Thèses sur le sujet « Parathyroid Hormone Receptor (PTH1R) »

Our first aim was to generate transgenic mice to express beta-galactosidase reporter gene under the control of the two promoters. The model would allow us to study the temporal and spatial expression of PTH1R during the onset of embryonic endochondral ossification, and in the adult. Our transgenic animals would allow us to identify regulatory elements that are essential for tissue specific PTH1R expression. We have cloned 11 kilobases of mouse PTH1R gene promoter sequence containing four untranslated exons U1, U2, U3 and SS, and fused this to a Lac Z reporter gene, which was in turn fused to a 250 by fragment containing the A-rich polyadenylylation signal. Three additional constructs were made with deletion of transcription start sites in exon U1 (DeltaU1), U3 (DeltaU3) and both (DeltaU1DeltaU3). Both the control and transgenic adult littermates showed high levels of beta-galactosidase-like activity in epiphyseal growth plate and kidney medulla. However, beta-galactosidase activity was not observed for fetuses aged post coital 14.5 and 15.5 days. We were unable to show tissue specific reporter activity in our transgenic animals.
In other study, we found that P2 is the predominant promoter controlling PTH1R gene expression in both bone and cartilage. (Abstract shortened by UMI.)

Parathyroid hormone (PTH) family members participate actively in various steps of endochondral bone development, in maintenance of bone health, and in mineral homeostasis. PTH type 2 receptor (PTH2R) and its ligand tuberoinfundibular peptide of 39 residues (TIP39) are recent additions to the PTH family of receptors and ligands. They are abundantly expressed in brain where it is proposed to possess neurotransmitter function.In this study, we demonstrate the presence of both TIP39 and PTH2R in the growth plate of murine long bones, and we show that the periarticular chondrocytes which express PTH2R are spatially separated from the hypertrophic chondrocytes expressing TIP39. Using CFK2 chondrocytic cells as an in vitro model, we demonstrate that TIP39/PTH2R signalling impairs cell proliferation through inhibition of progression at the G0/G1 phase of the cell cycle. We also show that this signalling inhibits cell differentiation through deregulated expression of the master transcription factor of chondrocyte function, Sox9.In order to investigate the in vivo functions of the TIP39/PTH2R signalling pathway, we generated transgenic mice overexpressing PTH2R in chondrocytes under the control of the Collagen type II promoter/enhancer. We observed that PTH2R overexpression reduced chondrocyte proliferation and impaired trabecular bone formation likely through alterations in Wnt signalling and β-catenin expression. In addition, PTH2R overexpression was observed to delay growth plate secondary ossification in postnatal development, thus retarding endochondral bone growth. We further observed a downregulation of GDF5, a member of the bone morphogeneticprotein family, and of WDR5, a transcriptional modulator of articular chondrocytes, two factors previously shown to influence the development of the secondary ossification centre.Taken together, our results indicate that TIP39 and PTH2R in growth plate chondrocytes exert a crucial signalling role during endochondral bone development. Further studies are needed to explore these functions in experimental pathological models such as chondrodysplasias and possibly osteoarthritis.
Les membres de la famille de l'hormone parathyroïdienne (PTH) participent au développement de l'os endochondral, et au maintien de la santé osseuse et de l'homéostasie minérale. Le récepteur de type 2 de la PTH (PTH2R), et son ligand le peptide tubéroinfundibulaire de 39 acides aminés (TIP39) sont des nouveaux membres de la famille des récepteurs de la PTH et ses ligands. Ils sont exprimés en abondance dans le cerveau où on leur attribue des fonctions de neurotransmetteurs.Dans la présente étude, nous démontrons chez la souris la présence du TIP39 et du PTH2R dans la plaque de croissance des os longs, et nous mettons en évidence la séparation spatiale des chondrocytes périarticulaires exprimant le PTH2R, et des chondrocytes hypertrophiques exprimant le TIP39. A l'aide du modèle in vitro de cellules chondrocytiques CFK2, nous démontrons que la signalisation du complexe TIP39/PTH2R ralentit la prolifération cellulaire en inhibant la progression au delà de la phase G0/G1 du cycle cellulaire. De plus, nous demontrons que la différenciation cellulaire est inhibée par la dérégulation de l'expression du Sox9, le principal facteur de transcription contrôlant la fonction chondrocytaire.Afin d'évaluer les fonctions in vivo de la voie de signalisation du complexe TIP39/PTH2R, nous avons géneré des souris transgéniques surexprimant le PTH2R dans les chondrocytes sous le contrôle du promoteur du gène du Collagène de type II. La surexpression du PTH2R a réduit la prolifération des chondrocytes et inhibé la formation de l'os trabéculaire, probablement par une modification de la signalisation du Wnt et de l'expression de la β-caténine. De plus, durant le développement post-natal, la surexpression du PTH2R a ralenti l'ossification de la plaque de croissancesecondaire, retardant ainsi la croissance de l'os endochondral. Nous avons de plus observé une diminution de l'activité de GDF5 et de WDR5 (respectivement membre de la famille des protéines morphogéniques de l'os, et modulateur transcriptionel des chondrocytes articulaires), deux facteurs connus pour influencer le développement du centre d'ossification secondaire.En conclusion, nos résultats indiquent que le TIP39 et le PTH2R jouent un rôle crucial de signalisation au cours du développement de l'os endochondral. Des études futures sont nécessaires afin d'explorer les fonctions du TIP39 et du PTH2R dans divers modèles expérimentaux, dont ceux de la chondrodysplasie et possiblement dans l'ostéoarthrite.

