Orientador: Lilia F. R. de Souza Li / Dissertação (mestrado) - Universidade Estadual de Campinas, Faculdade de Ciencias Medicas / Made available in DSpace on 2018-08-07T01:59:33Z (GMT). No. of bitstreams: 1 Andrade_SimoneCaixetade_M.pdf: 1991149 bytes, checksum: 4c5b783ab7776886c12977300fbb6af7 (MD5) Previous issue date: 2006 / Resumo: O CASR pertence à família C dos receptores que se acoplam à proteína G e é ativado quando interage com o cálcio extracelular, sendo responsável pelo ajuste do ¿set point¿ do cálcio extracelular por meio da regulação da secreção de PTH e excreção de cálcio. Mutações no Receptor Sensor de Cálcio (CASR) estão associadas a FHH (Hipercalcemia Hipocalciúrica Familiar) e NSHTP (Hiperparatireoidismo Neonatal Grave) quando inativadoras do receptor e ADH (Hipoparatireoidismo Autossômico Dominante) quando ativadoras. O Hiperparatireoidismo Neonatal Grave (NSHPT) é uma doença rara caracterizada por calcemias elevadas, próximas às consideradas incompatíveis à vida, associada ao aumento da concentração de PTH, desmineralização óssea grave e sintomas neonatais como hipotonia e baixo ganho ponderal. Trata-se de uma doença familiar, com pais portadores de Hipercalcemia Hipocalciúrica Familiar (FHH), uma doença autossômica dominante, geralmente assintomática, com calcemias elevadas ou no limite superior da normalidade, associada a concentrações de PTH normais, porém não suprimidas e hipocalciúria. ADH, por sua vez, cursam com desregulação no ajuste da concentração de cálcio extracelular, onde baixas concentrações de cálcio ativam o receptor e inibem a secreção de PTH pelas paratireóides e aumentam a excreção de cálcio pelos rins. Indivíduos afetados apresentam hipocalcemia, PTH no limite inferior ou abaixo do normal, hiperfosfatemia e hipercalciúria. O objetivo desse trabalho foi estudar duas famílias portadoras de NSHPT e FHH, identificar novas mutações e analisar o grau de expressão dos receptores mutados. Identificamos três mutações pontuais, nas posições c.1913.G>T, c.2.T>G e c.2244.C>G. Na família S encontramos a mutação c.1913.G>T que resulta em mudança de aminoácido Arginina por Leucina na posição 638 e a mutação silenciosa c.2244.C>G que não altera o aminoácido Prolina da posição 748. Na família J encontramos a mutação c.2.T>G que resulta em mudança do primeiro aminoácido Metionina e em perda da seqüência Kozak (AXXATGG). Um programa de análise para a previsão de seqüências utilizadas para início da tradução protéica, indicou que, na presença da mutação, o ATG com maior probabilidade de ser utilizado como o novo sítio de início de tradução localiza-se no exon 3, na mesma matriz de leitura original. Para análise da expressão do receptor, com a mutação no códon inicial de transcrição do receptor (p.M1?), inserimos no cDNA do CASR um fragmento correspondente à região -226 a 66 do CASR, contendo cinco potenciais seqüências Kozak. Para o estudo da expressão dos receptores mutados da família S inserimos as mutações no cDNA do CASR através de mutagênese sítio dirigida. Analisamos a expressão dos receptores mutados através do Western Blot. O receptor mutado p.R638L apresentou uma expressão similar ao receptor nativo e foram visualizadas as formas monoméricas correspondentes às bandas de 140kDa (forma imatura, parcialmente glicosilada) e 160kDa (forma madura e glicosilada) e bandas superiores maiores que 220kDa. A mutação c.2244.C>G é silenciosa e apresentou expressão similar à do receptor nativo. Em contraste, a expressão do receptor mutado p.M1? através do Western blot estava consideravelmente reduzida. Nos experimentos de imunocitoquímica, observamos que o receptor nativo foi bem expresso na superfície celular tanto em células não permeabilizadas, quanto permeabilizadas. Padrão semelhante foi observado para o receptor mutado p.R638L, indicando maturação e processamento apropriado no retículo endoplasmático enquanto que o receptor com a mutação p.M1? não foi visualizado na superfície celular de células não permeabilizadas e só foi identificado no interior de células permeabilizadas, sugerindo que o receptor mutado era retido no retículo endoplasmático não conseguindo se expressar na membrana plasmática / Abstract: The CASR belongs to family C of the G protein coupled receptors and it is activated by the interaction with extracellular calcium, which is responsible for adjusting extracellular calcium set point adjusting PTH and calcium excretion. Calcium Sensing Receptor mutations are related to Familial Hypocalciuric Hypercalcemia (FHH) and Neonatal Severe Hyperparathyroidism (NSHPT) when inactivating and to Autosomal Dominant Hypocalcemia (ADH) when activating. Neonatal Severe Hyperparathyroidism (NSHPT) is a rare disease characterized by hypercalcemia, calcium levels close to those incompatible with life, markedly elevated PTH levels, severe bone demineralization and neonatal symptoms as hypotonia and poor weight gain. Familial Hypocalciuric Hypercalcemia is a familial disease with Autosomal dominant inheritance, in which parents are usually affected, generally asymptomatic, mild ¿ to ¿ moderate hypercalcemia and normal PTH levels (but not suppressed) and hypocalciuria. In ADH, affected individuals¿ present hypocalcemia, PTH at the lower limit or normal range, hyperphosphatemia and hypercalciuria. The objective of this work was study of two families (S and J) with Neonatal Severe Hyperparathyroidism and Familial Hypocalciuric Hypercalcemia, search for new mutations and analyze the expression pattern of mutated receptors. Three new missense mutations were found: c.1913.G>T, c.2.T>G and c.2244.C>G. The mutation c.1913.G>T was identified at family S. and resulted in Arginine to Leucine change at codon 638. The silent mutation c.2244.C>G didn¿t change the amino acid Proline at codon 748. A novel mutation in exon 2, T to G transition at nucleotide 2, changing Metionine to Arginine was identified at family M. The mutation disrupts the original Kozak sequence (AXXATGG), altering the protein start site. Computational analysis using a program that predicts start sites showed that the putative new translation start site was in the exon 3 in frame. A portion of the gene containing the mutation and five cryptic Kozak sequences (-226 to 66) was used to analyze the expression of the mutant receptor (p.M1?). To analyze the expression pattern of Family S, the mutated cDNAs was inserted in a vector, using site direct mutagenesis. Western blot was performed to analyze the expression analysis of the mutated receptors. The p.R638L receptor showed similar expression pattern compared with the wild type receptor, presenting the monomeric forms of 140 (immature, partial glycosylated) and 160kDa (mature, glycosylated) and other forms higher than 200kDa. The mutation c.2244.C>G showed similar expression pattern compared with the wild type receptor. In contrast, Western blot expression levels of the mutant receptor p.M1? was dramatically reduced. Immunocytochemistry experiments showed strong staining at the cell surface of nonpermeabilized and permeabilized HEK293 cells expressing the wild type receptor. The same pattern was observed for the mutant receptor p.R638L, suggesting correct maturation and trafficking. While the mutant receptor p.M1? was not expressed on the cell surface and the staining was only identified inside permeabilized cells, suggesting that the mutant receptor was trapped within the endoplasmatic reticulum and was not expressed at the plasmatic membrane / Mestrado / Mestre em Farmacologia

Chau Shuk Ling. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2006. / Includes bibliographical references (leaves 103-115). / Abstracts in English and Chinese. / DECLARATION --- p.i / ACKNOWLEDGEMENT --- p.ii / ABBREVIATIONS --- p.iii / ABSTRACT IN ENGLISH --- p.v / ABSTRACT IN CHINESE --- p.viii / TABLE OF CONTENTS --- p.xi / Chapter CHAPTER I - --- INTRODUCTION / Chapter 1.1 --- Fluid Transport in Human Colon --- p.1 / Chapter 1.2 --- C1' Secretion across the Colonic Epithelium --- p.2 / Chapter 1.3 --- Properties of T84 Cells --- p.7 / Chapter 1.4 --- General Introduction on Extracellular Calcium Sensing Receptor (CaSR) --- p.8 / Chapter 1.5 --- Molecular Structure of CaSR --- p.9 / Chapter 1.6 --- CaSR-mediated Intracellular Signaling --- p.13 / Chapter 1.7 --- Agonists of CaSR --- p.16 / Chapter 1.8 --- Functions of CaSR --- p.18 / Chapter 1.8.1 --- Homeostatic Functions of CaSR --- p.18 / Chapter 1.8.2 --- Non-Homeostatic Functions of CaSR --- p.18 / Chapter 1.9 --- Molecular and Functional Study of CaSR Expressed in Colon --- p.19 / Chapter 1.10 --- Objectives of the Present Study --- p.21 / Chapter CHAPTER II - --- MATERIALS AND METHODS / Chapter 2.1 --- Solutions and Drugs --- p.22 / Chapter 2.2 --- Cell Culture --- p.23 / Chapter 2.3 --- Western Blot --- p.26 / Chapter 2.4 --- Simultaneous Measurement of Short-Circuit Current (Isc) and Intracellular Calcium Concentration ([Ca2+]i) --- p.27 / Chapter 2.4.1 --- Measurement of Isc and Transepithelial Resistance with Ussing Chamber --- p.27 / Chapter 2.4.2 --- Simultaneous Measurement of Isc and [Ca2+]i --- p.29 / Chapter 2.5 --- Measurement of Isc with Conventional Ussing Chamber --- p.32 / Chapter 2.6 --- Measurement of Intracellular cAMP Accumulation with Enzyme-Linked Immunosorbent Assay --- p.34 / Chapter 2.7 --- Data Analysis --- p.34 / Chapter CHAPTER III - --- RESULTS / Chapter 3.1 --- Expression of CaSR in T84 Cell Monolayers --- p.36 / Chapter 3.2 --- Poly L-arginine Induced an Increase in Isc --- p.38 / Chapter 3.2.1 --- Simultaneous Measurement of Isc and [Ca2+ ]i in Response to Poly L-arginine --- p.38 / Chapter 3.2.2 --- Interaction Between Poly L-arginine and Forskolin --- p.50 / Chapter 3.2.3 --- Ionic Mechanism of Isc Stimulated by Poly L-arginine --- p.58 / Chapter 3.2.3.1 --- Involvement of C1- in Isc Stimulated by Poly L-arginine --- p.58 / Chapter 3.2.3.2 --- Involvement of Basolateral K+ Channels in Isc Stimulated by Poly L-arginine --- p.61 / Chapter 3.2.4 --- Signaling Pathways Underlying the Isc Response to Poly L-arginine --- p.73 / Chapter CHAPTER IV - --- DISSCUSION / Chapter 4.1 --- Presence of CaSR in T84 Cell Monolayers --- p.78 / Chapter 4.2 --- Simultaneous Measurement of Isc and [Ca2+ ]i upon Application of Poly L-arginine --- p.82 / Chapter 4.3 --- Ionic Mechanism Underlying the Increase in Isc Stimulated by Poly L-arginine --- p.87 / Chapter 4.4 --- Signaling Pathway Underlying the Action of Poly L-arginine --- p.93 / Chapter 4.5 --- Does CaSR Mediate Poly L-arginine-evoked C1- Secretion across T84 Cell Monolayers? --- p.96 / Chapter 4.6 --- Future Study --- p.101 / REFERENCES --- p.103

Le ranelate de strontium (RS) est un traitement anti-ostéoporotique agissant sur la formation osseuse via les ostéoblastes et la résorption osseuse via les ostéoclastes grâce au Calcium Sensing Receptor (CaSR). L’activité physique (EXE) est bien connue pour améliorer les propriétés osseuses. Dans ce travail, nous avons étudié ① l’effet de différentes activités physiques (tapis roulant et impact). Nous avons observé que 10 impacts par jour pendant 8 semaines à 45cm de hauteur ont eu des effets bénéfiques sur l’os (paramètres de microarchitecture et biomécanique, marqueurs du remodelage). ② L’étude du RS et de l’EXE sur l’os de rate ovariectomisée a montré que le RS a des effets comparables à l’EXE et que le RS+EXE ont des effets cumulatifs sur l’os (paramètres de microarchitecture et biomécanique, marqueurs du remodelage). ③ Enfin, une étude in vivo sur des MLO-Y4 a montré la présence des CaSR sur la membrane des ostéocytes et leur nombre est modulé en fonction de la concentration en RS. De plus, le RS a un effet sur la différenciation des CSM lors d’une différenciation ostéogénique en favorisant la différenciation ostéocytaire et elle est modulée par la concentration en RS. En conclusion, ce travail a démontré l’importance d’une pratique d’un exercice physique et du traitement du RS contre l’ostéoporose : maintien de la balance du remodelage osseux du côté de la formation. L’effet cumulatif du RS+EXE s’explique par le fait que le RS agit sur les ostéoblastes, ostéocytes et ostéoclastes via les CaSR et l’EXE sur les mécanorécepteurs des ostéocytes. / Strontium ranelate (SR) is an anti-osteoroporotic treatment acting on bone formation via osteoblasts and bone resorption via osteoclasts thanks to Calcium Sensing Receptor (CaSR). Physical activity is well known to improve bone properties. In this work, we studied ① different physical acticities (treadmill and impact). We observed that 10 impacts per day during 8 weeks from 45cm of height had beneficial effects on bone (microarchitecture and biomechanical parameters, bone remodeling markers). ② The study of SR and EXE effects on bone ovariectomized rats showed that RS had similar effects to EXE and SR+EXE had cumulative effects on bone (microarchitecture and biomechanical parameters, bone remodeling markers). ③ Finally, an in vivo study on MLO-Y4 showed CaSR presence on osteocyte with their number depending on SR concentration. Moreover, RS had positive effects on CSM differentiation in favor of osteocyte differentiation and it is modulated by SR concentration. In conclusion, this work has demonstrated the importance of taking physical exercise as well as SR treatment for osteoporosis: maintaining the bone remodeling in favor of bone formation. The cumulative effect of SR+EXE is explained by the fact the SR acts on osteoblasts, osteocytes and osteoclasts via CaSR and the EXE on osteocyte mechanoreceptors.

