Orai1 expression pattern in tooth and craniofacial ectodermal tissues and potential functions during ameloblast differentiation

Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/113781/1/dvdy24307.pd

Inducible cAMP early repressor expression and function in parathyroid hormone-treated osteoblasts

Parathyroid hormone (PTH) induces expression of several early response genes in osteoblasts, including prostaglandin G/H synthase-2 (PGHS-2). Early response gene expression is rapid, transient and does not require new protein synthesis. While pretreatment of osteoblastic MC3T3-E1 cells with the protein synthesis inhibitor cycloheximide does not prevent PTH-induced PGHS-2 expression, it does inhibit attenuation of PGHS-2 transcription. This suggests that PTH simultaneously induces expression of both PGHS-2 and a transcriptional inhibitor. One likely candidate is inducible CAMP early repressor (ICER), a member of the CAMP response element modulator family of basic leucine zipper transcription factors. We hypothesized that PTH induces ICER in osteoblasts and that ICER represses PTH-mediated gene expression. Our specific aim was to determine if ICER is an inducible, cAMP-dependent, early response gene that acts as a transcriptional repressor in PTH-treated osteoblasts. ^ PTH induced ICER mRNA and protein in osteoblastic MC3T3-E1 cells and cultured neonatal mouse calvariae in both a time- and dose-dependent manner. PTH also induced ICER mRNA expression in ROS 17/2.8, UMR-106, and Py1a osteoblastic cell lines. In all cells tested, ICER induction was rapid, transient, and did not require new protein synthesis, thus classifying ICER as an early response gene in PTH-treated cells. PTH also induced ICER mRNA in neonatal mouse calvariae in vivo. Unexpectedly, ICER mRNA was detectable in control-treated animals. Handling stress did not appear to affect this response. ^ PTH\u27s cellular effects are transduced by the PTH/PTHrP receptor, which is coupled to the cAMP-protein kinase A (PKA), protein kinase C (PKC), and calcium signaling pathways. To determine which pathways mediate PTH-induced ICER expression, MC3T3-E1 cells were treated with selective signaling agonists and antagonists. ICER expression was significantly induced only after cAMP-PKA activation. ^ Dose-dependent overexpression of ICER produced biphasic effects on a PGHS-1(−368/+6)-luciferase reporter vector. Low ICER overexpression enhanced, while high ICER overexpression repressed, basal and induced promoter activity. Mutation of the AP-1 site mimicked ICER\u27s effect on the wild type. ^ Based on these studies, we conclude that ICER is a PTH-inducible, cAMP-dependent early response gene that represses PTH-mediated gene transcription in osteoblasts. However, low concentrations of ICER may also enhance PGHS-1 promoter activity, suggesting that ICER is not strictly a transcriptional repressor.

Three-Dimensional Force Measurements During Rapid Palatal Expansion in Sus scrofa

Rapid palatal expansion is an orthodontic procedure widely used to correct the maxillary arch. However, its outcome is significantly influenced by factors that show a high degree of variability amongst patients. The traditional treatment methodology is based on an intuitive and heuristic treatment approach because the forces applied in the three dimensions are indeterminate. To enable optimal and individualized treatment, it is essential to measure the three-dimensional (3D) forces and displacements created by the expander. This paper proposes a method for performing these 3D measurements using a single embedded strain sensor, combining experimental measurements of strain in the palatal expander with 3D finite element analysis (FEA). The method is demonstrated using the maxillary jaw from a freshly euthanized pig (Sus scrofa) and a hyrax-design rapid palatal expander (RPE) appliance with integrated strain gage. The strain gage measurements are recorded using a computer interface, following which the expansion forces and extent of expansion are estimated by FEA. A total activation of 2.0 mm results in peak total force of about 100 N—almost entirely along the direction of expansion. The results also indicate that more than 85% of the input activation is immediately transferred to the palate and/or teeth. These studies demonstrate a method for assessing and individualizing expansion magnitudes and forces during orthopedic expansion of the maxilla. This provides the basis for further development of smart orthodontic appliances that provide real-time readouts of forces and movements, which will allow personalized, optimal treatment

Different duration of parathyroid hormone exposure distinctively regulates primary response genes Nurr1 and RANKL in osteoblasts.

