Current Understanding of the Pathophysiology of Osteonecrosis of the Jaw.

PURPOSE OF REVIEW: Osteonecrosis of the jaw (ONJ) is a rare and severe necrotic bone disease reflecting a compromise in the body's osseous healing mechanisms and unique to the craniofacial region. Antiresorptive and antiangiogenic medications have been suggested to be associated with the occurrence of ONJ; yet, the pathophysiology of this disease has not been fully elucidated. This article raises the current theories underlying the pathophysiology of ONJ. RECENT FINDINGS: The proposed mechanisms highlight the unique localization of ONJ. The evidence-based mechanisms of ONJ pathogenesis include disturbed bone remodeling, inflammation or infection, altered immunity, soft tissue toxicity, and angiogenesis inhibition. The role of dental infections and the oral microbiome is central to ONJ, and systemic conditions such as rheumatoid arthritis and diabetes mellitus contribute through their impact on immune resiliency. Current experimental studies on mechanisms of ONJ are summarized. The definitive pathophysiology is as yet unclear. Recent studies are beginning to clarify the relative importance of the proposed mechanisms. A better understanding of osteoimmunology and the relationship of angiogenesis to the development of ONJ is needed along with detailed studies of the impact of drug holidays on the clinical condition of ONJ.

The skeletal impact of the chemotherapeutic agent etoposide.

Effects of the chemotherapeutic agent etoposide on the skeleton were determined in mice. Numbers of bone marrow cells were reduced and myeloid cells were increased. Bone volume was significantly decreased with signs of inhibition of bone formation. Etoposide after pre-treatment with zoledronic acid still reduced bone but overall bone volume was higher than with etoposide alone. INTRODUCTION: Chemotherapeutics target rapidly dividing tumor cells yet also impact hematopoietic and immune cells in an off target manner. A wide array of therapies have negative side effects on the skeleton rendering patients osteopenic and prone to fracture. This study focused on the pro-apoptotic chemotherapeutic agent etoposide and its short- and long-term treatment effects in the bone marrow and skeleton. METHODS: Six- to 16-week-old mice were treated with etoposide (20-25 mg/kg) or vehicle control in short-term (daily for 5-9 days) or long-term (3×/week for 17 days or 6 weeks) regimens. Bone marrow cell populations and their phagocytic/efferocytic functions were analyzed by flow cytometry. Blood cell populations were assessed by CBC analysis. Bone volume and area compartments and osteoclast numbers were measured by microCT, histomorphometry, and TRAP staining. Biomarkers of bone formation (P1NP) and resorption (TRAcP5b) were assayed from serum. Gene expression in bone marrow was assessed using qPCR. RESULTS: Flow cytometric analysis of the bone marrow revealed short-term etoposide reduced overall cell numbers and B220+ cells, with increased marrow apoptotic (AnnexinV+PI-) cells, mesenchymal stem-like cells, and CD68+, CD45+, and CD11b+ monocyte/myeloid cells (as a percent of the total marrow). After 6 weeks, the CD68+, Gr1+, CD11b+, and CD45+ cell populations were still relatively increased in etoposide-treated bone marrow. Skeletal phenotyping revealed etoposide decreased bone volume, trabecular thickness, and cortical bone volume. Gene expression in the marrow for the leptin receptor and CXCL12 were reduced with short-term etoposide, and an increased ratio of RANKL/OPG mRNA was observed. In whole bone, Runx2 and osteocalcin gene expressions were reduced, and in serum, P1NP was significantly reduced with etoposide. Treatment with the antiresorptive agent zoledronic acid prior to etoposide increased bone volume and improved the etoposide-induced decrease in skeletal parameters. CONCLUSIONS: These data suggest that etoposide induces apoptosis in the bone marrow and significantly reduces parameters of bone formation with rapid reduction in bone volume. Pre-treatment with an antiresorptive agent results in a preservation of bone mass. Preventive approaches to preserving the skeleton should be considered in human clinical studies.

Early effects of parathyroid hormone on bisphosphonate/steroid-associated compromised osseous wound healing.

