Differential effects of parathyroid hormone, parathyroid hormone-related protein, and abaloparatide on collagen 1 expression by mouse cementoblasts and mouse tooth root density.

INTRODUCTION: 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.

Regulation of transforming growth factor-ß1-stimulation of Runx2 acetylation for matrix metalloproteinase 13 expression in osteoblastic cells.

Transforming growth factor beta 1 (TGF-ß1) 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-ß1 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-ß1 might stabilize the phosphorylated Runx2 protein for its activity by other post-translational modification (PTM). This study demonstrated that TGF-ß1-stimulated Runx2 acetylation in rat osteoblastic cells. p300, a histone acetyltransferase interacted with Runx2, and it promoted Runx2 acetylation upon TGF-ß1-treatment in these cells. Knockdown of p300 decreased the TGF-ß1-stimulated Runx2 acetylation and MMP13 expression in rat osteoblastic cells. TGF-ß1-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-ß1 via Runx2 acetylation in rat osteoblastic cells, and these findings contribute to the knowledge of events presiding bone metabolism.

miR-873-3p targets HDAC4 to stimulate matrix metalloproteinase-13 expression upon parathyroid hormone exposure in rat osteoblasts.

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(1-34) and its analogs differentially modulate osteoblastic Rankl expression via PKA/SIK2/SIK3 and PP1/PP2A-CRTC3 signaling.

Osteoporosis can result from the loss of sex hormones and/or aging. Abaloparatide (ABL), an analog of parathyroid hormone-related protein (PTHrP(1-36)), is the second osteoanabolic therapy approved by the United States Food and Drug Administration after teriparatide (PTH(1-34)). All three peptides bind PTH/PTHrP receptor type 1 (PTHR1), but the effects of PTHrP(1-36) or ABL in the osteoblast remain unclear. We show that, in primary calvarial osteoblasts, PTH(1-34) promotes a more robust cAMP response than PTHrP(1-36) and ABL and causes a greater activation of protein kinase A (PKA) and cAMP response element-binding protein (CREB). All three peptides similarly inhibited sclerostin (Sost). Interestingly, the three peptides differentially modulated two other PKA target genes, c-Fos and receptor activator of NF-κB ligand (Rankl), and the latter both in vitro and in vivo Knockdown of salt-inducible kinases (SIKs) 2 and 3 and CREB-regulated transcription coactivator 3 (CRTC3), indicated that all three are part of the pathway that regulates osteoblastic Rankl expression. We also show that the peptides differentially regulate the nuclear localization of CRTC2 and CRTC3, and that this correlates with PKA activation. Moreover, inhibition of protein phosphatases 1 and 2A (PP1/PP2A) activity revealed that they play a major role in both PTH-induced Rankl expression and the effects of PTH(1-34) on CRTC3 localization. In summary, in the osteoblast, the effects of PTH(1-34), PTHrP(1-36), and ABL on Rankl are mediated by differential stimulation of cAMP/PKA signaling and by their downstream effects on SIK2 and -3, PP1/PP2A, and CRTC3.

Characterization of Runx2 phosphorylation sites required for TGF-ß1-mediated stimulation of matrix metalloproteinase-13 expression in osteoblastic cells.

Transforming growth factor-beta1 (TGF-ß1), a highly abundant growth factor in skeletal tissues, stimulates matrix metalloproteinase-13 (MMP-13) expression in osteoblastic cells. MMP-13 plays a critical role in bone remodeling. Runx2, a bone transcription factor, is required for TGF-ß1-mediated stimulation of MMP-13 expression in osteoblastic cells. In this study, the molecular mechanism responsible for TGF-ß1-stimulation of MMP-13 expression via Runx2 in osteoblastic cells was elucidated. TGF-ß1 stimulated the phosphorylation of Runx2 at serine amino acids, and ERK inhibition blocked this effect in rat (UMR106-01) and human (MG-63) osteoblastic cells. Pretreatment with okadaic acid, a serine-threonine phosphatase inhibitor, increased Runx2 serine phosphorylation in osteoblastic cells. When cells were pretreated with an ERK inhibitor, TGF-ß1-mediated stimulation of MMP-13 mRNA expression decreased. Nano-ESI/LC/MS analysis identified that TGF-ß1 stimulates Runx2 phosphorylation at three serine amino acids. Transient transfection of mouse mesenchymal stem cells (C3H10T1/2) with Runx2 serine mutant constructs decreased TGF-ß1-mediated Runx2 serine phosphorylation. A luciferase reporter assay identified that TGF-ß1 stimulated MMP-13 promoter activity in these cells only in the presence of the wild Runx2 construct, and not with mutant Runx2. Thus, TGF-ß1 stimulates the phosphorylation of Runx2 at three serine amino acids, and this event is required for MMP-13 expression in osteoblastic cells. Hence, this study contributes to the knowledge of events governing bone remodeling and bone-related diseases.

