RESUMEN
RANKL (receptor activator of NF-kappaB ligand) induces osteoclastogenesis by activating multiple signaling pathways in osteoclast precursor cells, chief among which is induction of long lasting oscillations in the intracellular concentration of Ca(2+) ([Ca(2+)](i)). The [Ca(2+)](i) oscillations activate calcineurin, which activates the transcription factor NFATc1. The pathway by which RANKL induces [Ca(2+)](i) oscillations and osteoclastogenesis is poorly understood. Here we report the discovery of a novel pathway induced by RANKL to cause a long lasting increase in reactive oxygen species (ROS) and [Ca(2+)](i) oscillations that is essential for differentiation of bone marrow-derived monocytes into osteoclasts. The pathway includes RANKL-mediated stimulation of Rac1 to generate ROS, which stimulate phospholipase Cgamma1 to evoke [Ca(2+)](i) oscillations by stimulating Ca(2+) release from the inositol 1,4,5-trisphosphate pool and STIM1-regulated Ca(2+) influx. Induction and activation of the pathway is observed only after 24-h stimulation with RANKL and lasts for at least 3 days. The physiological role of the pathway is demonstrated in mice with deletion of the Peroxiredoxin II gene and results in a mark increase is ROS and, consequently, a decrease in bone density. Moreover, bone marrow-derived monocytes in PrxII(-/-) primary culture show increased ROS and spontaneous [Ca(2+)](i) oscillations. These findings identify the primary RANKL-stimulated pathway to trigger the late stages of osteoclastogenesis and regulate bone resorption.
Asunto(s)
Señalización del Calcio/fisiología , Diferenciación Celular/fisiología , Osteoclastos/fisiología , Ligando RANK/metabolismo , Especies Reactivas de Oxígeno/metabolismo , Animales , Células de la Médula Ósea/citología , Células de la Médula Ósea/fisiología , Huesos/anatomía & histología , Huesos/fisiología , Células Cultivadas , Macrófagos/citología , Macrófagos/fisiología , Ratones , Ratones Noqueados , Osteoclastos/citología , Osteogénesis/fisiología , Peroxirredoxinas/genética , Peroxirredoxinas/metabolismo , Fosfolipasa C gamma/antagonistas & inhibidores , Fosfolipasa C gamma/metabolismo , Interferencia de ARN , Transducción de Señal/fisiología , Proteína de Unión al GTP rac1/genética , Proteína de Unión al GTP rac1/metabolismoRESUMEN
The effect of the potent soluble guanylyl cyclase (sGC) inhibitor 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ) on neurite outgrowth and retraction was investigated in PC12 cells and SH-SY5Y human neuroblastoma cells. ODQ inhibited neurite outgrowth and triggered neurite retraction in the cells stimulated with nerve growth factor (NGF), staurosporine, or Y-27632. The nitric oxide (NO) scavenger 2-(4-carboxyphenyl)-4,4,5,5-tetramethyl-imidazoline-1-oxyl-3-oxide (PTIO) had little effect on neurite outgrowth induced by Y-27632 or staurosporine. In the presence of ODQ, treatment of the cells with the cell-permeable cGMP analogue 8-bromo-cGMP failed to retrigger Y-27632- and staurosporine-induced neurite outgrowth. Furthermore, the depletion of sGC by RNA interference failed to prevent Y-27632- and staurosporine-induced neurite outgrowth. These results indicate that the NO/sGC/cGMP signaling cascade is not critically involved in ODQ-induced neurite remodeling. The MEK inhibitor PD98059 did not inhibit neurite outgrowth, and Y-27632 and staurosporine did not induce ERK phosphorylation, suggesting that the inhibitory effect of ODQ on neurite outgrowth is independent of the ERK signaling pathway. In contrast, pretreatment with dithionite or a hemin-glutathione mixture reversed the inhibitory effect of ODQ on Y-27632- and staurosporine-induced neurite outgrowth, indicating that ODQ might act on an intracellular redox-sensitive molecule. We conclude that ODQ inhibits Y-27632- and staurosporine-induced neurite outgrowth and triggers neurite retraction in an sGC-independent manner in neuronal cells and suggest that oxidation of unidentified redox-sensitive protein could be responsible for these effects.
