Human Anti-Mouse FZD1 monoclonal Antibody, clone SFB704 [APC]

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 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. (C) 2009 Elsevier Inc. All rights reserved.

PURPOSE. To investigate the biological functions of miR-204 in human trabecular meshwork (HTM) cells. METHODS. Changes in gene expression induced by miR-204 in HTM cells were evaluated by gene array analysis using arrays and confirmed by quantitative-PCR (Q-PCR). Direct targeting of miR-204 to 12 potential novel targets was confirmed using a luciferase system, and five of them were verified by Western blot analysis. Effects of miR-204 on apoptosis, cell viability, and accumulation of carbonylated proteins were evaluated in HTM cells treated with H2O2. Induction of endoplasmic reticulum (ER) stress markers by tunicamycin was analyzed by Q-PCR, and expression of IL-8 and IL-11 was analyzed by ELISA. RESULTS. MiR-204 decreased the expression of multiple genes in HTM cells. Twelve genes (AP1S2, Bcl2l2, BIRC2, EDEM1, EZR, FZD1, M6PR, RAB22A, RAB40B, SERP1, TCF12, and TCF4) were validated as direct targets of miR-204. Downregulation of expressions at protein levels of Bcl2l2, BIRC2, EZR, M6PR, and SERP1 were confirmed by Western blot analysis. HTM cells transfected with miR-204 showed increased levels of apoptosis, decreased viability, increased accumulation of oxidized proteins after H2O2 treatment, decreased induction of ER stress response markers, and reduced expression of inflammatory mediators IL-8 and IL-11. CONCLUSIONS. MiR-204 potentially plays an important role in the regulation of multiple functions in HTM cells including apoptosis, accumulation of damaged proteins, ER stress response, and expression of inflammatory mediators. (Invest Ophthalmol Vis Sci. 2011;52:2999-3007) DOI:10.1167/iovs.10-6708