SUMMARY: Administration of intermittent parathyroid hormone (PTH) promoted healing of tibial osseous defects and tooth extraction wounds and prevented the development of necrotic lesions in rats on a combined bisphosphonate and steroid regimen. INTRODUCTION: Osteonecrosis of the jaw (ONJ) has emerged in association with antiresorptive therapies. The pathophysiology of ONJ is unknown and no established cure currently exists. Our objective was to determine the effect of intermittent PTH administration on early osseous healing in the jaw and long bones of rats receiving bisphosphonate and steroid treatment. METHODS: Ovariectomized rats received the combination therapy of alendronate and dexamethasone (ALN/DEX) for 12 weeks. Osseous wounds were created in the jaw and tibia. PTH was administered intermittently and healing at 2 weeks post-op was compared between the jaw and tibia by microcomputed tomography and histomorphometric analyses. RESULTS: ALN/DEX treatment was associated with necrotic open wounds in the jaw but had no negative effects on healing and promoted bone fill in tibial defects. PTH therapy prevented the development of necrotic lesions in the jaw and promoted healing of the tibial defects. PTH therapy was associated with the promotion of osteocyte survival in osseous wounds both in the jaw and tibia. CONCLUSIONS: Wound healing was impaired in the jaw in rats on a combined bisphosphonate and steroid regimen, and PTH therapy rescued necrotic lesions. These findings suggest that PTH therapy could be utilized to prevent ONJ from occurring in patients on combination antiresorptive and steroid therapy.

Translating Science into Improved Health for All.

Our world is at a turning point with biological and social pathogens wreaking havoc at the same time that science and technology are exploding with new discoveries. It is a pivotal time for the new report Oral Health in America: Advances and Challenges to be released and a pivotal time for our profession to take action and lead. The art, science, and practice of dentistry is very different from 20 y ago when the original Surgeon General's report was released. We are on the precipice of individualized health care where providers will collaborate to deliver diagnostics and therapeutics that are data driven and inclusive of the social determinants of health. To move forward with alacrity requires a strong scientific foundation, effective educational approaches, an understanding of the upstream determinants of health, and partnerships across the health professions and beyond. Oral health has never been more important, and now is the time for our profession to further develop, elevate, and translate the science into practice and policy to improve the nation's health.

The Forefront of Dentistry-Promising Tech-Innovations and New Treatments.

KNOWLEDGE TRANSFER STATEMENT: This article discusses innovations in technology and treatments that have enormous potential to revolutionize our dental care, including novel concepts in electronic health records, communication between dentists and patients, biologics around diagnosis and treatment, digital dentistry, and, finally, the real-time optimization of information technology. The early implementation and validation of these innovations can drive down their costs and provide better dental and medical services to all members of our society.

Osteoclasts and odontoclasts: signaling pathways to development and disease.

Osteoclasts are cells essential for physiologic remodeling of bone and also play important physiologic and pathologic roles in the dentofacial complex. Osteoclasts and odontoclasts are necessary for tooth eruption yet result in dental compromise when associated with permanent tooth internal or external resorption. The determinants that separate their physiologic and pathologic roles are not well delineated. Clinical cases of primary eruption failure and root resorption are challenging to treat. Mineralized tissue resorbing cells undergo a fairly well characterized series of differentiation stages driven by transcriptional mediators. Signal transduction via cytokines and integrin-mediated events comprise the detailed pathways operative in osteo/odontoclastic cells and may provide insights to their targeted regulation. A better understanding of the unique aspects of osteoclastogenesis and osteo/odontoclast function will facilitate effective development of new therapeutic approaches. This review presents the clinical challenges and delves into the cellular and biochemical aspects of the unique cells responsible for resorption of mineralized tissues of the craniofacial complex.

Clodronate-Loaded Liposome Treatment Has Site-Specific Skeletal Effects.

Ineffective oral wound healing is detrimental to patients' oral health-related quality of life. Delineating the cellular mechanisms involved in optimal healing will elicit better approaches to treating patients with compromised healing. Osteal macrophages have recently emerged as important positive regulators of bone turnover. The contributions of macrophages to long bone healing have been studied, but their role in oral osseous wound healing following tooth extraction is less clear. Clodronate-loaded liposomes were used as a tool to deplete macrophages in C57BL/6J mice and assess oral osseous bone fill after extraction. In addition to macrophage ablation, osteoclast ablation occurred. Interestingly, depletion of macrophages and osteoclasts via clodronate treatment had differential effects based on skeletal location. In the nonwounded tibiae, clodronate treatment significantly increased CD68+ cells and decreased F4/80+ cells in the marrow, which correlated with increased trabecular bone volume fraction after 7 and 14 d. Serum formation and resorptive markers P1NP and TRAcP 5b were decreased as were tibial TRAP+ osteoclasts. In healing extraction sockets, clodronate treatment increased extraction socket trabecular bone thickness at 14 d, which correlated with decreased TRAP+ osteoclasts and F4/80+ macrophages. Conversely, nonwounded maxillary interseptal bone was unaffected by clodronate treatment. Furthermore, the increase in extraction socket bone fill with clodronate was less than the large increase in trabecular bone observed in a nonwounded long bone. These data suggest a temporal and spatial specificity in the roles of macrophages and osteoclasts in normal turnover and healing.

