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1.
Genetics ; 2021 Nov 22.
Artículo en Inglés | MEDLINE | ID: mdl-34850872

RESUMEN

Primary cilia are sensory and signaling hubs with a protein composition that is distinct from the rest of the cell due to the barrier function of the transition zone (TZ) at the base of the cilium. Protein transport across the TZ is mediated in part by the BBSome, and mutations disrupting TZ and BBSome proteins cause human ciliopathy syndromes. Ciliopathies have phenotypic variability even among patients with identical genetic variants, suggesting a role for modifier loci. To identify potential ciliopathy modifiers, we performed a mutagenesis screen on nphp-4 mutant Caenorhabditis elegans and uncovered a novel allele of bbs-5. Nphp-4;bbs-5 double mutant worms have phenotypes not observed in either individual mutant strain. To test whether this genetic interaction is conserved, we also analyzed zebrafish and mouse mutants. While Nphp4 mutant zebrafish appeared overtly normal, Bbs5 mutants exhibited scoliosis. When combined, Nphp4;Bbs5 double mutant zebrafish did not exhibit synergistic effects, but the lack of a phenotype in Nphp4 mutants makes interpreting these data difficult. In contrast, Nphp4;Bbs5 double mutant mice were not viable and there were fewer mice than expected carrying three mutant alleles. In addition, postnatal loss of Bbs5 in mice using a conditional allele compromised survival when combined with an Nphp4 allele. As cilia are still formed in the double mutant mice, the exacerbated phenotype is likely a consequence of disrupted ciliary signaling. Collectively, these data support an evolutionarily conserved genetic interaction between Bbs5 and Nphp4 alleles that may contribute to the variability in ciliopathy phenotypes.

2.
Front Cell Dev Biol ; 9: 705182, 2021.
Artículo en Inglés | MEDLINE | ID: mdl-34970537

RESUMEN

Atxn10 is a gene known for its role in cytokinesis and is associated with spinocerebellar ataxia (SCA10), a slowly progressing cerebellar syndrome caused by an intragenic pentanucleotide repeat expansion. Atxn10 is also implicated in the ciliopathy syndromes nephronophthisis (NPHP) and Joubert syndrome (JBTS), which are caused by the disruption of cilia function leading to nephron loss, impaired renal function, and cerebellar hypoplasia. How Atxn10 disruption contributes to these disorders remains unknown. Here, we generated Atxn10 congenital and conditional mutant mouse models. Our data indicate that while ATXN10 protein can be detected around the base of the cilium as well as in the cytosol, its loss does not cause overt changes in cilia formation or morphology. Congenital loss of Atxn10 results in embryonic lethality around E10.5 associated with pericardial effusion and loss of trabeculation. Similarly, tissue-specific loss of ATXN10 in the developing endothelium (Tie2-Cre) and myocardium (cTnT-Cre) also results in embryonic lethality with severe cardiac malformations occurring in the latter. Using an inducible Cagg-CreER to disrupt ATXN10 systemically at postnatal stages, we show that ATXN10 is also required for survival in adult mice. Loss of ATXN10 results in severe pancreatic and renal abnormalities leading to lethality within a few weeks post ATXN10 deletion in adult mice. Evaluation of these phenotypes further identified rapid epithelial-to-mesenchymal transition (EMT) in these tissues. In the pancreas, the phenotype includes signs of both acinar to ductal metaplasia and EMT with aberrant cilia formation and severe defects in glucose homeostasis related to pancreatic insufficiency or defects in feeding or nutrient intake. Collectively, this study identifies ATXN10 as an essential protein for survival.

3.
Dev Dyn ; 2021 Mar 16.
Artículo en Inglés | MEDLINE | ID: mdl-33728725

RESUMEN

BACKGROUND: Genetic tools to study gene function and the fate of cells in the anterior limb bud are very limited. RESULTS: We describe a transgenic mouse line expressing CreERT2 from the Aristaless-like 4 (Alx4) promoter that induces recombination in the anterior limb. Cre induction at embryonic day 8.5 revealed that Alx4-CreERT2 labeled cells using the mTmG Cre reporter contributed to anterior digits I to III as well as the radius of the forelimb. Cre activity is expanded further along the AP axis in the hindlimb than in the forelimb resulting in some Cre reporter cells contributing to digit IV. Induction at later time points labeled cells that become progressively restricted to more anterior digits and proximal structures. Comparison of Cre expression from the Alx4 promoter transgene with endogenous Alx4 expression reveals Cre expression is slightly expanded posteriorly relative to the endogenous Alx4 expression. Using Alx4-CreERT2 to induce loss of intraflagellar transport 88 (Ift88), a gene required for ciliogenesis, hedgehog signaling, and limb patterning, did not cause overt skeletal malformations. However, the efficiency of deletion, time needed for Ift88 protein turnover, and for cilia to regress may hinder using this approach to analyze cilia in the limb. Alx4-CreERT2 is also active in the mesonephros and nephric duct that contribute to the collecting tubules and ducts of the adult nephron. Embryonic activation of the Alx4-CreERT2 in the Ift88 conditional line results in cyst formation in the collecting tubules/ducts. CONCLUSION: Overall, the Alx4-CreERT2 line will be a new tool to assess cell fates and analyze gene function in the anterior limb, mesonephros, and nephric duct.

