RESUMEN
Heparan sulfate (HS) regulation of FGFR function, which is essential for salivary gland (SG) development, is determined by the immense structural diversity of sulfated HS domains. 3-O-sulfotransferases generate highly 3-O-sulfated HS domains (3-O-HS), and Hs3st3a1 and Hs3st3b1 are enriched in myoepithelial cells (MECs) that produce basement membrane (BM) and are a growth factor signaling hub. Hs3st3a1;Hs3st3b1 double-knockout (DKO) mice generated to investigate 3-O-HS regulation of MEC function and growth factor signaling show loss of specific highly 3-O-HS and increased FGF/FGFR complex binding to HS. During development, this increases FGFR-, BM- and MEC-related gene expression, while in adult, it reduces MECs, increases BM and disrupts acinar polarity, resulting in salivary hypofunction. Defined 3-O-HS added to FGFR pulldown assays and primary organ cultures modulates FGFR signaling to regulate MEC BM synthesis, which is critical for secretory unit homeostasis and acinar function. Understanding how sulfated HS regulates development will inform the use of HS mimetics in organ regeneration.
Asunto(s)
Membrana Basal , Diferenciación Celular , Células Epiteliales , Heparitina Sulfato , Ratones Noqueados , Glándulas Salivales , Transducción de Señal , Sulfotransferasas , Animales , Heparitina Sulfato/metabolismo , Membrana Basal/metabolismo , Glándulas Salivales/metabolismo , Glándulas Salivales/citología , Sulfotransferasas/metabolismo , Sulfotransferasas/genética , Ratones , Células Epiteliales/metabolismo , Células Epiteliales/citología , Receptores de Factores de Crecimiento de Fibroblastos/metabolismo , Receptores de Factores de Crecimiento de Fibroblastos/genética , Masculino , Factores de Crecimiento de Fibroblastos/metabolismoRESUMEN
Tight gas reservoirs are rich in microscale pores and fractures. The effect of microscale gas seepage in tight sandstone matrix on gas well productivity cannot be ignored. At present, the effect of microscale gas flow on the well testing model of fractured horizontal wells has not been systematically studied. In this paper, first, the coupling influence mechanism of "multicomponent gas effect-fracture geometry characteristics" is expounded, second, a seepage model coupling the flow in the fracture and the flow in the gas reservoir is established on the fracture wall surface, and third, an unsteady pressure analysis model for fractured horizontal wells in tight gas reservoirs is established. Results show that (a) when the microscale fracture is at a quite small level, the Knudsen diffusion plays a dominant role at a wide range of pressures; (b) the mass transfer rate of multicomponent shale gas through macrofractures increases with increasing CO2 fraction; (c) the unsteady flow process is divided into six stages in turn: microscale diffusion stage, linear flow stage in fractures, linear flow stage between fractures, early radial flow stage in gas reservoirs, linear flow stage in gas reservoirs, and late radial flow stage in gas reservoirs; and (d) with an increase of dimensionless bottom-hole storage coefficient, the second and third stages of flow are gradually covered up.
RESUMEN
Huangqin Su (HQS) tablet is mainly composed of baicalein which has been evaluated for its ability to inhibit influenza. The present study aimed to investigate the effect of HQS and oseltamivir phosphate (OS) (single or combination therapy) on influenza-induced acute pneumonia in male and female ICR mice. The regulatory effect of HQS on gut microbiota was also studied by using 16 s rDNA sequencing, and the targets and mechanisms of HQS against influenza were comprehensively analyzed by network pharmacology. Pharmacodynamic results, including lung index and pathological changes, showed that HQS exhibited significant anti-influenza efficacy and could improve the efficacy of low-dose OS (P < 0.05 and P < 0.01, respectively). The results of 16 s rDNA sequencing revealed that HQS modulated the gut microbiota and remarkably enriched the abundance of Lactobacillus. The findings of network pharmacology research suggested that the anti-influenza mechanism of HQS was related to TLRs, MAPK, and other signal transduction pathways. Taken together, this study identified the possibility of the combined use of HQS and OS and demonstrated the role of HQS in modulating the gut microbiota of mice against influenza. Network pharmacology studies also suggested that the anti-influenza effect of HQS was related to TLRs, MAPK, TNF, and other signaling pathways.