Trabectedin Reduces Skeletal Prostate Cancer Tumor Size in Association with Effects on M2 Macrophages and Efferocytosis.

Macrophages play a dual role in regulating tumor progression. They can either reduce tumor growth by secreting antitumorigenic factors or promote tumor progression by secreting a variety of soluble factors. The purpose of this study was to define the monocyte/macrophage population prevalent in skeletal tumors, explore a mechanism employed in supporting prostate cancer (PCa) skeletal metastasis, and examine a novel therapeutic target. Phagocytic CD68+ cells were found to correlate with Gleason score in human PCa samples, and M2-like macrophages (F4/80+CD206+) were identified in PCa bone resident tumors in mice. Induced M2-like macrophages in vitro were more proficient at phagocytosis (efferocytosis) of apoptotic tumor cells than M1-like macrophages. Moreover, soluble factors released from efferocytic versus nonefferocytic macrophages increased PC-3 prostate cancer cell numbers in vitro. Trabectedin exposure reduced M2-like (F4/80+CD206+) macrophages in vivo. Trabectedin administration after PC-3 cell intracardiac inoculation reduced skeletal metastatic tumor growth. Preventative pretreatment with trabectedin 7 days prior to PC-3 cell injection resulted in reduced M2-like macrophages in the marrow and reduced skeletal tumor size. Together, these findings suggest that M2-like monocytes and macrophages promote PCa skeletal metastasis and that trabectedin represents a candidate therapeutic target.

JunB as a potential mediator of PTHrP actions: new gene targets Ephrin B1 and VCAM-1.

Parathyroid hormone-related protein (PTHrP) is an integral mediator of physiologic and pathologic processes and has demonstrated actions in the periodontium. PTHrP functions via AP-1, and specifically through JunB. This study identified JunB-dependent downstream mediators of PTHrP using OCCM cementoblastic transfectants with JunB over- or reduced expression. Over-expressing cells showed an increase in proliferation, while the opposite was seen in siRNA transfected cells. Microarray analysis of over-expressing cells revealed more than 1000 regulated genes. Three genes were investigated in more detail. The PTH/PTHrP receptor (PTHR1) and ephrin B1 (EfnB1) were down-regulated, and vascular cell adhesion molecule-1 (VCAM-1) was up-regulated with JunB over-expression. JunB siRNA transfectants had increased PTHR1, but reduced ephrin B1 and unaltered VCAM-1 in vitro. To validate these targets, parental OCCM cells and primary osteoblasts were treated with PTHrP, resulting in reduced PTHR1 and ephrin B1, and increased VCAM-1. Cell transfectants were implanted subcutaneously in vivo, and microarray analysis and RT-PCR performed. Over-expression of JunB down-regulated PTHR1 and ephrin B1, and increased VCAM-1. JunB siRNA transfectant implants had increased PTHR1 and ephrin B1, but no altered VCAM-1. These data highlight new gene targets for PTHrP and indicate JunB is a critical mediator of PTHrP actions.

Regulation of SDF-1 (CXCL12) production by osteoblasts; a possible mechanism for stem cell homing.