Patients with Graves´ Disease (GD) have a higher risk of developing more severe and prolonged hypocalcaemia after total thyroidectomy (TT) than patients who undergo surgery for benign atoxic goitre. Since TT is the most effective treatment for GD, it is crucial to identify mechanisms for postoperative hypocalcaemia. The aim of this thesis was to study the mechanisms of calcium metabolism in patients with GD. It is safe to operate on GD patients with TT. Results in Paper I showed fewer recurrences and equal complication rates compared to patients who underwent subtotal thyroidectomy (ST). The transient lowering of PTH seen in the hypocalcaemic patients was fully restored one month after surgery (Papers II and V). The calcium-sensing receptor (CaSR) is crucial for maintaining plasma calcium, and single nucleotide polymorphisms (SNPs) in the gene may alter the sensing function. Thus, we analysed SNPs in CaSR in GD patients (Paper II) and showed that they had a more left-shifted calcium-PTH set-point compared to controls, implicating higher sensitivity. This is also supported by the results in the group of postoperatively hypocalcaemic patients. They already had lower plasma calcium preoperatively (Papers II, IV and V) and lacked the T/G G/A G/C, a haplotype shown in Paper III to have a close relationship to higher p-calcium levels. Moreover, a lack of the T allele in rs1801725 was seen in the group of patients needing permanent treatment with calcium and vitamin D, i.e. > 12 months, (paper V). Patients who became hypocalcaemic (p-calcium < 2.00 mmol/L) on day one postoperatively, had lower preoperative levels of thyroid stimulating hormone (TSH) and higher levels of  T3, this was also applied to the patient groups requiring temporary or permanent postoperative treatment (Papers II and V). In addition, hypocalcaemic patients treated for less than six months with anti-thyroid drugs had higher levels of bone metabolism markers CTX and P1NP than normocalcaemic patients (Paper V). In conclusion, the postoperative period of hypocalcaemia seen in patients with GD is a complex medical condition, caused by a combination of surgical trauma, different SNPs in CaSR, and high bone metabolism related to preoperative thyroid metabolism.

The calcium sensing receptor (CaSR) is a G-protein coupled receptor (GPCR) that plays a central role in calcium homeostasis. Loss-of-function mutations of the CaSR cause familial hypocalciuric hypercalcaemia type 1 (FHH1), whilst gain-of-function mutations are associated with autosomal dominant hypocalcaemia (ADH). However, 35% of cases of FHH and 60% of cases of ADH are not due to CaSR mutations. This thesis demonstrates that FHH type 2 (FHH2) and the new clinical disorder, ADH type 2 (ADH2), are due to loss- and gain-of-function mutations in the G-protein subunit, Gα11, respectively. The CaSR signals through Gα11 and FHH2-associated mutations are shown to exert their effects through haploinsufficiency. Three-dimensional modelling of ADH2-associated Gα11 mutations predicts impaired GTPase activity and increases in the rate of GDP/GTP exchange. Furthermore, mouse models of FHH2 and ADH2 have been identified and re-derived to enable in vivo studies of the role of Gα11 in calcium homeostasis. I also demonstrate that FHH3 is due to loss-of-function mutations in the adaptor protein 2 sigma subunit, AP2σ2, which exert dominant-negative effects. AP2σ2 is a component of the adaptor protein 2 (AP2), which is a crucial component of clathrin-coated vesicles (CCV) and facilitates clathrin-mediated endocytosis of plasma membrane components such as GPCRs. All of the identified FHH3-associated mutations affect the Arg15 residue of AP2σ2, which forms key polar contacts with CCV cargo proteins. This thesis proposes that FHH3-associated AP2σ2 mutations impair CaSR internalisation and thus negatively impact on CaSR signalling. In addition, these studies show that these signalling defects can be rectified by the use of the CaSR allosteric modulator cinacalcet, which may represent a useful therapeutic modality for FHH3 patients. In summary, FHH2 is due to loss-of-function mutations in Gα11 causing haploinsufficiency, whilst FHH3 is due to loss-of-function mutations in AP2σ2, which exert dominant-negative effects. In contrast, ADH2 is due to gain-of-function mutations in Gα11.