4.
Hum Mol Genet ; 30(3-4): 234-246, 2021 04 26.
Artículo en Inglés | MEDLINE | ID: mdl-33560420

RESUMEN

Primary cilia are critical sensory and signaling compartments present on most mammalian cell types. These specialized structures require a unique signaling protein composition relative to the rest of the cell to carry out their functions. Defects in ciliary structure and signaling result in a broad group of disorders collectively known as ciliopathies. One ciliopathy, Bardet-Biedl syndrome (BBS; OMIM 209900), presents with diverse clinical features, many of which are attributed to defects in ciliary signaling during both embryonic development and postnatal life. For example, patients exhibit obesity, polydactyly, hypogonadism, developmental delay and skeletal abnormalities along with sensory and cognitive deficits, but for many of these phenotypes it is uncertain, which are developmental in origin. A subset of BBS proteins assembles into the core BBSome complex, which is responsible for mediating transport of membrane proteins into and out of the cilium, establishing it as a sensory and signaling hub. Here, we describe two new mouse models for BBS resulting from a targeted LacZ gene trap allele (Bbs5-/-) that is a predicted congenital null mutation and conditional (Bbs5flox/flox) allele of Bbs5. Bbs5-/- mice develop a complex phenotype consisting of increased pre-weaning lethality craniofacial and skeletal defects, ventriculomegaly, infertility and pituitary anomalies. Utilizing the conditional allele, we show that the male fertility defects, ventriculomegaly and pituitary abnormalities are only present when Bbs5 is disrupted prior to postnatal day 7, indicating a developmental origin. In contrast, mutation of Bbs5 results in obesity, independent of the age of Bbs5 loss.


Asunto(s)
Síndrome de Bardet-Biedl/metabolismo , Proteínas del Citoesqueleto/genética , Modelos Animales de Enfermedad , Mutación , Proteínas de Unión a Fosfato/genética , Hipófisis/anomalías , Animales , Síndrome de Bardet-Biedl/genética , Síndrome de Bardet-Biedl/patología , Síndrome de Bardet-Biedl/fisiopatología , Proteínas del Citoesqueleto/metabolismo , Masculino , Ratones , Fenotipo , Proteínas de Unión a Fosfato/metabolismo , Hipófisis/crecimiento & desarrollo , Hipófisis/metabolismo
5.
PLoS One ; 14(1): e0204197, 2019.
Artículo en Inglés | MEDLINE | ID: mdl-30608923

RESUMEN

Bone remodeling involves the coordinated actions of osteoclasts, which resorb the calcified bony matrix, and osteoblasts, which refill erosion pits created by osteoclasts to restore skeletal integrity and adapt to changes in mechanical load. Osteoblasts are derived from pluripotent mesenchymal stem cell precursors, which undergo differentiation under the influence of a host of local and environmental cues. To characterize the autocrine/paracrine signaling networks associated with osteoblast maturation and function, we performed gene network analysis using complementary "agnostic" DNA microarray and "targeted" NanoString nCounter datasets derived from murine MC3T3-E1 cells induced to undergo synchronized osteoblastic differentiation in vitro. Pairwise datasets representing changes in gene expression associated with growth arrest (day 2 to 5 in culture), differentiation (day 5 to 10 in culture), and osteoblast maturation (day 10 to 28 in culture) were analyzed using Ingenuity Systems Pathways Analysis to generate predictions about signaling pathway activity based on the temporal sequence of changes in target gene expression. Our data indicate that some pathways involved in osteoblast differentiation, e.g. Wnt/ß-catenin signaling, are most active early in the process, while others, e.g. TGFß/BMP, cytokine/JAK-STAT and TNFα/RANKL signaling, increase in activity as differentiation progresses. Collectively, these pathways contribute to the sequential expression of genes involved in the synthesis and mineralization of extracellular matrix. These results provide insight into the temporal coordination and complex interplay between signaling networks controlling gene expression during osteoblast differentiation. A more complete understanding of these processes may aid the discovery of novel methods to promote osteoblast development for the treatment of conditions characterized by low bone mineral density.


Asunto(s)
Diferenciación Celular/genética , Osteoblastos/fisiología , Osteogénesis/genética , Transducción de Señal/genética , Transcriptoma/fisiología , Células 3T3 , Animales , Comunicación Autocrina/genética , Densidad Ósea/fisiología , Remodelación Ósea/genética , Conjuntos de Datos como Asunto , Matriz Extracelular/fisiología , Perfilación de la Expresión Génica , Redes Reguladoras de Genes/fisiología , Ratones , Análisis de Secuencia por Matrices de Oligonucleótidos , Comunicación Paracrina/genética
6.
Anat Rec (Hoboken) ; 302(1): 136-145, 2019 01.
Artículo en Inglés | MEDLINE | ID: mdl-30289203

