Jeanne M. Nervina

Nuclear orphan receptor Nurr1 directly transactivates the osteocalcin gene in osteoblasts.

Nurr1, an NGFI-B nuclear orphan receptor, which transactivates promoters through an NGFI-B response element (NBRE), is strongly induced by parathyroid hormone through the cAMP-protein kinase A signaling pathway in osteoblasts. Here, we demonstrate that multiple agents activating diverse signaling pathways in osteoblasts induce Nurr1. The strongest Nurr1 inducers were activators of cAMP-protein kinase A-coupled signaling, followed by protein kinase C- and calcium-coupled signaling activators. Receptor tyrosine kinase activators had minimal effect, whereas serine/threonine kinase activators had no effect on basal Nurr1 mRNA levels. Computer analysis of osteoblastic promoters indicated two potential NBREs in the rat osteocalcin (Ocn) promoter. Intriguingly, the proximal site maps to the cAMP-responsive cis-element. We tested whether Nurr1 induces Ocn expression through the NBRE-like site. Recombinant and endogenous Nurr1 protein from primary mouse osteoblasts bound to a consensus NBRE in EMSAs. Nurr1 induced a consensus 3×NBRE-luciferase reporter construct in mouse osteoblasts. Recombinant and endogenous Nurr1 protein bound to the proximal NBRE-like site in the Ocn promoter in EMSAs. Endogenous Nurr1 protein bound to this site as a monomer, because neither retinoid X receptor α nor retinoid X receptor β antibody supershifted the protein-DNA complex. Ocn promoter-luciferase constructs lacking or containing a mutated proximal NBRE-like site had markedly blunted responses to Nurr1 overexpression. Finally, adenovirally expressed Nurr1 protein bound to the proximal NBRE-like site in chromatin immunoprecipitation assays and induced Ocn mRNA in primary rat osteoblasts. We conclude that Ocn is a Nurr1 target gene, which positions Nurr1 in the core of transcriptional factors regulating osteoblastic gene expression.

CBCT in orthodontics: assessment of treatment outcomes and indications for its use

Since its introduction into dentistry in 1998, CBCT has become increasingly utilized for orthodontic diagnosis, treatment planning and research. The utilization of CBCT for these purposes has been facilitated by the relative advantages of three-dimensional (3D) over two-dimensional radiography. Despite many suggested indications of CBCT, scientific evidence that its utilization improves diagnosis and treatment plans or outcomes has only recently begun to emerge for some of these applications. This article provides a comprehensive and current review of key studies on the applications of CBCT in orthodontic therapy and for research to decipher treatment outcomes and 3D craniofacial anatomy. The current diagnostic and treatment planning indications for CBCT include impacted teeth, cleft lip and palate and skeletal discrepancies requiring surgical intervention. The use of CBCT in these and other situations such as root resorption, supernumerary teeth, temporomandibular joint (TMJ) pathology, asymmetries and alveolar boundary conditions should be justified on the basis of the merits relative to risks of imaging. CBCT has also been used to assess 3D craniofacial anatomy in health and disease and of treatment outcomes including that of root morphology and angulation; alveolar boundary conditions; maxillary transverse dimensions and maxillary expansion; airway morphology, vertical malocclusion and obstructive sleep apnoea; TMJ morphology and pathology contributing to malocclusion; and temporary anchorage devices. Finally, this article utilizes findings of these studies and current voids in knowledge to provide ideas for future research that could be beneficial for further optimizing the use of CBCT in research and the clinical practice of orthodontics.

The Rainbow Trout Pineal Organ: An Endocrine Photometer

Like the pineal organs of other vertebrates, those of teleost fish synthesize and secrete melatonin (N-acetyl-5-methoxytryptamine) (Fenwick, 1970; Gern et al., l978a,b; Falcon et al., 1989a; Kezuka et al., 1989; Zachmann et al., 1991). Unlike mammals, pineal organs in non-mammalian vertebrates are directly photoreceptive and these processes are involved in regulating melatonin synthesis. Pineal melatonin synthesis in chickens (Binkely et al., 1978; Kasal et al., 1979; Takahashi et al., 1980; Takahashi et al., 1989), anoline lizards (Menaker & Wisner, 1983) and probably many other nonmammalian vertebrates (Falcon et al., 1989b; Kezuka et al., 1989) is associated with an endogenous circadian clock. In mammals, experimental evidence does not indicate the presence of a clock within the pineal. Rather in mammals a clock, present in the suprachiasmatic nucleus, regulates pineal melatonin synthesis via neuronal connections from the SCN (Moore & Klein, 1974).

