The Hedgehog Receptor Patched Is Involved in Cholesterol Transport

International audienceBACKGROUND: Sonic hedgehog (Shh) signaling plays a crucial role in growth and patterning during embryonic development, and also in stem cell maintenance and tissue regeneration in adults. Aberrant Shh pathway activation is involved in the development of many tumors, and one of the most affected Shh signaling steps found in these tumors is the regulation of the signaling receptor Smoothened by the Shh receptor Patched. In the present work, we investigated Patched activity and the mechanism by which Patched inhibits Smoothened. METHODOLOGY/PRINCIPAL FINDINGS: Using the well-known Shh-responding cell line of mouse fibroblasts NIH 3T3, we first observed that enhancement of the intracellular cholesterol concentration induces Smoothened enrichment in the plasma membrane, which is a crucial step for the signaling activation. We found that binding of Shh protein to its receptor Patched, which involves Patched internalization, increases the intracellular concentration of cholesterol and decreases the efflux of a fluorescent cholesterol derivative (BODIPY-cholesterol) from these cells. Treatment of fibroblasts with cyclopamine, an antagonist of Shh signaling, inhibits Patched expression and reduces BODIPY-cholesterol efflux, while treatment with the Shh pathway agonist SAG enhances Patched protein expression and BODIPY-cholesterol efflux. We also show that over-expression of human Patched in the yeast S. cerevisiae results in a significant boost of BODIPY-cholesterol efflux. Furthermore, we demonstrate that purified Patched binds to cholesterol, and that the interaction of Shh with Patched inhibits the binding of Patched to cholesterol. CONCLUSION/SIGNIFICANCE: Our results suggest that Patched may contribute to cholesterol efflux from cells, and to modulation of the intracellular cholesterol concentration. This activity is likely responsible for the inhibition of the enrichment of Smoothened in the plasma membrane, which is an important step in Shh pathway activation

Presynaptic External Calcium Signaling Involves the Calcium-Sensing Receptor in Neocortical Nerve Terminals

Nerve terminal invasion by an axonal spike activates voltage-gated channels, triggering calcium entry, vesicle fusion, and release of neurotransmitter. Ion channels activated at the terminal shape the presynaptic spike and so regulate the magnitude and duration of calcium entry. Consequently characterization of the functional properties of ion channels at nerve terminals is crucial to understand the regulation of transmitter release. Direct recordings from small neocortical nerve terminals have revealed that external [Ca(2+)] ([Ca(2+)](o)) indirectly regulates a non-selective cation channel (NSCC) in neocortical nerve terminals via an unknown [Ca(2+)](o) sensor. Here, we identify the first component in a presynaptic calcium signaling pathway.By combining genetic and pharmacological approaches with direct patch-clamp recordings from small acutely isolated neocortical nerve terminals we identify the extracellular calcium sensor. Our results show that the calcium-sensing receptor (CaSR), a previously identified G-protein coupled receptor that is the mainstay in serum calcium homeostasis, is the extracellular calcium sensor in these acutely dissociated nerve terminals. The NSCC currents from reduced function mutant CaSR mice were less sensitive to changes in [Ca(2+)](o) than wild-type. Calindol, an allosteric CaSR agonist, reduced NSCC currents in direct terminal recordings in a dose-dependent and reversible manner. In contrast, glutamate and GABA did not affect the NSCC currents.Our experiments identify CaSR as the first component in the [Ca(2+)](o) sensor-NSCC signaling pathway in neocortical terminals. Decreases in [Ca(2+)](o) will depress synaptic transmission because of the exquisite sensitivity of transmitter release to [Ca(2+)](o) following its entry via voltage-activated Ca(2+) channels. CaSR may detects such falls in [Ca(2+)](o) and increase action potential duration by increasing NSCC activity, thereby attenuating the impact of decreases in [Ca(2+)](o) on release probability. CaSR is positioned to detect the dynamic changes of [Ca(2+)](o) and provide presynaptic feedback that will alter brain excitability