RESUMEN

Primary cilia are small organelles projecting from the cell surface of many cell types. They play a crucial role in the regulation of various signaling pathway. In this study, we investigated the importance of cilia for heart development by conditionally deleting intraflagellar transport protein Ift88 using the col3.6-cre mouse. Analysis of col3.6;Ift88 offspring showed a wide spectrum of cardiovascular defects including double outlet right ventricle and atrioventricular septal defects. In addition, we found that in the majority of specimens the pulmonary veins did not properly connect to the developing left atrium. The abnormal connections found resemble those seen in patients with total anomalous pulmonary venous return. Analysis of mutant hearts at early stages of development revealed abnormal development of the dorsal mesocardium, a second heart field-derived structure at the venous pole intrinsically related to the development of the pulmonary veins. Data presented support a crucial role for primary cilia in outflow tract development and atrioventricular septation and their significance for the formation of the second heart field-derived tissues at the venous pole including the dorsal mesocardium. Furthermore, the results of this study indicate that proper formation of the dorsal mesocardium is critically important for the development of the pulmonary veins. Anat Rec, 302:136-145, 2019. © 2018 Wiley Periodicals, Inc.


Asunto(s)
Cilios/patología , Modelos Animales de Enfermedad , Defectos del Tabique Interatrial/patología , Venas Pulmonares/anomalías , Síndrome de Cimitarra/patología , Animales , Colágeno Tipo III/fisiología , Factores de Transcripción MEF2/fisiología , Masculino , Ratones , Ratones Noqueados , Penetrancia , Proteínas Supresoras de Tumor/fisiología
7.
J Biol Chem ; 290(11): 6890-902, 2015 Mar 13.
Artículo en Inglés | MEDLINE | ID: mdl-25616662

RESUMEN

Microtubule-based centrioles in the centrosome mediate accurate bipolar cell division, spindle orientation, and primary cilia formation. Cellular checkpoints ensure that the centrioles duplicate only once in every cell cycle and achieve precise dimensions, dysregulation of which results in genetic instability and neuro- and ciliopathies. The normal cellular level of centrosomal protein 4.1-associated protein (CPAP), achieved by its degradation at mitosis, is considered as one of the major mechanisms that limits centriole growth at a predetermined length. Here we show that CPAP levels and centriole elongation are regulated by centrobin. Exogenous expression of centrobin causes abnormal elongation of centrioles due to massive accumulation of CPAP in the cell. Conversely, CPAP was undetectable in centrobin-depleted cells, suggesting that it undergoes degradation in the absence of centrobin. Only the reintroduction of full-length centrobin, but not its mutant form that lacks the CPAP binding site, could restore cellular CPAP levels in centrobin-depleted cells, indicating that persistence of CPAP requires its interaction with centrobin. Interestingly, inhibition of the proteasome in centrobin-depleted cells restored the cellular and centriolar CPAP expression, suggesting its ubiquitination and proteasome-mediated degradation when centrobin is absent. Intriguingly, however, centrobin-overexpressing cells also showed proteasome-independent accumulation of ubiquitinated CPAP and abnormal, ubiquitin-positive, elongated centrioles. Overall, our results show that centrobin interacts with ubiquitinated CPAP and prevents its degradation for normal centriole elongation function. Therefore, it appears that loss of centrobin expression destabilizes CPAP and triggers its degradation to restrict the centriole length during biogenesis.


Asunto(s)
Proteínas de Ciclo Celular/metabolismo , Centriolos/metabolismo , Proteínas Asociadas a Microtúbulos/metabolismo , Proteínas de Ciclo Celular/análisis , Proteínas de Ciclo Celular/genética , Línea Celular , Centriolos/ultraestructura , Eliminación de Gen , Humanos , Proteínas Asociadas a Microtúbulos/análisis , Complejo de la Endopetidasa Proteasomal/metabolismo , Proteolisis , Ubiquitinación , Regulación hacia Arriba
8.
Bone ; 69: 30-8, 2014 Dec.
Artículo en Inglés | MEDLINE | ID: mdl-25201465

RESUMEN

The extracellular matrix protein Fibulin-1 (Fbln1) has been shown to be involved in numerous processes including cardiovascular and lung development. Here we have examined the role of Fbln1 in bone formation. Alizarin red staining of skulls from Fbln1-deficient mice showed reduced mineralization of both membranous and endochondral bones. MicroCT (µCT) analysis of the calvarial bones (i.e., frontal, parietal and interparietal bones collectively) indicated that bone volume in Fbln1 nulls at neonatal stage P0 were reduced by 22% (p=0.015). Similarly, Fbln1 null frontal bones showed a 16% (p=0.035) decrease in bone volume, with a reduction in the interfrontal bone, and a discontinuity in the leading edge of the frontal bone. To determine whether Fbln1 played a role in osteoblast differentiation during bone formation, qPCR was used to measure the effects of Fbln1 deficiency on the expression of Osterix (Osx), a transcription factor essential for osteoblast differentiation. This analysis demonstrated that Osx mRNA was significantly reduced in Fbln1-deficient calvarial bones at developmental stages E16.5 (p=0.049) and E17.5 (p=0.022). Furthermore, the ability of Bmp-2 to induce Osx expression was significantly diminished in Fbln1-deficient mouse embryo fibroblasts. Together, these findings indicate that Fbln1 is a new positive modulator of the formation of membranous bone and endochondral bone in the skull, acting as a positive regulator of Bmp signaling.


