RESUMEN

The clinical management of bone defects caused by trauma or nonunion fractures remains a challenge in orthopedic practice due to the poor integration and biocompatibility properties of the scaffold or implant material. In the current work, the osteogenic properties of carboxyl-modified single-walled carbon nanotubes (COOH-SWCNTs) were investigated in vivo and in vitro. When human preosteoblasts and murine embryonic stem cells were cultured on coverslips sprayed with COOH-SWCNTs, accelerated osteogenic differentiation was manifested by increased expression of classical bone marker genes and an increase in the secretion of osteocalcin, in addition to prior mineralization of the extracellular matrix. These results predicated COOH-SWCNTs' use to further promote osteogenic differentiation in vivo. In contrast, both cell lines had difficulties adhering to multi-walled carbon nanotube-based scaffolds, as shown by scanning electron microscopy. While a suspension of SWCNTs caused cytotoxicity in both cell lines at levels >20 µg/mL, these levels were never achieved by release from sprayed SWCNTs, warranting the approach taken. In vivo, human allografts formed by the combination of demineralized bone matrix or cartilage particles with SWCNTs were implanted into nude rats, and ectopic bone formation was analyzed. Histological analysis of both types of implants showed high permeability and pore connectivity of the carbon nanotube-soaked implants. Numerous vascularization channels appeared in the formed tissue, additional progenitor cells were recruited, and areas of de novo ossification were found 4 weeks post-implantation. Induction of the expression of bone-related genes and the presence of secreted osteopontin protein were also confirmed by quantitative polymerase chain reaction analysis and immunofluorescence, respectively. In summary, these results are in line with prior contributions that highlight the suitability of SWCNTs as scaffolds with high bone-inducing capabilities both in vitro and in vivo, confirming them as alternatives to current bone-repair therapies.

Asunto(s)

Materiales Biocompatibles , Diferenciación Celular/efectos de los fármacos , Nanotubos de Carbono , Osteogénesis/efectos de los fármacos , Aloinjertos , Animales , Materiales Biocompatibles/química , Materiales Biocompatibles/farmacología , Materiales Biocompatibles/toxicidad , Línea Celular , Forma de la Célula/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Humanos , Nanotubos de Carbono/química , Nanotubos de Carbono/toxicidad , Osteoblastos/efectos de los fármacos , Ratas , Ratas Desnudas

RESUMEN

Sertoli cell secretory activities are highly dependent on ion channel functions and critical to spermatogenesis. The steroid hormone 1alpha,25(OH)2-vitamin D3 (1,25(OH)2-D3) stimulates exocytosis in different cell systems by activating a nongenotropic vitamin D receptor (VDR). Here, we described 1,25(OH)2-D3 stimulation of secretion via Cl(-) channel activation in the mouse immature Sertoli cell line TM4. 1,25(OH)2-D3 potentiation of chloride currents was dependent on hormone concentration, and correlated with a significant increase in whole-cell capacitance within 20-40 min. In addition, Cl(-) currents were potentiated by the nongenomic VDR agonist 1alpha,25(OH)2 lumisterol D3 (JN), while 1,25(OH)2-D3 potentiation of channels was suppressed by nongenomic VDR antagonist 1beta,25(OH)2-vitamin D3 (HL). Treatment of TM4 cells with PKC and PKA activators PMA and forskolin respectively, increased Cl(-) currents significantly, while PKC and PKA inhibitors Go6983 and H-89, respectively, abolished 1,25(OH)2-D3 stimulation of Cl(-) currents, suggesting phosphorylation pathways in 1,25(OH))2-D3 mediated channel responses. RT-PCR demonstrated the expression of outwardly rectifying ClC-3 channels in TM4 cells. Taken together, our results demonstrate a PKA/PKC-dependent 1,25(OH)2-D3/VDR nongenotropic pathway leading to Cl(-) channel and exocytosis activation in Sertoli cells. We conclude that 1,25(OH)2-D3 appears to be a modulator of male reproductive functions at least in part by stimulating Sertoli cell secretory functions.