Asunto(s)
Inhibidores Enzimáticos/farmacología , Guanilato Ciclasa/metabolismo , Neuritas/efectos de los fármacos , Oxadiazoles/farmacología , Quinoxalinas/farmacología , Receptores Citoplasmáticos y Nucleares/metabolismo , Animales , Línea Celular , Guanilato Ciclasa/antagonistas & inhibidores , Humanos , Factor de Crecimiento Nervioso/farmacología , Células PC12/citología , Ratas , Receptores Citoplasmáticos y Nucleares/antagonistas & inhibidores , Guanilil Ciclasa SolubleRESUMEN
Mammalian chitinase released by airway epithelia is thought to be an important mediator of disease manifestation in an experimental model of asthma. However, the intracellular signaling mechanisms engaged by exogenous chitinase in human airway epithelial cells are unknown. Here, we investigated the direct effects of exogenous chitinase from Streptomyces griseus on Ca(2+) signaling in human airway epithelial cells. Spectrofluorometry was used to measure intracellular Ca(2+) concentration ([Ca(2+)](i)) in fura-2-AM-loaded cells. S. griseus chitinase induced dose-dependent [Ca(2+)](i) increases in normal human bronchial epithelial cells and promoted [Ca(2+)](i) oscillations in H292 cells. Chitinase-induced [Ca(2+)](i) oscillations were independent of extracellular Ca(2+), suggesting that the observed [Ca(2+)](i) increases were due to Ca(2+) release from intracellular stores. Accordingly, after depleting endoplasmic reticulum (ER) Ca(2+) with the ER Ca(2+) ATPase inhibitor, thapsigargin, chitinase-mediated [Ca(2+)](i) increases were abolished. Treatment with the phospholipase C (PLC) inhibitor U73122 or the 1, 4, 5-trisinositolphosphate (IP(3)) receptor inhibitor 2-APB attenuated chitinase-induced [Ca(2+)](i) increases. Desensitization of protease-activated receptor-2 (PAR-2) by repetitive agonist stimulation or siRNA-mediated PAR-2 knock-down revealed that chitinase-mediated [Ca(2+)](i) increases were exclusively mediated by PAR-2 activation. Finally, chitinase was found to cleave a model peptide representing the cleavage site of PAR-2 and enhanced IL-8 production. These results indicate that exogenous chitinase is a potent proteolytic activator of PAR-2 that can directly induce PLC/IP(3)-dependent Ca(2+) signaling in human airway epithelial cells.
Asunto(s)
Bronquios/citología , Quitinasas/metabolismo , Células Epiteliales/metabolismo , Receptor PAR-2/metabolismo , Streptomyces griseus/enzimología , Bronquios/metabolismo , Calcio/metabolismo , Señalización del Calcio , Células Cultivadas , Humanos , Interleucina-8/biosíntesis , Cinética , Receptor PAR-2/genética , Especificidad por SustratoRESUMEN
PURPOSE: The sarco/endoplasmic reticulum Ca2+-ATPase (SERCA), encoded by ATP2A2, is an essential component for G-protein coupled receptor (GPCR)-dependent Ca2+ signaling. However, whether the changes in Ca2+ signaling and Ca2+ signaling proteins in parotid acinar cells are affected by a partial loss of SERCA2 are not known. MATERIALS AND METHODS: In SERCA2+/- mouse parotid gland acinar cells, Ca2+ signaling, expression levels of Ca2+ signaling proteins, and amylase secretion were investigated. RESULTS: SERCA2+/- mice showed decreased SERCA2 expression and an upregulation of the plasma membrane Ca2+ ATPase. A partial loss of SERCA2 changed the expression level of 1, 4, 5-tris-inositolphosphate receptors (IP3Rs), but the localization and activities of IP3Rs were not altered. In SERCA2+/- mice, muscarinic stimulation resulted in greater amylase release, and the expression of synaptotagmin was increased compared to wild type mice. CONCLUSION: These results suggest that a partial loss of SERCA2 affects the expression and activity of Ca2+ signaling proteins in the parotid gland acini, however, overall Ca2+ signaling is unchanged.
Asunto(s)
Señalización del Calcio/fisiología , Glándula Parótida/metabolismo , ATPasas Transportadoras de Calcio del Retículo Sarcoplásmico/metabolismo , Amilasas/metabolismo , Animales , Western Blotting , Calcio/metabolismo , Señalización del Calcio/efectos de los fármacos , Señalización del Calcio/genética , Carbacol/farmacología , Inmunohistoquímica , Receptores de Inositol 1,4,5-Trifosfato/metabolismo , Ratones , Ratones Noqueados , ATPasas Transportadoras de Calcio del Retículo Sarcoplásmico/genética , Transducción de Señal/efectos de los fármacos , Transducción de Señal/genética , Transducción de Señal/fisiologíaRESUMEN
PURPOSE: In non-excitable cells, which include parotid and pancreatic acinar cells, Ca(2+) entry is triggered via a mechanism known as capacitative Ca(2+) entry, or store-operated Ca(2+) entry. This process is initiated by the perception of the filling state of endoplasmic reticulum (ER) and the depletion of internal Ca(2+) stores, which acts as an important factor triggering Ca(2+) entry. However, both the mechanism of store-mediated Ca(2+) entry and the molecular identity of store-operated Ca(2+) channel (SOCC) remain uncertain. MATERIALS AND METHODS: In the present study we investigated the Ca(2+) entry initiation site evoked by depletion of ER to identify the localization of SOCC in mouse parotid and pancreatic acinar cells with microfluorometeric imaging system. RESULTS: Treatment with thapsigargin (Tg), an inhibitor of sarco/endoplasmic reticulum Ca(2+)-ATPase, in an extracellular Ca(2+) free state, and subsequent exposure to a high external calcium state evoked Ca(2+) entry, while treatment with lanthanum, a non-specific blocker of plasma Ca(2+) channel, completely blocked Tg-induced Ca(2+) entry. Microfluorometric imaging showed that Tg-induced Ca(2+) entry started at a basal membrane, not a apical membrane. CONCLUSION: These results suggest that Ca2+ entry by depletion of the ER initiates at the basal pole in polarized exocrine cells and may help to characterize the nature of SOCC.
