PSAT158 Dimeric [Cys25]PTH(1-34) and Monomeric PTH(1-34) Differentially Regulate the Expression of Genes Related to Calcium Homeostasis in Bone and Kidney

Abstract

Abstract Osteoporosis and its resulting fractures threaten health and life of many patients worldwide; therefore, the development of effective and safe drugs for osteoporosis is an urgent need. In our previous report, dimeric [Cys25]PTH(1-34) peptide with arginine-to-cysteine mutation at position 25 (Dimer) showed a substantial osteo-anabolic effect similar or superior to that of PTH(1-34) in several mouse models. Surprisingly, cAMP was not elevated in serum or urine the Dimer-treated group unlike in the PTH(1-34)-treated group. From this, we hypothesized that the Dimer stimulates anabolic responses through a signaling pathway different from the cAMP-PKA pathway. To test this hypothesis, we assessed responses down-stream of the activated signal cascade from PTHR1 using GPCR Phospho Antibody Array in cells, as well as the expression levels of mRNAs and proteins related to calcium metabolism in bones and kidneys of mice. Cell lysates obtained from GP2.3 cells (treated with PTH(1-34) and Dimer with PBS as a control) were applied to the GPCR-MAPK Pathway Phosphorylation Antibody Array (Full Moon BioSystems, USA), containing 193 antibodies. Each of the antibodies has 6 replicates that are printed on standard-size coated glass microscope slides. The results of the Phospho-antibody array were further confirmed by Western Blot assay. Eighteen CD1 mice of 9 WO were divided into three groups: control, PTH(1-34)-treated, and Dimer-treated groups. 40 µg/kg of PTH(1-34) and 80 µg/kg of the Dimer were subcutaneously injected for each of the experimental groups, respectively. Then, two hours after the injection, the animals were sacrificed, and their kidneys and tibiae were harvested. Total mRNA samples from kidneys and tibiae were extracted using trizol reagent. Protein samples were extracted via protein extraction buffer. We estimated mRNA and protein levels of biomarkers related to calcium regulation via RT-PCR and ELISA with harvested bone and kidney tissues. Several changes of phosphorylation of signaling proteins in GP2.3 cells were detected between Dimer-treated group and PTH(1-34)-treated group. Expression of mRNA and protein levels of RANKL was lower in the Dimer-treated group compared to the PTH(1-34)-treated group. In contrast, expression of mRNA level of OPG in tibia samples was lower in the Dimer-treated group. In kidney, the mRNA and protein levels of 1α(OH)ase in the Dimer-treated group were lower than in the PTH(1-34)-treated group. However, mRNA level of 24(OH)ase was higher in the Dimer-treated group than in the PTH(1-34)-treated group. Other parameters were not significantly different between the Dimer-treated and PTH(1-34)-treated groups. We speculate that the osteoanabolic effect of Dimer uses a signaling pathway different from the cAMP-PKA pathway, regulating different expression parameters related to calcium metabolism in both the bone and kidney. Further studies are required to define exact signaling pathway that Dimer acts on. Presentation: Saturday, June 11, 2022 1:00 p.m. - 3:00 p.m.