Protein kinase A (PKA) is involved in bone biology and is a key mediator of parathyroid hormone signaling in the osteoblast. However, the consequences of sustained PKA activation in bone are unclear. In this study, we inducibly activated PKA in osteoblasts by deleting its major regulatory subunit, Prkar1a , using a Col1α1-driven Cre system. Prkar1a ob-/- mice demonstrated rapid and profound bone pathologies in their femurs, lumbar and caudal vertebrae with cortical bone breakdown and cortical trabecularization. This phenotype was characterized by increased bone turnover and elevated osteoblastic and osteoclastic activities. Transcriptomic and qPCR analyses showed an impairment of osteoblast differentiation with a defect in ossification, expansion of stromal cells, and numbers of both osteoblastic and osteoclastic precursors. Moreover, there were alterations in gene expression of chemokines and Wnt members with enhanced osteoclastogenesis. Altogether, activation of PKA in osteoblasts by inducible deletion of Prkar1a causes a profound high bone turnover phenotype resembling several human bone diseases.

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.

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

Transforming growth factor-beta1 (TGF-β1) stimulates matrix metalloproteinase-13 (MMP-13, a bone-remodeling gene) expression, and this effect requires p300-mediated Runx2 (Runt-related transcription factor 2) acetylation in osteoblasts. p300 and Runx2 are transcriptional coactivator and bone transcription factor, respectively, which play key roles in the regulation of bone-remodeling genes. Non-coding ribonucleic acids (ncRNAs), such as long ncRNAs (lncRNAs) and microRNAs (miRNAs), have been linked to both physiological and pathological bone states. In this study, we proposed that TGF-β1-mediated stimulation of MMP-13 expression is due to the downregulation of p300 targeting miRNAs in osteoblasts. We identified miR-130b-5p as one of the miRNAs downregulated by TGF-β1 in osteoblasts. Forced expression of miR-130b-5p decreased p300 expression, Runx2 acetylation, and MMP-13 expression in these cells. Furthermore, TGF-β1 upregulated circ_ST6GAL1, (a circular lncRNA) in osteoblasts; circRNA directly targeted miR-130b-5p. Antisense-mediated knockdown of circ_ST6GAL1 restored the function of miR-130b-5p, resulting in downregulation of p300, Runx2, and MMP-13 in these cells. Hence, our results suggest that TGF-β1 influences circ_ST6GAL1 to sponge and degrade miR-130b-5p, thereby promoting p300-mediated Runx2 acetylation for MMP-13 expression in osteoblasts. Thus, the circ_ST6GAL1/miR-130b-5p/p300 axis has potential significance in the treatment of bone and bone-related disorders.

Teriparatide (PTH(1-34)) and its analogs, PTHrP(1-36) and abaloparatide (ABL) have been used for the treatment of osteoporosis, but their efficacy over long-term use is significantly limited. The 3 peptides exert time- and dose-dependent differential responses in osteoblasts, leading us to hypothesize that they may also differentially modulate the osteoblast transcriptome. We show that treatment of mouse calvarial osteoblasts with 1 nM of the 3 peptides for 4 h results in RNA-Seq data with PTH(1-34) regulating 367 genes, including 194 unique genes; PTHrP(1-36) regulating 117 genes, including 15 unique genes; and ABL regulating 179 genes, including 20 unique genes. There were 83 genes shared among all 3 peptides. Gene ontology analyses showed differences in Wnt signaling, cAMP-mediated signaling, bone mineralization, morphogenesis of a branching structure in biological processes; receptor ligand activity, transcription factor activity, cytokine receptor/binding activity and many other actions in molecular functions. The 3 peptides increased Vdr, Cited1 and Pde10a mRNAs in a pattern similar to Rankl , i.e., PTH(1-34) > ABL > PTHrP(1-36). mRNA abundance of other genes based on gene/pathway analyses, including Wnt4, Wnt7, Wnt11, Sfrp4, Dkk1, Kcnk10, Hdac4, Epha3, Tcf7, Crem, Fzd5, Pp2r2a , and Dvl3 showed that some genes were regulated similarly by all 3 peptides; others were not. Finally, siRNA knockdowns of SIK1/2/3 and CRTC1/2/3 in PTH(1-34)-treated cells revealed that Vdr and Wnt4 genes are regulated by SIKs and CRTCs, while others are not. Although many studies have examined PTH signaling in the osteoblast/osteocyte, ours is the first to examine the global effects of these peptides on the osteoblast transcriptome. Further delineation of which signaling events are attributable to PTH(1-34), PTHrP(1-36) or ABL exclusively and which are shared among all 3 will help improve our understanding of the effects these peptides have on the osteoblast and lead to the refinement of PTH-derived treatments for osteoporosis.