Asunto(s)
Calcio/metabolismo , Retículo Endoplásmico/metabolismo , Páncreas/metabolismo , Glándula Parótida/metabolismo , Animales , Canales de Calcio/efectos de los fármacos , Canales de Calcio/metabolismo , Células Cultivadas , Retículo Endoplásmico/efectos de los fármacos , Ratones , Ratones Endogámicos ICR , Microscopía Fluorescente , Páncreas/citología , Páncreas/efectos de los fármacos , Glándula Parótida/citología , Glándula Parótida/efectos de los fármacos , Tapsigargina/farmacologíaRESUMEN
OBJECTIVE: To test the antiadherent and antibacterial properties of surface modification of orthodontic wires with photocatalytic titanium oxide (TiO(2)). MATERIALS AND METHODS: TiO(2) was coated on the surface of the orthodontic wires by a sol-gel thin film dip-coating method. Bacterial adhesion to the wires was evaluated by the weight change of the wires. The antibacterial activity of the surface-modified orthodontic wires was demonstrated by the dilution agar plate method for Streptococcus mutans and spectrophotometry for Porphyromonas gingivalis. RESULTS: The orthodontic wires coated with the photocatalytic TiO(2) showed an antiadherent effect against S. mutans compared with the uncoated wires. The bacterial mass that bound to the TiO(2)-coated orthodontic wires remained unchanged, whereas that of the uncoated wires increased by 4.97%. Furthermore, the TiO(2)-coated orthodontic wires had a bactericidal effect on S. mutans and P. gingivalis, which cause dental caries and periodontitis, respectively. The antiadherent and antibacterial mechanisms of TiO(2) to break down the cell wall of those bacteria were revealed by scanning electron microscopy. CONCLUSION: The surface modification of orthodontic wires with photocatalytic TiO(2) can be used to prevent the development of dental plaque during orthodontic treatment.
Asunto(s)
Antibacterianos/farmacología , Adhesión Bacteriana/efectos de los fármacos , Materiales Biocompatibles Revestidos/farmacología , Alambres para Ortodoncia , Titanio/farmacología , Porphyromonas gingivalis/efectos de los fármacos , Porphyromonas gingivalis/fisiología , Streptococcus mutans/efectos de los fármacos , Streptococcus mutans/fisiología , Propiedades de SuperficieRESUMEN
Synaptotagmin is a Ca2+ sensing protein, which triggers a fusion of synaptic vesicles in neuronal transmission. Little is known regarding the expression of Ca2+-dependent synaptotagmin isoforms and their contribution to the release of secretory vesicles in mouse and rat parotid acinar cells. We investigated a type of Ca2+-dependent synaptotagmin and Ca2+ signaling in both rat and mouse parotid acinar cells using RT-PCR, microfluorometry, and amylase assay. Mouse parotid acinar cells exhibited much more sensitive amylase release in response to muscarinic stimulation than did rat parotid acinar cells. However, transient [Ca2+]i increases and Ca2+ influx in response to muscarinic stimulation in both cells were identical, suggesting that the expression or activity of the Ca2+ sensing proteins is different. Seven Ca2+-dependent synaptotagmins, from 1 to 7, were expressed in the mouse parotid acinar cells. However, in the rat parotid acinar cells, only synaptotagmins 1, 3, 4 and 7 were expressed. These results indicate that the expression of Ca2+-dependent synaptotagmins may contribute to the release of secretory vesicles in parotid acinar cells.
Asunto(s)
Calcio/fisiología , Glándula Parótida/metabolismo , Sinaptotagminas/metabolismo , Amilasas/metabolismo , Animales , Calcio/metabolismo , Carbacol/farmacología , Exocitosis/efectos de los fármacos , Exocitosis/fisiología , Ratones , Agonistas Muscarínicos/farmacología , Glándula Parótida/citología , Isoformas de Proteínas/metabolismo , Ratas , Transducción de SeñalRESUMEN
We previously reported that rebamipide (2-(4-chlorobenzoylamino)-3-[2(1H)-quinolinon-4-yl]-propionic acid) generated oscillations of intracellular Ca2+ concentration ([Ca2+]i) probably through the activation of cholecystokinin type 1 (CCK1) receptors in rat pancreatic acinar cells. Therefore, in the present study, we aimed to establish the pharmacological characteristics of rebamipide in rat pancreatic acinar cells. CCK-8S and rebamipide inhibited [125I]BH-CCK-8S binding to rat pancreatic acinar cell membranes with IC50 values of 3.13 nM and 37.7 microM, respectively. CCK-8S usually evoked [Ca2+]i oscillations at concentrations lower than 50 pM, and it induced biphasic [Ca2+]i increases at higher concentrations. In contrast to CCK-8S, rebamipide only induced [Ca2+]i oscillations at all the concentrations we used in this study. In addition, rebamipide was shown to inhibit high concentrations of CCK-8S-induced biphasic increases in [Ca2+]i, suggesting that rebamipide might be a partial agonist at cholecystokinin CCK1 receptors. Although rebamipide induced [Ca2+]i oscillations by activating the cholecystokinin CCK1 receptors, rebamipide did not cause amylase release and only inhibited CCK-stimulated amylase release reversibly and dose-dependently. However, rebamipide did not inhibit carbachol-, vasoactive intestinal polypeptide (VIP)-, and forskolin-induced amylase releases. These data indicate that rebamipide functions as a partial agonist for Ca2+ -mobilizing action, and it is also an antagonist for the amylase-releasing action of CCK.