Asunto(s)
Proteína Morfogenética Ósea 2/metabolismo , Proteínas de Unión al Calcio/metabolismo , Osteoblastos/metabolismo , Osteogénesis/fisiología , Factores de Transcripción/metabolismo , Animales , Diferenciación Celular , Inmunohistoquímica , Etiquetado Corte-Fin in Situ , Ratones , Ratones Noqueados , Osteoblastos/citología , Reacción en Cadena en Tiempo Real de la Polimerasa , Factor de Transcripción Sp7 , Transcriptoma
9.
Am J Physiol Lung Cell Mol Physiol ; 306(2): L162-9, 2014 Jan.
Artículo en Inglés | MEDLINE | ID: mdl-24213915

RESUMEN

The mechanisms for the development of bronchiectasis and airway hyperreactivity have not been fully elucidated. Although genetic, acquired diseases and environmental influences may play a role, it is also possible that motile cilia can influence this disease process. We hypothesized that deletion of a key intraflagellar transport molecule, IFT88, in mature mice causes loss of cilia, resulting in airway remodeling. Airway cilia were deleted by knockout of IFT88, and airway remodeling and pulmonary function were evaluated. In IFT88(-) mice there was a substantial loss of airway cilia on respiratory epithelium. Three months after the deletion of cilia, there was clear evidence for bronchial remodeling that was not associated with inflammation or apparent defects in mucus clearance. There was evidence for airway epithelial cell hypertrophy and hyperplasia. IFT88(-) mice exhibited increased airway reactivity to a methacholine challenge and decreased ciliary beat frequency in the few remaining cells that possessed cilia. With deletion of respiratory cilia there was a marked increase in the number of club cells as seen by scanning electron microscopy. We suggest that airway remodeling may be exacerbated by the presence of club cells, since these cells are involved in airway repair. Club cells may be prevented from differentiating into respiratory epithelial cells because of a lack of IFT88 protein that is necessary to form a single nonmotile cilium. This monocilium is a prerequisite for these progenitor cells to transition into respiratory epithelial cells. In conclusion, motile cilia may play an important role in controlling airway structure and function.


Asunto(s)
Hiperreactividad Bronquial/patología , Bronquiectasia/patología , Cilios/patología , Cilios/fisiología , Trastornos de la Motilidad Ciliar/patología , Animales , Hiperreactividad Bronquial/fisiopatología , Bronquiectasia/fisiopatología , Broncoconstrictores/farmacología , Trastornos de la Motilidad Ciliar/fisiopatología , Modelos Animales de Enfermedad , Cloruro de Metacolina/farmacología , Ratones , Ratones Noqueados , Depuración Mucociliar/fisiología , Mucosa Respiratoria/efectos de los fármacos , Mucosa Respiratoria/patología , Mucosa Respiratoria/fisiopatología , Proteínas Supresoras de Tumor/genética
10.
Cilia ; 2(1): 8, 2013 Jul 03.
Artículo en Inglés | MEDLINE | ID: mdl-23819925

RESUMEN

BACKGROUND: Cilia are found on nearly every cell type in the mammalian body, and have been historically classified as either motile or immotile. Motile cilia are important for fluid and cellular movement; however, the roles of non-motile or primary cilia in most tissues remain unknown. Several genetic syndromes, called the ciliopathies, are associated with defects in cilia structure or function and have a wide range of clinical presentations. Much of what we know about the formation and maintenance of cilia comes from model systems like C. elegans and Chalmydomonas. Studies of mammalian cilia in live tissues have been hampered by difficulty visualizing them. RESULTS: To facilitate analyses of mammalian cilia function we generated an inducible CiliaGFP mouse by targeting mouse cDNA encoding a cilia-localized protein somatostatin receptor 3 fused to GFP (Sstr3::GFP) into the ROSA26 locus. In this system, Sstr3::GFP is expressed from the ubiquitous ROSA26 promoter after Cre mediated deletion of an upstream Neo cassette flanked by lox P sites. Fluorescent cilia labeling was observed in a variety of live tissues and after fixation. Both cell-type specific and temporally regulated cilia labeling were obtained using multiple Cre lines. The analysis of renal cilia in anesthetized live mice demonstrates that cilia commonly lay nearly parallel to the apical surface of the tubule. In contrast, in more deeply anesthetized mice the cilia display a synchronized, repetitive oscillation that ceases upon death, suggesting a relationship to heart beat, blood pressure or glomerular filtration. CONCLUSIONS: The ability to visualize cilia in live samples within the CiliaGFP mouse will greatly aid studies of ciliary function. This mouse will be useful for in vivo genetic and pharmacological screens to assess pathways regulating cilia motility, signaling, assembly, trafficking, resorption and length control and to study cilia regulated physiology in relation to ciliopathy phenotypes.

