Immunohistochemical localization and mRNA expression of matrix Gla protein and fetuin-A in bone biopsies of hemodialysis patients

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Crowding Effects on the Structure and Dynamics of the Intrinsically Disordered Nuclear Chromatin Protein NUPR1

The intracellular environment is crowded with macromolecules, including sugars, proteins and nucleic acids. In the cytoplasm, crowding effects are capable of excluding up to 40% of the volume available to any macromolecule when compared to dilute conditions. NUPR1 is an intrinsically disordered protein (IDP) involved in cell-cycle regulation, stress-cell response, apoptosis processes, DNA binding and repair, chromatin remodeling and transcription. Simulations of molecular crowding predict that IDPs can adopt compact states, as well as more extended conformations under crowding conditions. In this work, we analyzed the conformation and dynamics of NUPR1 in the presence of two synthetic polymers, Ficoll-70 and Dextran-40, which mimic crowding effects in the cells, at two different concentrations (50 and 150 mg/ml). The study was carried out by using a multi-spectroscopic approach, including: site-directed spin labelling electron paramagnetic resonance spectroscopy (SDSL-EPR), nuclear magnetic resonance spectroscopy (NMR), circular dichroism (CD), small angle X-ray scattering (SAXS) and dynamic light scattering (DLS). SDSL-EPR spectra of two spin-labelled mutants indicate that there was binding with the crowders and that the local dynamics of the C and N termini of NUPR1 were partially affected by the crowders. However, the overall disordered nature of NUPR1 did not change substantially in the presence of the crowders, as shown by circular dichroism CD and NMR, and further confirmed by EPR. The changes in the dynamics of the paramagnetic probes appear to be related to preferred local conformations and thus crowding agents partially affect some specific regions, further pinpointing that NUPR1 flexibility has a key physiological role in its activity.This work was supported by Spanish Ministry of Economy and Competitiveness and European ERDF Funds (MCIU/AEI/FEDER, EU) (RTI 2018-097991-B-I00 to JN and PGC 2018-094548-B-I00 to AA and PM), and the Basque Government (IT1175-19 to AA and PM)

Sclérose tubéreuse de Bourneville : cible moléculaire de mTORC1 et le rôle des S6K