This thesis examines in vivo and in vitro the effects of prolonged exposure of target cells to PTH on PTH receptors and postreceptor components of the adenylate cyclase system. Using a vitamin D-deficient ($-$D) rat model, hyperparathyroidism in vivo resulted in a decreased number of PTH receptors in kidney and a decreased amount of G protein $ alpha$ subunits. The decreased amount of G$ sb{ rm s} alpha$ was shown to be specific for PTH target tissue and may have played a role in the heterologous desensitization of CT-stimulated adenylate cyclase, demonstrated in renal membranes from the $-$D rats. The study of the control of PTH receptors was pursued using an osteosarcoma clonal cell line, UMR-106, in vitro. Initial characterization of these cells revealed abundant, saturable, cell surface PTH receptors linked to the adenylate cyclase system. Demonstration that the majority of PTH binding was associated with morphologically distinct cells in the UMR-106 population indicated that PTH receptors may be maximally expressed during specific stages in the cell cycle. PTH receptors in UMR-106 cells were shown to be regulated by distinct homologous and heterologous mechanisms. PTH-mediated, homologous desensitization was associated with down-regulation of PTH receptors and loss of G$ sb{ rm s} alpha$ protein form the cell membrane. Heterologous desensitization of PTH responses by PGE$ sb2$ was shown to be cAMP mediated, resulting in a reversible modification of the PTH receptors. This work has demonstrated that multiple mechanisms exist for the regulation of PTH responses both in vivo and in vitro that involve modifications of both the PTH receptors and postreceptor components of the adenylate cyclase system.

Parathyroid hormone (PTH) is the major physiological regulator of the extracellular Ca2+ concentration ([Ca2+]o) in the body. The secretion of this hormone is suppressed at high [Ca2+]o. Previously this was thought to occur by intracellular degradation of the hormone in the secretory pathway of parathyroid (PT) cells but is now believed to result from extracellular Ca2+ stimulus-secretion coupling via the calcium sensing receptor (CaR). In contrast to the stimulation of PTH secretion upon inhibition of mature PTH proteolysis, inhibition of PT proteasomes caused the accumulation of PTH precursors and inhibited secretion of PTH. This suggests that PT proteasomes play a quality control function in the maturation of PTH but they do not directly participate in the [Ca2+]o-regulated secretion of the hormone. Treatment of PT cells with 12-O-tetradecanyolphorbol-13-acetate (TPA) blocks the high [Ca2+]o-induced CaR-mediated suppression of PTH secretion. To delineate the role of DAG-responsive protein kinase C (PKC) isoforms in this process, we complemented pharmacological modulation of PKC activity with physiological activation of the enzyme via the CaR. PKC-α was rapidly activated by high [Ca2+]o and was efficiently down-regulated by prolonged TPA treatment. In CaR-transfected HEK293 cells, TPA and high [Ca2+]o induced the activation of ERK1/2 but the TPA effect was CaR- and Ca2+-independent. The magnitude of neomycin-induced release of Ca2+ from intracellular stores following pharmacological modulation of PKC activity was opposite to that resulting from physiological activation/inhibition of the enzyme via the CaR. Influx of Ca2+ following activation of the receptor occurred by store-operated mechanisms. Over-expression of wt or DN PKC-α or-ε in PT cells using the Tet-On adenovirus gene delivery system revealed that the stimulatory effect of TPA on PTH secretion at high [Ca2+]o was enhanced in cells over-expressing wt PKC-α, but the coupling of the extracellular Ca2+ signal to PTH secretion was not dependent on the physiological activation of this PKC isoform via the CaR.