Previous studies in Dr. Yang’s laboratory have established a grafting, design, and subdomain approach in order to investigate the properties behind Ca2+-binding sites located in Ca2+-binding proteins by employing engineered proteins. These approaches have not only enabled us to isolate Ca2+-binding sites and obtain their Ca2+-binding affinities, but also to investigate conformational changes and cooperativity effects upon Ca2+ binding. The focus of my thesis pertains to optimizing the expression and purification of engineered proteins with tailored functions. Proteins were expressed in E. coli using different cell strains, vectors, temperatures, and inducer concentrations. After rigorous expression optimization procedures, proteins were further purified using chromatographic and/or refolding techniques. Expression and purification optimization of proteins is essential for further analyses, since the techniques used for these studies require high protein concentrations and purity. Evaluated proteins had yields between 5-70 mg/L and purities of 80-90% as confirmed by SDS-PAGE electrophoresis.

Ca2+, both as a first and a second messenger, is closely involved in the modulation and regulation of numerous important cellular events, such as cell proliferation, differentiation and cell death. Fine-tuned Ca2+ signaling is achieved by its reversible or irreversible binding to a repertoire of Ca2+ signaling molecules. Among them, the extracellular calcium sensing receptor (CaSR) senses Ca2+ concentration ([Ca2+]o) in the milieu outside of cells where Ca2+ serves as a first messenger. An array of naturally-occurring mutations in CaSR has been found in patients with inherited disorders of Ca2+ homeostasis, leading to abnormal intracellular responses toward [Ca2+]o. In the present study, we have computationally predicted and experimentally characterized the metal-binding properties of five Ca2+-binding sites within CaSR and the accompanying metal--induced conformational changes by using two complementary methods-the grafting approach and the subdomain approach. Based on our results, a model has been proposed to explain the distinct CaSR-mediated responses toward abnormally ¡°high¡± or ¡°low¡± extracellular Ca2+ levels. In addition, we predicted and verified the interaction between CaSR with the most ubiquitously expressed four EF-hand-containing intracellular Ca2+ sensor protein, calmodulin (CaM). Our results demonstrate that the C-terminal CaM-binding domain of the CaSR is essential for proper intracellular Ca2+ response to external signals. Furthermore, we have applied the grafting approach to study the metal-binding properties and oligomeric state of the single EF-hand containing protein, STIM1. Our studies confirmed that the single EF-hand motif in STIM1, which resides in an equilibratium between its monomeric and dimeric forms, was capable of binding Ca2+ with a dissociation constant comparable to the ER Ca2+ concentration, suggesting it could function as a ER Ca2+ sensor responsible for sensing the Ca2+ filling state of ER.

