Functional and association analysis of frizzled 1 (FZD1) promoter haplotypes with femoral neck geometry

Abstract

Frizzleds are receptors for Wnt signaling and are involved in skeletal morphogenesis. Little is known about the transcriptional regulation of frizzleds in bone cells. In the current study, we determined if two common and potentially functional genetic variants (rs2232157, rs2232158) in the frizzled-1 (FZD1) promoter region and their haplotypes influence FZD1 promoter activity in human osteoblast-like cells. We also determined if these variants are associated with femoral neck bone mineral density (BMD) and geometry in 1319 African ancestry men aged > or =40 years. Real-time quantitative PCR and western blot analysis demonstrated FZD1 mRNA and protein expression in the human osteoblast-like cell lines, MG63 and SaOS-2. Promoter activity was next assessed by transient expression of haplotype specific FZD1 promoter reporter plasmids in these cells. In comparison to the common GG haplotype, promoter activity was 3-fold higher for the TC haplotype in both cell lines (p<0.05). We previously demonstrated that rs2232158 is associated with differential FZD1 promoter activity and Egr1 binding and thus focused further functional analyses on the rs2232157 G-to-T polymorphism. Electrophoretic mobility shift assay demonstrated that distinct nuclear protein complexes were associated with rs2232157 in an allele specific manner. Bioinformatics analysis predicted that the G to T transversion creates an E2F1 binding site, further supporting the functional significance of rs2232157 in FZD1 promoter regulation. Individual SNPs and haplotypes were not associated with femoral neck BMD. The TC haplotype was associated with larger subperiosteal width and greater CSMI (p<0.05). These results suggest that FZD1 expression is regulated in a haplotype-dependent manner in osteoblasts and that these same haplotypes may be associated with biomechanical indices of bone strength.

Keywords: WNT; frizzled-1; haplotype; osteoblast; osteoporosis.

Figure 1

a) Schematic of the FZD1 5′ UTR region. The relative locations of rs2232157 and rs 2232158 are shown. b) Quantitative RT-PCR analysis of FZD1. Relative expression level (ΔCt) was calculated using ΔCt of GAPDH as a reference. c). Western blot analysis of FZD1 protein in MG63 and SaOS-2 cells. Beta-actin was used as an internal control to demonstrate protein loading. d) Activity of the haplotype specific FZD1 promoters. The promoter activity for different FZD1 haplotypes were assessed by transfecting MG63 and SaOS-2 cell lines with luciferase reporter plasmids. Differences in activity were tested using the Kruskal-Wallis and Wilcoxon rank sum test. e) Allele specific binding to the rs2232157 specific probes by nuclear proteins. EMSA was carried out using radio-labeled probes for the two alleles of rs2232157 with nuclear extracts from both MG63 and SaOS-2 cells. The two major complexes are labeled with A and B. To ensure specificity of binding, the reaction was incubated with 50-fold excess unlabeled G and T probes (denoted by g* and t*, respectively). As a control, binding reaction with radio-labeled probe without nuclear extract was conducted (denoted as C).