Physiological Bone Remodeling: Systemic Regulation and Growth Factor Involvement.

Bone remodeling is essential for adult bone homeostasis. It comprises two phases: bone formation and resorption. The balance between the two phases is crucial for sustaining bone mass and systemic mineral homeostasis. This review highlights recent work on physiological bone remodeling and discusses our knowledge of how systemic and growth factors regulate this process.

DMP-1-mediated Ghr gene recombination compromises skeletal development and impairs skeletal response to intermittent PTH.

Bone minerals are acquired during growth and are key determinants of adult skeletal health. During puberty, the serum levels of growth hormone (GH) and its downstream effector IGF-1 increase and play critical roles in bone acquisition. The goal of the current study was to determine how bone cells integrate signals from the GH/IGF-1 to enhance skeletal mineralization and strength during pubertal growth. Osteocytes, the most abundant bone cells, were shown to orchestrate bone modeling during growth. We used dentin matrix protein (Dmp)-1-mediated Ghr knockout (DMP-GHRKO) mice to address the role of the GH/IGF axis in osteocytes. We found that DMP-GHRKO did not affect linear growth but compromised overall bone accrual. DMP-GHRKO mice exhibited reduced serum inorganic phosphate and parathyroid hormone (PTH) levels and decreased bone formation indices and were associated with an impaired response to intermittent PTH treatment. Using an osteocyte-like cell line along with in vivo studies, we found that PTH sensitized the response of bone to GH by increasing Janus kinase-2 and IGF-1R protein levels. We concluded that endogenously secreted PTH and GHR signaling in bone are necessary to establish radial bone growth and optimize mineral acquisition during growth.

Sirtuin 1 is a negative regulator of parathyroid hormone stimulation of matrix metalloproteinase 13 expression in osteoblastic cells: role of sirtuin 1 in the action of PTH on osteoblasts.

Parathyroid hormone (PTH) is the only current anabolic treatment for osteoporosis in the United States. PTH stimulates expression of matrix metalloproteinase 13 (MMP13) in bone. Sirtuin 1 (SIRT1), an NAD-dependent deacetylase, participates in a variety of human diseases. Here we identify a role for SIRT1 in the action of PTH in osteoblasts. We observed increased Mmp13 mRNA expression and protein levels in bone from Sirt1 knock-out mice compared with wild type mice. PTH-induced Mmp13 expression was significantly blocked by the SIRT1 activator, resveratrol, in osteoblastic UMR 106-01 cells. In contrast, the SIRT1 inhibitor, EX527, significantly enhanced PTH-induced Mmp13 expression. Two h of PTH treatment augmented SIRT1 association with c-Jun, a component of the transcription factor complex, activator protein 1 (AP-1), and promoted SIRT1 association with the AP-1 site of the Mmp13 promoter. This binding was further increased by resveratrol, implicating SIRT1 as a feedback inhibitor regulating Mmp13 transcription. The AP-1 site of the Mmp13 promoter is required for PTH stimulation of Mmp13 transcriptional activity. When the AP-1 site was mutated, EX527 was unable to increase PTH-stimulated Mmp13 promoter activity, indicating a role for the AP-1 site in SIRT1 inhibition. We further showed that SIRT1 deacetylates c-Jun and that the cAMP pathway participates in this deacetylation process. These data indicate that SIRT1 is a negative regulator of MMP13 expression, SIRT1 activation inhibits PTH stimulation of Mmp13 expression, and this regulation is mediated by SIRT1 association with c-Jun at the AP-1 site of the Mmp13 promoter.

The Histone-Deacetylase-Inhibitor Suberoylanilide Hydroxamic Acid Promotes Dental Pulp Repair Mechanisms Through Modulation of Matrix Metalloproteinase-13 Activity.

