Human pth 1 34

Four-day old CaSR pups were injected with either 50 μg/kg/d human PTH (1–34) (Bachem, Torrance, CA) or vehicle (saline) subcutaneously for 17 days, as previously described [30]. Mouse weight was measured daily to determine the injection dose. Animals were sacrificed one day after the last injection (day 18), and blood and bones were collected. Bones were imaged using x-ray radiography after fixation.

Mice heterozygous for Pappa (C57BL/6, 129) were bred as previously described to produce wild-type (WT) and PAPP-A knock-out (KO) littermates for these experiments [16 (link)]. Genotypes were determined before assignment to groups and confirmed at harvest. Three-month-old female WT and PAPP-A KO mice (n = 10 per group) were treated with human PTH 1-34 (Bachem Americas), at a dose of 80 μg/kg, or with vehicle (0.9% acidified saline with 2% heat-inactivated WT mouse serum) by subcutaneous injection five days per week for six weeks. This study was approved by the Institutional Animal Care and Use Committee of Mayo Clinic.

Human PTH(1–34), human PTH(1–31), and bovine PTH(3–34) were purchased from Bachem, reconstituted at 100 µM stock, aliquoted and frozen at −80 °C. The Rho inhibitor exoenzyme C3 transferase and the Rho Activator II (CN-03) were each purchased from Cytoskeleton. 8-bromo-cAMP was from Calbiochem, and geldanamycin was from Sigma.

Lrp6^f/f mice were obtained from Van Andel Research Institute. ^{60 (link), 61 (link)} Transgenic mice expressing the Cre recombinase under the control of a 3.9-kb fragment of the human osteocalcin promoter (OC-Cre) were obtained from T. Clemens (Baltimore, MD).^{62 (link)} The generation of homozygous deletion Cre^+/−;Lrp6^f/f mice (LRP6-KO hereafter) and control Cre^−/−;Lrp6^f/f (WT hereafter) were described previously.^{23 (link)} Mice were maintained on a mixed background of C57Bl/6J, 129, and FVB/N. All animals were maintained in the Animal Facility of the Johns Hopkins University School of Medicine. The experimental protocol was reviewed and approved by the Institutional Animal Care and Use Committee of the Johns Hopkins University, Baltimore, MD. Genomic DNA extraction and genotyping of the animals were prepared as described previously.^{23 (link)} Primers for Cre recombinase and the loxP sites were used for PCR. For PTH treatment, two month-old male WT mice and KO mice were randomized into four groups: WT-vehicle, WT-PTH, KO-vehicle, and KO-PTH. Six mice of each treatment group were used. Mice were subcutaneously injected with either Human PTH1–34 or vehicle (1 mM acetic acid in phosphate buffered saline (PBS)) at a dosage of 80 μg/kg daily, five days per week, for 4 weeks. Human PTH1–34 was purchased from Bachem Bioscience Inc. (King of Prussia, PA).