Faculty Bibliography

INTRODUCTION/BACKGROUND:Parathyroid hormone (PTH) plays an important role in maintaining mineral homeostasis by regulating 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 ± 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 revealed that after intermittent treatment for 3 days, PTH (1-34) caused an increase in COL1A1 protein immediately 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. Abaloparatide may be a potential therapeutic approach for achieving improved cementogenesis.

Abaloparatide 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.

TGF-β1 (transforming growth factor-beta1), a secreted polypeptide cytokine, stimulates ATF-3 (activating transcription factor-3) expression in a sustained and prolonged manner in human breast cancer cells (MDA-MB231), but not in normal human mammary epithelial cells (MCF-10A). Cyclin A (cell proliferation gene), Runx2 (metastasis gene), and MMP-13 (matrix metalloproteinase-13; invasive gene) were identified as ATF-3 target genes in these cells. Because ATF-3 has very few druggable sites, its direct targeting is difficult. Recent evidence has indicated that microRNAs (miRNAs) are key players in the post-transcriptional modulation of gene expression under several conditions. Bioinformatic analysis suggested a list of putative miRNAs that target ATF-3. Therefore, we hypothesized that TGF-β1 downregulates the miRNAs that target ATF-3, resulting in the activation of genes that participate in breast cancer progression and skeletal metastasis. Our findings indicate that TGF-β1 downregulated the expression of miR-4638-3p in MDA-MB231 cells. At the molecular level, forced expression of miR-4638-3p reduced the expression of ATF-3 and its downstream targets, Runx2 and MMP-13, in these cells. At the cellular level, overexpression of miR-4638-3p reduced proliferation, invasion, and migration, and induced G0/G1 cell cycle arrest and apoptosis in MDA-MB231 cells. Overall, this study highlights the possibility of utilizing miR-4638-3p as a therapeutic molecule to curb skeletal metastasis of breast cancer cells.

A paper by Sun et al. identified the Ca²⁺-activated Cl^- channel anoctamin 1 or ANO1 (TMEM16A) as an important regulator of osteoclast function by interacting with RANKL activating signaling pathways involved in bone resorption. Although Cl^- transporters (e.g. ClC7, CLIC5) have been known to be involved in the active process of bone resorption, ANO1 appears to control osteoclast differentiation and function to levels beyond those of other Cl^-transporters. Regulating ANO1 function might be a useful target for therapeutics in osteoporosis.

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.

Transforming growth factor beta 1 (TGF-β1) functions as a coupling factor between bone development and resorption. Matrix metalloproteinase 13 (MMP13) is important in bone remodeling, and skeletal dysplasia is caused by a deficiency in MMP13 expre-ssion. Runx2, a transcription factor is essential for bone development, and MMP13 is one of its target genes. TGF-β1 promoted Runx2 phosphorylation, which was necessary for MMP13 production in osteoblastic cells, as we previously shown. Since the phosphorylation of some proteins causes them to be degraded by the ubiquitin/proteasome pathway, we hypothesized that TGF-β1 might stabilize the phosphorylated Runx2 protein for its activity by other post-translational modification (PTM). This study demonstrated that TGF-β1-stimulated Runx2 acetylation in rat osteoblastic cells. p300, a histone acetyltransferase interacted with Runx2, and it promoted Runx2 acetylation upon TGF-β1-treatment in these cells. Knockdown of p300 decreased the TGF-β1-stimulated Runx2 acetylation and MMP13 expression in rat osteoblastic cells. TGF-β1-treatment stimulated the acetylated Runx2 bound at the MMP13 promoter, and knockdown of p300 reduced this effect in these cells. Overall, our studies identified the transcriptional regulation of MMP13 by TGF-β1 via Runx2 acetylation in rat osteoblastic cells, and these findings contribute to the knowledge of events presiding bone metabolism.

Matrix-metalloproteinase-13 (MMP13) is important for bone formation and remodeling; however, its role in tooth development remains unknown. To investigate this, MMP13-knockout (Mmp13

Parathyroid hormone (PTH) is necessary for the regulation of calcium homeostasis and PTH (1-34) was the first approved osteoanabolic therapy for osteoporosis. It is well established that intermittent PTH increases bone formation and that bone remodeling and several cytokines and chemokines play an essential role in this process. Earlier, we had established that the chemokine, monocyte chemoattractant protein-1 (MCP-1/CCL2), was the most highly stimulated gene in rat bone after intermittent PTH injections. Nevertheless, MCP-1 function in bone appears to be complicated. To identify the primary cells expressing MCP-1 in response to PTH, we performed in situ hybridization of rat bone sections after hPTH (1-34) injections and showed that bone-lining osteoblasts are the primary cells that express MCP-1 after PTH treatment. We previously demonstrated MCP-1's importance by showing that PTH's anabolic effects are abolished in MCP-1 null mice, further implicating a role for the chemokine in this process. To establish whether rhMCP-1 peptide treatment could rescue the anabolic effect of PTH in MCP-1 null mice, we treated 4-month-old wild-type (WT) mice with hPTH (1-34) and MCP-1-/- mice with rhMCP-1 and/or hPTH (1-34) for 6 weeks. Micro-computed tomography (μCT) analysis of trabecular and cortical bone showed that MCP-1 injections for 6 weeks rescued the PTH anabolic effect in MCP-1-/- mice. In fact, the combination of rhMCP-1 and hPTH (1-34) has a synergistic anabolic effect compared with monotherapies. Mechanistically, PTH-enhanced transforming growth factor-β (TGF-β) signaling is abolished in the absence of MCP-1, while MCP-1 peptide treatment restores TGF-β signaling in the bone marrow. Here, we have shown that PTH regulates the transcription of the chemokine MCP-1 in osteoblasts and determined how MCP-1 affects bone cell function in PTH's anabolic actions. Taken together, our current work indicates that intermittent PTH stimulates osteoblastic secretion of MCP-1, which leads to increased TGF-β signaling, implicating it in PTH's anabolic actions.

Matrix metalloproteinase-13 (MMP-13) plays a predominant role in endochondral bone formation and bone remodeling. Parathyroid hormone (PTH) stimulates the expression of MMP-13 via Runx2, a bone transcription factor in rat osteoblastic cells (UMR106-01), and histone deacetylase 4 (HDAC4) acts as a corepressor of Runx2. Moreover, microRNAs (miRNAs) play an important role in regulating genes posttranscriptionally. Here, we hypothesized that PTH upregulates the miRNAs targeting HDAC4, which could lead to increased Runx2 activity and MMP-13 expression in rat osteoblastic cells. We identified several miRNAs that putatively target rat HDAC4 using bioinformatics tools. miR-873-3p was significantly upregulated by PTH in rat osteoblasts. miR-873-3p overexpression downregulated HDAC4 protein expression, increased Runx2 binding at the MMP-13 promoter, and increased MMP-13 messenger RNA expression in UMR106-01 cells. A luciferase reporter assay identified the direct targeting of miR-873-3p at the 3'-untranslated region of HDAC4. Thus, miR-873-3p targeted HDAC4 and relieved the corepressor effect of HDAC4 on Runx2 for MMP-13 expression in rat osteoblasts. This study advances our knowledge of posttranscriptional gene regulation occurring in bone and bone-related diseases and clarifies the role of miRNAs as diagnostic biomarkers.