Stromal derived factor-1 (SDF-1 or CXCL12) controls many aspects of stem cell function including trafficking and proliferation. Previously, it was demonstrated that DNA-damaging agents such as irradiation, cyclophosphamide or 5-fluorouracil increase the expression of SDF-1 by osteoblasts in murine marrow. Here, the production of SDF-1 by osteoblasts in vitro in response to cytokines known to be particularly important in bone physiology was examined using primary human osteoblasts (HOBs), mixed marrow stromal cells (BMSCs), and by, mouse, rat and human osteoblast-like cell lines. From these studies, it was determined that the expression of SDF-1 is an early feature of osteoblastic induction that may be modulated by IL-1beta, PDGF-BB, VEGF, TNF-alpha and PTH. Each of these factors increased SDF-1 synthesis, while TGF-beta1 decreased SDF-1 secretion. Of note, the biodistribution of SDF-1 in culture was equally distributed between the medium and detergent-soluble and -insoluble fractions of the cultures. Immunohistochemistry of developing bones demonstrated that SDF-1 was also a feature of early bone development first beginning in the perichondrium and moving into the marrow cavity of the developing bone analogue. As SDF-1 expression increases in response to PTH in vitro, animals were treated with an anabolic regime of PTH for 21 days. Under these conditions, significant increases in SDF-1 mRNA expression were observed near the growth plate and epiphysis regions of the long bones. Yet, in serum, immunodetectable SDF-1 levels were significantly reduced (24%) in the PTH-treated animals (Vehicle: 408 +/- 25 vs. PTH 308 +/- 20 SDF-1 pg/ml). Together, these data suggest a possible mechanism for localizing stem cells into a developing marrow where increased expression of SDF-1 in the local marrow environment along with decreased SDF-1 in the serum may create a homing gradient.

Parathyroid hormone-related protein as a growth regulator of prostate carcinoma.

Parathyroid hormone-related protein (PTHrP) is produced by prostate carcinoma cells and tumors, but little is known of its role in prostate carcinogenesis. The goal of this study was to evaluate PTHrP expression in the regulation of prostate carcinoma growth using human and animal models. PTHrP expression was assessed in prostate cancer cell lines in vitro. Seven of nine cell lines produced PTHrP, and increased expression was seen during cell proliferation. The MatLyLu rat prostate carcinoma model was used to determine the effects of PTHrP overexpression on prostate tumor growth. PTHrP overexpression did not alter proliferation of the cells in vitro. However, when PTHrP-overexpressing cells were injected into rat hind limbs, primary tumor growth and tumor size were significantly enhanced as compared with control cells. To evaluate PTHrP in human prostate carcinoma patients, immunohistochemistry was performed on metastatic bone lesions. Immunolocalization of PTHrP protein was found in the cytoplasm and nucleus of cancer cells in the bone microenvironment. Because nuclear localization of PTHrP has been associated with an inhibition of apoptosis, the ability of full-length PTHrP to protect prostate cancer cells from apoptotic stimuli was examined. Cells transfected with full-length PTHrP showed significantly increased cell survival after exposure to apoptotic agents as compared with cells producing no PTHrP (plasmid control) or cells transfected with PTHrP lacking its nuclear localization signal. To determine the mechanism of action of PTHrP in prostate cancer cells, the parathyroid hormone/PTHrP receptor status of the cells was determined. These cell lines did not demonstrate parathyroid hormone/PTHrP receptor-mediated binding of iodinated PTHrP or steady-state receptor message by Northern blot analysis, but they did have a detectable receptor message by reverse transcription-PCR analysis. In summary, PTHrP is expressed in many prostate cancer cell lines in vitro and in metastatic bone lesions in vivo. PTHrP expression positively influences primary tumor size in vivo and protects cells from apoptotic stimuli. These data suggest that PTHrP plays an important role in the promotion of prostate tumor establishment and/or progression.

PTH and Vitamin D Repress DMP1 in Cementoblasts.

A complex feedback mechanism between parathyroid hormone (PTH), 1,25(OH)2D3 (1,25D), and fibroblast growth factor 23 (FGF-23) maintains mineral homeostasis, in part by regulating calcium and phosphate absorption/reabsorption. Previously, we showed that 1,25D regulates mineral homeostasis by repressing dentin matrix protein 1 (DMP1) via the vitamin D receptor pathway. Similar to 1,25D, PTH may modulate DMP1, but the underlying mechanism remains unknown. Immortalized murine cementoblasts (OCCM.30), similar to osteoblasts and known to express DMP1, were treated with PTH (1-34). Real-time quantitative polymerase chain reaction (PCR) and Western blot revealed that PTH decreased DMP1 gene transcription (85%) and protein expression (30%), respectively. PTH mediated the downregulation of DMP1 via the cAMP/protein kinase A (PKA) pathway. Immunohistochemistry confirmed the decreased localization of DMP1 in vivo in cellular cementum and alveolar bone of mice treated with a single dose (50 µg/kg) of PTH (1-34). RNA-seq was employed to further identify patterns of gene expression shared by PTH and 1,25D in regulating DMP1, as well as other factors involved in mineral homeostasis. PTH and 1,25D mutually upregulated 36 genes and mutually downregulated 27 genes by ≥2-fold expression (P ≤ 0.05). Many identified genes were linked with the regulation of bone/tooth homeostasis, cell growth and differentiation, calcium signaling, and DMP1 transcription. Validation of RNA-seq results via PCR array confirmed a similar gene expression pattern in response to PTH and 1,25D treatment. Collectively, these results suggest that PTH and 1,25D share complementary effects in maintaining mineral homeostasis by mutual regulation of genes/proteins associated with calcium and phosphate metabolism while also exerting distinct roles on factors modulating mineral metabolism. Furthermore, PTH may modulate phosphate homeostasis by downregulating DMP1 expression via the cAMP/PKA pathway. Targeting genes/proteins mutually governed by PTH and 1,25D may be a viable approach for designing new therapies for preserving mineralized tissue health.