The calcium-sensing receptor (CaR) is a G-protein coupled receptor that controls mammalian extracellular calcium (Ca2+o) homeostasis. CaR downstream signalling involves intracellular calcium (Ca2+i) mobilisation which can be negatively modulated by protein kinase C (PKC)-mediated phosphorylation of CaR residue Thr-888 (CaRT888). The nature of this regulation was investigated here using siRNA-based knockdown of individual PKC isotypes. Knocking down PKCα expression increased CaR-induced Ca2+i mobilisation in CaR-HEK cells, significantly lowering the EC50 for Ca2+o relative to control siRNA-transfected cells. In accordance, PKCα knockdown also decreased CaRT888 phosphorylation which also permitted the triggering of Ca2+i mobilisation in CaR-HEK cells at sub-threshold Ca2+o concentrations. Interestingly, PKCε knockdown attenuated CaR-induced Ca2+i mobilisation in CaR-HEK cells, significantly increasing the EC50 for Ca2+o. However, this knockdown was also also found to inhibit CaRT888 phosphorylation and this is the first time that CaRT888 phosphorylation has been shown to be dissociate from Ca2+i mobilisation. The results show the complexity of the interactions that potentially underlie the CaR’s pleiotropic signalling and provides novel targets for examining signal bias. Classically an increase in cAMP is known to trigger PTH seceretion. The observation in this study shows that raising intracellular cAMP levels with forskolin also decreased CaRT888 phosphorylation permitting increased Ca2+i mobilisation. This suggests that cAMP may stimulate the phosphatase (most likely protein phosphatase 2A (PP2A)). Nevertheless, knocking down Gα12, which has been shown to activate PP2A, resulted in increased CaRT888 phosphorylation and lower Ca2+i mobilisation (increased EC50 for Ca2+o). This suggests the possibility of CaR as a cAMP sensor that can detect an increase in intracellular cAMP in order to stop PTH serection. Three novel CaR effectors, P70 ribosamal protein S6 kinase, insulin-like growth factor receptor-1 and nuclear factor of kappa light polypeptide gene enhancer in B-cells inhibitor, were identified in CaR-HEK cells. It was shown that a) high Ca2+o stimulated the activation of these effectors and b) each effector was inhibited by knockdown of PKCα and Gα12, which further confirmed the association of these signals with CaR. These data show that CaR also plays an important role outside Ca2+o homeostasis, such as growth and inflammation. Finally, five CaR mutations associated with autosomal dominant hypocalcaemia (ADH) were found to increase Ca2+o-induced Ca2+i mobilisation, as well as ERK and p38MAPK activation, when transfected stably in HEK-293 cells. Cotreatment with the calcilytic NPSP795 inhibited ERK and p38MAPK phosphorylation in all 5 gain-of-function mutants and in the wild type CaR cells, with IC50s for the compound in the nanomolar range. These data highlight the potential utility of CaR negative allosteric modulators in the treatment of gain-of-function CaR mutations. Together these data enhance our understanding of CaRT888 phosphorylation and CaR signalling.

Parathyroid hormone (PTH) secretion maintains free-ionised extracellular calcium (Ca2+o) homeostasis under the control of the calcium-sensing receptor (CaR). In humans and dogs, blood acidosis and alkalosis is associated with increased or suppressed PTH secretion respectively. Furthermore, large (1.0 pH unit) changes in extracellular pH (pHo) alter Ca2+o sensitivity of the CaR in CaR-transfected HEK-293 cells (CaR-HEK). Indeed, it has been found in this laboratory that even pathophysiological acidosis (pH 7.2) renders CaR less sensitive to Ca2+o while pathophysiological alkalosis (pH 7.6) increases its Ca2+o sensitivity, both in CaR-HEK and parathyroid cells. If true in vivo, then CaR’s pHo sensitivity might represent a mechanistic link between metabolic acidosis and hyperparathyroidism in ageing and renal disease. However, in acidosis one might speculate that the additional H+ could displace Ca2+ bound to plasma albumin, thus increasing free-Ca2+ concentration and so compensating for the decreased CaR responsiveness. Therefore, I first demonstrated that a physiologically-relevant concentration of albumin (5% w/v) failed to overcome the inhibitory effect of pH 7.2 or stimulatory effect of pH 7.6 on CaR-induced intracellular Ca2+ (Ca2+i) mobilisation. Determining the molecular basis of CaR pHo sensitivity would help explain cationic activation of CaR and permit the generation of experimental CaR models that specifically lack pHo sensitivity. With extracellular histidine and free cysteine residues the most likely candidates for pHo sensing (given their sidechains’ pK values), all 17 such CaR residues were mutated to non-ionisable residues. However, none of the resulting CaR mutants exhibited significantly decreased CaR pHo sensitivity. Even co-mutation of the two residues whose individual mutation appeared to elicit modest reductions (CaRH429V and CaRH495V) failed to exhibit any change in CaR pHo sensitivity. I conclude therefore, that neither extracellular histidine nor free cysteine residues account for CaR pHo sensitivity. Next, it is known that cytosolic cAMP drives PTH secretion in vivo and that cAMP potentiates Ca2+o-induced Ca2+i mobilisation in CaR-HEK cells. Given the physiological importance of tightly controlled PTH secretion and Ca2+o homeostasis, here I investigated the influence of cAMP on CaR signalling in CaR-HEK cells. Agents that increase cytosolic cAMP levels such as forskolin and isoproterenol potentiated Ca2+o-induced Ca2+i mobilisation and lowered the Ca2+o threshold for Ca2+i mobilisation. Indeed, forskolin lowered the EC50 for Ca2+o on CaR (2.3 ± 0.1 vs. 3.0 ± 0.1 mM control, P<0.001). Forskolin also potentiated CaR-induced ERK phosphorylation; however protein kinase A activation appeared uninvolved in any of these effects. Pertussis toxin, used to block CaR-induced suppression of cAMP accumulation, also lowered the Ca2+o threshold for Ca2+i mobilisation though appeared to do so by increasing efficacy (Emax). Furthermore, mutation of the CaR’s two putative PKA consensus sequences (CaRS899 and CaRS900) to a non-phosphorylatable residue (alanine) failed to alter the potency of Ca2+o for CaR or attenuate the forskolin response. In contrast, phosphomimetic mutation of CaRS899 (to aspartate) did increase CaR sensitivity to Ca2+o. Together this suggests that PKA-mediated CaRS899 phosphorylation could potentiate CaR activity but that this does not occur following Ca2+o treatment in CaR-HEK cells. Together, these data show that cAMP regulates the Ca2+o threshold for Ca2+i mobilisation, thus helping to explain differential efficacy between CaR downstream signals. If true in vivo, this could help explain how multiple physiological signal inputs may be integrated in parathyroid cells.