Parathyroid hormone induces the nuclear orphan receptor NOR-1 in osteoblasts

Parathyroid hormone (PTH) significantly affects osteoblast function by altering gene expression. We have identified neuron-derived orphan receptor-1 (NOR-1) as a PTH-induced primary gene in osteoblastic cells. NOR-1, Nurr1, and Nur77 comprise the NGFI-B nuclear orphan receptor family and Nurr1 and Nur77 are PTH-induced primary osteoblastic genes. Ten nM PTH maximally induced NOR-1 mRNA at 2h in primary mouse osteoblasts and at 1h in mouse calvariae. Cycloheximide pretreatment did not inhibit PTH-induced NOR-1 mRNA. PTH activates cAMP-protein kinase A (PKA), protein kinase C (PKC), and calcium signaling. Forskolin (PKA activator) and PMA (PKC activator) mimicked PTH-induced NOR-1 mRNA. Ionomycin (calcium ionophore) and PTH(3-34), which do not activate PKA, failed to induce NOR-1 mRNA. PKA inhibition with H89 blocked PTH- and FSK-induced NOR-1 mRNA. PMA pretreatment to deplete PKC inhibited PMA-induced, but not PTH-induced, NOR-1 mRNA. We conclude that NOR-1 is a PTH-regulated primary osteoblastic gene that is induced mainly through cAMP-PKA signaling.

Parathyroid hormone induces expression of the inducible cAMP early repressor in osteoblastic MC3T3-E1 cells and mouse calvariae.

Parathyroid hormone (PTH) regulates gene expression in skeletal osteoblasts mainly through the cAMP–protein kinase A (PKA) pathway. In neuroendocrine cells, activation of the cAMP–PKA signaling pathway leads to induction of the inducible cAMP early repressor (ICER), which is transcribed from an intronic promoter of the CREM gene and acts as a transcriptional repressor. To investigate whether PTH induces ICER expression in osteoblastic cells, RNA from MC3T3‐E1 cells was subjected to reverse transcriptase‐polymerase chain reaction using primers spanning the ICER sequence. Amplified products were subcloned, sequenced, and used as a probe for Northern blot analysis. In MC3T3‐E1 cells, PTH induced ICER mRNA levels, which peaked at 2 h and declined to baseline by 8 h. Cycloheximide caused superinduction of ICER mRNA in response to PTH. In cultured mouse calvariae, PTH also induced ICER mRNA accumulation, which peaked at 2 h and returned almost to baseline by 10 h. Overexpression of ICER IIγ decreased both baseline and PTH‐stimulated prostaglandin G/H synthase 2 promoter activity in MC3T3‐E1 cells. The induction of ICER represents a novel mechanism by which PTH regulates gene expression in osteoblastic cells.

Parathyroid hormone induces RGS-2 expression by a cyclic adenosine 3′,5′-monophosphate-mediated pathway in primary neonatal murine osteoblasts

