PTH stimulation of Rankl transcription is regulated by SIK2 and 3 and mediated by CRTC2 and 3 through action of protein phosphatases 1, 2, 4, and 5.

Osteoporosis is characterized by a decrease in the density and quality of bone tissue and is associated with substantial morbidity/mortality. Homeostatic processes that form new and remove old/damaged bone are dysregulated, with resultant net bone resorption. Parathyroid hormone (PTH) is a key regulator of this homeostasis and along with its analogs has been used to treat osteoporosis, however its use is limited to an "anabolic window". PTH stimulates both formation and resorption, the latter largely due to increased receptor activator of nuclear factor kappa-β ligand (RANKL). Our laboratory has found a cascade of messengers, Salt-inducible kinases (SIKs) and protein phosphatases (PPs), regulate nuclear translocation of CREB-regulated transcriptional coactivators (CRTCs), but the individual and/or combined contributions of these factors has not yet been established in osteoblasts. In this study, we reveal precise mechanisms involved in CRTC1/2/3 nuclear translocation and delineate their roles as co-activators of Tnfsf11 (RANKL gene name) transcription throughout osteoblast differentiation using a primary mouse calvarial osteoblast model. By performing a series of siRNA knockdowns of CRTC1/2/3, SIK1/2/3, and PP1/2/3/4/5/6/7, we determined the regulation of CRTCs upon PTH-stimulation via qPCR, quantitative immunofluorescence, Western blotting, and co-immunoprecipitation. CRTC2 is determined to be the primary co-activator of Tnfsf11 transcription with SIK2/3 inhibition upon PTH-stimulation making CRTC2 available for nuclear translocation by PP1/2/4/5 action. Understanding the mechanisms involved in this cascade may reveal novel targets in the treatment of osteoporosis and allow researchers a new line of approach for drug design that could overcome the "anabolic window" limiting current PTH-derived treatments.