Projects

Hypothesis: The pathways activated by CaSR ligands present in the gut (e.g. aromatic amino acids or polyamines) are distinct from those in other tissues, such as parathyroid.

Objectives: To identify colon-specific signalling pathways driven by the CaSR and to study the consequences of losing CaSR expression in the colon. ESR_MUW1 will 1) Examine how “intestine-specific” ligands (L-Phe, poly-Arg or spermine) affect Ca²⁺-driven CaSR signalling pathways; 2) Test the impact of calcimimetics/calcilytics on the choice of downstream pathways: such as intracellular Ca²⁺ oscillations, cAMP or IP3 accumulation using the HTRF technology, impact on protein kinase activation by reverse-phase protein array or in-cell western blot;. 3) Study the effect of the New Western Diet (NWD): high in fat low in fibre, folate, calcium and vitamin D on the development of premalignant lesions in mice lacking the CaSR in the intestine.

Hypothesis: Colorectal tumours expressing the CaSR are less aggressive, form fewer metastases, and are more susceptible to treatment and to the chemopreventive effect of Ca²⁺.

Objectives:  ESR_MUW2 will 1) test several substances such as epigenetic modifiers (DNA methyltransferase inhibitors and/or histone deacetylase inhibitors) and allosteric modulators of the CaSR are able to restore CaSR expression in vitro; 2) He will analyse potential synergies between DNMTi/HDACi and calcimimetics, regarding the expression of the CaSR and antitumourigenic properties. 3) and validate this in a colon cancer model in vivo; 4) ESR_MUW2 will test the effect of CaSR-targeting drugs in AOM/DSS induced tumours in a model lacking the CaSR in the intestine. 5) In case of promising outcome, ESR_MUW2 will test the effect of CaSR-targeting drugs on xenografts obtained from primary human colon tumours.

Hypothesis: CaSR activation in the airways leads to airway hyperresponsiveness, remodeling and inflamation, which can be prevented by pharmacological ablation of the CaSR, suggesting a role for the CaSR in the pathogenesis of asthma and possibly COPD and other inflammatory lung diseases. Topically delivered calcilytics can be repurposed as anovel asthma/COPD treatment.

Objectives: ESR_CU will 1) Characterise the ability of inhaled calcilytics to ameliorate airway function in established pre-clinical models of asthma and COPD; 2) Characterise the interplay between the smooth muscle and epithelium CaSR; 3) Determine the effects of cronic administartion of topically delivered calcilytics in vivo.

Hypothesis: Allosteric activators of the CaSR inhibit NB tumour growth by inducing cytodifferentiation and/or cell death and by targeting the CaSR expressed in tumour cells and/or vessels. CaSR signalling in NB cells is not dependent on the origin of the expression (native versus ectopic/transfection mediated expression).

Objectives: ESR_FSJD will 1) Determine the effect of calcimimetics on NB tumour growth in vitro and in vivo; 2) Assess the molecular mechanisms responsible for the observed phenotypes: 3) Analyse ligand- and cell-type biased signalling (comparing pathways activated by calcimimetics in NB versus non-neoplastic cells; and in NB cells with ectopic versus native expression); 4) Explore the effect of calcimimetics on metastases of NB cells to the bone marrow.

Hypothesis: The CaSR signalling depends on the cellular context it is expressed in.

Objectives: ESR_UCPH will 1) perform pharmacological testing of various ortho- and allosteric ligands on primary cells and cell lines of relevance to the Consortium such as (neuroblastoma, colon cancer, breast cancer cells) and map their signalling pathways and potential for biased signalling. ESR_UCPH will test the ligands using dynamic mass redistribution assays on the Corning Epic BT system. Due to the label-free technology it is possible to study the cell response in a more natural setting, closer to physiology; thus we expect to have greater translational value. 2) ESR_UCPH will investigate the most promising ligand-cell combinations in more detail using specific signalling pathway inhibitors to identify the signalling pathway(s) that contribute to the observed signalling fingerprint. 3) Observations from objective 2 will be further analysed with assays for the anticipated pathways (e.g. measurement of inositol monophosphate, cAMP and pERK using time-resolved FRET assays). 4) ESR_UCPH will correlate the observed signalling profiles with physiological outputs e.g. apoptosis in primary neuroblastoma cells.

Hypothesis: The CaSR is a determinant of pancreatic beta-cell function and peripheral insulin action.

Objectives: ESR_UL will 1) Investigate role of CaSR in beta-cell electrophysiology, and the development and maintenance of beta-cell mass, using islets harvested from Nuf mice with a gain-of-function CaSR mutation; 2) Characterise effects of CaSR on the peripheral actions of insulin in wild-type and affected Nuf mice.

Hypothesis: CaSR signalling involves pleiotropic interactions between multiple G protein pathways with the potential for cell-specific competition between these effectors known as conditional efficacy.

Objectives:To determine the relative stoichiometry of CaSR partner/effector proteins between different cell-types and then relate these to their differential responsiveness. ESR_UM will use HEK-293, endothelial (HUVEC), parathyroid (PTH-C1), and colonic cells to compare CaSR signalling responses among cell types [Ca²⁺i and a panel of kinase effectors]. Phosphorylation of the CaSR on T888 is a crucial signalling control. Therefore, ESR_UM will compare the relative activities of gain-of-function (T888A) and loss-of-function (T888D) CaSR mutants between the 4 cell types in order to identify cell-specific differences in the consequence of CaSRT888 phosphorylation. Finally, ESRUM will examine the relative effects of altering intracellular cAMP concentration or relative expression of particular effector proteins (G_q/11, G_i/o, G_12/13, rho & rho-GEF) in the different cells to show how these alter CaSR signalling dynamics.