Parathyroid hormone (PTH) is a promising anabolic agent for the treatment of osteoporosis. However, PTH is also potently catabolic. To help delineate the molecular mediators of PTHs opposing effects on skeletal metabolism, we have examined PTH-induced regulator of G-protein signaling-2 (RGS-2) expression and function in murine osteoblasts. RGS proteins are GTPase-activating proteins (GAPs) that regulate GTP-binding protein-coupled receptor (GPCR) signaling by enhancing the intrinsic GTPase activity of Galpha subunits. We found that 10 nmol/L PTH maximally induced RGS-2 mRNA in murine MC3T3-E1 cells, rat Py1a and ROS-17/2.8 cells, primary mouse osteoblasts (MOB cells), and mouse calvariae organ culture at 1-2 h posttreatment. PTH signaling through its receptor, PTHR1, is coupled to cAMP-protein kinase A (PKA), protein kinase C (PKC), and calcium signaling pathways. We examined the effect of selective signaling agonists and antagonists on RGS-2 expression in MOB cells to determine which pathway(s) mediates PTH-induced RGS-2 expression. Although selective activation of all three pathways led to RGS-2 expression, cAMP-PKA activation with 10 nmol/L PTH and 10 micromol/L forskolin elicited the strongest induction. Similarly, RGS-2 mRNA expression was most strongly inhibited by the PKA inhibitor, H89 (10-30 micromol/L). The phorbol ester, PMA (1 micromol/L), which activates the PKC pathway, and ionomycin (1 micromol/L), which activates the calcium pathway, produced small but detectable elevations in RGS-2 mRNA levels. Overnight treatment with 1 micromol/L PMA to deplete PKC did not affect subsequent RGS-2 induction by PTH, but significantly inhibited PMA-induced RGS-2 expression. Treatment with 1-100 nmol/L PTH(3-34), which does not activate cAMP-PKA signaling, did not induce RGS-2 expression. MOB cells pretreated with 3 microg/mL cycloheximide produced sustained RGS-2 mRNA levels 2 h after 10 nmol/L PTH treatment. Actinomycin D (5 microg/mL) completely blocked 10 nmol/L PTH-induced RGS-2 expression. Finally, we tested the effect of RGS-2 overexpression on PTH- and fluprostenol-induced interleukin (IL)-6 promoter activity in MOB cells. PTH induces IL-6 through PKA activation, whereas fluprostenol induces IL-6 through PKC activation. We found that RGS-2 overexpression significantly inhibited IL-6 promoter activity following fluprostenol treatment, but not following PTH treatment. We conclude that RGS-2 is a PTH-induced primary response gene in murine osteoblasts that is induced mainly through the cAMP-PKA pathway and specifically inhibits Galphaq-coupled receptors.

PGC-1α is induced by parathyroid hormone and coactivates Nurr1-mediated promoter activity in osteoblasts

Parathyroid hormone (PTH) potently activates cAMP-protein kinase A (PKA)-driven molecular cascades in osteoblasts. The NR4A/NGFI-B orphan nuclear receptor (NR) Nurr1 is a PTH-induced, cAMP-responsive primary response gene (PRG) that transactivates osteocalcin (Ocn) expression through a putative NGFI-B response element (NBRE) in the proximal promoter. As a true orphan NR, Nurr1s expression level and coactivator recruitment regulate its transactivation capacity. We postulated that Nurr1s induction through cAMP-PKA signaling might favor a coactivator that is likewise cAMP-dependent. A possible candidate is the cAMP-inducible coactivator PPARgamma coactivator-1alpha (PGC-1alpha). We hypothesize that PGC-1alpha is a PTH-induced PRG that synergizes with Nurr1 to induce target gene transcription in osteoblasts. We show that 10 nM PTH for 2 h maximally induced PGC-1alpha mRNA in primary mouse osteoblasts (MOBs) and calvariae. Selective signaling agonists and antagonists demonstrated that PTH induced PGC-1alpha mRNA primarily through the cAMP-PKA pathway. Protein synthesis inhibition sustained PTH-induced PGC-1alpha expression. PGC-1alpha enhanced Nurr1-induced transactivation of a consensus 3xNBRE-luciferase construct and the rat (-1050)Ocn promoter-luciferase construct from 3.7- to 9.6- and 10.1-fold, respectively. This synergy required Nurr1-DNA binding, since a mutation of the Ocn promoter NBRE abolished both Nurr1- and Nurr1-PGC-1alpha-induced transactivation. Using GST pull-down assays, PGC-1alpha directly interacted with in vitro-generated and nuclear Nurr1. We conclude that PGC-1alpha is a PTH-induced, cAMP-dependent PRG that directly synergizes with Nurr1 to transactivate target genes in osteoblasts. Taken together with published data, our findings suggest that Nurr1 and PGC-1alpha may be pivotal mediators of cAMP-induced osteoblast gene expression and osteoblast function.