An Inhibitory Effect of Extracellular Ca2+ on Ca2+-Dependent Exocytosis

Aim: Neurotransmitter release is elicited by an elevation of intracellular Ca 2+ concentration ([Ca 2+] i). The action potential triggers Ca 2+ influx through Ca 2+ channels which causes local changes of [Ca 2+] i for vesicle release. However, any direct role of extracellular Ca 2+ (besides Ca 2+ influx) on Ca 2+-dependent exocytosis remains elusive. Here we set out to investigate this possibility on rat dorsal root ganglion (DRG) neurons and chromaffin cells, widely used models for studying vesicle exocytosis. Results: Using photolysis of caged Ca 2+ and caffeine-induced release of stored Ca 2+, we found that extracellular Ca 2+ inhibited exocytosis following moderate [Ca 2+]i rises (2–3 mM). The IC50 for extracellular Ca 2+ inhibition of exocytosis (ECIE) was 1.38 mM and a physiological reduction (,30%) of extracellular Ca 2+ concentration ([Ca 2+]o) significantly increased the evoked exocytosis. At the single vesicle level, quantal size and release frequency were also altered by physiological [Ca 2+] o. The calcimimetics Mg 2+,Cd 2+, G418, and neomycin all inhibited exocytosis. The extracellular Ca 2+-sensing receptor (CaSR) was not involved because specific drugs and knockdown of CaSR in DRG neurons did not affect ECIE. Conclusion/Significance: As an extension of the classic Ca 2+ hypothesis of synaptic release, physiological levels of extracellular Ca 2+ play dual roles in evoked exocytosis by providing a source of Ca 2+ influx, and by directly regulatin

Caracterisation biochimique et localisation des recepteurs H_1 et H_2 de l'histamine

SIGLEINIST T 70629 / INIST-CNRS - Institut de l'Information Scientifique et TechniqueFRFranc

Pharmacologie du récepteur aux ions calcium extracellulaires

Le récepteur aux ions calcium extracellulaires (CaSR) appartient à la famille 3 des récepteurs couplés aux protéines G. Il contrôle la sécrétion de l'hormone parathyroïdienne (PTH) et joue un rôle clef dans la régulation de l'homéostasie calcique. Son activité est modulée par de faibles variations de la concentration des ions calcium (Ca2+) et magnésium (Mg2+) extracellulaires conduisant à la stimulation de la voie des phospholipases C et A2. Le développement récent de molécules de synthèse capables de potentialiser (calcimimétiques) les actions du Ca2+ sur ce récepteur et de réduire la concentration de PTH circulant in vivo, suggère qu'il constitue une nouvelle cible thérapeutique pour le traitement des hyperparathyroïdies primaires et secondaires. Son blocage par un antagoniste (calcilytique) stimule la sécrétion de PTH et pourrait être mis à profit pour le traitement de l'ostéoporose. Ce récepteur est aussi exprimé dans d'autres tissus tels que la thyroïde, les reins, l'os ou encore dans des populations neuronales et gliales, où il participerait aux réponses cellulaires complexes induites par les ions Ca2+ et Mg2+ présents dans les fluides extracellulaires

Pharmacologie du récepteur aux ions calcium extracellulaires. [Pharmacology of the extracellular calcium ion receptor]

ReviewThe calcium sensing receptor (CaSR) belongs to family 3 of G-protein coupled receptors. The CaSR, expressed at the surface of the parathyroid cells, controls parathyroid hormone (PTH) secretion and is the main regulator of calcium homeostasis. Its activity is regulated by small changes in the physiological concentrations of calcium and magnesium ions present in the serum and extracellular fluids, leading to the stimulation of the phospholipases C and A2. Molecules that potentiate the effect of extracellular calcium are called calcimimetics. They reduce the PTH level in vivo and have been proposed to be of therapeutic benefit for the treatment of both primary and secondary hyperparathyroidism. The blocking of CaSR by a calcilytic molecule results in the increase in serum PTH and might be of interest in the treatment of osteoporosis. The CaSR is also expressed in the thyroid, kidney, bone and in neuronal and glial cell populations, where it should be involved in the complex responses associated with calcium and magnesium ions present in the extracellular fluids

Caractérisation pharmacologique et fonctionnelle du récepteur smoothened

LE KREMLIN-B.- PARIS 11-BU Méd (940432101) / SudocPARIS-BIUP (751062107) / SudocSudocFranceF

