TY - JOUR
T1 - Functional relevance of the BMD-associated polymorphism rs312009: Novel Involvement of RUNX2 in LRP5 transcriptional regulation
AU - Agueda, Lídia
AU - Velázquez-Cruz, Rafael
AU - Urreizti, Roser
AU - Yoskovitz, Guy
AU - Sarriõn, Patricia
AU - Jurado, Susana
AU - Güerri, Roberto
AU - Garcia-Giralt, Natàlia
AU - Nogués, Xavier
AU - Mellibovsky, Leonardo
AU - Díez-Pérez, Adolfo
AU - Marie, Pierre J.
AU - Balcells, Susana
AU - Grinberg, Daniel
PY - 2011/5/1
Y1 - 2011/5/1
N2 - LRP5 is an osteoporosis susceptibility gene. Association analyses reveal that individual single-nucleotide polymorphisms (SNPs) determine variation in bone mineral density (BMD) among individuals as well as fracture risk. In a previous study, we identified a lumbar spine BMD-associated SNP, rs312009, located in the LRP5 5' region. A RUNX2 binding site was identified in this region by gel-shift experiments. Here we test the functionality of this SNP and examine whether RUNX2 is indeed a regulator of LRP5 expression. Gene reporter assays were used to test rs312009 functionality. Bioinformatic predictive tools and gel-shift and gene reporter assays were used to identify and characterize additional RUNX2 binding elements in the 3.3-kb region upstream of LRP5. Allelic differences in the transcriptional activity of rs312009 were observed in two osteoblastic cell lines, the T allele being a better transcriber than the C allele. RUNX2 cotransfection in HeLa cells revealed that the LRP5 5' region responded to RUNX2 in a dose-dependent manner and that the previously identified RUNX2 binding site participated in this response. Also, RUNX2 inhibition by RNAi led to nearly 60% reduction of endogenous LRP5 mRNA in U-2 OS cells. Four other RUNX2 binding sites were identified in the 5' region of LRP5. Luciferase experiments revealed the involvement of each of them in the RUNX2 response. The allelic differences observed point to the involvement of rs312009 as a functional SNP in the observed association. To our knowledge, this is the first time that the direct action of RUNX2 on LRP5 has been described. This adds evidence to previously described links between two important bone-regulating systems: the RUNX2 transcription-factor cascade and the Wnt signaling pathway. © 2011 American Society for Bone and Mineral Research.
AB - LRP5 is an osteoporosis susceptibility gene. Association analyses reveal that individual single-nucleotide polymorphisms (SNPs) determine variation in bone mineral density (BMD) among individuals as well as fracture risk. In a previous study, we identified a lumbar spine BMD-associated SNP, rs312009, located in the LRP5 5' region. A RUNX2 binding site was identified in this region by gel-shift experiments. Here we test the functionality of this SNP and examine whether RUNX2 is indeed a regulator of LRP5 expression. Gene reporter assays were used to test rs312009 functionality. Bioinformatic predictive tools and gel-shift and gene reporter assays were used to identify and characterize additional RUNX2 binding elements in the 3.3-kb region upstream of LRP5. Allelic differences in the transcriptional activity of rs312009 were observed in two osteoblastic cell lines, the T allele being a better transcriber than the C allele. RUNX2 cotransfection in HeLa cells revealed that the LRP5 5' region responded to RUNX2 in a dose-dependent manner and that the previously identified RUNX2 binding site participated in this response. Also, RUNX2 inhibition by RNAi led to nearly 60% reduction of endogenous LRP5 mRNA in U-2 OS cells. Four other RUNX2 binding sites were identified in the 5' region of LRP5. Luciferase experiments revealed the involvement of each of them in the RUNX2 response. The allelic differences observed point to the involvement of rs312009 as a functional SNP in the observed association. To our knowledge, this is the first time that the direct action of RUNX2 on LRP5 has been described. This adds evidence to previously described links between two important bone-regulating systems: the RUNX2 transcription-factor cascade and the Wnt signaling pathway. © 2011 American Society for Bone and Mineral Research.
KW - LRP5
KW - LUCIFERASE
KW - OSTEOPOROSIS
KW - RUNX2
KW - SNP
U2 - https://doi.org/10.1002/jbmr.293
DO - https://doi.org/10.1002/jbmr.293
M3 - Article
VL - 26
SP - 1133
EP - 1144
JO - Journal of Bone and Mineral Research
JF - Journal of Bone and Mineral Research
SN - 0884-0431
ER -