Effects of deferoximine on chondrocyte alkaline phosphatase activity: Proxidant role of deferoximine in thalassemia

The homozygous form of β-thalassemia, the most common single gene disorder, is treated by red cell transfusion therapy. Following transfusion, the chelator, deferoximine, is administered to patients to remove excess iron. However, when this drug is given to young children, metaphyseal dysplasia and abnormalities of linear growth are frequently observed. To explore the notion that deferoximine mine interferes with endochondral growth by chelating zinc, we examined the effect of the drug on chondrocytes maintained in long-term culture. We found that deferoximine caused a dose-dependent inhibition of a wide range of functions including cell proliferation, protein synthesis (and possibly under-hydroxylation of type X collagen), and mineral deposition. Directly relevant to the mineralization process was the observation that the drug dramatically lowered the activity of alkaline phosphatase, a zinc-requiring enzyme. To test the hypothesis that enzyme inhibition was due to chelation of zinc by deferoximine, the cell culture medium was supplemented with excess zinc. However, this treatment did not overcome the deferoximine-dependent change in enzyme activity. We next examined the possibility that deferoximine, in the presence of ascorbate, could form a free radical system that would serve to inactivate the enzyme. Using alkaline phosphatase extracted from chick cartilage, we noted that the activity of the phosphatase was markedly reduced in the presence of deferoximine and ascorbate. These effects were consistant with the notion that deferoximine and ascorbate can act as a prooxidant couple. This conclusion was confirmed when we measured the oxidative activities of the system using nitroblue tetrazolium and cytochrome c. Indeed, we noted that deferoximine markedly activates the autocatalytic oxidation of ascorbate. We next investigated the possibility that the change in alkaline phosphatase activity was due to the formation of reactive oxygen radicals. Though oxygen radical scavengers and disproportionating agents did not change the activity of the enzyme, α-tocopherol provided complete protection. In conclusion, the deferoximine-ascorbate couple inactivates chondrocyte alkaline phosphatase probably by generation of free radicals. As free radicals can damage cartilage as well as other tissues, clinical regimens that are directed at elevating ascorbate levels in thalassemia need to be carefully reviewed.

Cone beam computed tomography use in orthodontics.

Cone beam computed tomography (CBCT) is widely used by orthodontists to obtain three-dimensional (3-D) images of their patients. This is of value as malocclusion results from discrepancies in three planes of space. This review tracks the use of CBCT in orthodontics, from its validation as an accurate and reliable tool, to its use in diagnosing and treatment planning, and in assessing treatment outcomes in orthodontics.

Expression of inducible cAMP early repressor is coupled to the cAMP-protein kinase A signaling pathway in osteoblasts

We previously showed that parathyroid hormone (PTH) induces inducible cAMP early repressor (ICER) in osteoblastic cells and mouse calvariae. PTH signaling in osteoblastic cells is transduced by PTH receptor 1, which is coupled to cAMP-protein kinase A (PKA), protein kinase C (PKC), and calcium signaling pathways. In the present study, we examined the role of these pathways in mediating PTH-induced ICER mRNA and protein expression in osteoblastic MC3T3-E1 cells. Using RT-PCR, we found that PTH(1-34), forskolin (FSK), and 8-bromo-cAMP (8Br-cAMP) induced ICER expression, while phorbol myristate acetate (PMA), ionomycin, and PTH(3-34) did not. Similar results were found for the induction of ICER protein. PKA inhibition by H89 markedly reduced PTH- and FSK-induced ICER expression, while PKC depletion by PMA had little effect. We also tested ICER induction by other osteotropic signaling agonists. Other cAMP-PKA pathway activators, such as PTH-related protein (PTHrP), induced ICER expression, while agents that signal through other pathways did not. PTHrP maximally induced ICER mRNA at 2-4 h, which then returned to baseline by 10 h. Finally, PTH, FSK, and PTHrP induced ICER in cultured mouse calvariae and osteoblastic ROS 17/2.8, UMR-106, and Pyla cells. We conclude that ICER expression in osteoblasts requires activation of the cAMP-PKA signaling pathway.