title: Functional relevance of the BMD-associated polymorphism rs312009: novel involvement of RUNX2 in LRP5 transcriptional regulation.
abstract: 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.
authors.affiliation: Department of Genetics, Faculty of Biology, University of Barcelona, IBUB, CIBERER, Barcelona, Spain.
authors.forename: Lídia
authors.initials: L
authors.lastname: Agueda
authors.forename: Rafael
authors.initials: R
authors.lastname: Velázquez-Cruz
authors.forename: Roser
authors.initials: R
authors.lastname: Urreizti
authors.forename: Guy
authors.initials: G
authors.lastname: Yoskovitz
authors.forename: Patricia
authors.initials: P
authors.lastname: Sarrión
authors.forename: Susana
authors.initials: S
authors.lastname: Jurado
authors.forename: Roberto
authors.initials: R
authors.lastname: Güerri
authors.forename: Natàlia
authors.initials: N
authors.lastname: Garcia-Giralt
authors.forename: Xavier
authors.initials: X
authors.lastname: Nogués
authors.forename: Leonardo
authors.initials: L
authors.lastname: Mellibovsky
authors.forename: Adolfo
authors.initials: A
authors.lastname: Díez-Pérez
authors.forename: Pierre J
authors.initials: PJ
authors.lastname: Marie
authors.forename: Susana
authors.initials: S
authors.lastname: Balcells
authors.forename: Daniel
authors.initials: D
authors.lastname: Grinberg
mesh_terms: D020029:Base Pairing / Q000235:genetics; D001483:Base Sequence; D001665:Binding Sites; D015519:Bone Density / Q000235:genetics*; D002460:Cell Line; D050659:Core Binding Factor Alpha 1 Subunit / Q000235:genetics* / Q000378:metabolism; D005786:Gene Expression Regulation; D006801:Humans; D026502:LDL-Receptor Related Proteins / Q000235:genetics* / Q000378:metabolism; D060492:Low Density Lipoprotein Receptor-Related Protein-5; D008969:Molecular Sequence Data; D009154:Mutation / Q000235:genetics; D010006:Osteoblasts / Q000378:metabolism; D020641:Polymorphism, Single Nucleotide / Q000235:genetics*; D011485:Protein Binding; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D020218:Response Elements / Q000235:genetics; D014158:Transcription, Genetic; D014162:Transfection
chemical_list: D050659:Core Binding Factor Alpha 1 Subunit; D026502:LDL-Receptor Related Proteins; C555482:LRP5 protein, human; D060492:Low Density Lipoprotein Receptor-Related Protein-5; D012333:RNA, Messenger; C493709:RUNX2 protein, human