A prognostic factor is a marker or a feature that can be used to estimate the risk of recurrence of disease, metastatic spread and clinical outcome. Despite intensive search for more sophisticated markers in renal cell carcinoma (RCC), few have added prognostic information to earlier described factors like stage of disease, nuclear grade, tumour type, and in metastatic disease; performance status, anaemia, hypercalcaemia and increased erythrocyte sedimentation. In the dominating tumour type, clear cell renal RCC (cRCC), hypoxia is common, leading to an up-regulation of hypoxia inducible factor (HIF). The majority of cRCC have a mutation in the von Hippel Lindau gene (VHL-gene), which regulates HIF and in turn leads to up-regulation of a number of target genes for potential growth factors. The aim of the study was to evaluate the possible prognostic information of a few factors associated to pVHL/HIF, anemia and/or hypercalcaemia in RCC; erythropoietin (EPO) and it´s receptor (EPO-R), carbonic anhydrase IX (CA IX), parathyroid hormone-related protein (PTHrP) and osteopontin (OPN). Patients diagnosed with RCC between 1982-2007 were included in the studies. The tumour tissue expressions of EPO, EPO-R and PTHrP were assessed using immunohistochemistry. Serum/plasma levels of EPO, CA IX, PTHrP and OPN were also analyzed using immunometric methods. Our study demonstrated that the expression of EPO and EPO-R were related, and the expressions differed significantly between RCC types. The serum EPO levels did not associate to the tumour expression of EPO or EPO-R, indicating that circulating EPO derives from other sources than tumour cells. Erythropietin receptor expression was more frequent in advanced stages of disease, but neither EPO, nor EPO-R, were independent prognostic factors for survival. Serum CA IX levels were higher in cRCC compared to papillary RCC (pRCC). In cRCC, the CA IX serum levels correlated positively to TNM stage, but serum CA IX did not add independent prognostic information. Parathyroid hormone-related protein is a cause of hypercalcaemia in malignancy, and we observed that circulating PTHrP related to hypercalcaemia in RCC. The tumour expression of PTHrP associated positively to serum PTHrP, but not to serum calcium. We found an association between PTHrP and OPN in plasma, and both plasma PTHrP and OPN were positively associated to TNM stage.  Neither serum/plasma PTHrP nor tumour expression of PTHrP were independent prognostic factors for survival. The serum OPN levels were higher in pRCC but no impact on survival was observed in this RCC type. In contrast, plasma/serum OPN was an independent prognostic factor for disease-specific survival in cRCC. Our results support a role for these factors in RCC. The expressions vary between tumour types, which can be explained by different gene aberrations. Some of the factors have a close relation to para-malignant symptoms like hypercalcaemia. Most of the factors correlate positively to TNM-stage, reflecting a relation to advanced disease. Although expression of EPO, EPO-R, PTHrP and CA IX did not add independent prognostic information, the results might contribute to greater understanding of important mechanisms and associations in RCC. Osteopontin is a strong independent prognostic factor in cRCC, and should be further evaluated as a tool in the clinic when treating RCC patients.