Parathyroid hormone-related protein regulates extracellular matrix gene expression in cementoblasts and inhibits cementoblast-mediated mineralization in vitro.

Parathyroid hormone-related protein (PTHrP) has been implicated in regulating tooth eruption and/or development. Formation of cementum, a mineralized tissue covering the tooth root surface, is a critical biological event for tooth root development. To test the hypothesis that PTHrP targets cementoblasts (CMs) and acts to regulate cementogenesis, CM cell lines were established and their responsiveness to PTHrP stimulation was determined, in vitro. First, subclones were derived from two immortalized murine cell populations that contained CMs; SV-CM/periodontal ligament (PDL) cells were obtained from the root surface of first mandibular molars of CD-1 mice and immortalized with SV40 T-antigen (TAg), and OC-CM cell population was established from OC-TAg transgenic mice in which their cells harbor an osteocalcin (OC and/or OCN) promoter-driving immortal gene SV40 TAg. Based on our previous in situ studies, CM subclones were identified as cells expressing bone sialoprotein (BSP) and OCN transcripts, while PDL cell lines were designated as cells lacking BSP and OCN messenger RNA (mRNA). CMs exhibited a cuboidal appearance and promoted biomineralization, both in vitro and in vivo. In contrast, PDL cells (PDL subclones) displayed a spindle-shaped morphology and lacked the ability to promote mineralized nodule formation, both in vitro and in vivo. Next, using these subclones, the effect of PTHrP on cementogenesis was studied. CMs, not PDL cells, expressed PTH/PTHrP receptor mRNA and exhibited PTHrP-mediated elevation in cyclic adenosine monophosphate (cAMP) levels and c-fos gene induction. PTHrP stimulation repressed mRNA expression of BSP and OCN in CMs and blocked CM-mediated mineralization, in vitro. Collectively, these data suggest that CMs possess PTH/PTHrP receptors and, thus, are direct targets for PTHrP action during cementogenesis and that PTHrP may serve as an important regulator of cementogenesis.

Effects of differentiation and transforming growth factor beta 1 on PTH/PTHrP receptor mRNA levels in MC3T3-E1 cells.

TGF beta has opposing effects on osteoblasts which are thought to be differentiation stage dependent; however, little is known concerning the effects of TGF beta on osteoblastic characteristics at different stages of maturation. The purpose of this study was to characterize the pattern of mRNA expression for the PTH/PTHrP receptor during normal osteoblastic differentiation in vitro, and evaluate the effects of TGF beta 1 on PTH/PTHrP receptor and osteocalcin (OCN) steady-state mRNA at different stages of osteoblastic differentiation. MC3T3-E1 preosteoblasts were plated at low density and induced to differentiate with ascorbic acid and beta-glycerophosphate. The first group served as a vehicle control and the remaining five groups received a single 48 h TGF beta 1 (3.0 ng/ml)-pulse staggered on a weekly basis for 30 days. Cell cultures were harvested weekly and evaluated for: steady-state PTH/PTHrP receptor and OCN mRNA levels via northern analysis, calcium and phosphorous levels, bone nodules via Von Kossa staining, alkaline phosphatase enzyme levels, and hydroxyproline levels. Group 1 (control) samples followed a normal pattern of proliferation, extracellular matrix deposition, and mineralization. PTH/PTHrP receptor and OCN mRNA expression increased 8-fold and 10-fold respectively, over the collection periods. When TGF beta 1 was administered during the first 48 h period (group 2) while cells were rapidly proliferating, there was a persistent inhibition of PTH/PTHrP receptor expression and a striking reduction in OCN mRNA expression at all time points. There was also a down-regulation of PTH/PTHrP receptor and OCN expression when TGF beta 1 was administered later during osteoblast differentiation (groups 3-6); however, these effects were not persistent. In addition there was a total lack of bone nodule formation in group two cultures, whereas groups 3-6 had increasing bone nodule formation because the TGF beta 1 was administered later in the culture period. These studies indicate that expression of the PTH/PTHrP receptor increases with osteoblastic differentiation and suggest that TGF beta 1 inhibits osteoblastic maturation with more persistent effects found in less differentiated osteoblastic cells.