La Sclérose tubéreuse de Bourneville (STB) est une maladie génétique responsable de la formation de tumeurs bénignes dans de nombreux organes.Les kystes rénaux sont des facteurs de morbidité et de mortalité importants chez les patients STB.TSC1 et TSC2 sont les gènes mutés chez ces patients dont les protéines codées forment un complexe qui inhibe la voie de signalisation du mTORC1 (mechanistic target of rapamycin complexe 1), qui contrôle la croissance et la division cellulaires.Par conséquent, la perte d'activité du complexe TSC1/2 est associée à une activation incontrôlée de mTORC1. En cas de TSC, la S6 Kinase 1 (S6K1) joue un rôle prédominant sur les autres kinases de sa famille.S6K1 est une Ser/Thr kinase directement activée par mTORC1.Des études d'épistasie génétique chez Drosophila ont montré que la suppression de S6k rétablissait le phénotype de prolifération des mutants Tsc2 à un niveau normal.Pourtant, l'effet de S6K1 dans la pathogenèse de la TSC n'a pas encore été découvert. Pendant mon doctorat, je me suis demandé comment l'axe mTORC1/S6K1 induisait la cystogenèse rénale. Nous avons caractérisé un modèle murin qui inactive Tsc1 dans les tubules rénaux et entraîne une hypertrophie rénale et une cystogenèse.Nous avons ensuite combiné l'inactivation de S6k1 et observé une diminution importante du poids des reins et de la surface kystique et une amélioration de la fonction rénale.De façon intéressante, nous n'avons pas observé aucune différence en termes de prolifération entre les mutants Tsc1 en présence ou en absence de la S6K1.La division cellulaire orientée (OCD) est un processus qui régule la morphogenèse du tubule du néphron.Au cours de la croissance tubulaire normale, la division se produit alignée avec l'axe du tubule, ce qui permet l'élongation.Une perturbation de l'OCD a été associée au développement de kystes.Nous avons mesuré l'orientation de la division cellulaire dans les tubules rénaux dans nos modèles et nous avons constaté que l'inactivation de Tsc1 déclenchait une désorientation de la mitose dépendante de S6K1, vu que les angles mitotiques du double mutant Tsc1/S6K1 étaient corrigés.Le fuseau mitotique est organisé et orienté par les centrosomes, organelles qui, dans les cellules rénales, se trouvent à la surface apicale pendant l'interphase et qui migrent aux deux pôles latéraux pendant la mitose.Nous avons pu observer que l'hyperactivation de la S6K1 provoquait un mauvais positionnement des centrosomes en mitose et en interphase. Des cultures 3D de cellules rénales mutées en Tsc1 ou Tsc1/S6k1 ont confirmé que l'inactivation de S6K1 suffit pour rétablir une orientation correcte de la division cellulaire et un positionnement apical du centrosome.Nous nous sommes demandé quel substrat de S6K1 pourrait être responsable de ce phénotype.Nous avons donc réalisé un criblage phosphoprotéomique à l'aide des lignées cellulaires mutées, afin de trouver des protéines phosphorylées par S6K1 dans le contexte de la perte de Tsc1.Dans la liste des protéines différemment phosphorylées en l'absence de S6K1, nous avons observé un enrichissement en protéines du cytosquelette et des jonctions intercellulaires.Parmi ces protéines, nous nous sommes concentrés sur Afadin, une protéine qui relie le cortex d'actine aux jonctions adhérentes.Afadin est connue pour se lier à la machinerie du fuseau pendant la mitose et sa présence est importante pour assurer une orientation correcte de la division cellulaire dans les tubules rénaux.Nous avons validé Afadin en tant que substrat de S6K1.Enfin, nous avons invalidé Afadin dans la lignée cellulaire mutante Tsc1 et avons pu observer une correction du positionnement du centrosome uniquement lorsqu'une forme non phosphorylable d'Afadin était ré-exprimée dans ces cellules.En conclusion, notre travail démontre qu'une dérégulation de l'orientation du fuseau mitotique est importante pour un déclanchement de la cystogenèse rénale et que cela est sous le contrôle de la voie mTORC1/S6K1.Tuberous sclerosis complex (TSC) is a genetic disorder that causes benign tumor formation in many different organs, such as in the brain, kidney, skin, lung, heart, and eyes. Renal cysts are important morbidity and mortality factors in TSC patients. Renal cysts are progressive enlargements of the renal tubules that eventually compromise the filtration capacity of the organ. TSC1 and TSC2 are the genes mutated in the patients and code for a protein complex that functions as an inhibitor of the mechanistic target of rapamycin complex 1 (mTORC1) signaling, a master regulator of cell growth. In case of TSC, S6 Kinase 1 (S6K1) has a predominant role on the other kinases of its family, the AGC kinases, known to be involved in many pathologies. S6K1 is a serine/threonine kinase directly activated by mTORC1. Genetic epistasis studies in Drosophila showed that deletion of S6k reverts the overgrowth phenotype of Tsc2 mutants to normal levels. However, the effect of S6K1 in TSC pathogenesis has not been uncovered yet. During my Ph.D., I asked how mTORC1/S6K1 drives cystogenesis in the kidney. We characterized a murine model that inactivates Tsc1 in the renal tubules. The inactivation of Tsc1 led to kidney hypertrophy and cystogenesis. We then combined the inactivation of S6k1, and we observed an extensive decrease of the kidney weight and cystic surface and an improvement of renal function. We first quantified the proliferation rate and, interestingly, we could not observe any difference between the Tsc1 mutants in presence or absence of S6K1 activity. Oriented cell division (OCD) is a process that regulates the nephron tubule morphogenesis during kidney development. During normal tubular growth, the division usually occurs aligned with the tubule axis, which allows the elongation of the tubule during the proliferative phase, to maintain the correct diameter. Very often a perturbation of OCD has been connected to cyst development. We measured the orientation of cell division in the renal tubules in our models and we found that inactivation of Tsc1 triggered an S6K1-dependent misorientation of the mitosis and that the mitotic angles in Tsc1/S6K1 double mutant were corrected as in the controls. The mitotic spindle is organized and oriented by the centrosomes, organelles that, in renal cells, are at the apical surface during interphase and that migrate at the two lateral poles during mitosis to nucleate the spindle microtubules. We could observe that hyperactive S6K1 caused a mispositioning of the centrosomes both in mitosis and in interphase. 3D cultures of renal cells that we mutated for Tsc1 or Tsc1/S6k1 confirmed that is sufficient to inactivate S6K1 to restore a correct orientation of cell division and an apical positioning of the centrosome. We asked which substrate of S6K1 could be responsible for this phenotype. We therefore carried out a phosphoproteomic screening using the cell lines we generated, to find proteins phosphorylated by S6K1 in context of TSC1 loss. In the list of proteins differentially phosphorylated in Tsc1 and Tsc1/S6k1 mutant conditions, we found a significant enrichment in proteins of the cytoskeleton and of the cell-to-cell junctions. Among these proteins, we focused on Afadin, protein that connects the actin cortex to the Nectin-based adherens junctions. Afadin is known to bind the spindle machinery during mitosis and its presence has been shown to be important to assure a correct orientation of cell division in renal tubules. We validated Afadin as an S6K1 substrate both in vivo and in culture and using an in vivo kinase assay. Finally, we invalidated Afadin in the Tsc1-mutant cell line and we could observe a correction of the centrosome positioning only when an unphosphorylateble form of Afadin was re-expressed in these cells. To conclude, our work shows that a misorientation of the mitotic division occurs for the development of cysts associated with TSC and is under the control of the mTORC1/S6K1 axis