Asunto(s)

Calcitriol/farmacología , Canales de Cloruro/metabolismo , Células de Sertoli/efectos de los fármacos , Células de Sertoli/metabolismo , Adenilil Ciclasas/metabolismo , Animales , Calcifediol/farmacología , Calcitriol/análogos & derivados , Calcitriol/metabolismo , Línea Celular , Membrana Celular/efectos de los fármacos , Membrana Celular/metabolismo , Canales de Cloruro/antagonistas & inhibidores , Canales de Cloruro/genética , AMP Cíclico/agonistas , Proteínas Quinasas Dependientes de AMP Cíclico/antagonistas & inhibidores , Proteínas Quinasas Dependientes de AMP Cíclico/metabolismo , Relación Dosis-Respuesta a Droga , Activadores de Enzimas/farmacología , Exocitosis/efectos de los fármacos , Expresión Génica , Activación del Canal Iónico/efectos de los fármacos , Masculino , Ratones , Isoformas de Proteínas/antagonistas & inhibidores , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Proteína Quinasa C/antagonistas & inhibidores , Proteína Quinasa C/metabolismo , ARN Mensajero/metabolismo , Receptores de Calcitriol/agonistas , Receptores de Calcitriol/antagonistas & inhibidores , Receptores de Calcitriol/genética , Receptores de Calcitriol/metabolismo , Células de Sertoli/patología

RESUMEN

Titanium alloy, Ti6Al4V, is widely used in dental and orthopedic implants. Despite its excellent biocompatibility, Ti6Al4V releases toxic Al and V ions into the surrounding tissue after implantation. In addition, the elastic modulus of Ti6Al4V ( approximately 110GPa) is significantly higher than that of bone (10-40GPa), leading to a modulus mismatch and consequently implant loosening and deosteointegration. Zeolite coatings are proposed to prevent the release of the toxic ions into human tissue and enhance osteointegration by matching the mechanical properties of bone. Zeolite MFI coatings are successfully synthesized on commercially pure titanium and Ti6Al4V for the first time. The coating shows excellent adhesion by incorporating titanium from the substrate within the zeolite framework. Higher corrosion resistance than the bare titanium alloy is observed in 0.856M NaCl solution at pHs of 7.0 and 1.0. Zeolite coatings eliminate the release of cytotoxic Al and V ions over a 7 day period. Pluripotent mouse embryonic stem cells show higher adhesion and cell proliferation on the three-dimensional zeolite microstructure surface compared with a two-dimensional glass surface, indicating that the zeolite coatings are highly biocompatible.

Asunto(s)

Materiales Biocompatibles Revestidos/química , Materiales Biocompatibles Revestidos/farmacología , Fibroblastos/citología , Prótesis e Implantes , Titanio/química , Zeolitas/química , Zeolitas/farmacología , Aleaciones , Animales , Técnicas de Cultivo de Célula/métodos , Línea Celular , Supervivencia Celular/efectos de los fármacos , Corrosión , Fibroblastos/efectos de los fármacos , Humanos , Ensayo de Materiales , Ratones , Diseño de Prótesis , Propiedades de Superficie , Ingeniería de Tejidos/métodos

RESUMEN

In the absence of mechanical stimulation, brief exposure of osteoblasts to 1alpha,25(OH)(2)vitamin D(3) (1,25D) triggers plasma membrane electrical responses that couple to exocytosis. Here we describe for the first time 1,25D induction of exocytotic ATP release in static ROS 17/2.8 and SAOS-2 cells and primary calvarial osteoblasts expressing a vitamin D receptor (VDR). We found that 10 nM 1,25D optimally induced 45 +/- 1% and 40 +/- 1% of partial and complete exocytotic events, respectively, from a 1,25D-sensitive pool of ATP-containing secretory vesicles within 60 s. We measured a dose-dependent 1,25D induction of ATP secretion, with maximal response of approximately 6.2-fold (16.93 +/- 1.82 nM for SAOS-2) and 3.1-fold (18.89 +/- 1.39 nM for ROS 17/2.8) obtained with 10 nM 1,25D compared with basal ATP levels (2.75 +/- 0.39 nM, SAOS-2; 6.09 +/- 0.58 nM, ROS 17/2.8 cells). The natural metabolite 25(OH)vitamin D(3) (25D, 10 nM) induced a significant 3.6-fold increase of ATP release in ROS 17/2.8 cells, but there was no induction with the antagonist 1beta,25(OH)(2)vitamin D(3) (1beta,25D, 10 nM) or the steroid 17beta-estradiol (10 nM). 1,25D-induced ATP secretion was abolished when cells were preincubated with inhibitors of vesicular exocytosis. siRNA VDR silencing prevented 1,25D stimulation of ATP exocytosis in ROS 17/2.8 and SAOS-2 cells. Similarly, 1,25D failed to stimulate ATP exocytosis in primary osteoblasts from a VDR knockout mouse. ATP secretion coupled to 1,25D induction of cytosolic calcium and chloride channel potentiation. Rapid 1,25D stimulation of ATP secretion involving nontranscriptional VDR functions in osteoblasts may help explain 1,25D bone anabolic properties.