Parathyroid hormone-related protein binding to human T-cell lymphotropic virus type I-infected lymphocytes.

An HTLV-I-infected human lymphocyte line (MT-2) was evaluated for 1) the presence of receptors for PTH-related protein (PTHrP), 2) cell proliferation in response to PTHrP, and 3) adrenylate cyclase and intracellular calcium response to PTHrP. PTHrP-(1-36) was labeled with 125I, purified, and used to detect binding to MT-2 cells. Specific binding ranged between 4-9% of the total radioactivity. Specific binding increased with increasing cell number, was maximal within 30-60 min, and was highest at 37 C. Scatchard analysis revealed a one-binding site fit, with a Kd of 14.5 nM. Binding was not competed for by calcitonin, calcitonin gene-related peptide, or interleukin-1 beta. PTHrP at 1.0 and 0.1 microM inhibited proliferation in MT-2 cells. PTHrP did not alter adenylate cyclase stimulation in MT-2 cells, but did cause an increase in intracellular calcium. These findings indicate that MT-2 cells have receptors for PTHrP and are consistent with a potential autocrine role of PTHrP in HTLV-I-infected lymphoid cells.

Matrix gamma-carboxyglutamic acid protein is a key regulator of PTH-mediated inhibition of mineralization in MC3T3-E1 osteoblast-like cells.

As part of its overall function as a major regulator of calcium homeostasis, PTH stimulates bone resorption and inhibits osteoblast-mediated biomineralization. To determine the basis for the inhibitory actions of this hormone, we compared the time course of PTH-dependent inhibition of mineralization in MC3T3-E1 osteoblast-like cells with changes in mRNA levels for several extracellular matrix proteins previously associated either with induction or inhibition of mineralization. Mineralizing activity was rapidly lost in PTH-treated cells ( approximately 30% inhibition after 3 h, 50% inhibition at 6 h). Of the proteins examined, changes in matrix gamma-carboxyglutamic acid protein were best correlated with PTH-dependent inhibition of mineralization. Matrix gamma-carboxyglutamic acid protein mRNA was rapidly induced 3 h after PTH treatment, with a 6- to 8-fold induction seen after 6 h. Local in vivo injection of PTH over the calvaria of mice also induced a 2-fold increase in matrix gamma-carboxyglutamic acid protein mRNA. Warfarin, an inhibitor of matrix gamma-carboxyglutamic acid protein gamma-carboxylation, reversed the effects of PTH on mineralization in MC3T3-E1 cells, whereas vitamin K enhanced PTH activity, as would be expected if a gamma-carboxyglutamic acid-containing protein were required for PTH activity. Levels of the other mRNAs examined were not well correlated with the observed changes in mineralization. Osteopontin, an in vitro inhibitor of mineralization, was induced approximately 4-fold 12 h after PTH addition. Bone sialoprotein mRNA, which encodes an extracellular matrix component most frequently associated with mineral induction, was inhibited by 50% after 12 h of PTH treatment. Osteocalcin mRNA, encoding the other known gamma-carboxyglutamic acid protein in bone, was also inhibited by PTH, but, again, with a significantly slower time course than was seen for mineral inhibition. Taken together, these results show that the rapid inhibition of osteoblast mineralization induced by in vitro PTH treatment is at least in part explained by induction of matrix gamma-carboxyglutamic acid protein.

Parathyroid hormone-related protein down-regulates bone sialoprotein gene expression in cementoblasts: role of the protein kinase A pathway.