OPG and RANKL mRNA and protein expressions in the primary and secondary metaphyseal trabecular bone of PTH-treated rats are independent of that of SOST

Sclerostin, encoded by the SOST gene, is a recently identified protein which seems to affect bone remodeling by inhibiting bone formation via Wnt pathways. A previous study on OPG and RANKL, two cytokines involved in the control of osteoclastogenesis, showed that the anabolic effect produced by intermittent treatment with parathyroid hormone was characterized by an increase in OPG/RANKL mRNA ratio in the primary spongiosa of metaphyseal bone of rat femur, and by its falling in the secondary spongiosa, in comparison to controls (Silvestrini et al. (2007a)). Considering that Wnt pathway components seem to regulate osteoclast formation and bone resorption by repression of RANKL transcription and by positive regulation of OPG gene in osteoblastic cells, we have evaluated, in the same rats, whether and how SOST mRNA and protein in the primary and secondary metaphyseal bone are affected by PTH. SOST mRNA and protein significantly fell in both primary and secondary spongiosa where only a few osteocytes were positive to sclerostin. These data show that in the two metaphyseal areas no relationship does exist between the trends of OPG and RANKL mRNA and that of SOST, suggesting that there are no direct links between the effects induced by PTH on these molecules, at least in terms of gene expression

(Di) Segni di pietra sul duomo di Terni. Dai tracciati di cantiere ai modelli digitali

Gli antichi tracciati, realizzati in scala 1:1 su parti di strutture esistenti, svolgevano un ruolo fondamentale nelle diverse fasi del cantiere edile, dalla progettazione all’esecuzione. Lo studio di tali segni, condotto integrando le tecniche tradizionali di rilevamento a quelle per l’acquisizione massiva e alle metodologie digitali per la costruzione di modelli, ha l’obiettivo di comprendere le relazioni che intercorrono tra idea e realizzazione attraverso il continuo confronto tra modelli reali e modelli ideali, ampliando la conoscenza e la documentazione di antichi manufatti

Effects of intermittent parathyroid hormone (PTH) administration on SOST mRNA and protein in rat bone

Sclerostin, the secreted protein product of the SOST gene, which is mainly expressed by osteocytes, has recently been proposed as a negative regulator of bone osteoblastogenesis. Chronic elevation of PTH reduces SOST expression by osteocytes, while controversial results have been obtained by intermittent PTH administration. We have investigated the effects of intermittently administered PTH on SOST expression and sclerostin localization, comparing them with those of controls, as they appeared in three different bone segments of rat tibia: secondary trabecular metaphyseal and epiphyseal bone, and cortical diaphyseal bone. The histomorphometric results demonstrate that PTH enhances bone turnover through anabolic effects, as shown by the association of increased bone resorption variables with a significant rise in BV/TV, Tb.Th and Tb.N and a fall in Tb.Sp. PTH induces a SOST mRNA and protein fall in secondary metaphyseal trabeculae, diaphyseal bone and in epiphyseal trabeculae. Numbers of sclerostin immunopositive osteocytes/mm2 show no change, compared with controls; there are fewer sclerostin-positive osteocytes in secondary metaphyseal trabeculae than in the other two bone areas, both in the control and PTH groups. The low numbers of sclerostin-positive osteocytes in the metaphyseal trabecular bone seem to be directly related to the fact that this area displays a high remodeling rate. The anabolic effects of PTH are in line with the fall of SOST mRNA and protein in all the three bone segments examined; the rise of bone turnover supports a negative role of SOST in bone formation. © 2007 Springer Science+Business Media B.V