Monocytes, as innate immune cells, are recruited to sites of infection or injury to modulate immune responses and tissue repair. Ca²⁺, which is released from dying cells, could act as an attractant for monocytes eliciting chemokinetic responses. Triggering further the production and release of pro-inflammatory cytokines, Ca²⁺ mimics a danger signal on monocytes in terms of sterile inflammation. This extracellular signal is transduced through calcium-sensing receptor (CaSR) into immunomodulatory responses, more specifically into the assembly of the inflammasome platform, which promotes the maturation of pro-interleukine-1β and pro-interleukine-18 and subsequently their release (Rossol et al., 2012). Given the ubiquitous presence of Ca²⁺ in the human body and additional microenvironments of elevated [Ca²⁺], e.g., during cell signaling, at erosion zones of osteoclasts, but also in non-normal states like inflammation (e.g. gingivitis), skin injury, and cell death, one may ask how a cell could discriminate between physiological and pathological calcium concentrations. The present study unraveled the mechanism of Ca²⁺-induced inflammasome activation in human monocytes. Calcium and phosphate or rather calciprotein particles were uncovered as the main instigators of inflammation in the present in vitro setup. The results demonstrated that strong Ca²⁺-mediated pro-inflammatory responses of monocytes are only present under increased extracellular phosphate concentrations. Such cell culture conditions are prone to the formation of calciprotein particles mainly consisting of calcium, phosphate, and fetuin-A. Due to the presence of fetuin-A in fetal (bovine) serum, crystallization of calcium and phosphate is prevented, and the size of the formed amorphous particles remains around 100 nm. The simultaneous existence of Ca²⁺ facilitates the activation of CaSR, which in turn boosts constitutive macropinocytosis. This mechanism is at least partially mediated by Gαq protein-dependent pathways. The increased uptake of extracellular fluid and thereby uptake of calciprotein particles lead to enhanced lysosomal cathepsin B activity, but obviously not to lysosomal membrane leakage, as it is known for crystalline structures. Despite lysosomal membrane integrity, a yet unknown mechanism drives inflammasome activation along with Interleukine-1β (IL-1β) release and cell death. A further role of phosphate in CaSR signaling under the present conditions is discussed, as anion binding sites are already described for this receptor.:I LIST OF ABBREVIATIONS 7 II LIST OF FIGURES 11 III LIST OF TABLES 13 1 INTRODUCTION 14 1.1 Ca²⁺ and Pi, an indispensable, but dangerous team 14 Physiological relevance 14 Regulation of Ca²⁺ homeostasis 14 Regulation of [Pi] homeostasis 15 Clinical relevance 16 Calciprotein particle (CPP) 17 1.2 The extracellular calcium ion-sensing receptor - CaSR 19 General overview 19 CaSR-dependent macropinocytosis 21 1.3 Monocytes – mononuclear phagocytes 23 1.4 The inflammasome, activation and physiological function 25 NLRP3 inflammasome 25 Ca²⁺-/CPP-triggered activation of NLRP3 inflammasome 27 1.5 Objective 29 2 MATERIAL AND METHODS 30 2.1 Material 30 2.1.1 Human material 30 2.1.2 Cell lines 30 2.1.3 Media, buffer, chemicals, and consumables 30 2.1.4 Antibodies 31 2.1.5 Assay Kits 31 2.2 Methods 32 2.2.1 Isolation and culture of monocytes from human blood 32 2.2.2 Cell culture and transfection