Direct application of histone-deacetylase-inhibitors (HDACis) to dental pulp cells (DPCs) induces chromatin changes, promoting gene expression and cellular-reparative events. We have previously demonstrated that HDACis (valproic acid, trichostatin A) increase mineralization in dental papillae-derived cell-lines and primary DPCs by stimulation of dentinogenic gene expression. Here, we investigated novel genes regulated by the HDACi, suberoylanilide hydroxamic acid (SAHA), to identify new pathways contributing to DPC differentiation. SAHA significantly compromised DPC viability only at relatively high concentrations (5 µM); while low concentrations (1 µM) SAHA did not increase apoptosis. HDACi-exposure for 24 h induced mineralization-per-cell dose-dependently after 2 weeks; however, constant 14d SAHA-exposure inhibited mineralization. Microarray analysis (24 h and 14 days) of SAHA exposed cultures highlighted that 764 transcripts showed a significant >2.0-fold change at 24 h, which reduced to 36 genes at 14 days. 59% of genes were down-regulated at 24 h and 36% at 14 days, respectively. Pathway analysis indicated SAHA increased expression of members of the matrix metalloproteinase (MMP) family. Furthermore, SAHA-supplementation increased MMP-13 protein expression (7 d, 14 days) and enzyme activity (48 h, 14 days). Selective MMP-13-inhibition (MMP-13i) dose-dependently accelerated mineralization in both SAHA-treated and non-treated cultures. MMP-13i-supplementation promoted expression of several mineralization-associated markers, however, HDACi-induced cell migration and wound healing were impaired. Data demonstrate that short-term low-dose SAHA-exposure promotes mineralization in DPCs by modulating gene pathways and tissue proteases. MMP-13i further increased mineralization-associated events, but decreased HDACi cell migration indicating a specific role for MMP-13 in pulpal repair processes. Pharmacological inhibition of HDAC and MMP may provide novel insights into pulpal repair processes with significant translational benefit. J. Cell. Physiol. 231: 798-816, 2016. © 2015 Wiley Periodicals, Inc.

Parathyroid hormone regulates histone deacetylase (HDAC) 4 through protein kinase A-mediated phosphorylation and dephosphorylation in osteoblastic cells.

Histone deacetylases (HDACs) are crucial regulators of gene expression in transcriptional co-repressor complexes. Previously, we reported that HDAC4 was a basal repressor of matrix metalloproteinase-13 (MMP-13) transcription and parathyroid hormone (PTH) regulates HDAC4 to control MMP-13 promoter activity through dissociation from Runx2. Here, we show that PTH induces the protein kinase A (PKA)-dependent phosphorylation of HDAC4 in the nucleus of the rat osteoblastic cell line, UMR 106-01. We demonstrate that PKA-dependent phosphorylated HDAC4 is released from Runx2 bound to the MMP-13 promoter in these cells. Point mutation of Ser-740 in rHDAC4 prevents the release of HDAC4 from Runx2 on the MMP-13 promoter and also prevents the PTH stimulation of MMP-13 transcription. Thus, PTH-induced phosphorylation of rHDAC4 at Ser-740 is crucial for regulating MMP-13 transcription in osteoblasts. PTH causes degradation of HDAC4, and this product appears in the cytoplasm. The cytoplasmic degradation of HDAC4 is blocked by PKA and lysosomal inhibitors, but is not affected by proteasome, caspase-3, or serine and aspartic protease inhibitors. In addition, the phosphatase inhibitor, okadaic acid, prevents degradation indicating that dephosphorylation is associated with degradation. These mechanisms regulating HDAC4 and their roles in such processes are crucial for bone and chondrocyte development. Our data support a link between PTH regulating HDAC4 phosphorylation by PKA, trafficking, partial degradation, and the control of MMP-13 transcription through association with Runx2.

A positive role of microRNA-15b on regulation of osteoblast differentiation.