Asunto(s)

Adenosina Trifosfato/metabolismo , Calcitriol/farmacología , Osteoblastos/efectos de los fármacos , Animales , Exocitosis/efectos de los fármacos , Técnica del Anticuerpo Fluorescente , Técnicas de Silenciamiento del Gen , Ratones , Ratones Noqueados , Osteoblastos/metabolismo , Ratas , Receptores de Calcitriol/genética , Receptores de Calcitriol/metabolismo

RESUMEN

Osteoblast apoptosis plays a crucial role in bone remodeling. Physiological doses of 1 alpha,25(OH)(2)-vitamin D(3) (1,25D) protect osteoblasts against apoptosis by means of mechanisms only partially understood. We studied activation of an Akt survival cascade downstream of 1,25D nongenomic stimulation of phosphatidylinositide-3'-kinase (PI3K) in osteoblastic cells. We measured a dose- and time-dependent 1,25D induction of Akt phosphorylation (p-Akt) in cultured osteoblastic cells. Maximal response was achieved with 10 nM 1,25D after 5 min. We found that staurosporine (STSP)-induced apoptosis was significantly reduced in 1,25D-pretreated osteoblasts. 1,25D prosurvival effects were abolished when cells were preincubated with inhibitors of PI3K activation. By means of siRNA silencing, we proved that 1,25D induction of p-Akt requires a classic vitamin D receptor (VDR) in osteoblasts. Furthermore, non-osteoblastic CV-1 cells transfected with an enhanced green fluorescent protein (EGFP)-VDR construct responded to 1,25D treatment with a rapid p-Akt response associated with increased cell survival not detected in native, nontransfected cells. We measured increased levels of p-Akt substrates p-Bad and p-FKHR and significantly reduced activity of caspases 8 and 3/7 after 1,25D treatment. In addition, 1,25D-induced protection against apoptosis was abolished when osteoblasts were preincubated with pertussis toxin. We conclude that anti-apoptotic effects of 1,25D in osteoblasts occur through nongenomic activation of a VDR/PI3K/Akt survival pathway that includes phosphorylation of multiple p-Akt substrates and reduction of caspase activities.

Asunto(s)

Apoptosis/efectos de los fármacos , Calcitriol/farmacología , Osteoblastos/enzimología , Fosfatidilinositol 3-Quinasas/metabolismo , Proteínas Proto-Oncogénicas c-akt/metabolismo , Receptores de Calcitriol/metabolismo , Transducción de Señal/efectos de los fármacos , Animales , Inhibidores de Caspasas , Línea Celular Tumoral , Relación Dosis-Respuesta a Droga , Activación Enzimática/efectos de los fármacos , Factores de Transcripción Forkhead/metabolismo , Modelos Biológicos , Proteínas del Tejido Nervioso/metabolismo , Osteoblastos/citología , Osteoblastos/efectos de los fármacos , Toxina del Pertussis/farmacología , Fosforilación/efectos de los fármacos , Ratas , Estaurosporina/farmacología , Factores de Tiempo , Proteína Letal Asociada a bcl/metabolismo