of cell lines 33 2.2.3 [Ca2+] measurement in cell culture medium 33 2.2.4 Dynamic mass redistribution (DMR) measurement 33 2.2.5 CPP preparation and stimulation of monocytes with CPPs 34 2.2.6 Detection of macropinocytosis 35 2.2.7 Detection of cathepsin activity 35 2.2.8 Detection of lysosomal leakage 36 2.2.9 Detection of depolarization of mitochondria 36 2.2.10 Detection of cell death 36 2.2.11 Live-imaging confocal Raman microspectroscopy 37 2.2.12 Transmission electron microscopy - electron dispersive x-ray analysis 37 2.2.13 CPP analysis via dynamic light scattering (DLS) 39 2.2.14 Detection of IL-1β via ELISA 39 2.2.15 Detection and quantification of intracellular cAMP 40 2.2.16 Western Blot 41 2.2.17 Statistics 41 3 RESULTS 42 3.1 Ca²⁺ stimulates NLRP3 inflammasome activation through CaSR signaling only at elevated [Pi] 42 3.1.1 [Pi] > 3 mM is necessary for Ca²⁺-triggered IL-1β release 42 3.1.2 NLRP3 is necessary for Ca²⁺-triggered IL-1β release 43 3.1.3 CaSR partially mediates Ca²⁺-triggered NLRP3 inflammasome activation 44 3.1.4 Pi is not replaceable during Ca²⁺-triggered IL-1β release 45 3.2 Ca²⁺-activated GPCR signaling is affected by Pi 46 3.2.1 Ca²⁺-triggered signaling differs between high and low [Pi] cell culture conditions in monocytes 46 3.2.2 CaSR-mediated DMR responses are affected by Pi 51 3.3 Addition of Ca²⁺ to serum-containing RPMI1640 cell culture medium results in spontaneous formation of nanoparticles 55 3.4 Ca²⁺ stimulation of monocytes enhances macropinocytosis 60 3.4.1 Visualization of CPP uptake 60 3.4.2 CPPs were taken up by an actin- and CaSR-dependent mechanism 64 3.5 Actin remodeling is necessary for IL-1β production after stimulation with [Ca²⁺] in high [Pi] medium 69 3.5.1 Actin remodeling is necessary for Ca²⁺-triggered IL-1β production 69 3.5.2 CPPs and elevated extracellular [Ca²⁺] are responsible for IL-1β release of human monocytes 70 3.5.3 Sr²⁺ prevents Ca²⁺-triggered inflammasome activation 75 3.6 Formation and uptake of CPPs results in increased cathepsin B activity and non-apoptotic cell death 78 3.6.1 Elevated [Ca²⁺] and [Pi] lead to increased cathepsin B activity 78 3.6.2 Ca²⁺-induced cell death strongly depends on elevated [Pi] 81 3.6.3 Cell death (LDH release) partially depends on NLRP3 and CaSR 84 3.7 Summary and graphical overview of results 85 4 DISCUSSION 86 4.1 CaSR is involved in Ca²⁺-mediated NLRP3 inflammasome activation 88 4.2 [Pi] influences Ca²⁺-induced cellular reactions 90 4.2.1 Effects of [Pi] on extracellular calcium ion-sensing receptor signaling 90 4.3 Uptake of CPPs triggers inflammasome activation and is accompanied by cell death 94 4.3.1 Fetuin-A, [Ca²⁺], and [Pi] determine CPP formation and pro-inflammatory responses 94 4.3.2 Effects of CaSR agonists and Gαq-activating GPCR ligands on macropinocytosis and inflammasome activation 97 4.3.3 The inhibitory effects of Sr²⁺ in Ca²⁺-mediated inflammasome activation 98 4.3.4 Uptake of CPPs is accompanied by increased lysosomal activity 100 4.3.5 IL-1β release and cell death are mediated by lysosomal Ca²⁺ efflux 101 4.3.6 Pi fluxes are potentially involved in inflammasome activation 102 4.3.7 Potential mediators of cell death 103 4.4 Clinical relevance of CPPs 105 4.5 Outlook 108 5 SUMMARY 110 6 REFERENCES 114 7 SUPPLEMENTARY 131 7.1 Supplementary figures 131 7.2 Supplementary tables 135 8 DECLARATION OF AUTHORSHIP 146 LIST OF PUBLICATIONS AND TALKS 147 ACKNOWLEDGEMENT 148