Hypothesis: therapeutic solutions to sarcopenia involve activation of myogenesis. As the CaSR is expressed in myocytes, selective CaSR modulators will impact the onset of sarcopenia.

Objectives: Sarcopenia is an age-related process characterized by loss of skeletal muscle mass and function and it causes physical disability. ESR_UNIFI will 1) develop an in vitro model of primary cultures of human skeletal muscle myocytes and will evaluate which proteins, including receptors, modulating signals and ion channels, are involved in myogenesis; 2) assess the role of the CaSR in differentiation of adult mesenchymal stem cells into myocytes and in myoblasts differentiated from adipose mesenchymal stem cells (hAMSCs); 3) test selective CaSR modulators for their benefits in skeletal muscle differentiation.

Hypothesis: Selective CaSR antagonists improve insulin secretion and glucose tolerance in diabetes models.

Objectives: 1) Characterise effects of CaSR antagonists (calcilytics) on islet function and insulin secretion; 2) Evaluate glucose-lowering effect of calcilytic drugs in mouse models with impaired insulin secretory responses.

Hypothesis: CaSR is a determinant of osteolytic breast cancer metastases. Activation of the CaSR in breast cancer cells is facilitated by high Ca²⁺_o and /or other molecules which are present in bone microenvironment.

Objectives: ESR_UPJV will 1) Investigate the role of CaSR in breast cancer cell migration in vitro, using MDA-MB-231 and MCF-7 breast cancer cells overexpressing the wild-type form of the CaSR or a dominant negative mutant of the receptor. Ca²⁺_o as well as hydroxyapatite will be used as chemoattractant factor. 2) Characterise the role of the CaSR in early bone metastatic event (metastatic stem cells) ex vivo, following bone marrow invasion of zeocin resistant MDA-MB-231 and MCF-7 cells after intracardiac and fat pad injection. 3) Investigate bone targeting potential of chemical modulators of the CaSR in nude mice using MALDI-Mass Spectrometry Imaging after intracardiac injection of breast cancer cell lines.

Hypothesis: CaSR is believed to activate three different G-proteins in a ligand-combination dependent manner. So far, this has been indirectly inferred from downstream signalling activation, such as ERK, cAMP, and intracellular calcium oscillations. A second hypothesis is that the intracellular signalling state can (de-)sensitize CaSR for extracellular signals, for instance via its phosphorylation by PKC.

Objectives: To study ligand-biased activation of G_q and G_i by the CaSR as function of Ca²⁺_o, and allosteric modifiers at the level of single cells using G-protein sensors (G_q and G_i) to track receptor decisions at the membrane (AIM 1). These experiments will be carried out as function of the modulation of intracellular signalling processes, such as PKC and PP2A that are known to be involved in the phosphorylation of CaSR, to investigate whether the cellular signalling state can (de-)sensitize the receptor (AIM 2). To reach those objectives, ESR_VU will perform experiments using G_q & G_i FRET sensors using the same cell lines as ESR_UM. Mathematical modelling of CaSR signalling based on the results of ESR_VU, ESR_UM, ESR_MUW1 will be done to integrate data.

Hypothesis: CaSR activation is a key event in AD associated amyloid-beta induced neuronal death.

Objectives: Alzheimer’s disease (AD) is the most prevalent neurodegenerative disease affecting nearly 30 million people worldwide. Amyloid-beta induces cell loss in the nervous system both in vitro and in vivo. Amyloid-beta binds to and activates CaSR, which leads to the generation of Reactive Oxygen Species and Peroxynitrite, which subsequently induce oxidative damage and cell death. Blockers of CaSR prevent amyloid-beta toxicity. To identify the role of CaSR in pathomechanism of AD ESRBIOT will 1) Develop hIPSC derived neuronal cell cultures from at least 2 clinically identified AD patients and an age-matched control. In these cell cultures ESR_BIOT will determine the expression level of CaSR and CaSR-mediated signalling by using functional readouts (electrophysiology, stimulated Ca2+ influx assay), ESR_BIOT will also test whether blocking CaSR will normalise AD-related in vitro phenotypes. 2) Determine the effect of amyloid-beta challenge on the AD and control hIPSC derived neuronal cell cultures by using functional readouts and toxicity assays. ESR_BIOT will test the effect of CaSR blockers on prevention of amyloid-beta induced toxicity.

Hypothesis: CaSR ligands could become therapeutic agents for treating calciotropic disorders such as hyperparathyroidism and non-calciotropic disorders such as cardiovascular disease, diabetes and cancer.

Objectives: Identifying and evaluating the ligand specificity of CaSR receptor modulators. ESR_SAFAN will 1) Profile small molecule libraries in silico in close collaboration with the Novartis group. 2) Build a homology model for the CaSR receptor. 3) Evaluate by molecular docking the binding modes of the compounds that result from in silico profiling. 4) Analyse by molecular dynamic simulation techniques the dynamic behaviours of the receptor and of the complexes resulting from molecular docking experiments.

Hypothesis: Pancreatic islands, colon tumours and vascular endothelial cells can be automatically segmented by image cytometry.

Objectives: Automated image cytometry enables identification and quantitative measurement of cell and tissue characteristics (cell number & size, tissue area, cell-cell distances), cellular/sub-cellular localization and quantitative in situ analysis of protein expression in the natural tissue context.
ESR_TG will 1) Define (in collaboration with ESR_MUW2, ES_RUOXF, ESR_UL and ESR_CU) the cell and tissue structures as well as molecules of interest; and perform standardized sampling, staining and microscope-based processing of relevant tissue samples; 2) Develop and validate of In-Vitro Diagnostics (IVD)-compliant adaptive segmentation algorithms; 3) Apply the algorithms (with ESR_MUW2, ESR_UOXF, ESR_UL and ESR_CU) and integrate into the TissueFAXS/StrataQuest workflow.