Introdução - O receptor de vitamina D (VDR) é expresso em vários tecidos e quando este se encontra na sua forma ativada, modula a expressão de diversos genes. Esses incluem variações dos níveis circulantes de 1,25(OH) 2 D , variações na densidade mineral óssea, secreção e sensibilidade à insulina em resposta à glicose, suscetibilidade à diabetes tipo 1 e 2, obesidade, dislipidemias e hipertensão arterial. Atualmente, evidências têm sugerido o envolvimento da vitamina D com a síndrome metabólica. Objetivo - Investigar a concentração sérica da vitamina D e sua relação com a síndrome metabólica e avaliar a potencial associação entre estes fatores com a presença de polimorfismos no gene do receptor de vitamina D (VDR) em indivíduos adultos. Métodos - Trata-se de um estudo transversal, onde foram avaliados 372 indivíduos adultos. Foram coletadas amostras sanguíneas para dosagens laboratoriais da 25(OH)D 3 , PTH e exames bioquímicos relacionados à SM, além disso foram realizadas avaliações antropométricas (peso, altura, IMC). A síndrome metabólica (SM) foi classificada usando o critério proposto pelo National Cholesterol Education Program-Adult Treatment Panel III (NCEP-ATP III). A resistência a insulina foi estimada pelo cálculo de HOMA IR e a função da célula pelo cálculo de HOMA . A 25(OH)D foi dosada por HPLC e a insuficiência foi determinada pelo ponto de corte da curva Roc (52,6nmol/l). Foram avaliados também PTH intacto e cálcio sérico. Os polimorfismos BsmI e FokI foram detectados através da digestão das enzimas de restrições específicas para cada polimorfismo e confirmados através da técnica PCR alelo específico (ASPCR) ou amplificação de mutação refratária (ARMs) nos indivíduos com e sem SM (52 por cento vs. 48 por cento , respectivamente). A análise estatística inclui construção da curva ROC, teste T de Student, testes de correlação, teste de equilíbrio de Hardy-Weinberg, ANOVA, regressão logística binária (Odds Ratio). Estas análises foram conduzidas no software SPSS para Windows, versão 18 e p < 0,05 foi considerado significante. Resultados - A idade média dos participantes foi 51(15) anos, o IMC médio 29(6) kg/m 3 2 e 48 por cento apresentaram SM. Como esperado, os 3 indivíduos com SM apresentaram maiores valores de idade 57(12) anos, IMC 32(6) Kg/m , circunferência de cintura 103(13) cm, pressão sistólica 138(17) mmHg e diastólica 83(10) mmHg, glicemia de jejum 98(12) mg/dl, triglicérides 165(76) mg/dl, índices HOMA-IR 2.2(1.7) e 116(95), e menores valores de colesterol HDL colesterol 41(11) mg/dl. Com relação às concentrações séricas de 25(OH)D propostas pela análise da curva ROC, 43 por cento dos indivíduos com SM e 57 por cento dos indivíduos sem SM apresentam insuficiência desta vitamina. Correlações entre 25(OH)D 3 3 com PTH (r = -0.153; p = 0.005) e com circunferência da cintura (r = -0.106; p = 0.05) foram observada em todos os participantes. Considerando os polimorfismos do gene VDR, nos pacientes com SM, não houve associação entre o polimorfismo BsmI e os componentes da SM, HOMA e IR, 25(OH)D e PTH. No entanto, indivíduos sem SM, mas com homozigose para polimorfismo BsmI (genótipo recessivo bb ), apresentaram concentrações mais baixas de 25(OH)D 3 3 do que aqueles com o genótipo BB normal. Além disso, os indivíduos com SM e heterozigose para o polimorfismo FokI (genótipo Ff) têm maiores concentrações de PTH e HOMA do que aqueles com genótipo normal FF. Nesse mesmo grupo, os indivíduos com o genótipo recessivo ff têm maior resistência à insulina do que aqueles com genótipo Ff. Por outro lado, os pacientes sem SM, mas carregando o genótipo Ff, apresentaram maiores concentrações de triglicerídeos e baixos níveis de HDL do que aqueles com genótipo FF. A presença de um alelo f no genótipo (Ff ou ff) é, aparentemente, o suficiente para aumentar os níveis de triglicérides e resistência à insulina, quando comparados ao genótipo normal FF. Conclusão - Os resultados demonstram que o polimorfismo FokI no gene VDR associa-se a resistência à insulina e maiores concentrações de PTH em pacientes que apresentam SM. Além disso, o polimorfismo BsmI associa-se a menores concentrações de 25(OH)D em indivíduos sem SM. Portanto, esses novos dados indicam que polimorfismos no gene do VDR estão associados a diferentes fenótipos dos componentes da SM
Introduction - The vitamin D receptor (VDR) is expressed in many tissues and when it is in its activated form modulates the expression of several genes. These include changes in circulating levels of 1,25(OH)2D3, variations in bone mineral density, sensitivity and secretion of insulin in response to glucose, susceptibility to type 1 and 2 diabetes mellitus, obesity, dyslipidemia and hypertension. Currently, evidences have suggested the involvement of vitamin D with the metabolic syndrome. Objective - To investigate the serum concentrations of vitamin D and its relationship with metabolic syndrome (MS) and to evaluate the potential association between these factors with the presence of polymorphisms in vitamin D receptor gene in individuals adults. Methods - This is a cross-sectional study, which evaluated 243 adults and elderly. We collected blood samples for measurements