Secreted frizzled-related protein 1 (sFRP1) is an antagonist of Wnt PIK-294

Secreted frizzled-related protein 1 (sFRP1) is an antagonist of Wnt PIK-294 signaling an important pathway in maintaining bone homeostasis. for 2 weeks increased trabecular bone volume in WT mice (females: +30% to 50%; males: +35% to 150%) compared with mice (females: +5%; males: +18% to 54%). Percentage increases in bone formation also were lower in PTH-treated mice compared with PTH-treated WT mice. In conclusion overexpression of sFRP1 inhibited bone formation as well as attenuated PTH anabolic action on bone. The gender differences in the bone phenotype of the animal warrants further investigation. ? 2010 American Society for Bone and Mineral Research gene). The sFRPs consist of at least seven users in vertebrates (sFRP1 to sFRP5 crescent and sizzled) and contain a frizzled (Fz)-related cysteine-rich domain name that can bind to both Wnt proteins and Fz but lack the intracellular sequence found in the Fz receptors. The sFRPs antagonize Wnt function by binding to the Wnt proteins and preventing Wnt/receptor activation. There is evidence to support the ability of sFRPs to inhibit both canonical and noncanonical Wnt signaling.(9) Overexpression of sFRP1 in human osteoblasts has been reported to accelerate the rate of osteoblast and osteocyte death.(10) Adult mice deficient in sFRP1 showed resistance to age-related bone loss.(11) The lack of sFRP1 in mice resulted in decreases in osteoblast and osteocyte apoptosis.(10) knockout mice also have reduced bone anabolic responses to parathyroid hormone (PTH) treatment.(12) In addition to osteoblastogenesis sFRP1 affects osteoclastogenesis because sFRP1 can directly bind RANKL(13) and/or inhibit the fusion of mononuclear cells (14) thereby inhibiting osteoclast formation. Despite broad tissue distribution ablation of in mice did not affect blood and urine markers of bone resorption or organ function in most nonskeletal tissues.(11) Recently a small molecule inhibitor (diarylsulfone sulfonamide) that PIK-294 can bind and inhibit sFRP1 was shown to stimulate bone formation (15) suggesting that inhibition of sFRP1 may be a potential target to stimulate Wnt signaling to increase bone PIK-294 formation. Since Wnt signaling is critical for osteoblast maturation skeletal acquisition and maintenance we hypothesized that overexpression of sFRP1 in transgenic mice (mice. Materials and Methods Generation of the mice and experimental protocol The mice used for this research project STOCK Tg(sFRP1-EGFP)142Gsat/Mmcd and identification number 011017-UCD were obtained from the Mutant Mouse BCL3 Regional Resource Center (MMRRC) a National Center for Research Resource (NCRR)-National Institutes of Health (NIH)-funded strain repository and were donated to the MMRRC by the National Institute of Neurological Disorders and Stroke (NINDS)-funded Gene Expression Nervous System ATLas (GENSAT) engineering bacterial artificial chromosomes (BAC) transgenic project. The genotype has been modified to contain multiple copies of a modified BAC in which an reporter gene is usually inserted immediately upstream of the coding sequence of the gene generating site-specific expression of green fluorescent protein (GFP) in the gene of interest (mouse was an FVB/N-Swiss Webster hybrid.(16) Mice genotypes were confirmed by a PCR protocol with a GFP R2 primer (TAGCGG CTGAAGCACTGC A). An actin primer was included in all PCR for genotyping. Electrophoresis was performed in 2% agarose gel at 90 V for 90 moments. Wild-type (WT) mice experienced one band at 1000 bp for actin whereas mice experienced two bands at 1000 bp (actin) and at about 300 bp (sFRP1-EGFP fuse protein) respectively (Fig. 1transgenic mice (Tg) PCR product gives two bands at 1 kb (with 0.95% calcium and 0.67% phosphate. Mice were housed in a room that was managed at 70 °F with a 12 hour light/dark cycle. All animals PIK-294 were treated according to the US Department of Agriculture (USDA) animal care guidelines with the approval of the UC Davis Committee on Animal Research. At 12 weeks of age both female and male WT or mice were treated subcutaneously with either vehicle [phosphate-buffered daline (PBS)] (= 10 to 11) or 40 PIK-294 μg/kg human PTH(1-34) (= 12) (Bachem Bioscience Inc. Torrance CA USA) 5×/week for 2.