RESUMEN

The molecular mechanisms underlying antiproliferative actions of the steroid 1alpha,25-dihydroxy vitamin D(3) (1,25D) in human osteosarcoma cells are known only partially. To better understand the signaling involved in 1,25D anti-tumorigenic properties in bone, we stably silenced vitamin D receptor (VDR) expression in the human osteosarcoma SaOS-2 cell line. We found that 1,25D treatment reduced cell proliferation by approximately 25% after 3 days only in SaOS-2 cells expressing native levels of VDR protein, and involved activation of MAPK/AP-1/p21(waf1) pathways. Both sustained (3 days) and transient (15min) 1,25D treatment activated JNK and ERK1/2 MAPK signaling in a nongenomic VDR-dependent manner. However, only sustained exposure to hormone led to upregulation of p21 and subsequent genomic control of the cell cycle. Specific blockade of MEK1/MEK2 cascade upstream from ERK1/2 abrogated 1,25D activation of AP-1 and p21, and subsequent antiproliferative effects, even in the presence of a nuclear VDR. We conclude that 1,25D-induced inhibition of human osteosarcoma cell proliferation occurs via sustained activation of JNK and MEK1/MEK2 pathways downstream of nongenomic VDR signaling that leads to upregulation of a c-Jun/c-Fos (AP-1) complex, which in turn modulates p21(waf1) gene expression. Our results demonstrate a cross-talk between 1,25D/VDR nongenomic and genomic signaling at the level of MAP kinase activation that leads to reduction of cell proliferation in human osteosarcoma cells.

Asunto(s)

Proliferación Celular/efectos de los fármacos , Inhibidor p21 de las Quinasas Dependientes de la Ciclina/metabolismo , Proteínas Quinasas Activadas por Mitógenos/metabolismo , Receptores de Calcitriol/metabolismo , Factor de Transcripción AP-1/metabolismo , Vitamina D/farmacología , Western Blotting , Butadienos/farmacología , Línea Celular Tumoral , Inhibidor p21 de las Quinasas Dependientes de la Ciclina/genética , Activación Enzimática/efectos de los fármacos , Inhibidores Enzimáticos/farmacología , Humanos , Proteínas Quinasas JNK Activadas por Mitógenos/metabolismo , Sistema de Señalización de MAP Quinasas/efectos de los fármacos , Proteínas Quinasas Activadas por Mitógenos/antagonistas & inhibidores , Modelos Biológicos , Nitrilos/farmacología , Proteínas Proto-Oncogénicas c-fos/metabolismo , ARN Interferente Pequeño/genética , Receptores de Calcitriol/genética , Factores de Tiempo , Factor de Transcripción AP-1/genética , Transfección , Regulación hacia Arriba/efectos de los fármacos , Vitamina D/análogos & derivados

RESUMEN

1alpha,25(OH)(2)-vitamin D(3) (1,25D) is considered a bone anabolic hormone. 1,25D actions leading to bone formation involve gene transactivation, on one hand, and modulation of cytoplasmic signaling, on the other. In both cases, a functional vitamin D receptor (VDR) appears to be required. Here we study 1,25D-stimulated calcium signaling that initiates at the cell membrane and leads to exocytosis of bone materials and increased osteoblast survival. We found that rapid 1,25D-induction of exocytosis couples to cytoplasmic calcium increase in osteoblastic ROS 17/2.8 cells. In addition, we found that elevation of cytoplasmic calcium concentration is involved in 1,25D anti-apoptotic effects via Akt activation in ROS 17/2.8 cells and non-osteoblastic CV-1 cells. In both cases, 1,25D-stimulated elevation of intracellular calcium is due in part to activation of L-type Ca(2+) channels. We conclude that 1,25D bone anabolic effects that involve increased intracellular Ca(2+) concentration in osteoblasts can be explained at two levels. At the single-cell level, 1,25D promotes Ca(2+)-dependent exocytotic activities. At the tissue level, 1,25D protects osteoblasts from apoptosis via a Ca(2+)-dependent Akt pathway. Our studies contribute to the understanding of the molecular basis of bone diseases characterized by decreased bone formation and mineralization.

Asunto(s)

Calcitriol/metabolismo , Señalización del Calcio , Exocitosis , Animales , Línea Celular Tumoral , Supervivencia Celular , Ratas

RESUMEN

We demonstrate for the first time the expression of 14.3.3sigma, an epithelial cell differentiation marker, in human corneal epithelium. 14.3.3sigma appeared at 30 kDa, pI 4-5, in 2D gels of corneal extracts. We found no significant differences in 14.3.3sigma levels between healthy corneas and corneas from keratoconus, corneal dystrophy, and corneal edema patients. 14.3.3sigma immunofluorescence was observed in the cytoplasm and nucleus of epithelial cells and colocalized with cyclin-B1. 14.3.3sigma was secreted by HCE-2 cells; HCE-2-conditioned medium induced matrix metalloproteinase-1 in cultured keratocytes. In summary, our work presents evidence of 14.3.3sigma expression in corneal epithelium and elaborates over its possible implications in corneal pathologic conditions.