Mise en évidence de l'effet chemoattractif de sonic hedgehog sur les precurseurs neuraux dérivés de la zone sous-ventriculaire chez le rongeur

LE KREMLIN-B.- PARIS 11-BU Méd (940432101) / SudocSudocFranceF

Découverte et caractérisation pharmacologique de nouveaux antagonistes du récepteur smoothened (les composés mrt)

La voie de signalisation Sonic Hedgehog (Shh) joue un rôle fondamental au cours de l embryogenèse pour la mise en place de nombreux tissus. Elle persiste à l âge adulte et régulerait notamment le contrôle de fonctions cérébrales. Son activation requiert la liaison d un peptide Shh sur le récepteur Patched (Ptc) qui réprime l activité constitutive de Smoothened (Smo), un récepteur apparenté à la famille des récepteurs couplés aux protéines G (RCPG). Récemment, des essais cliniques pour le traitement de médulloblastomes et de diverses tumeurs solides chez l Homme ont été menés avec des antagonistes de Smo. Cependant, ces molécules ont révélé des limitations à leur utilisation puisque des résistances au traitement sont apparues. Le travail de cette thèse a conduit au développement d un modèle pharmacophorique des antagonistes de Smo qui a ensuite permis le criblage virtuel d une banque de molécules et l identification de nouvelles familles d antagonistes de Smo. L acylthiourée MRT-10 et l acylurée MRT-14 ont été les deux premiers composés caractérisés. Des études de relations structure-activité ont permis l identification d une nouvelle famille d inhibiteurs du récepteur Smo de haute affinité à laquelle l acylguanidine MRT-83 appartient. Ce composé s adapte parfaitement au modèle pharmacophorique des antagonistes de Smo. Les modifications structurales que MRT-83 présentes en comparaison avec les deux têtes de séries précédemment caractérisées sont à l origine du gain d activité de MRT-83 sur de nombreux tests cellulaires mettant en jeu l activation de la voie Shh. Le composé MRT-83 inhibe la liaison de la BODIPY-cyclopamine sur le récepteur Smo humain et bloque la prolifération des précurseurs des cellules granulaires de rat avec une affinité de l ordre du nanomolaire, comparable à celle des antagonistes de référence de Smo tels que le GDC-0449 et le LDE-225. Malgré l homologie de séquence entre Smo et la famille des récepteurs Frizzled impliqués dans la signalisation Wnt, le composé MRT-83 ne présente aucun effet sur la voie Wnt. MRT-83 bloque la translocation de Smo dans le cil primaire induite par l activation de la voie Shh dans les cellules NT2, une lignée issue d un tératocarcinome humain, contrairement à l antagoniste de Smo de référence, la cyclopamine qui induit l adressage du récepteur dans le cil primaire. L injection stéréotaxique dans le ventricule latéral de cerveau de souris adulte de MRT-83, contrairement à celle d un composé de structure analogue, dépourvu d activité sur Smo, inhibe l expression des transcrits de Ptc induite par l injection de Shh dans la zone sous-ventriculaire, l une des deux principales aires de neurogenèse adulte. Ces résultats démontrent que les dérivés MRT bloquent également la signalisation Shh in vivo. Ainsi, les composés MRT-10, MRT-14, MRT-83 et les molécules de structure analogues caractérisées sont de puissants antagonistes de Smo. Ces molécules constituent de nouveaux outils pharmacologiques qui pourraient permettre d améliorer notre compréhension des mécanismes moléculaires et biochimiques régulant la signalisation Hh et permettre le développement de nouvelles molécules en clinique pour le traitement des tumeurs Hh-dépendantes.The Sonic Hedgehog (Shh) signaling pathway is implicated in multiple physiological responses including the control of brain functions. In mammals, the Shh pathway is expressed at the primary cilium and its activation requires the binding of a Shh peptide to the Patched (Ptc) receptor which represses the constitutive activity of Smoothened (Smo), a proposed member of the G-protein-coupled receptor (GPCR) family. Recently, clinical trials for treating medulloblastoma and various solid tumors in human have been conducted with Smo antagonists such as GDC-0449 or LDE-225. Such molecules may have some limitations leading to treatment resistance. In the present work, the development of a pharmacophoric model of Smo antagonists allowed a virtual screening strategy to identify novel Smo inhibitors. The acylthiourea MRT-10 and the acylurea MRT-14 were the two first leads identified. Structure-activity relationship experiments led to the discovery of new series of Smo inhibitors with high potency and to which the acylguanidine MRT-83 belongs. This inhibitor perfectly fits with the proposed pharmacophoric model for Smo antagonists. The discrete structural differences between MRT-83 and the original leads may account for the increased potency of MRT-83 observed in various in vitro Shh-based assays. MRT-83 inhibits BODIPY-cyclopamine binding to human Smo and Shh-mediated proliferation of rat granule cell proliferation with nanomolar potency similar to GDC-0449 or LDE-225. Despite significant homology of Smo with the Frizzled family of receptors which are involved in the Wnt signaling pathway, MRT-83 displays no significant effect on this pathway. MRT-83 blocks Smo translocation induced by Shh pathway activation to the primary cilia of NT2 cells that derive from a pluripotent testicular carcinoma whereas cyclopamine, a reference Smo antagonist, induces Smo accumulation of Smo signals at the primary cilium. Therefore, it might be anticipated that MRT-83, like GDC-0449 and LDE-225, interacts with Smo in a manner different from that of cyclopamine, suggesting that while their binding sites are overlapping, they are not identical. Stereotaxic injection of MRT-83 into the lateral ventricle of adult mice but not of a structurally-related compound inactive at Smo, abolished upregulation of Ptc transcription induced by Shh in the neighboring subventricular zone, one of the two main neurogenic areas of the adult brain. These data demonstrate that MRT derivatives efficiently antagonize Shh signaling in vivo. Thus, MRT-10, MRT-14, MRT-83 and structurally-related molecules are potent Smo antagonists. These compounds should be useful for clarifying the molecular and biochemical mechanisms underlying the resistance of Smo inhibitors in brain cancer cells and may help develop new therapies against Shh pathway-related brain diseases.PARIS11-SCD-Bib. électronique (914719901) / SudocSudocFranceF