of 25(OH)D3, iPTH, biochemical tests related to MS, and anthropometric evaluation (weight, height, BMI) were also assessed. MS was classified using the criteria proposed by the National Cholesterol Education Program-Adult Treatment Panel III (NCEP-ATP III). Insulin resistance and cell secretion were estimated by calculating HOMA IR and HOMA , respectively. The 25(OH)D3 was measured by HPLC and insufficiency was determined by the Roc curve cut-off (52.6 nmol/L). Intact PTH and serum calcium were also evaluated. The BsmI and FokI polymorphisms were detected by enzymatic digestion with specific enzymes and confirmed by allele specific PCR (ASPCR) or amplification of refractory mutation (ARM) in individuals with or without MS (52 per cent vs. 48 per cent , respectively). Statistical analyses include construction of Roc curves, Student T test, correlation tests, Hardy-Weinberg test, ANOVA, binary logistic regression (odds ratio), and TwoStep Cluster. These analyses were conducted with SPSS for Windows, version 18 and p < 0.05 was considered significant. Results - The mean age of participants was 51(15) years, mean BMI was 29(6) kg/m2, and 48 per cent of individuals presented MS. As expected, subjects with MS showed higher values of age (57(12) years), BMI was 32(6) kg/m2, waist circumference was 103(13) cm, systolic blood pressure was 138(17) mmHg, diastolic was 83(10) mmHg, fasting glucose was 98(12) mg/dl, triglycerides was 165(76) mg/dl, HOMA-IR was 2.2(1.7), HOMA was 116(95), and lower levels of HDL cholesterol was observed (41 mg/dl(11)). With respect to serum 25(OH)D3 proposed by ROC curve analysis, 43 per cent of individuals with MS and 57 per cent of individuals without MS presented insufficiency of this vitamin. Correlations between 25(OH)D3, iPTH (r = -0,153, p = 0.005), and waist circumference (r = -0,106, p = 0.05) were observed in all participants. Considering the VDR gene polymorphisms, in patients with MetSyn, there is no association among BsmI polymorphism and components of MetSyn, HOMA IR and , 25(OH)D3, and PTH. However, subjects without MetSyn, but with homozygosis for BsmI polymorphism (recessive bb genotype), presented lower levels of 25(OH)D3 than those with normal BB genotype. In addition, individuals with MetSyn and heterozygosis for FokI polymorphism (Ff genotype) have higher concentrations of PTH and HOMA than those with normal FF genotype. In this same group, subjects with the recessive ff genotype have higher insulin resistance than those with Ff genotype. On the other hand, patients without MetSyn, but carrying the Ff genotype, have higher concentration of triglycerides and lower levels of HDL than those with FF genotype. Interestingly, the presence of one allele f in the (Ff or ff) genotype is apparently enough to increase triglycerides levels and insulin resistance, when compared to the normal FF genotype. Conclusion - The results show that FokI polymorphism in the VDR gene is associated to insulin resistance and higher concentrations of PTH in patients with MetSyn. Moreover, BsmI polymorphism is related to a lower concentration of 25(OH)D3 in individuals without MetSyn. Therefore, the results indicated that VDR gene polymorphisms are associated to different phenotypes of MetSyn components

碩士
國立臺灣大學
土木工程學研究所
104
Parathyroid hormone/ parathyroid hormone-related protein type 1 receptor, known as PTHR1, is a family of B class G protein couple receptors, its structure contains 7 α helix. PTHR1 regulates skeletal development, bone turnover and calcium ion concentration. Binding of PTHR1 and the ligand of Parathyroid hormone, PTH and Parathyroid hormone-related protein changes the conformations of PTHR1 and plays important role in transferring the signal for crucial biochemical reactions. However, the transmembrane domain structure of PTHR1.is still unclear. Only the structure of extracellular domain of PTHR1 has been revealed by the experiments. The conformation of the transmembrane domain of PTHR1 is known to be playing important role in the signaling mechanism. On the other hand, PTHR1 is associated with many diseases, such as Osteoporosis, Hypoparathyroidism, Brachydactyl type E and Eiken syndrome. Therefore understanding and developing the full atomistic and conformation of the full length PTHR1, can provide the new insights into these diseases and help the design of drugs. The transmembrane domains of Glucagon receptor and corticotropin-releasing factor receptor 1 have been revealed by the experiments. On account of the sequence similarities, we use the comparative protein structure modelling software, Modeller to predict the transmembrane structure of PTHR1. In this thesis, we combined the molecular dynamics and Modeller to solve the structure of full length PTHR1. The hydrogen bonds, hydrophobic interactions and the disulfide bonds are analyzed to provide fundamental insights into the structure and binding sites of the PTHR1 This study also provides a framework to study the structure of PTHR1 in membrane. In the future, we can use this model to investigate the ligand binding mechanisms of PTHR1, enabling the design of new treatments with the disease for PTHR1 related diseases.