Asunto(s)

Biomarcadores de Tumor/metabolismo , Epitelio Corneal/metabolismo , Exonucleasas/metabolismo , Proteínas de Neoplasias/metabolismo , Proteínas 14-3-3 , Western Blotting , Técnicas de Cultivo de Célula , Distrofias Hereditarias de la Córnea/metabolismo , Edema Corneal/metabolismo , Ciclina B/metabolismo , Ciclina B1 , Electroforesis en Gel Bidimensional , Exorribonucleasas , Fibroblastos/enzimología , Técnica del Anticuerpo Fluorescente Indirecta , Humanos , Immunoblotting , Queratocono/metabolismo , Metaloproteinasa 1 de la Matriz/metabolismo

RESUMEN

We explored the use of carbon nanotubes (CNTs) as suitable scaffold materials for osteoblast proliferation and bone formation. With the aim of controlling cell growth, osteosarcoma ROS 17/2.8 cells were cultured on chemically modified single-walled (SW) and multiwalled (MW) CNTs. CNTs carrying neutral electric charge sustained the highest cell growth and production of plate-shaped crystals. There was a dramatic change in cell morphology in osteoblasts cultured on MWNTs, which correlated with changes in plasma membrane functions.

Asunto(s)

Huesos/citología , Nanotubos de Carbono , Animales , Huesos/fisiología , Canales de Calcio/metabolismo , Línea Celular , Proliferación Celular , Canales de Cloruro/metabolismo , Electrofisiología , Microscopía Electrónica de Rastreo , Nanotubos de Carbono/química , Osteoblastos/citología , Osteoblastos/fisiología , Ratas

RESUMEN

Membrane-initiated cellular responses to steroids include modulation of ion channel activities via signal transduction pathways. However, the molecular mechanisms involved in nongenomic actions remain only partially understood. Our research has focused on the rapid effects of 1alpha,25(OH)(2) Vitamin D(3) [1,25D] on L-type Ca(2+) [L-Ca] and DIDS-sensitive Cl(-) channels in osteoblasts. Physiological nanomolar concentrations of hormonally active 1,25D promote rapid (1-5 min) potentiation of outward Cl(-) currents in osteosarcoma ROS 17/2.8 cells and mouse primary osteoblasts. In addition, 1,25D increases inward barium currents through L-Ca channels at low depolarizing potentials within seconds in a fashion similar to the 1,4-dihydropyridine [DHP] agonist Bay K8644. We found that second messenger cAMP is involved in 1,25D potentiation of Cl(-) and Ca(2+) channels. Nongenomic 1,25D effects on ion channel activities in osteoblasts appear to involve different mechanisms that include a possible direct interaction with the L-Ca channel molecule, on one hand, and signaling through the cAMP pathway, on the other. Rapid 1,25D actions on Cl(-) and Ca(2+) currents seem to couple to secretory activities in osteoblasts, thus contributing to bone mass formation.

Asunto(s)

Calcitriol/fisiología , Canales de Calcio Tipo L/metabolismo , Canales de Cloruro/metabolismo , Osteoblastos/metabolismo , Animales , Línea Celular , Ratones , Técnicas de Placa-Clamp

RESUMEN

Structural and molecular studies have shown that the vitamin D receptor (VDR) mediates 1alpha,25(OH)2-vitamin D3 gene transactivation. Recent evidence indicates that both VDR and the estrogen receptor are localized to plasma membrane caveolae and are required for initiation of nongenomic (NG) responses. Computer docking of the NG-specific 1alpha,25(OH)2-lumisterol to the VDR resulted in identification of an alternative ligand-binding pocket that partially overlaps the genomic pocket described in the experimentally determined x-ray structure. Data obtained from docking five different vitamin D sterols in the genomic and alternative pockets were used to generate a receptor conformational ensemble model, providing an explanation for how VDR and possibly the estrogen receptor can have genomic and NG functionality. The VDR model is compatible with the following: (i) NG chloride channel agonism and antagonism; (ii) variable ligand-stabilized trypsin digest banding patterns; and (iii) differential transcriptional activity, employing different VDR point mutants and 1alpha,25(OH)2-vitamin D3 analogs.