Osteoblast differentiation is tightly regulated by several factors including microRNAs (miRNAs). In this paper, we report that pre-mir-15b is highly expressed in differentiated osteoblasts. The functional role of miR-15b in osteoblast differentiation was determined using miR-15b mimic/inhibitor and the expression of osteoblast differentiation marker genes such as alkaline phosphatase (ALP), type I collagen genes was decreased by miR-15b inhibitor. Runx2, a bone specific transcription factor is generally required for expression of osteoblast differentiation marker genes and in response to miR-15b inhibitor treatment, Runx2 mRNA expression was not changed; whereas its protein expression was decreased. Even though Smurf1 (SMAD specific E3 ubiquitin protein ligase 1), HDAC4 (histone deacetylase 4), Smad7, and Crim1 were found to be few of miR-15b's putative target genes, there was increased expression of only Smurf1 gene at mRNA and protein levels by miR-15b inhibitor. miR-15b mimic treatment significantly increased and decreased expressions of Runx2 and Smurf1 proteins, respectively. We further identified that the Smurf1 3'UTR is directly targeted by miR-15b using the luciferase reporter gene system. This is well documented that Smurf1 interacts with Runx2 and degrades it by proteasomal pathway. Hence, based on our results we suggest that miR-15b promotes osteoblast differentiation by indirectly protecting Runx2 protein from Smurf1 mediated degradation. Thus, this study identified that miR-15b can act as a positive regulator for osteoblast differentiation.

Abaloparatide Has the Same Catabolic Effects on Bones of Mice When Infused as PTH (1-34).

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.

Differential effects of PTH (1-34), PTHrP (1-36) and abaloparatide on the murine osteoblast transcriptome.

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.

Differential Effects of PTH (1-34), PTHrP (1-36), and Abaloparatide on the Murine Osteoblast Transcriptome.

Teriparatide (PTH (1-34)), PTHrP (1-36), and abaloparatide (ABL) have been used for the treatment of osteoporosis, but their efficacy long term is significantly limited. The 3 peptides exert time- and dose-dependent differential responses in osteoblasts, leading us to hypothesize they may also differentially modulate the osteoblast transcriptome. Treatment of mouse calvarial osteoblasts with 1 nM of the peptides for 4 hours results in RNA sequencing 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 similarities in Wnt signaling, cAMP-mediated signaling, ossification, but differences in morphogenesis of a branching structure in biological processes; receptor ligand activity, transcription factor activity, and cytokine receptor/binding activity in molecular functions. The peptides increased Vdr, Cited1, and Pde10a messenger RNAs (mRNAs) in a pattern similar to Rankl, that is, PTH (1-34) greater than ABL greater than PTHrP (1-36). mRNA abundance of other genes, including Wnt4, Wnt7, Wnt11, Sfrp4, Dkk1, Kcnk10, Hdac4, Epn3, Tcf7, Crem, Fzd5, Ppp2r2a, and Dvl3, showed that some genes were regulated similarly by all 3 peptides; others were not. Finally, small interfering RNA knockdowns of SIK1/2/3 and CRTC1/2/3 in PTH (1-34)-treated cells revealed that Vdr and Wnt4 genes are regulated by salt-inducible kinases (SIKs) and CREB-regulated transcriptional coactivators (CRTCs), while others are not. Although many studies have examined PTH signaling in the osteoblast/osteocyte, ours is the first to compare the global effects of these peptides on the osteoblast transcriptome or to analyze the roles of the SIKs and CRTCs.

Ca2+-activated chloride channel ANO1: A new regulator of osteoclast function.

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.

Bioactive, full-length parathyroid hormone delivered using an adeno-associated viral vector.

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.

The Critical Role of MMP13 in Regulating Tooth Development and Reactionary Dentinogenesis Repair Through the Wnt Signaling Pathway.

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 -/- ) mice were used to analyze phenotypic changes in the dentin-pulp complex, mineralization-associated marker-expression, and mechanistic interactions. Immunohistochemistry demonstrated high MMP13-expression in pulp-tissue, ameloblasts, odontoblasts, and dentin in developing WT-molars, which reduced in adults, with human-DPC cultures demonstrating a >2000-fold increase in Mmp13-expression during mineralization. Morphologically, Mmp13 -/- molars displayed critical alterations in the dentin-phenotype, affecting dentin-tubule regularity, the odontoblast-palisade and predentin-definition with significantly reduced dentin volume (∼30% incisor; 13% molar), and enamel and dentin mineral-density. Reactionary-tertiary-dentin in response to injury was reduced at Mmp13 -/- molar cusp-tips but with significantly more dystrophic pulpal mineralization in MMP13-null samples. Odontoblast differentiation-markers, nestin and DSP, reduced in expression after MMP13-loss in vivo, with reduced calcium deposition in MMP13-null DPC cultures. RNA-sequencing analysis of WT and Mmp13 -/- pulp highlighted 5,020 transcripts to have significantly >2.0-fold change, with pathway-analysis indicating downregulation of the Wnt-signaling pathway, supported by reduced in vivo expression of the Wnt-responsive gene Axin2. Mmp13 interaction with Axin2 could be partly responsible for the loss of odontoblastic activity and alteration to the tooth phenotype and volume which is evident in this study. Overall, our novel findings indicate MMP13 as critical for tooth development and mineralization processes, highlighting mechanistic interaction with the Wnt-signaling pathway.

