SUN-218 Parathyroid Hormone Stimulates Rankl Expression Through Nuclear Translocation of CRTC2 and 3 in Mouse Differentiating Osteoblasts but Not in a Rat Osteosarcoma Line

Abstract Disclosure: N.C. Partridge: None. J. Joseph: None. W. Petrosky: None. C. Le henaff: None. Parathyroid hormone (PTH) is a regulator of bone homeostasis and is used to treat osteoporosis, however, prolonged treatment leads to excessive bone resorption through receptor activator of nuclear factor kappa-Β ligand (RANKL). PTH and its analogs act through increased cAMP to activate protein kinase A (PKA). This enzyme controls gene expression in part through inhibition of salt-inducible kinases (SIKs). Current models suggest nuclear translocation of CREB-regulated transcription coactivators (CRTC1/2/3) are responsible for Rankl transcription in osteoblasts after PTH-treatment. SIKs phosphorylate CRTCs preventing their translocation to the nucleus. To determine the role of CRTCs in Rankl transcription in these cells, primary mouse calvarial osteoblasts were differentiated in culture or the rat osteosarcoma cell line, UMR 106-01, was plated, the cells treated with PTH and qPCR, quantitative immunofluorescence and ELISAs performed. We have previously found that CRTC2 is the primary co-activator with CRTC3 acting as a compensatory factor in undifferentiated and early-stage osteoblasts, with CRTC2 and 3 being equally important in late stage (mineralized) osteoblasts. CRTC1 was not involved at any stage. Here, we found that PTH (rat 1-34,10-8 M) increased nuclear translocation of CRTC2 and 3 in a time-dependent manner in the differentiated mouse osteoblasts, with 22% of nuclei showing immunofluorescence at 30 min for CRTC2, 88% at 45 min, and 82% at 60 min. In contrast, CRTC3 showed 91% at all three times after PTH treatment. Control levels varied from 0-13%. No translocation into the nucleus of either CRTC2 or 3 occurred in the UMR 106-01 cells after PTH treatment. Measurement of Rankl mRNA abundance showed that PTH stimulated its expression by 15-20-fold at 4 h in the differentiated mouse osteoblasts while there was only a 2-3-fold stimulation in the UMR cells. In contrast, the UMR cells showed a 20-30-fold stimulation by PTH of Mmp13 expression and an 80-90% decrease in Sost expression (an alternative pathway of SIK action through HDAC4/5). ELISA of RANKL proteins showed no PTH stimulation after 24-72 h of the intracellular or secreted proteins in the differentiated mouse osteoblasts suggesting that PTH regulation of this transmembrane protein is extremely complex, while its transcription is regulated by CRTC2 and 3 in these cells. The lack of translocation of CRTCs into the nucleus of the rat osteosarcoma cells explains why there is so little stimulation of Rankl in these cells while transcription of genes regulated by HDAC4/5, Mmp13 and Sost, are highly modified. As a result, the normal differentiated mouse osteoblasts likely serve as a better model for PTH action that more closely mimics the in vivo situation than the osteosarcoma cells, although the latter have given us data to help understand PTH signaling. Presentation: Sunday, July 13, 2025