6, Col5a1, Col6a2, Eno1, Myof, Nfib and Scara5, were significantly altered in the in vitro and in vivo osteoporosis models.

Lastly, to determine whether the genetic variations of these 7 genes were associated with bone density and osteoporosis phenotypes in humans, we performed the quantitative bone density analysis and osteoporosis case-control analysis of 116 ingle nucleotide polymorphism (SNPs) in these 7 genes in the Korean Women’s Cohort (3570 subjects). There was a significant association between the SNPs in the 5 genes, ANXA6, COL5A1, ENO1, MYOF and SCARA5, and bone density and/or osteoporosis. Especially, the SNPs in the ANXA6 gene showed a highly significant association with bone density and their p-values satisfied the Bonferroni-corrected significance level. These results indicate that genetic variation in the ANXA6 gene is significantly associated with bone density and osteoporosis. This study may provide insight into the genetic basis of osteoporosis.