Estrogen receptor alpha and NFATc1 bind to a bone mineral density-associated SNP to repress WNT5B in osteoblasts

Genetic factors and estrogen deficiency contribute to the development of osteoporosis. The single-nucleotide polymorphism (SNP) rs2887571 is predicted from genome-wide association studies (GWASs) to associate with osteoporosis but has had an unknown mechanism. Analysis of osteoblasts from 110 different individuals who underwent joint replacement revealed that the genotype of rs2887571 correlates with WNT5B expression. Analysis of our ChIP-sequencing data revealed that SNP rs2887571 overlaps with an estrogen receptor alpha (ER alpha) binding site. Here we show that 17 beta-estradiol (E2) suppresses WNT5B expression and further demonstrate the mechanism of ER alpha binding at the enhancer containing rs2887571 to suppress WNT5B expression differentially in each genotype. ER alpha interacts with NFATc1, which is predicted to bind directly at rs2887571. CRISPR-Cas9 and ChIP-qPCR experiments confirm differential regulation of WNT5B between each allele. Homozygous GG has a higher binding affinity for ERs than homozygous AA and results in greater suppression of WNT5B expression. Functionally, WNT5B represses alkaline phosphatase expression and activity, decreasing osteoblast differentiation and mineralization. Furthermore, WNT5B increases interleukin-6 expression and suppresses E2-induced expression of alkaline phosphatase during osteoblast differentiation. We show that WNT5B suppresses the differentiation of osteoblasts via receptor tyrosine kinase-like orphan receptor 1/2 (ROR1/2), which activates DVL2/3/RAC1/CDC42/JNK/SIN3A signaling and inhibits beta-catenin activity. Together, our data provide mechanistic insight into how ER alpha and NFATc1 regulate the non-coding SNP rs2887571, as well as the function of WNT5B on osteoblasts, which could provide alternative therapeutic targets for osteoporosis..

Automated summary

Genetic factors and estrogen deficiency contribute to the development of osteoporosis. The SNP rs2887571 correlates with WNT5B expression, with different mechanisms in each genotype. WNT5B suppresses alkaline phosphatase expression and activity, inhibiting osteoblast differentiation and mineralization. WNT5B inhibits osteoblast differentiation by activating the ROR1/2 signaling pathway and suppressing beta-catenin activity.