Asunto(s)
Alanina/análogos & derivados , Alanina/farmacología , Amilasas/metabolismo , Calcio/metabolismo , Páncreas/efectos de los fármacos , Quinolonas/farmacología , Receptor de Colecistoquinina A/metabolismo , Sincalida/análogos & derivados , Alanina/metabolismo , Animales , Antioxidantes/metabolismo , Antioxidantes/farmacología , Unión Competitiva , Células Cultivadas , Colecistoquinina/farmacología , Relación Dosis-Respuesta a Droga , Radioisótopos de Yodo , Cinética , Páncreas/citología , Páncreas/metabolismo , Quinolonas/metabolismo , Ratas , Receptor de Colecistoquinina A/agonistas , Sincalida/metabolismo , Sincalida/farmacología , Succinimidas/metabolismoRESUMEN
Protein kinases, such as protein kinase C, have been shown to be associated with secretory granules and to regulate the event of exocytosis in various tissues including parotid salivary acinar cells. However, in submandibular acinar cells that play an important role in the secretion of proteins into the oral cavity, kinase activity on the granule membrane has not been explored. Therefore, in the present study, we isolated the secretory granules from rat submandibular acinar cells and investigated the localisation of protein kinases on the granule membrane. Initially, we isolated and purified secretory granules from rat submandibular acinar cells. Addition of [gamma-32P] ATP to granule-membrane lysate phosphorylated the granule-membrane-associated 26, 32, 55 and 58kDa proteins, suggesting the presence of endogenous kinase activity on the membrane. Moreover, the phosphorylation of 26 and 32kDa proteins was inhibited by staurosporine and K252a, both non-specific protein kinase C inhibitors. However, the phosphorylation of 26 and 32kDa proteins was not inhibited by other protein kinase C inhibitors, such as calphostin C, GF109203X and chelerythrine, indicating that protein kinase C was not responsible for the phosphorylation. In addition, H-89, ML-9, KN-62 and genistein did not appear to inhibit this phosphorylation, indicating that protein kinase A, myosin light chain kinase (MLCK), Ca2+/calmodulin-dependent protein kinase II (CAMKII) and tyrosine kinase were not involved in the phosphorylation of 26 and 32kDa proteins. Moreover, Ca2+ had no effect on the kinase activity. Therefore, our results suggest that an unidentified, staurosporine-inhibitable protein kinase activity is associated with the secretory granule membrane of rat submandibular acinar cells.
Asunto(s)
Inhibidores Enzimáticos/farmacología , Proteínas Quinasas/metabolismo , Vesículas Secretoras/enzimología , Estaurosporina/farmacología , Glándula Submandibular/enzimología , Animales , Calcio/farmacología , Carbazoles/farmacología , Relación Dosis-Respuesta a Droga , Alcaloides Indólicos , Proteínas de la Membrana/metabolismo , Fosforilación/efectos de los fármacos , Proteína Quinasa C/antagonistas & inhibidores , Proteína Quinasa C/metabolismo , Inhibidores de Proteínas Quinasas , Ratas , Ratas Sprague-Dawley , Vesículas Secretoras/ultraestructuraRESUMEN
The human dental follicle partially differentiates into the periodontal ligament (PDL), but their biological functions are different. The gene-expression profiles of the dental follicle and PDL were compared using the cDNA microarray technique. Microarray analysis identified 490 genes with a twofold or greater difference in expression, 365 and 125 of which were more abundant in the dental follicle and PDL, respectively. The most strongly expressed genes in the dental follicle were those related to bone development and remodeling (EGFL6, MMP8, FRZB, and NELL1), apoptosis and chemotaxis (Nox4, CXCL13, and CCL2), and tooth and embryo development (WNT2, PAX3, FGF7, AMBN, AMTN, and SLC4A4), while in the PDL it was the tumor-suppressor gene WIF1. Genes related to bone development and remodeling (STMN2, IBSP, BMP8A, BGLAP, ACP5, OPN, BMP3, and TM7SF4) and wound healing (IL1, IL8, MMP3, and MMP9) were also more strongly expressed in the PDL than in the dental follicle. In selected genes, a comparison among cDNA microarray, real-time reverse-transcription polymerase chain reaction, and immunohistochemical staining confirmed similar relative gene expressions. The gene-expression profiles presented here identify candidate genes that may enable differentiation between the dental follicle and PDL.
