Faculty Bibliography

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.

Parathyroid hormone (PTH) was the first osteoanabolic hormone for treating osteoporosis. We have previously shown that PTH acts through PTHR1 and protein kinase A (PKA) activation to regulate osteoblastic gene expression. Our study aimed to elucidate the effects of increased PKA activity and better understand the actions of PTH (1-34) in bone. Tamoxifen (1mg/10g) was injected weekly to 1 or 5 month-old C57Bl/6J male col1CRE^ERT/Prkar1a^fl/fl mice or Prkar1a^fl/fl mice as controls for 3-4 weeks to delete the PKA regulatory subunit 1A in osteoblasts and increase PKA activity. At both ages, col1CRE^ERT/Prkar1a^fl/fl mice demonstrated bone pathologies in their skulls, femurs and vertebrae and tumors in their tails (Figure 1). MicroCT showed cortical bone breakdown with apparent trabecular bone in the cortical area in femurs and vertebrae and expansion of bone in skulls. Deletion of Prkar1a increased bone turnover with a huge increase in osteoblast activity shown by serum-P1NP levels (6.5-13 fold), only single fluorescent labeling and a substantial increase in osteoclast activity shown by CTX levels (4.4-12 fold) and TRAP staining. Surprisingly, the col1CRE^ERT/Prkar1a^fl/fl skulls showed thicker calvariae, shown by alizarin red staining and muCT but no changes in mandibles or teeth. Col1CRE^ERT/Prkar1a^fl/fl mice had tumors in their tails evident by an invasion of stromal and osteoclastic cells but with intact growth plate, cartilage and intervertebral discs. In conclusion, high PKA activity in osteoblasts appears to be involved with high bone turnover and pathological events mimicking hyperparathyroidism, Jansen's metaphyseal chondrodysplasia or McCune-Albright syndrome. [Formula presented]
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Abaloparatide, a novel analog of parathyroid hormone-related protein (PTHrP 1-34) became, in 2017, the second osteoanabolic therapy for the treatment of osteoporosis. This study aims to compare the effects of PTH (1-34), PTHrP (1-36), and abaloparatide on bone remodeling in male mice. Intermittent daily subcutaneous injections of 80 μg/kg/day were administered to four-month-old C57Bl/6J male mice for six weeks. During treatment, mice were followed by DEXA-Piximus to assess changes in bone mineral density (BMD) in the whole body, femur and tibia. At either four or eighteen hours after the final injection, femurs were harvested for μCT analyses and histomorphometry, sera were assayed for bone turnover marker levels, and tibiae were separated into cortical, trabecular, and bone marrow fractions for gene expression analyses. Our results showed that, compared with PTH (1-34), abaloparatide resulted in a similar increase in BMD at all sites, while no changes were seen with PTHrP (1-36). With both PTH (1-34) and abaloparatide, μCT and histomorphometry analyses revealed similar increases in bone volume associated with an increased trabecular thickness, in bone formation rate as shown by P1NP serum level and in vivo double labeling, and in bone resorption as shown by CTX levels and osteoclast number. Gene expression analyses of trabecular and cortical bone showed that PTH (1-34) and abaloparatide led to different actions in osteoblast differentiation and activity, with increased Runx2, Col1A1, Alpl, Bsp, Ocn, Sost, Rankl/Opg and c-fos at different time points. Abaloparatide seems to generate a faster response on osteoblastic gene expression than PTH (1-34). Taken together, abaloparatide at the same dose is as effective as PTH (1-34) as an osteoanabolic, with an increase in bone formation but also an increase in bone resorption in male mice. This article is protected by copyright. All rights reserved.

Parathyroid hormone (PTH) plays a central role in regulation of calcium metabolism and is an osteoanabolic treatment for osteoporosis. We hypothesized that similar to PTH (1-34) treatment, an increase in PKA activity in osteoblasts will cause an increase in bone accrual. Our study aims to elucidate the effects of increased PKA activity in bone. Weekly injections of tamoxifen were administered to 1 month-old or 5 month-old C57Bl/6 male and female col1CRE^ERT/Prkar1a^fl/fl mice, or Prkar1a^fl/fl mice as controls, for 3-4 weeks to delete the PKA regulatory subunit 1A and increase PKA activity. This resulted in a decrease of whole body (-6%), femoral (-24%), and tibial BMD (-22%) in 2 month-old col1CRE^ERT/Prkar1a^fl/fl mice. lCT showed dramatic excess trabecular area and disappearance of cortical bone in vertebrae and femurs. Surprisingly, 6 month-old mice developed tumors in their tails. By lCT, at both ages, col1CRE^ERT/Prkar1a^fl/fl mice showed decreases in cortical thickness and cortical BMD and increases in cortical porosity. Only 2 month-old col1CRE^ERT/Prkar1a^fl/fl mice showed drastically decreased BV/TV (-64%), Tb.N (-49%) and trabecular BMD(-30%). At both ages, deletion of Prkar1a dramatically increased bone turnover with a huge increase in osteoblast activity shown by serum-P1NP levels (6.5-to 13-fold) only single fluorescent labeling and a substantial increase in osteoclast activity shown by CTX levels (4.4-to 12-fold) and TRAP staining. In both age groups, cortical and trabecular bone showedasubstantial increaseinbone sialoprotein mRNA levels (3-to 6-fold) with a shutdown in osteocalcin mRNA (0.2-to 0.4-fold). Furthermore, PTH-regulated genes were also significantly changed: SOST expression (0.1-to 0.2-fold), RANKL (2-to 3-fold) and MMP13 ([3fold). The overall data showagreatincreaseintrabecular bone mass with breakdown of cortical bone. In conclusion, high PKA activity in osteoblasts appears to be involved in increasing immature trabecular bone and resorbing cortical bone and mimics hyperparathyroidism