Asunto(s)

Calcitriol/metabolismo , Núcleo Celular/química , Receptores de Calcitriol/química , Transducción de Señal/fisiología , Sitios de Unión , Núcleo Celular/metabolismo , Canales de Cloruro/metabolismo , Ligandos , Modelos Moleculares , Mutación Puntual , Conformación Proteica , Receptores de Calcitriol/genética , Receptores de Calcitriol/metabolismo

RESUMEN

Osteoblasts are a main target for the steroid 1alpha,25(OH)2-Vitamin D3 (1,25D3), where a major outcome is the modulation of the bone remodeling process. 1,25D3 deficiency leads to clinical disorders such as osteomalacia and osteoporosis, characterized by a state of insufficiently calcified tissue and bone loss, respectively. In the osteoblast nucleus, 1,25D3 modulates gene transcription for the synthesis of bone matrix proteins via the Vitamin D receptor (VDR). At the plasma membrane level, 1,25D3 potentiates ion channel functions, activates signal transduction pathways, and increases cytoplasmic calcium concentrations. So far, no clear physiological significance has been attributed to membrane-initiated 1,25D3 actions in single cells. To investigate if (a) 1,25D3 is a modulatory agent of secretion in osteoblasts and (b) the classical VDR is involved in rapid electrical events in the cell membrane, we studied hormone effects on ion channel activities in relation to exocytosis in osteoblasts isolated from VDR knockout (KO) and wild-type (WT) mice. This paper is a retrospect of the electrophysiological studies done in our laboratory to date. We found that 1,25D3-promoted ion channel responses are coupled to secretion in calvarial osteoblasts, and develop only in the presence of a functional nuclear steroid VDR. This 1,25D3-regulated exocytosis in osteoblasts, which takes place within minutes of hormone application, seems to be the natural complement of genomic actions that evolve at a longer time scale. The absence of both 1,25D3 membrane and nuclear effects in VDR KO osteoblasts may explain bone abnormalities typically found in VDR KO mice.

Asunto(s)

Calcitriol/farmacología , Calcitriol/fisiología , Canales Iónicos/efectos de los fármacos , Osteoblastos/efectos de los fármacos , Osteoblastos/fisiología , Animales , Calcitriol/deficiencia , Electrofisiología , Exocitosis/efectos de los fármacos , Exocitosis/fisiología , Canales Iónicos/fisiología , Ratones , Ratones Noqueados , Osteoblastos/metabolismo , Receptores de Calcitriol/fisiología , Transducción de Señal/efectos de los fármacos , Transcripción Genética/efectos de los fármacos

RESUMEN

The steroid hormone 1 alpha,25(OH)(2)-vitamin D(3) (1,25D) regulates gene transcription through a nuclear receptor [vitamin D receptor (VDR)] and initiation of rapid cellular responses through a putative plasma membrane-associated receptor (VDR(mem)). This study characterized the VDR(mem) present in a caveolae-enriched membrane fraction (CMF), a site of accumulation of signal transduction agents. Saturable and specific [(3)H]-1,25D binding in vitro was found in CMF of chick, rat, and mouse intestine; mouse lung and kidney; and human NB4 leukemia and rat ROS 17/2.8 osteoblast-like cells; in all cases the 1,25D K(D) binding dissociation constant = 1-3 nM. Our data collectively support the classical VDR being the VDR(mem) in caveolae: 1) VDR antibody immunoreactivity was detected in CMF of all tissues tested; 2) competitive binding of [(3)H]-1,25D by eight analogs of 1,25D was significantly correlated between nuclei and CMF (r(2) = 0.95) but not between vitamin D binding protein (has a different ligand binding specificity) and CMF; 3) confocal immunofluorescence microscopy of ROS 17/2.8 cells showed VDR in close association with the caveolae marker protein, caveolin-1, in the plasma membrane region; 4) in vivo 1,25D pretreatment reduced in vitro [(3)H]-1,25D binding by 30% in chick and rat intestinal CMF demonstrating in vivo occupancy of the CMF receptor by 1,25D; and 5) comparison of [(3)H]-1,25D binding in VDR KO and WT mouse kidney tissue showed 85% reduction in VDR KO CMF and 95% reduction in VDR KO nuclear fraction. This study supports the presence of VDR as the 1,25D-binding protein associated with plasma membrane caveolae.