Asunto(s)
Saco Dental/metabolismo , Regulación del Desarrollo de la Expresión Génica/fisiología , Ligamento Periodontal/metabolismo , Diferenciación Celular/fisiología , Perfilación de la Expresión Génica , Regulación del Desarrollo de la Expresión Génica/genética , Humanos , Inmunohistoquímica , Análisis de Secuencia por Matrices de Oligonucleótidos , Reacción en Cadena en Tiempo Real de la Polimerasa , Reacción en Cadena de la Polimerasa de Transcriptasa InversaRESUMEN
Regulators of G-protein signaling (RGS) proteins are regulators of Ca(2+) signaling that accelerate the GTPase activity of the G-protein α-subunit. RGS1, RGS2, RGS4, and RGS16 are expressed in the pancreas, and RGS2 regulates G-protein coupled receptor (GPCR)-induced Ca(2+) oscillations. However, the role of RGS4 in Ca(2+) signaling in pancreatic acinar cells is unknown. In this study, we investigated the mechanism of GPCR-induced Ca(2+) signaling in pancreatic acinar cells derived from RGS4(-/-) mice. RGS4(-/-) acinar cells showed an enhanced stimulus intensity response to a muscarinic receptor agonist in pancreatic acinar cells. Moreover, deletion of RGS4 increased the frequency of Ca(2+) oscillations. RGS4(-/-) cells also showed increased expression of sarco/endoplasmic reticulum Ca(2+) ATPase type 2. However, there were no significant alterations, such as Ca(2+) signaling in treated high dose of agonist and its related amylase secretion activity, in acinar cells from RGS4(-/-) mice. These results indicate that RGS4 protein regulates Ca(2+) signaling in mouse pancreatic acinar cells.
RESUMEN
A mutation of Atp2a2 gene encoding the sarco/endoplasmic reticulum Ca(2+)-ATPase 2 (SERCA2) causes Darier's disease in human and null mutation in one copy of Atp2a2 leads to a high incidence of squamous cell tumor in a mouse model. In SERCA2 heterozygote (SERCA2(+/-)) mice keratinocytes, mechanisms involved in partial depletion of SERCA2 gene and its related tumor induction have not been studied. In this study, we investigated Ca(2+) signaling and differential gene expression in primary cultured keratinocytes from SERCA2(+/-) mice. SERCA2(+/-) keratinocytes showed reduced initial increases in intracellular concentration of calcium in response to ATP, a G-protein coupled receptor agonist, and higher store-operated Ca(2+) entry with the treatment of thapsigargin, an inhibitor of SERCA, compared to wild type kerationcytes. Protein expressions of plasma membrane Ca(2+) ATPases, NFATc1, phosphorylated ERK, JNK, and phospholipase gamma1 were increased in SERCA2(+/-) keratinocytes. Using the gene fishing system, we first found in SERCA2(+/-) keratinocytes that gene level of tumor-associated calcium signal transducer 1, crystalline alphaB, procollagen XVIII alpha1, and nuclear factor I-B were increased. Expression of involucrin, a marker of keratinocyte differentiation, was decreased in SERCA2(+/-) keratinocytes. These results suggest that the alterations of Ca(2+) signaling by SERCA2 haploinsufficiency alternate the gene expression of tumor induction and differentiation in keratinocytes.
Asunto(s)
Carcinoma de Células Escamosas/metabolismo , Heterocigoto , Queratinocitos/metabolismo , ATPasas Transportadoras de Calcio del Retículo Sarcoplásmico/genética , ATPasas Transportadoras de Calcio del Retículo Sarcoplásmico/metabolismo , Animales , Biomarcadores , Señalización del Calcio/fisiología , Carcinoma de Células Escamosas/genética , Humanos , RatonesRESUMEN
The hyperosmotic stimulus is regarded as a mechanical factor for bone remodeling. However, whether the hyperosmotic stimulus affects 1alpha, 25-dihydroxyvitamin D(3) (1alpha,25(OH)(2)D(3))-induced osteoclastogenesis is not clear. In the present study, the effect of the hyperosmotic stimulus on 1alpha,25(OH)(2)D(3)-induced osteoclastogenesis was investigated in an osteoblast-preosteoclast co-culture system. Serial doses of sucrose were applied as a mechanical force. These hyperosmotic stimuli significantly evoked a reduced number of 1alpha,25(OH)(2)D(3)-induced tartrate-resistant acid phosphatase-positive multinucleated cells and 1alpha,25(OH)(2)D(3)-induced bone-resorbing pit area in a co-culture system. In osteoblastic cells, receptor activator of nuclear factor kappaB ligand (RANKL) and Runx2 expressions were down-regulated in response to 1alpha,25(OH)(2)D(3). Knockdown of Runx2 inhibited 1alpha,25(OH)(2)D(3)-induced RANKL expression in osteoblastic cells. Finally, the hyperosmotic stimulus induced the overexpression of TonEBP in osteoblastic cells. These results suggest that hyperosmolarity leads to the down-regulation of 1alpha,25(OH)(2)D(3)-induced osteoclastogenesis, suppressing Runx2 and RANKL expression due to the TonEBP overexpression in osteoblastic cells.