11.
BMC Biol ; 11: 27, 2013 Mar 28.
Artículo en Inglés | MEDLINE | ID: mdl-23537390

RESUMEN

BACKGROUND: The pituitary gland is formed by the juxtaposition of two tissues: neuroectoderm arising from the basal diencephalon, and oral epithelium, which invaginates towards the central nervous system from the roof of the mouth. The oral invagination that reaches the brain from the mouth is referred to as Rathke's pouch, with the tip forming the adenohypophysis and the stalk disappearing after the earliest stages of development. In tetrapods, formation of the cranial base establishes a definitive barrier between the pituitary and oral cavity; however, numerous extinct and extant vertebrate species retain an open buccohypophyseal canal in adulthood, a vestige of the stalk of Rathke's pouch. Little is currently known about the formation and function of this structure. Here we have investigated molecular mechanisms driving the formation of the buccohypophyseal canal and their evolutionary significance. RESULTS: We show that Rathke's pouch is located at a boundary region delineated by endoderm, neural crest-derived oral mesenchyme and the anterior limit of the notochord, using CD1, R26R-Sox17-Cre and R26R-Wnt1-Cre mouse lines. As revealed by synchrotron X-ray microtomography after iodine staining in mouse embryos, the pouch has a lobulated three-dimensional structure that embraces the descending diencephalon during pituitary formation. Polaris(fl/fl); Wnt1-Cre, Ofd1(-/-) and Kif3a(-/-) primary cilia mouse mutants have abnormal sonic hedgehog (Shh) signaling and all present with malformations of the anterior pituitary gland and midline structures of the anterior cranial base. Changes in the expressions of Shh downstream genes are confirmed in Gas1(-/-) mice. From an evolutionary perspective, persistence of the buccohypophyseal canal is a basal character for all vertebrates and its maintenance in several groups is related to a specific morphology of the midline that can be related to modulation in Shh signaling. CONCLUSION: These results provide insight into a poorly understood ancestral vertebrate structure. It appears that the opening of the buccohypophyseal canal depends upon Shh signaling and that modulation in this pathway most probably accounts for its persistence in phylogeny.


Asunto(s)
Proteínas Hedgehog/metabolismo , Boca/embriología , Boca/metabolismo , Hipófisis/embriología , Hipófisis/metabolismo , Transducción de Señal , Vertebrados/embriología , Animales , Proteínas de Ciclo Celular/deficiencia , Proteínas de Ciclo Celular/metabolismo , Cilios/metabolismo , Ectodermo/embriología , Ectodermo/metabolismo , Extinción Biológica , Peces/embriología , Fósiles , Proteínas Ligadas a GPI/deficiencia , Proteínas Ligadas a GPI/metabolismo , Regulación del Desarrollo de la Expresión Génica , Proteínas Hedgehog/genética , Maxilares/embriología , Ratones , Boca/anatomía & histología , Mutación/genética , Filogenia , Hipófisis/anatomía & histología , Cráneo/anatomía & histología , Cráneo/embriología
12.
PLoS One ; 7(3): e33350, 2012.
Artículo en Inglés | MEDLINE | ID: mdl-22413019

RESUMEN

BACKGROUND: The disintegrin and metalloenzyme ADAM17 participates in numerous inflammatory and proliferative diseases, and its pathophysiological role was implicated in kidney fibrosis, polycystic kidney disease and other chronic kidney diseases. At present, we have little understanding how the enzyme activity is regulated. In this study we wanted to characterize the role of α5ß1 integrin in ADAM17 activity regulation during G protein-coupled receptor (GPCR) stimulation. METHODOLOGY/PRINCIPAL FINDINGS: We showed previously that the profibrotic GPCR agonist serotonin (5-HT) induced kidney mesangial cell proliferation through ADAM17 activation and heparin-binding epidermal growth factor (HB-EGF) shedding. In the present studies we observed that in unstimulated mesangial cell lysates α5ß1 integrin co-precipitated with ADAM17 and that 5-HT treatment of the cells induced dissociation of α5ß1 integrin from ADAM17. Using fluorescence immunostaining and in situ proximity ligation assay, we identified the perinuclear region as the localization of the ADAM17/α5ß1 integrin interaction. In cell-free assays, we showed that purified α5ß1 integrin and ß1 integrin dose-dependently bound to and inhibited activity of recombinant ADAM17. We provided evidence that the conformation of the integrin determines its ADAM17-binding ability. To study the effect of ß1 integrin on ADAM17 sheddase activity, we employed alkaline phosphatase-tagged HB-EGF. Overexpression of ß1 integrin lead to complete inhibition of 5-HT-induced HB-EGF shedding and silencing ß1 integrin by siRNA significantly increased mesangial cells ADAM17 responsiveness to 5-HT. CONCLUSIONS/SIGNIFICANCE: Our data show for the first time that ß1 integrin has an important physiological role in ADAM17 activity regulation. We suggest that regulating α5ß1 integrin binding to ADAM17 could be an attractive therapeutic target in chronic kidney diseases.