Parathyroid hormone (PTH) exerts dual effects, anabolic or catabolic, on bone when administrated intermittently or continuously, via mechanisms that remain largely unknown. PTH binding to cells induces PTH-responsive genes including primary response genes (PRGs). PRGs are rapidly induced without the need for de novo protein synthesis, thereby playing pivotal roles in directing subsequent molecular responses. In this study, to understand the role of PRGs in mediating osteoblastic cellular responses to PTH, we investigated whether various durations of PTH differentially induce PRGs in primary osteoblasts and MC3T3-E1. Nurr1 and RANKL, PRGs known for their anabolic and catabolic roles in bone metabolism respectively, presented distinctive transient vs. sustained induction kinetics. Corroborating their roles, maximum induction of Nurr1 was sufficiently achieved by brief PTH in as little as 30 minutes and continued beyond that, while maximum induction of RANKL was achieved only by prolonged PTH over 4 hours. Our data suggested distinctive regulatory mechanisms for Nurr1 and RANKL: PKA-mediated chromatin rearrangement for transcriptional regulation of both PRGs and ERK-mediated transcriptional regulation for RANKL but not Nurr1. Lastly, we classified PRGs into two groups based on the induction kinetics: The group that required brief PTH for maximum induction included Nur77, cox-2, and Nurr1, all of which are reported to play roles in bone formation. The other group that required prolonged PTH for maximum induction included IL-6 and RANKL, which play roles in bone resorption. Together, our data suggested the crucial role of PRG groups in mediating differential osteoblastic cellular responses to intermittent vs. continuous PTH. Continued research into the regulatory mechanisms of PKA and ERK for PRGs will help us better understand the molecular mechanisms underlying the dual effects of PTH, thereby optimizing the current therapeutic use of PTH for osteoporosis

Different duration of parathyroid hormone exposure distinctively regulates primary response genes Nurr1 and RANKL in osteoblasts.

Parathyroid hormone (PTH) exerts dual effects, anabolic or catabolic, on bone when administrated intermittently or continuously, via mechanisms that remain largely unknown. PTH binding to cells induces PTH-responsive genes including primary response genes (PRGs). PRGs are rapidly induced without the need for de novo protein synthesis, thereby playing pivotal roles in directing subsequent molecular responses. In this study, to understand the role of PRGs in mediating osteoblastic cellular responses to PTH, we investigated whether various durations of PTH differentially induce PRGs in primary osteoblasts and MC3T3-E1. Nurr1 and RANKL, PRGs known for their anabolic and catabolic roles in bone metabolism respectively, presented distinctive transient vs. sustained induction kinetics. Corroborating their roles, maximum induction of Nurr1 was sufficiently achieved by brief PTH in as little as 30 minutes and continued beyond that, while maximum induction of RANKL was achieved only by prolonged PTH over 4 hours. Our data suggested distinctive regulatory mechanisms for Nurr1 and RANKL: PKA-mediated chromatin rearrangement for transcriptional regulation of both PRGs and ERK-mediated transcriptional regulation for RANKL but not Nurr1. Lastly, we classified PRGs into two groups based on the induction kinetics: The group that required brief PTH for maximum induction included Nur77, cox-2, and Nurr1, all of which are reported to play roles in bone formation. The other group that required prolonged PTH for maximum induction included IL-6 and RANKL, which play roles in bone resorption. Together, our data suggested the crucial role of PRG groups in mediating differential osteoblastic cellular responses to intermittent vs. continuous PTH. Continued research into the regulatory mechanisms of PKA and ERK for PRGs will help us better understand the molecular mechanisms underlying the dual effects of PTH, thereby optimizing the current therapeutic use of PTH for osteoporosis

Deletion of Orai1 leads to bone loss aggravated with aging and impairs function of osteoblast lineage cells

Osteoblast lineage cells, a group of cells including mesenchymal progenitors, osteoblasts, and osteocytes, are tightly controlled for differentiation, proliferation and stage-specific functions in processes of skeletal development, growth and maintenance. Recently, the plasma membrane calcium channel Orai1 was highlighted for its role in skeletal development and osteoblast differentiation. Yet the roles of Orai1 in osteoblast lineage cells at various stages of maturation have not been investigated. Herein we report the severe bone loss that occurred in Orai1−/− mice, aggravated by aging, as shown by the microcomputed tomography (mCT) and bone histomorphometry analysis of 8-week and 12-week old Orai1−/− mice and sex-matched WT littermates. We also report that Orai1 deficiency affected the differentiation, proliferation, and type I collagen secretion of primary calvarial osteoblasts, mesenchymal progenitors, and osteocytes in Orai1−/− mice; specifically, our study revealed a significant decrease in the expression of osteocytic genes Fgf23, DMP1 and Phex in the cortical long bone of Orai1−/− mice; a defective cellular and nuclear morphology of Orai1−/− osteocytes; and defective osteogenic differentiation of Orai1−/− primary calvarial osteoblasts (pOBs), including a decrease in extracellular-secretion of type I collagen. An increase in the mesenchymal progenitor population of Orai1−/− bone marrow cells was indicated by a colony forming unit-fibroblasts (CFU-F) assay, and the increased proliferation of Orai1−/− pOBs was indicated by an MTT assay. Notably, Orai1 deficiency reduced the nuclear localization and transcription activity of the Nuclear Factor of Activated T-cell c1 (NFATc1), a calcium-regulated transcription factor, in pOBs. Altogether, our study demonstrated the crucial role of Orai1 in bone development and maintenance, via its diverse effects on osteoblast lineage cells from mesenchymal progenitors to osteocytes. Keywords: Orai1, Osteoblast lineage cells, Bone, Knockout mic

Therapeutic effect of localized vibration on alveolar bone of osteoporotic rats.