Parathyroid hormone activation of matrix metalloproteinase-13 transcription requires the histone acetyltransferase activity of p300 and PCAF and p300-dependent acetylation of PCAF.

Parathyroid hormone (PTH) regulates the transcription of many genes involved in bone remodeling in osteoblasts. One of these genes is matrix metalloproteinase-13 (MMP-13), which is involved in bone remodeling and early stages of endochondral bone formation. We have previously shown that Mmp-13 gene expression is highly induced by PTH treatment in osteoblastic UMR 106-01 cells, as well as primary osteoblasts. Here, we show that p300/CBP-associated factor (PCAF), in addition to p300 and Runx2, is required for PTH activation of Mmp-13 transcription. PCAF was increasingly recruited to the MMP-13 proximal promoter region after PTH treatment, and this was associated with an increase in RNA polymerase II recruitment and histone acetylation. In addition, PTH treatment increased the acetylation of PCAF, a process that required p300. Knockdown of PCAF, p300, or Runx2 by siRNA decreased Mmp-13 mRNA expression after PTH treatment in both UMR 106-01 cells and primary osteoblasts. We found that there is a mutual dependence between p300 and PCAF to be recruited to the Mmp-13 promoter after PTH treatment. In promoter-reporter assays, p300 and PCAF had an additive effect on PTH stimulation of MMP-13 promoter activity, and this required their histone acetyltransferase activity. Our findings demonstrate that PCAF acts downstream of PTH signaling as a transcriptional coactivator that is required for PTH stimulation of MMP-13 transcription. PCAF cooperates with p300 and Runx2 to mediate PTH activation of MMP-13 transcription.

Cellular and molecular mechanisms of bone remodeling.

Physiological bone remodeling is a highly coordinated process responsible for bone resorption and formation and is necessary to repair damaged bone and to maintain mineral homeostasis. In addition to the traditional bone cells (osteoclasts, osteoblasts, and osteocytes) that are necessary for bone remodeling, several immune cells have also been implicated in bone disease. This minireview discusses physiological bone remodeling, outlining the traditional bone biology dogma in light of emerging osteoimmunology data. Specifically discussed in detail are the cellular and molecular mechanisms of bone remodeling, including events that orchestrate the five sequential phases of bone remodeling: activation, resorption, reversal, formation, and termination.

HDAC4 represses matrix metalloproteinase-13 transcription in osteoblastic cells, and parathyroid hormone controls this repression.

Parathyroid hormone (PTH) is a hormone regulating bone remodeling through its actions on both bone formation and bone resorption. Previously we reported that PTH induces matrix metalloproteinase-13 (MMP-13) transcription in osteoblastic cells. Here, we show that histone deacetylase 4 (HDAC4) interacts with Runx2, binds the MMP-13 promoter, and suppresses MMP-13 gene transcription in the rat osteoblastic cell line, UMR 106-01. PTH induces the rapid cAMP-dependent protein kinase-dependent release of HDAC4 from the MMP-13 promoter and subsequent transcription of MMP-13. Knock-out of HDAC4 either by siRNA in vitro or by gene deletion in vivo leads to an increase in MMP-13 expression, and overexpression of HDAC4 decreases the PTH induction of MMP-13. All of these observations indicate that HDAC4 represses MMP-13 gene transcription in bone. Moreover, PTH stimulates HDAC4 gene expression and enzymatic activity at times corresponding to the reassociation of HDAC4 with the MMP-13 promoter and a decline in its transcription. Thus, HDAC4 is a basal repressor of MMP-13 transcription, and PTH regulates HDAC4 to control MMP-13 promoter activity. These data identify a novel and discrete mechanism of regulating HDAC4 levels and, subsequently, gene expression.