ABSTRACTAbaloparatide is a peptide analog of parathyroid hormone‐related protein (PTHrP 1–34) and was approved in 2017 as the second osteoanabolic peptide for treating osteoporosis. We previously showed that intermittent abaloparatide is equally as effective as PTH (1–34). This study was designed to compare the catabolic effects of PTH (1–34) and abaloparatide on bone in young female wild‐type mice. Two‐month‐old C57Bl/6J female mice were continuously infused with human PTH (1–34) or abaloparatide at 80 μg/kg BW/day or vehicle for 2 weeks. At euthanasia, DEXA‐PIXImus was performed to assess bone mineral density (BMD) in the whole body, femurs, tibiae, and vertebrae. Bone turnover marker levels were measured in sera, femurs were harvested for micro–computer tomography (μCT) analyses and histomorphometry, and tibiae were separated into cortical and trabecular fractions for gene expression analyses. Our results demonstrated that the infusion of abaloparatide resulted in a similar decrease in BMD as infused PTH (1–34) at all sites. μCT and histomorphometry analyses showed similar decreases in cortical bone thickness and BMD associated with an increase in bone turnover from the increased bone formation rate found by in vivo double labeling and serum P1NP and increased bone resorption as shown by osteoclast numbers and serum cross‐linked C‐telopeptide. Trabecular bone did not show major changes with either treatment. Osteoblastic gene expression analyses of trabecular and cortical bone revealed that infusion of PTH (1–34) or abaloparatide led to similar and different actions in genes of osteoblast differentiation and activity. As with intermittent and in vitro treatment, both infused PTH (1–34) and abaloparatide similarly regulated downstream genes of the PTHR1/SIK/HDAC4 pathway such as Sost and Mmp13 but differed for those of the PTHR1/SIK/CRTC pathway. Taken together, at the same dose, infused abaloparatide causes the same high bone turnover as infused PTH (1–34) with a net resorption in female wild‐type mice. © 2023 The Authors. JBMR Plus published by Wiley Periodicals LLC on behalf of American Society for Bone and Mineral Research.

Introduction: Parathyroid hormone (PTH) plays an important role in maintaining mineral homeostasis by regu-lating calcium and phosphate levels. Clinical trials have shown that peptides of PTH (1-34), PTH-related protein (PTHrP 1-36), and the new peptide modeled on PTHrP, abaloparatide, can have different anabolic effects on osteoporotic subjects, but the underlying mechanisms are still unclear. The prevalence of moderate and major gingival recession has been shown to be higher in postmenopausal women with osteoporosis. In addition, there is a significant association between osteoporosis and tooth loss. Methods: We investigated the actions of these peptides on the cementoblasts and teeth of mice. The murine cementoblast line, OCCM-30, known to express collagen I (Col1a1), was treated with intermittent PTH (1-34), PTHrP (1-36), or abaloparatide for 6 h/d for 3 days. Microcomputed tomography was performed on the teeth of mice receiving daily injections of phosphate-buffered saline, PTH (1-34), or abaloparatide. Statistical differences were analyzed by a 2-way or 1-way analysis of variance followed by a Tukey's post-hoc test. Results are expressed as mean 6 standard deviation, and P \0.05 was considered significant. Results: Gene expression showed regulation of Bsp, Col1a1, Opg, Rankl, and Mmp13 by the 3 peptides in these cells. Western blots re-vealed that after intermittent treatment for 3 days, PTH (1-34) caused an increase in COL1A1 protein immedi-ately after treatment. In contrast, abaloparatide showed a latent effect in increasing COL1A1 protein 18 hours after treatment. PTHrP had no effect on COL1A1 expression. Immunofluorescence confirmed the same result as the Western blots. Microcomputed tomography of teeth showed PTH (1-34) injections increased molar root mineral density in mice, whereas abaloparatide increased density in roots of incisors and molars. Conclusions: This study reveals the differential anabolic effects of intermittent PTH (1-34), PTHrP (1-36), and abaloparatide on cementoblasts, as revealed by COL1A1 expression and root mineral density. Abalopara-tide may be a potential therapeutic approach for achieving improved cementogenesis. (Am J Orthod Dentofacial Orthop 2023;163:378-89)