Asunto(s)

Calcitriol/metabolismo , Caveolas/química , Receptores de Calcitriol/análisis , Receptores de Calcitriol/metabolismo , Animales , Unión Competitiva , Calcitriol/análisis , Caveolas/metabolismo , Caveolina 1 , Caveolinas/análisis , Membrana Celular/química , Membrana Celular/metabolismo , Núcleo Celular/química , Núcleo Celular/metabolismo , Pollos , Humanos , Ratones , Ratas , Distribución Tisular

RESUMEN

1alpha,25(OH)(2)-Vitamin D(3) (1,25D) modulates osteoblast gene expression of bone matrix proteins via a nuclear vitamin D receptor (VDR) and also modifies the electrical state of the plasma membrane through rapid nongenomic mechanisms still not fully understood. The physiological significance of 1,25D membrane-initiated effects remains unclear. To elucidate whether the VDR is required for 1,25D-promoted electrical responses, we studied 1,25D modulation of ion channel activities in calvarial osteoblasts isolated from VDR knockout (KO) and WT mice. At depolarizing potentials, Cl(-) currents were significantly potentiated (13.5 +/- 1.6-fold increase, n = 12) by 5 nM 1,25D in VDR WT but not in KO (0.96 +/- 0.3 fold increase, n = 11) osteoblasts. L-type Ca(2+) currents significantly shift their peak activation by -9.3 +/- 0.7 mV (n = 10) in the presence of 5 nM 1,25D in VDR WT but not in KO cells, thus facilitating Ca(2+) influx. Furthermore, we found that 1,25D significantly increased whole-cell capacitance in VDR WT (DeltaCap = 2.3 +/- 0.4 pF, n = 8) but not in KO osteoblasts (DeltaCap = 0.3 +/- 0.1 pF, n = 8); this corresponds to a rapid (1-2 min) fusion in WT of 71 +/- 33 versus in KO only 9 +/- 6 individual secretory granules. We conclude that, in calvarial osteoblasts, 1,25D modulates ion channel activities only in cells with a functional VDR and that this effect is coupled to exocytosis. This is a demonstration of the requirement of a functional classic steroid receptor for the rapid hormonal modulation of electric currents linked to secretory activities in a target cell.

Asunto(s)

Calcitriol/farmacología , Canales Iónicos/efectos de los fármacos , Osteoblastos/efectos de los fármacos , Receptores de Calcitriol/fisiología , Animales , Ratones , Ratones Noqueados , Osteoblastos/metabolismo , Reacción en Cadena de la Polimerasa , Receptores de Calcitriol/genética

RESUMEN

Rapid nongenomic responses to steroids include modulation of ion channel activities on the cell membrane of target cells, but little is known about the molecular mechanisms involved. In this paper we investigate the mechanisms underlying the combined action of the secosteroid hormone 1alpha,25-dihydroxyvitamin D3 [1alpha,25(OH)(2)D3] on three different ion channel types in rat osteoblasts, which include a voltage-gated L-type Ca(2+) channel, a mechanosensitive Cl(-) channel, and a stretch-activated cation (SA-Cat) channel. We found that physiological nanomolar concentrations of 1alpha,25(OH)(2)D3 rapidly modify the overall electrical activity of the membrane in ROS 17/2.8 cells. 1alpha,25(OH)(2)D3 increases the osteoblast L-type Ca(2+) channel activity at low depolarizing voltages in a fashion similar to the 1,4-dihydropyridine (DHP) agonist Bay K8644. At highly depolarizing potentials 1alpha,25(OH)(2)D3 potentiates volume-sensitive Cl(-) currents through mechanisms that may involve a putative membrane receptor. We show for the first time that 1alpha,25(OH)(2)D3 also increases inward currents through SA-Cat channels at positive membrane voltages in a dose-dependent manner. Contrary to our expectations, the stereoisomer 1beta,25(OH)(2)D3, which suppresses 1alpha,25(OH)(2)D3 activation of osteoblast Cl(-) currents, mimicked 1alpha,25(OH)(2)D3 agonist effects on Ca(2+) and SA-Cat channel activities. Cyclic AMP is involved in 1alpha,25(OH)(2)D3 effects on both Ca(2+) and SA-Cat channels, but not in Cl(-) channels. We conclude that 1alpha,25(OH)(2)D3 rapid effects on ion channel activities in ROS 17/2.8 cells occur through multiple mechanisms that, on the one hand, involve a possible direct interaction with the L-type Ca(2+) channel molecule and, on the other hand, molecular pathways that may include a putative membrane receptor.