The superfamily of G protein-coupled receptors (GPCR) regulates numerous physiological and pathophysiological processes. Hence GPCRs are of significant interest for pharmacological therapy. Embedded into cytoplasmic membranes, GPCRs represent the core of large signaling complexes, which are critical for transduction of exogenous stimuli towards activation of downstream signaling pathways. As a member of the GPCR family B, the parathyroid hormone receptor (PTHR) activates adenylyl cyclases, phospholipases C β as well as mitogen-activated protein kinase-dependent signaling pathways, thereby mediating endocrine and paracrine effects of parathyroid hormone (PTH) and parathyroid hormone-related peptide (PTHrP), respectively. This regulates, calcium homeostasis, bone metabolism and bone development. Paradoxically, PTH is able to induce both catabolic and anabolic bone metabolism. The anabolic effect of PTH is successfully applied in the therapy of severe osteoporosis. Domination of anabolic or catabolic bone-metabolism is entailed by temporal and cell-type specific determinants. The molecular bases are presumably differential arrangements of adaptor proteins within large signaling complexes that may lead to differential activation of signaling pathways, thereby regulating physiological effects. The molecular mechanisms are largely unclear; thus, there is significant interest in revealing a better understanding of PTHR-related adaptor proteins. To identify novel adaptor proteins which direct PTHR signaling pathways, a proteomic screening approach was developed. In this screening, vav2, a guanine-nucleotide exchange factor (GEF) for small GTPases which regulates cytoskeleton reorganization, was found to interact with intracellular domains of PTHR. Evidence is provided that vav2 impairs PTH-mediated phospholipase C β (PLCβ) signaling pathways by competitive interactions with G protein αq subunits. Vice versa, PTH was shown to regulate phosphorylation and subsequent GEF activity of vav2. These findings may thus shed new light on the molecular mechanisms underlying the effects of PTH on bone metabolism by PLC-signaling, cell migration and cytoskeleton organization. In addition to the understanding of intracellular molecular signaling processes, screening for ligands is a fundamental and demanding prerequisite for modern drug development. To this end, ligand binding assays represent a fundamental technique. As a substitution for expensive and potentially harmful radioligand binding, fluorescence-based ligand-binding assays for PTHR were developed in this work. Based on time-resolved fluorescence, several assay variants were established to facilitate drug development for the PTHR
Die Superfamilie der G-Protein-gekoppelten Rezeptoren (GPCRs) reguliert eine Vielzahl von physiologischen und pathophysiologischen Prozessen, was sie bedeutend für die Pharmakotherapie macht. Eingebettet in die Zytoplasmamembran sind GPCRs das Zentrum von Signalkomplexen, die eine Transduktion äußerer Stimuli zur Aktivierung von nachgeschalteten Signalwegen ermöglichen. Der zur Familie B der GPCRs gehörige Parathormon-Rezeptor (PTHR) aktiviert Adenylyl-Zyklasen-, Phospholipasen Cβ- und Mitogen-aktivierte Proteinkinase (MAPK)-abhängige Signalwege, wodurch endokrine und parakrine Wirkungen des Parathormons (PTH) und des Parathormon-ähnlichen Peptides (PTHrP) vermittelt werden. Dies ermöglicht die Regulation der Calcium-Homöostase, des Knochenmetabolismus und der Knochenentwicklung. Paradoxerweise kann PTH sowohl katabole als auch anabole Effekte auf den Knochenstoffwechsel induzieren. Den anabolen Effekt von PTH nutzt man erfolgreich in der Therapie der schweren Osteoporose. Ob ein anaboler oder kataboler Knochenmetabolismus überwiegt, wird durch zeitliche und Zelltyp-spezifische Faktoren bestimmt. Dem zugrunde liegt vermutlich unter anderem eine differenzielle Anordnung verschiedener Adapterproteine innerhalb der Signalkomplexe, die zur differenziellen Aktivierung von