RESUMEN
RANKL is essential for the terminal differentiation of monocytes/macrophages into osteoclasts. RANKL induces long-lasting oscillations in the intracellular concentration of Ca(2+) ([Ca(2+)](i)) only after 24 h of stimulation. These Ca(2+) oscillations play a switch-on role in NFATc1 expression and osteoclast differentiation. Which Ca(2+) transporting pathway is induced by RANKL to evoke the Ca(2+) oscillations and its specific role in RANKL-mediated osteoclast differentiation is not known. This study examined the effect of a partial loss of sarco/endoplasmic reticulum Ca(2+) ATPase type 2 (SERCA2) on osteoclast differentiation in SERCA2 heterozygote mice (SERCA2(+/-)). The BMD in the tibias of SERCA2(+/-) mice increased >1.5-fold compared with wildtype mice (WT). RANKL-induced [Ca(2+)](i) oscillations were generated 48 h after RANKL treatment in the WT mice but not in the SERCA2(+/-) bone marrow-derived macrophages (BMMs). Forty-eight hours after RANKL treatment, there was a lower level of NFATc1 protein expression and markedly reduced translocation of NFATc1 into the nucleus during osteoclastogenesis of the SERCA2(+/-) BMMs. In addition, RANKL treatment of SERCA2(+/-) BMMs incompletely induced formation of multinucleated cells, leading to reduced bone resorption activity. These results suggest that RANKL-mediated induction of SERCA2 plays a critical role in the RANKL-induced [Ca(2+)](i) oscillations that are essential for osteoclastogenesis.
Asunto(s)
Osteoclastos/efectos de los fármacos , Osteoclastos/enzimología , Osteogénesis/efectos de los fármacos , Ligando RANK/farmacología , ATPasas Transportadoras de Calcio del Retículo Sarcoplásmico/deficiencia , Animales , Densidad Ósea/efectos de los fármacos , Células de la Médula Ósea/citología , Señalización del Calcio/efectos de los fármacos , Células Cultivadas , Ratones , Monocitos/efectos de los fármacos , Monocitos/enzimología , Monocitos/patología , Factores de Transcripción NFATC/metabolismo , Osteoclastos/patología , Osteopetrosis/enzimología , Osteopetrosis/patología , Osteopetrosis/fisiopatología , Transporte de Proteínas/efectos de los fármacos , ATPasas Transportadoras de Calcio del Retículo Sarcoplásmico/metabolismoRESUMEN
Sphingosine-1-phosphate (S1P), a bioactive sphingolipid metabolite, regulates multiple cellular responses such as Ca(2+) signaling, growth, survival, and differentiation. Because sphingosine kinase (SphK) is the enzyme directly responsible for production of S1P, many factors have been identified that regulate its activity and subsequent S1P levels. Here we synthesized a previously unidentified SphK activator, K6PC-5, and have studied its effects on intracellular Ca(2+) signaling in HaCaT cells and epidermal differentiation in murine skin. K6PC-5, a hydrophobic compound chemically named N-(1,3-dihydroxyisopropyl)-2-hexyl-3-oxo-decanamide, activated SphK (obtained from C57BL/6 murine blood and F9-12 cell lysates) in a dose-dependent manner. K6PC-5 induced both intracellular Ca(2+) concentration ([Ca(2+)](i)) oscillations in HaCaT cells and Ca(2+) mobilization in hairless mouse epidermis. Both dimethylsphingosine (DMS) and dihydroxysphingosine (DHS), SphK inhibitors, and transfection of SphK1-siRNA blocked K6PC-5-induced increases in [Ca(2+)](i). The K6PC-5-induced [Ca(2+)](i) oscillations were dependent on thapsigargin-sensitive Ca(2+) stores and Ca(2+) entry, but independent of the classical phospholipase C-mediated pathway. In addition, K6PC-5 enhanced the expression of involucrin and filaggrin, specific differentiation-associated marker proteins in HaCaT cells, whereas transfection of SphK1-siRNA blocked the increase of involucrin. Topical K6PC-5 also enhanced the expression of involucrin, loricrin, filaggrin, and keratin 5 in intact murine epidermis. Finally, topical K6PC-5 inhibited epidermal hyperplasia by exerting antiproliferative effects on keratinocytes in murine epidermis. These results suggest that K6PC-5 acts to regulate both differentiation and proliferation of keratinocytes via [Ca(2+)](i) responses through S1P production. Thus, regulation of S1P levels may represent a novel approach for treatment of skin disorders characterized by abnormal differentiation and proliferation, such as atopic dermatitis and psoriasis.
Asunto(s)
Amidas/farmacología , Señalización del Calcio/fisiología , Diferenciación Celular/efectos de los fármacos , Activadores de Enzimas/farmacología , Epidermis/metabolismo , Lisofosfolípidos/metabolismo , Fosfotransferasas (Aceptor de Grupo Alcohol)/metabolismo , Esfingosina/análogos & derivados , Animales , Calcio/metabolismo , Señalización del Calcio/efectos de los fármacos , Línea Celular , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Epidermis/efectos de los fármacos , Epidermis/patología , Femenino , Proteínas Filagrina , Humanos , Hiperplasia , Proteínas de Filamentos Intermediarios/metabolismo , Queratina-5/metabolismo , Queratinocitos/efectos de los fármacos , Queratinocitos/metabolismo , Queratinocitos/patología , Proteínas de la Membrana/metabolismo , Ratones , Ratones Pelados , Ratones Endogámicos C57BL , Neoplasias de Células Germinales y Embrionarias/patología , Precursores de Proteínas/metabolismo , Esfingosina/metabolismo , Teratocarcinoma/patologíaRESUMEN
The development of gustatory papillae in mammalian embryos requires the coordination of a series of morphological events, such as proliferation, differentiation and innervation. In mice, the circumvallate papilla (CVP) is a specialized structure that develops in a characteristic spatial and temporal pattern in the posterior region of the tongue dorsal surface. The distinct expression patterns of Shh and Ptc, which play important roles in the development of other epithelial appendages, have been localized in the trench wall that gives rise to von Ebner's gland (VEG). To define the cellular mechanisms responsible for morphogenesis and differentiation during early development of CVP and VEG, the localization patterns of keratins (cytokeratins) K7, K8, K18, K19, K14 and connexin-43, which are dependent on Shh expression in other developmental systems, have been examined in detail. The distinct localization of keratins K7, K8, K18, K19, K14 and connexin-43 in the epithelium giving rise to the CVP and VEG suggests that cytodifferentiation is established prior to morphological changes. Interestingly, the localization of proliferating cell nuclear antigen, a marker for cell proliferation, is similar to that of Shh. An understanding of the regulatory roles of cell-cell interactions and signalling molecules in orchestrating a mutual network will bring us nearer to defining the molecular and cellular mechanisms underlying morphogenesis in mammalian taste bud development.