Asunto(s)

Canales Iónicos/metabolismo , Osteoblastos/metabolismo , Vitamina D/análogos & derivados , Vitamina D/farmacología , Animales , Línea Celular , Membrana Celular/efectos de los fármacos , Membrana Celular/metabolismo , Potenciales de la Membrana/efectos de los fármacos , Potenciales de la Membrana/fisiología , Osteoblastos/efectos de los fármacos , Ratas , Vitamina D/química

RESUMEN

The steroid hormone 1 alpha,25(OH)(2)-vitamin D(3) [1 alpha,25(OH)(2)D(3)] mediates through its widely distributed nuclear receptor (VDR(nuc)) regulation of gene transcription (genomic responses) and through a putative membrane receptor (VDR(mem)) a variety of rapid responses. Rapid responses studied in our laboratories include opening of voltage-gated calcium and chloride channels in ROS 17/2.8 osteoblast cells, activation of MAP-kinase in human leukemia NB4 cells and chick intestinal cells, release of insulin by rat pancreatic beta-cells, and in chick duodena transcaltachia (the rapid hormonal stimulation of intestinal Ca(2+) transport). 1 alpha,25(OH)(2)D(3) is conformationally flexible (side chain, seco B-ring and A-ring) and accordingly is able to generate a large array of different shapes to serve as ligands for available receptors (VDR(nuc) and VDR(mem)) in the vitamin D endocrine system. Our laboratories have utilized a number of conformationally restricted analogs of 1 alpha,25(OH)(2)D(3) (from a library of several hundred analogs) to evaluate the preferred shape of the ligands for rapid and genomic responses. The determination of the X-ray structure of the 1 alpha,25(OH)(2)D(3)-occupied VDR(nuc) revealed that the preferred ligand shape was a twisted 6-s-trans bowl shape [Molecular Cell 5 (2000) 173-179]. Optimal agonists for genomic responses include 1 alpha,25(OH)(2)D(3) and other side chain conformationally flexible analogs such as 20-epi-1 alpha,25(OH)(2)D(3) [approximately equal to 200-500-fold more potent than 1 alpha,25(OH)(2)D(3)] and 21-(3'-hydroxy-3-methylbutyl)-1 alpha,25(OH)(2)D(3) [an analog with two side chains] all which can achieve the preferred VDR(nuc) shape. In contrast, rapid responses require a 6-s-cis shape of the agonist ligand such as can be achieved by the natural hormone 1 alpha,25(OH)(2)D(3) or by analogs permanently locked in the 6-s-cis shape such as 1 alpha,25(OH)(2)lumisterol(3) or 1 alpha,25(OH)(2)-7-dehydrocholesterol. Additionally, we have discovered analogs that are specific in their antagonist properties for either rapid or genomic responses. Thus, 1 beta,25(OH)(2)D(3) is an antagonist of only rapid responses [via the VDR(mem)], while 23S-25-dehydro-1 alpha,25(OH)D(3)-26,23-lactone is an antagonist of only nuclear responses [via the VDR(nuc)]. In conclusion, we have presented evidence that 1 alpha,25(OH)(2)D(3) mediated rapid response and genomic response signal transduction pathways utilize differing shapes of ligand, both as agonists and antagonists.

Asunto(s)

Lactonas/farmacología , Receptores de Calcitriol/metabolismo , Transducción de Señal/efectos de los fármacos , Activación Transcripcional/efectos de los fármacos , Vitamina D/análogos & derivados , Vitamina D/química , Animales , Cristalografía por Rayos X , Humanos , Conformación Molecular , Receptores de Calcitriol/genética , Receptores Citoplasmáticos y Nucleares/metabolismo , Vitamina D/metabolismo , Vitamina D/farmacología