Asunto(s)
Proteínas ADAM/metabolismo , Integrina alfa5beta1/metabolismo , Células Mesangiales/enzimología , Proteína ADAM17 , Animales , Células Cultivadas , Activación Enzimática , Factor de Crecimiento Similar a EGF de Unión a Heparina , Integrina beta1/metabolismo , Péptidos y Proteínas de Señalización Intercelular/metabolismo , Células Mesangiales/metabolismo , Complejos Multiproteicos/metabolismo , Unión Proteica/efectos de los fármacos , Transporte de Proteínas , Ratas , Ratas Sprague-Dawley , Receptores Acoplados a Proteínas G/agonistas , Tiofenos/farmacología
13.
FASEB J ; 26(4): 1663-71, 2012 Apr.
Artículo en Inglés | MEDLINE | ID: mdl-22223751

RESUMEN

We investigated the role of the chondrocyte primary cilium in mechanotransduction events related to cartilage extracellular matrix synthesis. We generated conditionally immortalized wild-type (WT) and IFT88(orpk) (ORPK) mutant chondrocytes that lack primary cilia and assessed intracellular Ca(2+) signaling, extracellular matrix synthesis, and ATP release in response to physiologically relevant compressive strains in a 3-dimensional chondrocyte culture system. All conditions were compared to unloaded controls. We found that cilia were required for compression-induced Ca(2+) signaling mediated by ATP release, and an associated up-regulation of aggrecan mRNA and sulfated glycosaminosglycan secretion. However, chondrocyte cilia were not the initial mechanoreceptors, since both WT and ORPK cells showed mechanically induced ATP release. Rather, we found that primary cilia were required for downstream ATP reception, since ORPK cells did not elicit a Ca(2+) response to exogenous ATP even though WT and ORPK cells express similar levels of purine receptors. We suggest that purinergic Ca(2+) signaling may be regulated by polycystin-1, since ORPK cells only expressed the C-terminal tail. This is the first study to demonstrate that primary cilia are essential organelles for cartilage mechanotransduction, as well as identifying a novel role for primary cilia not previously reported in any other cell type, namely cilia-mediated control of ATP reception.


Asunto(s)
Adenosina Trifosfato/metabolismo , Señalización del Calcio/fisiología , Calcio/metabolismo , Condrocitos/fisiología , Cilios/metabolismo , Mecanotransducción Celular/fisiología , Animales , Células Cultivadas , Condrocitos/citología , Fuerza Compresiva , Matriz Extracelular/metabolismo , Ratones , Ratones Transgénicos , Estrés Mecánico
14.
J Cell Biochem ; 113(4): 1274-81, 2012 Apr.
Artículo en Inglés | MEDLINE | ID: mdl-22105856

RESUMEN

Receptor activator of NF-κB ligand (RANKL)-RANK receptor signaling to induce NFATc1 transcription factor is critical for osteoclast differentiation and bone resorption. RANK adaptor proteins, tumor necrosis factor receptor-associated factors (TRAFs) play an essential role in RANKL signaling. Evidence indicates that NIP45 (NFAT interacting protein) binds with TRAFs and NFATc2. We therefore hypothesized that NIP45 regulates RANKL induced osteoclast differentiation. In this study, we demonstrate that RANKL treatment down regulates NIP45 expression in mouse bone marrow derived pre-osteoclast cells. Lentiviral (pGIPZ) mediated shRNA knock-down of NIP45 expression in RANKL stimulated pre-osteoclast cells resulted in increased levels of NFATc1, NFATc2, and TRAF6 but not TRAF2 expression compared to control shRNA transduced cells. Also, NIP45 suppression elevated p-IκB-α levels and NF-κB-luciferase reporter activity. Confocal microscopy demonstrated NIP45 colocalized with TRAF6 in the cytosol of osteoclast progenitor cells. In contrast, RANKL stimulation induced NIP45 nuclear translocation and colocalization with NFATc2 in these cells. Coimmuneprecipitation assay demonstrated NIP45 binding with NFATc2 but not NFATc1. We further show that shRNA knock-down of NIP45 expression in pre-osteoclast cells significantly increased RANKL induced osteoclast differentiation and bone resorption activity. Taken together, our results indicate that RANKL signaling down regulates NIP45 expression and that NIP45 is a negative regulator of osteoclast differentiation.


Asunto(s)
Diferenciación Celular/fisiología , Péptidos y Proteínas de Señalización Intracelular/fisiología , Proteínas Nucleares/fisiología , Osteoclastos/citología , Ligando RANK/fisiología , Animales , Western Blotting , Células Cultivadas , Regulación hacia Abajo , Inmunoprecipitación , Ratones , Microscopía Confocal , Factores de Transcripción NFATC/metabolismo , Transducción de Señal/fisiología , Factor 6 Asociado a Receptor de TNF/metabolismo
15.
J Am Soc Nephrol ; 22(5): 839-48, 2011 May.
Artículo en Inglés | MEDLINE | ID: mdl-21493775