PTH-related protein (PTHrP) acts as a paracrine and/or autocrine regulator of cell proliferation, apoptosis, and differentiation and is implicated in tooth development. The current studies employed cementoblasts to determine the role(s) and mechanisms of PTHrP in regulating cementum formation. Results demonstrated that PTHrP repressed gene expression and protein synthesis of bone sialoprotein (BSP) and abolished cementoblast-mediated biomineralization in vitro. The BSP gene inhibition required protein synthesis. The PTHrP analog (1-31) and other activators of the PKA pathway (3-isobutyl-1-methylxathine (IBMX), forskolin (FSK) and Sp-Adenosine-3', 5'-cyclic monophosphorothioate (Sp-cAMPss) also down-regulated BSP gene expression and blocked cementoblast-mediated biomineralization. In contrast, the PTHrP analog (7-34), a PTHrP antagonist, and the activators of the PKC pathway [phorbol 12-myristate 13-acetate (PMA) and phorbol 12, 13-dibutyrate (PDBu)] promoted BSP gene expression. In addition, the PKA pathway inhibitor (9-(2-tetrahydrofuryl) adenine (THFA) partially, but significantly reversed the PTHrP-mediated down-regulation of BSP gene expression. Furthermore, THFA alone significantly increased BSP messenger RNA (mRNA) expression in cementoblasts. In contrast, the inhibitor of the PKC pathway (GF109203X) did not reverse the PTHrP inhibitory effect on BSP gene expression. Furthermore, GF109203X alone dramatically reduced the BSP transcript levels. These data indicate that the cAMP/PKA pathway mediates the PTHrP-mediated down-regulation of BSP mRNA expression in cementoblasts; and furthermore, this pathway may, through an intrinsic inhibition mechanism, regulate the basal level of BSP mRNA expression. In contrast, the activation of PKC promotes BSP gene expression. These data provide new insights into the molecular mechanisms involved in PTHrP regulation of cementogenesis.

Proto-oncogene c-fos is transcriptionally regulated by parathyroid hormone (PTH) and PTH-related protein in a cyclic adenosine monophosphate-dependent manner in osteoblastic cells.

PTH and PTH-related protein (PTHrP) bind to the PTH-1 (PTH/PTHrP) receptor and produce anabolic and catabolic effects in bone. To investigate postreceptor mechanisms of action, MC3T3-E1 cells were induced to differentiate to optimize PTH-1 receptor expression, and differentiated MC3T3-E1 cells were treated with varying doses of PTH (1-34) for 1 h. Northern blot analysis revealed a dose-dependent stimulation of steady state c-fos messenger RNA (mRNA), with measurable expression at doses as low as 1 pM PTH. The time course of c-fos mRNA induction was rapid, with peak levels detected at 30-45 min. Increased steady state c-fos mRNA was due to increased transcription of the c-fos gene as demonstrated by nuclear run-on assays and was dependent on the temporal differentiation state of the MC3T3-E1 cells. Stimulation of c-fos mRNA was induced exclusively by N-terminal PTH and PTHrP (which is also responsible for cAMP activation), and did not occur with PTH (7-34), (53-84), or PTHrP (107-139). The effects of PTH (1-34) on c-fos stimulation were dependent on intracellular cAMP. Forskolin [a guanine-nucleotide-binding protein (G(alpha)) agonist] stimulated c-fos mRNA, whereas 9-(tetrahydro-2-furyl) adenine (THFA) (a cAMP antagonist), 1,9 dideoxyforskolin (a cAMP independent analog of forskolin), and phorbol 12-myristate 13-acetate (a protein kinase C activator) did not. Furthermore, THFA inhibited the ability of PTH (1-34) to stimulate c-fos mRNA in a time-dependent manner. These findings indicate that c-fos is transcriptionally regulated by PTH (1-34) in osteoblastic cells, and that cAMP is a mediator of PTH-stimulated c-fos induction. Several known bone-associated proteins contain DNA binding sites in their promoter regions that recognize c-fos in conjunction with c-jun (AP-1 sites). Consequently, the induction of c-fos by PTH (1-34) in osteoblastic cells may be a sensitive indicator of PTH effects in vitro and in vivo, and provide valuable information regarding mechanisms of PTH action in bone.

3',5'-Cyclic adenosine monophosphate activation in osteoblastic cells: effects on parathyroid hormone-1 receptors and osteoblastic differentiation in vitro.