Asunto(s)
Proteínas Hedgehog/biosíntesis , Queratinas/biosíntesis , Receptores de Superficie Celular/biosíntesis , Glándulas Salivales Menores/metabolismo , Papilas Gustativas/metabolismo , Lengua/metabolismo , Animales , Antígenos de Diferenciación/biosíntesis , Diferenciación Celular , Proliferación Celular , Conexina 43/biosíntesis , Embrión de Mamíferos/citología , Femenino , Inmunohistoquímica , Ratones , Ratones Endogámicos ICR , Morfogénesis , Receptores Patched , Receptor Patched-1 , Antígeno Nuclear de Célula en Proliferación/biosíntesis , Glándulas Salivales Menores/citología , Glándulas Salivales Menores/embriología , Transducción de Señal , Papilas Gustativas/citología , Papilas Gustativas/embriología , Lengua/citología , Lengua/embriologíaRESUMEN
Reactive oxygen species, such as the superoxide anion, H2O2, and the hydroxyl radical, have been considered as cytotoxic by-products of cellular metabolism. However, recent studies have provided evidence that H2O2 serves as a signaling molecule modulating various physiological functions. Here we investigated the effect of H2O2 on the regulation of intracellular Ca2+ signaling in rat cortical astrocytes. H2O2 triggered the generation of oscillations of intracellular Ca2+ concentration ([Ca2+]i) in a concentration-dependent manner over the range 10-100 microM. The H2O2-induced [Ca2+]i oscillations persisted in the absence of extracellular Ca2+ and were prevented by depletion of intracellular Ca2+ stores with thapsigargin. The H2O2-induced [Ca2+]i oscillations were not inhibited by pretreatment with ryanodine but were prevented by 2-aminoethoxydiphenyl borate and caffeine, known antagonists of inositol 1,4,5-trisphosphate receptors. H2O2 activated phospholipase C (PLC) gamma1 in a dose-dependent manner, and U73122, an inhibitor of PLC, completely abolished the H2O2-induced [Ca2+]i oscillations. In addition, RNA interference against PLCgamma1 and the expression of the inositol 1,4,5-trisphosphate-sequestering "sponge" prevented the generation of [Ca2+]i oscillations. H2O2-induced [Ca2+]i oscillations and PLC1 phosphorylation were inhibited by pretreatment with dithiothreitol, a sulfhydryl-reducing agent. Finally, epidermal growth factor induced H2O2 production, PLCgamma1 activation, and [Ca2+]i increases, which were attenuated by N-acetylcysteine and diphenyleneiodonium and by the overexpression of peroxiredoxin type II. Therefore, we conclude that low concentrations of exogenously applied H2O2 generate [Ca2+]i oscillations by activating PLCgamma1 through sulfhydryl oxidation-dependent mechanisms. Furthermore, we show that this mechanism underlies the modulatory effect of endogenously produced H2O2 on epidermal growth factor-induced Ca2+ signaling in rat cortical astrocytes.
Asunto(s)
Astrocitos/efectos de los fármacos , Astrocitos/metabolismo , Señalización del Calcio/efectos de los fármacos , Calcio/metabolismo , Corteza Cerebral/citología , Peróxido de Hidrógeno/farmacología , Fosfolipasa C gamma/metabolismo , Animales , Células Cultivadas , Relación Dosis-Respuesta a Droga , Activación Enzimática , Factor de Crecimiento Epidérmico , Estrenos , Inositol 1,4,5-Trifosfato/metabolismo , Fosfolipasa C gamma/antagonistas & inhibidores , Pirrolidinonas , Ratas , Ratas Wistar , TapsigarginaRESUMEN
The morphogenesis of fungiform papillae occurs in a stereotyped pattern on the dorsal surface of the mammalian tongue via epithelial-mesenchymal interactions. These interactions are thought to be achieved via intercellular communication. Gap junctions can be observed in many developing tissues and have been suggested to participate in a variety of functions, including the regulation of cell proliferation, differentiation, and apoptosis. Here, we demonstrate that the expression of Connexin 43 (Cx43), a gap junction protein, is correlated significantly with the development of fungiform papillae, which exhibit a pattern formation and morphogenesis similar to the development of other epithelial appendages. Antisense-oligodeoxynucleotide (AS-ODN) against Cx43 was used to assess the developmental functions of Cx43. The expression patterns of the signaling molecules were disrupted by Cx43 inhibition. Interestingly, the expression patterns of Shh, a key molecule in the determination of the spacing patterns of fungiform papillae, were disturbed after treatment with Cx43 AS-ODN. We have also attempted to determine the functions of Bmp-2 by applying NOGGIN protein to tongue cultures. Our results indicate that upstream regulation via Cx43 controls the Shh and Bmp-2 pathways for the morphogenesis and pattern formation of fungiform papillae.