RESUMEN

Primary cilia dysfunction alters renal tubular cell proliferation and differentiation and associates with accelerated cyst formation in polycystic kidney disease. However, the mechanism leading from primary ciliary dysfunction to renal cyst formation is unknown. We hypothesize that primary cilia prevent renal cyst formation by suppressing pathologic tubular cell hypertrophy and proliferation. Unilateral nephrectomy initiates tubular cell hypertrophy and proliferation in the contralateral kidney and provides a tool to examine primary cilia regulation of renal hypertrophy. Conditional knockout of the primary cilia ift88 gene leads to delayed, adult-onset renal cystic disease, which provides a window of opportunity to conduct unilateral nephrectomy and examine downstream kinetics of renal hypertrophy and cyst formation. In wild-type animals, unilateral nephrectomy activated the mTOR pathway and produced appropriate structural and functional hypertrophy without renal cyst formation. However, in ift88 conditional knockout animals, unilateral nephrectomy triggered increased renal hypertrophy and accelerated renal cyst formation, leading to renal dysfunction. mTOR signaling also increased compared with wild-type animals, suggesting a mechanistic cascade starting with primary ciliary dysfunction, leading to excessive mTOR signaling and renal hypertrophic signaling and culminating in cyst formation. These data suggest that events initiating hypertrophic signaling, such as structural or functional loss of renal mass, may accelerate progression of adult polycystic kidney disease toward end-stage renal disease.


Asunto(s)
Cilios/fisiología , Enfermedades Renales Quísticas/etiología , Riñón/patología , Transducción de Señal/fisiología , Animales , Proliferación Celular , Femenino , Tasa de Filtración Glomerular , Hipertrofia , Masculino , Ratones , Serina-Treonina Quinasas TOR/fisiología , Proteínas Supresoras de Tumor/genética , Proteínas Supresoras de Tumor/fisiología , Regulación hacia Arriba
16.
Dev Dyn ; 240(3): 723-36, 2011 Mar.
Artículo en Inglés | MEDLINE | ID: mdl-21337470

RESUMEN

We describe the localization of the golgin GMAP210 and the intraflagellar protein IFT88 in the Golgi of spermatids and the participation of these two proteins in the development of the acrosome-acroplaxome complex, the head-tail coupling apparatus (HTCA) and the spermatid tail. Immunocytochemical experiments show that GMAP210 predominates in the cis-Golgi, whereas IFT88 prevails in the trans-Golgi network. Both proteins colocalize in proacrosomal vesicles, along acrosome membranes, the HTCA and the developing tail. IFT88 persists in the acrosome-acroplaxome region of the sperm head, whereas GMAP210 is no longer seen there. Spermatids of the Ift88 mouse mutant display abnormal head shaping and are tail-less. GMAP210 is visualized in the Ift88 mutant during acrosome-acroplaxome biogenesis. However, GMAP210-stained vesicles, mitochondria and outer dense fiber material build up in the manchette region and fail to reach the abortive tail stump in the mutant. In vitro disruption of the spermatid Golgi and microtubules with Brefeldin-A and nocodazole blocks the progression of GMAP210- and IFT88-stained proacrosomal vesicles to the acrosome-acroplaxome complex but F-actin distribution in the acroplaxome is not affected. We provide the first evidence that IFT88 is present in the Golgi of spermatids, that the microtubule-associated golgin GMAP210 and IFT88 participate in acrosome, HTCA, and tail biogenesis, and that defective intramanchette transport of cargos disrupts spermatid tail development.


Asunto(s)
Acrosoma/metabolismo , Aparato de Golgi/metabolismo , Proteínas Nucleares/metabolismo , Espermátides/metabolismo , Proteínas Supresoras de Tumor/metabolismo , Acrosoma/ultraestructura , Actinas/metabolismo , Animales , Brefeldino A/farmacología , Proteínas del Citoesqueleto , Técnica del Anticuerpo Fluorescente Indirecta , Aparato de Golgi/ultraestructura , Immunoblotting , Inmunohistoquímica , Masculino , Ratones , Microscopía Electrónica , Microtúbulos/metabolismo , Microtúbulos/ultraestructura , Nocodazol/farmacología , Proteínas Nucleares/genética , Ratas , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Espermátides/ultraestructura , Proteínas Supresoras de Tumor/genética
17.
Am J Physiol Renal Physiol ; 299(6): F1507-14, 2010 Dec.
Artículo en Inglés | MEDLINE | ID: mdl-20926633

RESUMEN

Recently, the use of overexpression of telomerase reverse transcriptase (TERT) has led to the generation of immortalized human cell lines. However, this cell immortalization approach has not been reported in well-differentiated mouse cells, such as renal epithelial cells. We sought to establish and then characterize a mouse collecting duct cell line, using ectopic expression of mTERT. Isolated primary cortical collecting duct (CCD) cell lines were transduced with mouse (m)TERT, using a lentiviral vector. mTERT-negative cells did not survive blasticidin selection, whereas mTERT-immortalized cells proliferated in selection media for over 40 subpassages. mTERT messenger RNA and telomerase activity was elevated in these cells, compared with an SV40-immortalized cell line. Flow cytometry with Dolichos biflorus agglutinin was used to select the CCD principal cells, and we designated this cell line mTERT-CCD. Cells were well differentiated and exhibited morphological characteristics typically found in renal epithelial cells, such as tight junction formation, microvilli, and primary cilia. Further characterization using standard immunofluorescence revealed abundant expression of aquaporin-2 and the vasopressin type 2 receptor. mTERT-CCD cells exhibited cAMP-stimulated/benzamil-inhibited whole cell currents. Whole cell patch-clamp currents were also enhanced after a 6-day treatment with aldosterone. In conclusion, we have successfully used mTERT to immortalize mouse collecting duct cells that retain the basic in vivo phenotypic characteristics of collecting duct cells. This technique should be valuable in generating cell lines from genetically engineered mouse models.