Comment moduler l’activité du récepteur du calcium extracellulaire ?

Le récepteur du Ca2+ extracellulaire (CaR), localisé à la membrane de la cellule parathyroïdienne, répond aux fluctuations des ions Ca2+ sériques et contrôle l’homéostasie calcique. Il appartient à la classe III des récepteurs couplés aux protéines G. Son activité est contrôlée par divers ions divalents et trivalents, mais aussi par des molécules endogènes telles que les acides aminés aromatiques de forme L ou la spermine. Le développement de molécules calcimimétiques capables de potentialiser les actions du Ca2+ sur le CaR et de réduire la concentration de la parathormone circulante in vivo, ainsi que des études cliniques récentes, suggèrent qu’il constitue une nouvelle cible thérapeutique pour le traitement des hyperparathyroïdies primaires et secondaires. Les calcilytiques bloquent le CaR et stimulent la sécrétion de parathormone. Ces nouvelles molécules, qui agissent au niveau des sept domaines transmembranaires du CaR, devraient permettre de caractériser les fonctions physiologiques associées au CaR et aux ions Ca2+ extracellulaires dans divers tissus tels que le rein, l’os ou le cerveau.The extracellular calcium-sensing receptor (CaR) belongs to class III of G-protein coupled receptors. The CaR is expressed at the surface of the parathyroid cells and plays an essential role in the regulation of Ca2+ homeostasis through the control of parathyroid secretion. The CaR is activated by Ca2+ and Mg2+ present in the extracellular fluids, various di- and trivalent cations, L-aminoacids and charged molecules including several antibiotics. Calcimimetics potentiate the effect of Ca2+ and are proposed to be of therapeutic benefit for the treatment of both primary and secondary hyperparathyroidism. Calcilytics block the Ca2+-induced activation of the CaR. Three-dimensional models of the seven transmembrane domains of the human CaR have been used to identify specific residues implicated in the recognition of calcimimetics and calcilytics. These molecules should be useful for delineating the physiological roles played by the CaR in several tissues and for clarifying the direct effects attributed to extracellular Ca2+