Delivering the parathyroid hormone (PTH) gene has been attempted preclinically in a handful of studies, but delivering full-length PTH (1-84) using adeno-associated viral (AAV) vectors has not. Given the difficulty in achieving therapeutic levels of secreted proteins using gene therapy, this study seeks to determine the feasibility of doing so with PTH. An AAV vector was used to deliver human PTH driven by a strong promoter. We demonstrate the ability to secrete full-length PTH from various cell types in vitro. PTH secretion from hepatocytes was measured over time and a fluorescent marker was used to compare the secretion rate of PTH in various cell types. Potency was measured by the ability of PTH to act on the PTH receptors of osteosarcoma cells and induced proliferation. PTH showed potency in vitro by inducing proliferation in two osteosarcoma cell lines. In vivo, AAV was administered systemically in immunocompromised mice which received xenografts of osteosarcoma cells. Animals that received the highest dose of AAV-PTH had higher liver and plasma concentrations of PTH. All dosing groups achieved measurable plasma concentrations of human PTH that were above the normal range. The high-dose group also had significantly larger tumors compared to control groups on the final day of the study. The tumors also showed dose-dependent differences in morphology. When looking at endocrine signaling and endogenous bone turnover, we observed a significant difference in tibial growth plate width in animals that received the high-dose AAV as well as dose-dependent changes in blood biomarkers related to PTH. This proof-of-concept study shows promise for further exploration of an AAV gene therapy to deliver full-length PTH for hypoparathyroidism. Additional investigation will determine efficacy in a disease model, but data shown establish bioactivity in well-established models of osteosarcoma.

Bone is a dynamic tissue which is constantly renewed by a precise balance between osteoblastic bone formation and osteoclastic bone resorption. This process is important for normal bone mass and strength, and for mineral homeostasis. Bone remodeling is stringently regulated by communication between bone cells such as osteoclasts, osteoblasts and osteocytes and by systemic factors. An imbalance can result in bone diseases including osteoporosis. Resorption by osteoclasts precedes bone formation by osteoblasts. The osteocytes, within the bone matrix, contribute to the control of bone remodeling. In this article, we present the current knowledge of the structure, characteristics and functions of bone cells.

Abstract Osteoporosis is a prevalent disease with substantial morbidity/mortality among the aging population. Due to gaps in knowledge, current therapeutics are limited in their ability to prevent degeneration of bone while also stimulating formation of new bone. Teriparatide (PTH (1-34)) and its analogs PTHrP (1-36) and abaloparatide (ABL), have been utilized for treatment of osteoporosis but have significant limitations in efficacy over long-term use. Research from our laboratory has shown PTH (1-34), PTHrP (1-36), and ABL exert time and dose-dependent differential responses in the osteoblast, leading us to hypothesize they may also differentially modulate the osteoblast transcriptome. In this study we show that treatment of mouse calvarial osteoblasts with 1 nM of these peptides for 4 h results in differing effects on the osteoblast transcriptome by performing gene enrichment analysis of RNA-Seq data. Genes were selected with a Log2 fold change >1 and a false discovery rate <0.05. These data were analyzed and compiled into heat maps for each peptide and smear/volcano plots. RNA-Sequencing revealed that PTH (1-34) regulated 367 genes, 194 were unique; PTHrP (1-36) regulated 117 genes, 15 were unique; ABL regulated 179 genes, 20 were unique. There were 74 genes shared only among PTH(1-34) and ABL; 16 genes shared only among PTH (1-34) and PTHrP; and 83 genes shared among all three peptides. Data collected show pathway-specific differences including, 1) cAMP/PKA, 2) Wnt/β-catenin, 3) Transcriptional regulation 4) Inflammatory response 5) Transmembrane transport, 6) Metabolism, and 7) NF-kB. Further analysis of the data illuminated that the three peptides increased Vitamin D receptor (VDR) and Cbp/p300-interacting transactivator 1 (CITED1) mRNAs similar to RankL expression. These findings were confirmed via qRT-PCR of additional cultured samples of mouse calvarial osteoblasts, treated with 1 nM of PTH (1-34), PTHrP (1-36), and ABL for 4 h prior to harvest. RankL mRNA, VDR mRNA, and CITED1 mRNA were measured for each sample and statistical differences were analyzed via Kruskal Wallis p<0.05. Additionally, we analyzed mRNA levels of several genes of interest, including WNT7b, WNT11, TCF7, SFRP4, FZD5, PP2R2A, and DVL3 mRNA. Pathway analysis and subsequent qRT-PCR confirmation has shown that PTH (1-34) and ABL lead to a significant increase in Wnt11 mRNA, while PTHrP (1-36) does not. Our findings highlight the complexity of the genetic and functional events triggered by PTH (1-34) and its analogs. Many studies have examined PTH signaling in the osteoblast/osteocyte; ours is the first to examine global effects of these peptides on the osteoblast transcriptome. Further delineation of which signaling events are attributable to PTH (1-34), PTHrP (1-36), and ABL exclusively and which are shared among all three will further our understanding of the effects these peptides have on the osteoblast and lead to refinement of PTH-derived treatments for osteoporosis. Presentation: Monday, June 13, 2022 11:45 a.m. - 12:00 p.m.