OBJECTIVES:Vibration, in the form of high frequency acceleration (HFA), stimulates alveolar bone formation under physiologic conditions and during healing after dental extractions. It is not known if HFA has an anabolic effect on osteoporotic alveolar bone. Our objective is to determine if HFA has a regenerative effect on osteoporotic alveolar bone. METHODS AND MATERIALS:Adult female Sprague-Dawley rats were divided into five groups: 1) Ovariectomized Group (OVX), 2) Sham-OVX Group that received surgery without ovariectomy, 3) OVX-HFA Group that was ovariectomized and treated daily with HFA, 4) OVX+Static Force Group that was ovariectomized and received the same force as HFA, but without vibration, and 5) Control Group that did not receive any treatment. All animals were fed a low mineral diet for 3 months. Osteoporosis was confirmed by micro-CT of the fifth lumbar vertebra and femoral head. HFA was applied to the maxillary first molar for 5 minutes/day for 28 and 56 days. Maxillae were collected for micro-CT, histology, fluorescent microscopy, protein and RNA analysis, and three-point bending mechanical testing. RESULTS:Micro-CT analysis revealed significant alveolar bone osteoporosis in the OVX group. Vibration restored the quality and quantity of alveolar bone to levels similar to the Sham-OVX group. Animals exposed to HFA demonstrated higher osteoblast activity and lower osteoclast activity. Osteogenic transcription factors (RUNX2, Foxo1, Osterix and Wnt signaling factors) were upregulated following vibration, while RANKL/RANK and Sclerostin were downregulated. HFA did not affect serum TRAcP-5b or CTx-1 levels. The osteogenic effect was highest at the point of HFA application and extended along the hemimaxillae this effect did not cross to the contra-lateral side. CONCLUSIONS:Local application of vibration generated gradients of increased anabolic metabolism and decreased catabolic metabolism in alveolar bone of osteoporotic rats. Our findings suggest that HFA could be a predictable treatment for diminished alveolar bone levels in osteoporosis patients

Osteoclast markers and number of osteoclasts increase in response to HFA mechanical stimulation.

<p><b>(A)</b> Mean concentration of RANKL protein in the maxillary right alveolar bone after 1, 3, 7 and 14 days as measured by ELISA. Data expressed as the mean ± SEM of RANKL concentration in picograms per 100 mg tissue of 5 samples. (* Significantly different from control; **significantly different from OTM group). <b>(B)</b> Light microphotographs of Cathepsin K–positive osteoclasts in immunohistochemically stained sections of mesiopalatal root of the maxillary first molar. Images were collected close to the alveolar crest 28 days after application of force. Osteoclasts are stained as brown multinucleated cells <i>(arrow heads</i>) in sections from different groups and in the detail higher magnification. <b>(C)</b> Mean number of osteoclasts at different time points, in PDL and adjacent alveolar bone of mesiopalatal root of maxillary first molar. Each value represents the mean ± SEM of four animals (*Significantly different from control; **significantly different from OTM group). <b>(D)</b> Representative axial μCT sections showing decrease in bone density in OTM+ HFA at 14 days. Arrow demonstrates the direction of force application. <b>(E)</b> Average bone volume fraction (bone volume/total volume [BV/TV]) was calculated for Control, OTM and OTM+HFA group for the alveolar bone in the area of the maxillary first molar. Each value represents the mean ± SEM of four animals. (* Significantly different from control; ** significantly different from OTM group).</p

Vibration paradox in orthodontics: Anabolic and catabolic effects

<div><p>Vibration in the form of High Frequency Acceleration (HFA) is anabolic on the craniofacial skeleton in the absence of inflammation. Orthodontic forces trigger an inflammation-dependent catabolic cascade that is crucial for tooth movement. It is unknown what effect HFA has on alveolar bone if applied during orthodontic treatment. The objectives of this study are to examine the effect of HFA on the rate of tooth movement and alveolar bone, and determine the mechanism by which HFA affects tooth movement. Adult Sprague Dawley rats were divided to control, orthodontic force alone (OTM), and different experimental groups that received the same orthodontic forces and different HFA regimens. Orthodontic tooth movement was assessed when HFA parameters, frequency, acceleration, duration of exposure, and direct or indirect application were varied. We found that HFA treatment significantly enhanced the inflammation-dependent catabolic cascade during orthodontic tooth movement. HFA treatment increased inflammatory mediators and osteoclastogenesis, and decreased alveolar bone density during orthodontic tooth movement. Each of the HFA variables produced significant changes in the rate of tooth movement and the effect was PDL-dependent. This is the first report that HFA enhances inflammation-dependent catabolic cascades in bone. The clinical implications of our study are highly significant, as HFA can be utilized to enhance the rate of orthodontic tooth movement during the catabolic phase of treatment and subsequently be utilized to enhance retention during the anabolic remodeling phase after orthodontic forces are removed.</p></div