Asunto(s)
Proteínas Morfogenéticas Óseas/biosíntesis , Conexina 43/antagonistas & inhibidores , Uniones Comunicantes/metabolismo , Lengua/metabolismo , Transactivadores/biosíntesis , Factor de Crecimiento Transformador beta/biosíntesis , Animales , Proteína Morfogenética Ósea 2 , Proteínas Morfogenéticas Óseas/antagonistas & inhibidores , Proteínas Portadoras/biosíntesis , Conexina 43/genética , Conexina 43/metabolismo , Regulación del Desarrollo de la Expresión Génica , Proteínas Hedgehog , Hibridación in Situ , Ratones , Ratones Endogámicos ICR , Oligonucleótidos Antisentido/farmacología , Técnicas de Cultivo de Órganos , Lengua/embriología , Factor de Crecimiento Transformador beta/antagonistas & inhibidoresRESUMEN
BACKGROUND: The German cockroach has been reported to act as an allergen that might be associated with a protease reaction in asthma. However, the molecular identities of the antigens in German cockroach extract (GCE) with protease activity and the protease-activated receptors (PARs) that are activated by GCE in human airway epithelial cells have not been characterized. OBJECTIVE: We investigated the direct effect of GCE on Ca(2+) signaling in human airway epithelial cells and the type of PARs activated by GCE. METHODS: The Ca(2+)-sensitive dye Fura2 was used to determine intracellular Ca(2+) concentration ([Ca(2+)](i)) by means of spectrofluorometry. RESULTS: GCE induced a baseline type of [Ca(2+)](i) oscillations in a dose-dependent manner. The oscillations persisted for long periods of time in the absence of Ca(2+) entry across the plasma membrane, suggesting that the observed [Ca(2+)](i) increases were due to Ca(2+) release from intracellular stores. Accordingly, after depleting endoplasmic reticulum Ca(2+) with thapsigargin, an endoplasmic reticulum Ca(2+) ATPase inhibitor, the GCE-mediated [Ca(2+)](i) signals were abolished. Whereas desensitization of PAR-1, PAR-3, and PAR-4 had no effect on GCE-mediated Ca(2+) mobilization, no GCE-mediated [Ca(2+)](i) increase was observed after desensitization of PAR-2. CONCLUSIONS: These results indicate that GCE has a direct effect on human airway epithelial cells, in particular generating [Ca(2+)](i) oscillations through Ca(2+) release from thapsigargin-sensitive Ca(2+) stores through activation of PAR-2.
Asunto(s)
Bronquios/inmunología , Señalización del Calcio , Cucarachas/inmunología , Endopeptidasas/inmunología , Células Epiteliales/inmunología , Receptor PAR-2/metabolismo , Alérgenos/inmunología , Animales , Bronquios/citología , Señalización del Calcio/efectos de los fármacos , Línea Celular , Cucarachas/enzimología , Endopeptidasas/farmacología , Humanos , Tapsigargina/metabolismoRESUMEN
AlF4-is known to generate oscillations in intracellular Ca2+ concentration ([Ca2+]i) by activating G proteins in many cell types. However, in rat pancreatic acinar cells, AlF4--evoked [Ca2+]i oscillations were reported to be dependent on extracellular Ca2+, which contrasts with the [Ca2+]i oscillations induced by cholecystokinin (CCK). Therefore, we investigated the mechanisms by which AlF4- generates extracellular Ca2+-dependent [Ca2+]i oscillations in rat pancreatic acinar cells. AlF4(-)-induced [Ca2+]i oscillations were stopped rapidly by the removal of extracellular Ca2+ and were abolished on the addition of 20 mM caffeine and 2 microM thapsigargin, indicating that Ca2+ influx plays a crucial role in maintenance of the oscillations and that an inositol 1,4,5-trisphosphate-sensitive Ca2+ store is also required. The amount of Ca2+ in the intracellular Ca2+ store was decreased as the AlF4--induced [Ca2+]i oscillations continued. Measurement of 45Ca2+ influx into isolated microsomes revealed that AlF4-directly inhibited sarco/endoplasmic reticulum Ca2+-ATPase (SERCA). The activity of plasma membrane Ca2+-ATPase during AlF4- stimulation was not significantly different from that during CCK stimulation. After partial inhibition of SERCA with 1 nM thapsigargin, 20 pM CCK-evoked [Ca2+]i oscillations were dependent on extracellular Ca2+. This study shows that AlF4- induces [Ca2+]i oscillations, probably by inositol 1,4,5-trisphosphate production via G protein activation but that these oscillations are strongly dependent on extracellular Ca2+ as a result of the partial inhibition of SERCA.