Asunto(s)
Microbioma Gastrointestinal , Gripe Humana , Neumonía , Animales , Femenino , Masculino , Ratones , ADN Ribosómico/farmacología , Ratones Endogámicos ICR , Farmacología en Red , Oseltamivir/farmacología , Scutellaria baicalensisRESUMEN
Store-operated calcium entry (SOCE) is triggered by assembly of Orai1 with STIM proteins in ER-PM junctions. Plasma membrane PIP2 as well as PIP2-binding protein, SEPT4, significantly impact Orai1-STIM1 interaction. While septins and PIP2 can organize the actin cytoskeleton, it is unclear whether the status of actin within the junctions contributes to SOCE. We report herein that actin remodeling modulates STIM1 clustering. Our findings show that a PIP2- and SEPT4-dependent mechanism involving CDC42, WASP/WAVE, and ARP2 regulates actin remodeling into a ring-like structure around STIM1 puncta. CDC42 localization in the ER-plasma membrane region is enhanced following ER-Ca2+ store depletion. PIP2 depletion or knockdown of SEPT4 attenuate the recruitment of CDC42 to the ER-PM region. Importantly, knockdown of SEPT4, or CDC42+ARP2, disrupts the organization of actin as well as STIM1 clustering. Consequently, Orai1 recruitment to STIM1 puncta, SOCE, and NFAT translocation to the nucleus are all attenuated. Ca2+ influx induced by STIM1-C terminus is not affected by CDC42 knockdown. In aggregate, our findings reveal that PIP2 and SEPT4 affect Orai1/STIM1 clustering by coordinating actin remodeling within ER-PM junctions. This dynamic reorganization of actin has an important role in regulation of SOCE and downstream Ca2+-dependent effector functions.
Asunto(s)
Complejo 2-3 Proteico Relacionado con la Actina , Septinas , Citoesqueleto de Actina/metabolismo , Calcio/metabolismo , Membrana Celular/metabolismo , Proteína ORAI1/genética , Molécula de Interacción Estromal 1RESUMEN
A severe consequence of radiation therapy in patients with head and neck cancer is persistent salivary gland hypofunction which causes xerostomia and oral infections. We previously showed that irradiation (IR) of salivary glands in mice triggers initial transient increases in mitochondrial reactive oxygen species (ROSmt), mitochondrial [Ca2+] ([Ca2+]mt), and activated caspase-3 in acinar cells. In contrast, loss of salivary secretion is persistent. Herein we assessed the role of ROSmt in radiation-induced irreversible loss of salivary gland function. We report that treatment of mice with the mitochondrial-targeted antioxidant, MitoTEMPO, resulted in almost complete protection of salivary gland secretion following either single (15 Gy) or fractionated (5 × 3 Gy) doses of irradiation. Salivary gland cells isolated from MitoTEMPO-treated, irradiated, mice displayed significant attenuation of the initial increases in ROSmt, ([Ca2+]mt, and activated caspase-3 as compared to cells from irradiated, but untreated, animals. Importantly, MitoTEMPO treatment prevented radiation-induced decrease in STIM1, consequently protecting store-operated Ca2+ entry which is critical for saliva secretion. Together, these findings identify the initial increase in ROSmt, that is induced by irradiation, as a critical driver of persistent salivary gland hypofunction. We suggest that the mitochondrially targeted antioxidant, MitoTEMPO, can be potentially important in preventing IR-induced salivary gland dysfunction.