Asunto(s)
Técnicas de Cultivo de Célula/métodos , Línea Celular , Túbulos Renales Colectores/citología , Telomerasa/genética , Animales , Túbulos Renales Colectores/metabolismo , Ratones , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa
18.
Curr Biol ; 19(13): R526-35, 2009 Jul 14.
Artículo en Inglés | MEDLINE | ID: mdl-19602418

RESUMEN

Respect for the primary cilium has undergone a remarkable renaissance over the past decade, and it is now thought to be an essential regulator of numerous signaling pathways. The primary cilium's functions range from the movement of cells and fluid, to sensory inputs involved with olfaction and photoreception. Disruption of cilia function is involved in multiple human syndromes collectively called 'ciliopathies'. The cilium's activities are mediated by targeting of receptors, channels, and their downstream effector proteins to the ciliary or basal body compartment. These combined properties of the cilium make it a critical organelle facilitating the interactions between the cell and its environment. Here, we review many of the recent advances contributing to the ascendancy of the primary cilium and how the extraordinary complexity of this organelle inevitably assures many more exciting future discoveries.


Asunto(s)
Cilios/metabolismo , Transducción de Señal/fisiología , Animales , Cilios/ultraestructura , Proteínas Hedgehog/metabolismo , Humanos , Mecanotransducción Celular/fisiología , Neuronas Receptoras Olfatorias/citología , Células Fotorreceptoras/citología , Sensación/fisiología , Proteínas Wnt/metabolismo
19.
Development ; 136(6): 897-903, 2009 Mar.
Artículo en Inglés | MEDLINE | ID: mdl-19211681

RESUMEN

Primary cilia mediate Hh signalling and mutations in their protein components affect Hh activity. We show that in mice mutant for a cilia intraflagellar transport (IFT) protein, IFT88/polaris, Shh activity is increased in the toothless diastema mesenchyme of the embryonic jaw primordia. This results in the formation of ectopic teeth in the diastema, mesial to the first molars. This phenotype is specific to loss of polaris activity in the mesenchyme since loss of Polaris in the epithelium has no detrimental affect on tooth development. To further confirm that upregulation of Shh activity is responsible for the ectopic tooth formation, we analysed mice mutant for Gas1, a Shh protein antagonist in diastema mesenchyme. Gas1 mutants also had ectopic diastema teeth and accompanying increased Shh activity. In this context, therefore, primary cilia exert a specific negative regulatory effect on Shh activity that functions to repress tooth formation and thus determine tooth number. Strikingly, the ectopic teeth adopt a size and shape characteristic of premolars, a tooth type that was lost in mice around 50-100 million years ago.


Asunto(s)
Proteínas Hedgehog/metabolismo , Diente/metabolismo , Animales , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Cilios/metabolismo , Diastema , Proteínas Ligadas a GPI , Regulación del Desarrollo de la Expresión Génica , Proteínas Hedgehog/genética , Proteínas de la Membrana/genética , Proteínas de la Membrana/metabolismo , Ratones , Ratones Transgénicos , Mutación/genética , Fenotipo , Diente/crecimiento & desarrollo
20.
Dev Dyn ; 237(8): 2053-60, 2008 Aug.
Artículo en Inglés | MEDLINE | ID: mdl-18629867

RESUMEN

Ift88 is a component of the intraflagellar transport complex required for formation and maintenance of cilia. Disruption of Ift88 results in depletion of cilia. The goal of the current study was to determine the role of primary cilia in ovarian function. Deletion of Ift88 in ovary using Cre-Lox recombination in mice resulted in a severe delay in mammary gland development including lack of terminal end bud structures, alterations in the estrous cycle, and impaired ovulation. Because estrogen drives the formation of end buds and Cre was expressed in the granulosa cells of the ovary, we tested the hypothesis that addition of estradiol to the mutant mice would compensate for defects in ovarian function and rescue the mammary gland phenotype. Mammary gland development including the formation of end bud structures resumed in mutant mice that were injected with estradiol. Together the results suggest that cilia are required for ovarian function.


Asunto(s)
Cilios/fisiología , Células de la Granulosa/patología , Células de la Granulosa/fisiología , Proteínas Supresoras de Tumor/genética , Proteínas Supresoras de Tumor/metabolismo , Animales , Transporte Biológico/fisiología , Cilios/patología , Estradiol/farmacología , Estrógenos/farmacología , Ciclo Estral/efectos de los fármacos , Ciclo Estral/fisiología , Femenino , Células de la Granulosa/efectos de los fármacos , Proteínas de Homeodominio/genética , Proteínas de Homeodominio/metabolismo , Integrasas/genética , Glándulas Mamarias Animales/efectos de los fármacos , Glándulas Mamarias Animales/crecimiento & desarrollo , Ratones , Ratones Mutantes , Ovulación/efectos de los fármacos , Ovulación/fisiología , Fenotipo
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