PTH has anabolic and catabolic effects in bone through activation of the PTH-1 (PTH/PTHrP) receptor and the cAMP/protein kinase A pathway. The effects of agents that regulate cAMP in nontransformed osteoblasts in relation to cell differentiation have not been described. The purpose of this study was to determine the effects of PTH fragments with differing cAMP-stimulating activity, and nonPTH cAMP regulators on PTH-1 receptor expression and activity, and osteoblast differentiation in vitro using MC3T3-E1 and primary rat calvarial cells. PTH (1-34), but not PTH (53-84), (7-34), or PTHrP (107-139) treatment (24 h) resulted in down-regulation of steady-state messenger RNA for the PTH-1 receptor. Forskolin (a stimulator of cAMP accumulation) also down regulated the PTH-1 receptor, whereas 9-(tetrahydro-2-furyl) adenine (THFA) (an inhibitor of adenylyl cyclase) had no effect. Similarly, PTH (1-34) treatment for 48 h abolished PTHrP binding to cell surface receptors; however, neither the PTH analogs nor the cAMP regulating agents altered PTH binding or numbers of binding sites on osteoblastic cells. Basal levels of cAMP were reduced in cultured cells treated for 6 days with PTH (7-34) or THFA compared with controls. In contrast, PTH-stimulated cAMP levels were significantly increased in cultures treated with PTH (7-34) and THFA for 6 days during osteoblast differentiation and were decreased in cultures treated with PTH (1-34) and forskolin compared with controls. To evaluate effects of the cAMP pathway on osteoblast differentiation, cultures were treated continuously with PTH analogs and cAMP regulators during an 18-day differentiation regime, total RNA was isolated at multiple time points, and Northern blot analysis for osteocalcin (OCN) was performed. THFA and PTH (7-34)-treated cultures had increased OCN expression; whereas, PTH (1-34) and forskolin reduced OCN expression. Interestingly, PTH (7-34) and THFA-treated cultures had increased mineralized nodule formation, in contrast to PTH (1-34) and forskolin treatment, which reduced nodule formation. Similarly, calcium accumulation in cultures was significantly increased in the PTH (7-34) and THFA-treated cultures and reduced in the PTH (1-34) and forskolin-treated cultures. These data demonstrate that agents that increase cAMP down regulate PTH-1 receptor messenger RNA and inhibit osteoblast differentiation in vitro. Agents that reduce or block adenylyl cyclase or cAMP activity do not alter PTH-1 receptor expression or binding, but have striking effects on promoting osteoblast differentiation. We conclude that many effects of PTH on osteoblasts may be mimicked or antagonized by agents that alter cAMP activity and bypass the PTH-1 receptor.

Parathyroid hormone stimulates fra-2 expression in osteoblastic cells in vitro and in vivo.

PTH and PTH-related protein (PTHrP) are key mediators of skeletal development and homeostasis through their activation of the PTH-1 receptor. Previous studies have found that several AP-1 family members are regulated by PTH, such as c-fos, fra-1, and c-jun. There are numerous genes in the bone microenvironment that contain AP-1 sites, and different Fos family members are reported to have opposing transcriptional activities at AP-1 sites. The purpose of this study was to identify the effects of PTH on expression of the AP-1 protein complex member, fra-2, to extend our understanding of transcriptional regulators of PTH action. PTH induction of fra-2 messenger RNA (mRNA) levels in MC3T3-E1 preosteoblastic cells was maximal with 0.1 microM PTH (1-34). The expression in vitro was greatest 1 h after treatment and was present with N-terminal PTH but not PTH (7-34) or (53-84). Cycloheximide treatment induced fra-2 expression, and actinomycin D inhibited basal and PTHrP-induced expression. AP-1 protein in nuclear extracts of MC3T3-E1 cells was increased with PTH treatment at 3 h and consisted of high levels of Fra-2 protein, as evidenced by a supershift in an electrophoretic mobility shift assay and Western blot analysis. Up-regulation of steady-state fra-2 mRNA was also noted in vivo, where injection of PTH (1-34) (20 microgram) resulted in a more-than-7-fold maximal increase in fra-2 mRNA expression in the calvaria of mice, after 1 h of treatment. These data add to the transcriptional mediators induced by PTH and suggest that the interplay of AP-1 family members will provide insight into regulatory pathways of PTH and PTHrP for their anabolic and catabolic actions in bone.