RESUMEN
alpha-klotho was identified as a gene associated with premature aging-like phenotypes characterized by short lifespan. In mice, we found the molecular association of alpha-Klotho (alpha-Kl) and Na+,K+-adenosine triphosphatase (Na+,K+-ATPase) and provide evidence for an increase of abundance of Na+,K+-ATPase at the plasma membrane. Low concentrations of extracellular free calcium ([Ca2+]e) rapidly induce regulated parathyroid hormone (PTH) secretion in an alpha-Kl- and Na+,K+-ATPase-dependent manner. The increased Na+ gradient created by Na+,K+-ATPase activity might drive the transepithelial transport of Ca2+ in cooperation with ion channels and transporters in the choroid plexus and the kidney. Our findings reveal fundamental roles of alpha-Kl in the regulation of calcium metabolism.
Asunto(s)
Calcio/metabolismo , Glucuronidasa/fisiología , Homeostasis , Animales , Calcio/líquido cefalorraquídeo , Membrana Celular/enzimología , Membrana Celular/metabolismo , Plexo Coroideo/metabolismo , Citoplasma/enzimología , Citoplasma/metabolismo , Retículo Endoplásmico/metabolismo , Endosomas/metabolismo , Inhibidores Enzimáticos/farmacología , Retroalimentación Fisiológica , Glucuronidasa/genética , Glucuronidasa/metabolismo , Aparato de Golgi/metabolismo , Células HeLa , Humanos , Transporte Iónico , Riñón/enzimología , Riñón/metabolismo , Proteínas Klotho , Ratones , Ouabaína/farmacología , Glándulas Paratiroides/enzimología , Glándulas Paratiroides/metabolismo , Hormona Paratiroidea/metabolismo , ATPasa Intercambiadora de Sodio-Potasio/antagonistas & inhibidores , ATPasa Intercambiadora de Sodio-Potasio/metabolismoRESUMEN
The concept of fractal geometry is useful for the analysis of irregular and complex structures often seen in nature. Here we apply this concept to investigate the structural mechanism of the development of pulmonary emphysema in the klotho mouse, which, after milk feeding, exhibits characteristics resembling aging and develops emphysema. We calculated the relationships between perimeter and size characterizing shape and between cumulative frequency and size of the terminal air spaces identified from histologic slides and found that both relations followed a power law with fractal properties. However, the fractal dimensions related to the shape and size (Dsn) in the klotho mice were significantly lower than in controls. Additionally, in the klotho mice, Dsn decreased with age without significant change in mean linear intercept. These abnormal morphological changes were restored when the klotho mice were fed with a vitamin D-deficient diet. Previously undescribed morphological model simulations showed that a random destruction, in which the destruction process occurs homogeneously in the lungs, was more consistent with the data than a correlated destruction that is usually seen in smoking-related human emphysema. These results suggest that the pathological changes in the lungs of the klotho mice are derived not from localized causes, but from systemic causes that are related to abnormal activation of vitamin D. The morphogenesis of emphysema in the klotho mice and morphological analyses using fractal geometry may contribute to the understanding of the progressive nature and cause of parenchymal destruction in human emphysema.
Asunto(s)
Glucuronidasa/deficiencia , Enfisema Pulmonar/patología , Envejecimiento , Animales , Modelos Animales de Enfermedad , Progresión de la Enfermedad , Fractales , Proteínas Klotho , Pulmón/patología , Ratones , Modelos Biológicos , Enfisema Pulmonar/etiología , Vitamina DRESUMEN
Klotho mutant mice exhibit a set of phenotypes resembling human ageing. Although the function of Klotho remains unclear, mediation of its pleiotropic functions by putative humoral factor(s) has been presumed. Newly established antibodies against Klotho allowed the detection of secreted Klotho, a candidate for the putative humoral factor, in sera and cerebrospinal fluid. Surprisingly the secreted Klotho was 130 kDa, in contrast to the 70 kDa predicted form from klotho gene transcripts. The secreted as well as the membrane-bound Klotho proteins were suggested to form oligomerized complex. These results delineate post-translation processing of Klotho and possible regulatory mechanisms for secretion of Klotho in vivo.
Asunto(s)
Membrana Celular/metabolismo , Proteínas de la Membrana/sangre , Proteínas de la Membrana/líquido cefalorraquídeo , Procesamiento Proteico-Postraduccional , Empalme Alternativo , Animales , Anticuerpos Monoclonales/química , Western Blotting , Células CHO , Cromatografía , Cromatografía en Gel , Cricetinae , Electroforesis en Gel de Poliacrilamida , Glucuronidasa , Glicosilación , Proteínas Klotho , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Fenotipo , Plásmidos/metabolismo , Pruebas de Precipitina , ARN Mensajero/metabolismo , Ratas , Ratas Sprague-Dawley , TransfecciónRESUMEN
klotho mutant mice provide a unique model to analyze mechanisms of aging because their phenotypes resemble those of human aging-associated disorders. The klotho gene encodes Klotho, a type I membrane protein that shares sequence similarity with members of the glycosidase family 1. Because Klotho lacks the glutamic acid residues that have been shown to be involved in the catalytic activity of this family of enzymes, the function of this protein was unknown. Here, we have studied the biochemical characteristics of recombinant Klotho. The purified chimeric Klotho-human IgG1 Fc protein (KLFc) was assayed with a series of 4-methylumbelliferyl (4Mu) beta-glycosides as potential substrates. An enzymatic activity of Klotho was observed only with 4-methylumbelliferyl beta-D-glucuronide in contrast to bovine liver beta-glucuronidase, which exhibits a rather wide substrate specificity. Furthermore, the enzymatic activity of KLFc was reduced by the addition of specific inhibitors of beta-glucuronidase. A number of natural beta-glucuronides were screened by competitive inhibition for KLFc beta-glucuronidase. We found that steroid beta-glucuronides such as beta-estradiol 3-beta-D-glucuronide, estrone 3-beta-D-glucuronide, and estriol 3beta-D-glucuronide were hydrolyzed by KLFc. The artificial fluorescent substrate and the steroid conjugates share a common phenolic structure. Collectively, these data suggest that Klotho functions as a novel beta-glucuronidase and that steroid beta-glucuronides are potential candidates for the natural substrate(s) of Klotho.