Signalwegen führen und so eine Steuerung bestimmter physiologischer Effekte ermöglichen. Die molekularen Mechanismen sind jedoch noch weitgehend unklar, weshalb großes Interesse besteht, ein besseres Verständnis über die PTHR-assoziierten Adapterproteine zu entwickeln. Zur Identifizierung neuer Adapterproteine, die PTHR-Signalwege beeinflussen, wurde in dieser Arbeit ein auf dem Proteom-basierender Screening-Ansatz entwickelt. Dieser führte zur Entdeckung einer Interaktion von intrazellulären Domänen des PTHR mit vav2, einem Guanin-Nukleotid Austauschfaktor (GEF) für kleine GTPasen, der die Zytoskelett-Reorganisation steuert. Des Weiteren wurde nachgewiesen, dass vav2 über kompetitive Interaktionen mit G Protein αq Untereinheiten PTH-vermittelte Phospholipase Cβ (PLCβ)-abhängige Signalwege beeinflusst. Umgekehrt wurde gezeigt, dass PTH die Phosphorylierung und damit die GEF Aktivität von vav2 reguliert. Diese Befunde können Aufschluss über molekulare Mechanismen geben, die den Wirkungen von PTH auf den Knochenstoffwechsel durch PLC-Signalwege, Zellmigration und Zytoskelett-Reorganisation zugrunde liegen. Neben dem Verständnis über molekulare Prozesse der intrazellulären Signalgebung ist die Suche nach Liganden eine herausfordernde Grundvoraussetzung für die aktuelle Arzneistoffentwicklung. Liganden-Bindungs-Experimente stellen dafür elementare Techniken dar. Zur Substitution kostenintensiver und potentiell gesundheitsschädlicher Radioliganden-Bindungen, wurden in dieser Arbeit Fluoreszenz-basierte Liganden-Bindungs-Experimente für den PTHR entwickelt. Basierend auf Zeit-aufgelöster Fluoreszenz wurden mehrere Varianten dieser Experimente etabliert, um die Arzneistoffentwicklung am PTHR zu unterstützen

Indiana University-Purdue University Indianapolis (IUPUI)
PTH increases osteoclasts by upregulating RANKL in cells of the osteoblastic lineage, but the precise differentiation stage of the PTH target cell remains undefined. Recent findings demonstrate that PTH regulates gene expression in osteocytes and that these cells are an important source of RANKL. We therefore investigated whether direct regulation of the RANKL gene by PTH in osteocytes is required to stimulate osteoclastic bone resorption. To address this question, we examined bone resorption and RANKL expression in transgenic mice in which PTH receptor signaling is activated only in osteocytes (DMP1-caPTHR1) crossed with mice lacking the distal control region regulated by PTH in the RANKL gene (DCR -/-). Longitudinal analysis of circulating C-terminal telopeptide (CTX) in male mice showed elevated resorption in growing mice that progressively decreased to plateau at 3-5 month of age. Resorption was significantly higher (~100%) in DMP1-caPTHR1 mice and non-significantly lower (15-30%) in DCR -/-mice, versus wild type littermates (WT) across all ages. CTX in compound DMP1-caPTHR1; DCR -/-mice was similar to DMP1-caPTHR1 mice at 1 and 2 months of age, but by 3 months of age, was significantly lower compared to DMP1-caPTHR1 mice (50% higher than WT), and by 5 months, it was undistinguishable from WT mice. Micro-CT analysis revealed lower tissue material density in the distal femur of DMP1-caPTHR1 mice, indicative of high remodeling, and this effect was partially corrected in compound vi mice. The increased resorption exhibited by DMP1-caPTHR1 mice was accompanied by elevated RANKL mRNA in bone at 1 and 5 months of age. RANKL expression levels displayed similar patterns to CTX levels in DMP1-caPTHR1; DCR -/-compound mice at 1 and 5 month of age. The same pattern of expression was observed for M-CSF. We conclude that resorption induced by PTH receptor signaling requires direct regulation of the RANKL gene in osteocytes, but this dependence is age specific. Whereas DCR-independent mechanisms involving gp130 cytokines or vitamin D 3 might operate in the growing skeleton, DCR-dependent, cAMP/PKA/CREB-activated mechanisms mediate resorption induced by PTH receptor signaling in the adult skeleton.