Asunto(s)
Antioxidantes/farmacología , Mitocondrias/efectos de los fármacos , Glándulas Salivales/efectos de los fármacos , Glándulas Salivales/efectos de la radiación , Animales , Calcio/metabolismo , Caspasa 3/metabolismo , Fraccionamiento de la Dosis de Radiación , Activación Enzimática , Ratones , Mitocondrias/enzimología , Mitocondrias/metabolismo , Compuestos Organofosforados/farmacología , Piperidinas/farmacología , Radiación Ionizante , Especies Reactivas de Oxígeno/metabolismo , Saliva/metabolismo , Glándulas Salivales/metabolismo , Glándulas Salivales/fisiopatología , Molécula de Interacción Estromal 1/metabolismoRESUMEN
The intracellular calcium signaling processes are tightly regulated to ensure the generation of calcium signals with the specific spatiotemporal characteristics required for regulating various cell functions. Compartmentalization of the molecular components involved in the generation of these signals at discrete intracellular sites ensures the signaling specificity and transduction fidelity of the signal for regulating downstream effector processes. Store-operated calcium entry (SOCE) is ubiquitously present in cells and is critical for essential cell functions in a variety of tissues. SOCE is mediated via plasma membrane Ca2+ channels that are activated when luminal [Ca2+] of the endoplasmic reticulum ([Ca2+]ER) is decreased. The ER-resident stromal interaction molecules, STIM1 and STIM2, respond to decreases in [Ca2+]ER by undergoing conformational changes that cause them to aggregate at the cell periphery in ER-plasma membrane (ER-PM) junctions. At these sites, STIM proteins recruit Orai1 channels and trigger their activation. Importantly, the two STIM proteins concertedly modulate Orai1 function as well as the sensitivity of SOCE to ER-Ca2+ store depletion. Another family of plasma membrane Ca2+ channels, known as the Transient Receptor Potential Canonical (TRPC) channels (TRPC1-7) also contribute to sustained [Ca2+]i elevation. Although Ca2+ signals generated by these channels overlap with those of Orai1, they regulate distinct functions in the cells. Importantly, STIM1 is also required for plasma membrane localization and activation of some TRPCs. In this review, we will discuss various molecular components and factors that govern the activation, regulation and modulation of the Ca2+ signal generated by Ca2+ entry pathways in response to depletion of ER-Ca2+ stores. This article is part of a Special Issue entitled: ECS Meeting edited by Claus Heizmann, Joachim Krebs and Jacques Haiech.
Asunto(s)
Señalización del Calcio/fisiología , Calcio/metabolismo , Membrana Celular/metabolismo , Retículo Endoplásmico/metabolismo , Proteína ORAI1/metabolismo , Canales Catiónicos TRPC/metabolismo , Animales , Humanos , Proteínas de Neoplasias/metabolismo , Molécula de Interacción Estromal 1/metabolismo , Molécula de Interacción Estromal 2/metabolismoRESUMEN
Salivary glands secrete saliva, a mixture of proteins and fluids, which plays an extremely important role in the maintenance of oral health. Loss of salivary secretion causes a dry mouth condition, xerostomia, which has numerous deleterious consequences including opportunistic infections within the oral cavity, difficulties in eating and swallowing food, and problems with speech. Secretion of fluid by salivary glands is stimulated by activation of specific receptors on acinar cell plasma membrane and is mediated by an increase in cytosolic [Ca2+] ([Ca2+]i). The increase in [Ca2+]i regulates a number of ion channels and transporters that are required for establishing an osmotic gradient that drives water flow via aquaporin water channels in the apical membrane. The Store-Operated Ca2+ Entry (SOCE) mechanism, which is regulated in response to depletion of ER-Ca2+, determines the sustained [Ca2+]i increase required for prolonged fluid secretion. Core components of SOCE in salivary gland acinar cells are Orai1 and STIM1. In addition, TRPC1 is a major and non-redundant contributor to SOCE and fluid secretion in salivary gland acinar and ductal cells. Other TRP channels that contribute to salivary flow are TRPC3 and TRPV4, while presence of others, including TRPM8, TRPA1, TRPV1, and TRPV3, have been identified in the gland. Loss of salivary gland function leads to dry mouth conditions, or xerostomia, which is clinically seen in patients who have undergone radiation treatment for head-and-neck cancers, and those with the autoimmune exocrinopathy, Sjögren's syndrome (pSS). TRPM2 is a unique TRP channel that acts as a sensor for intracellular ROS. We will discuss recent studies reported by us that demonstrate a key role for TRPM2 in radiation-induced salivary gland dysfunction. Further, there is increasing evidence that TRPM2 might be involved in inflammatory processes. These interesting findings point to the possible involvement of TRPM2 in Sjögren's Syndrome, although further studies will be required to identify the exact role of TRPM2 in this disease.
RESUMEN
Ca2+ entry mediated by the calcium channel, Orai1, provides critical Ca2+ signals that regulate cell function. The ER-Ca2+ sensor protein, STIM1, recruits and strongly activates Orai1 within ER-PM junctions. STIM2 is a poor activator of Orai1, and its physiological role is not well understood. Herein, we report a crucial function for STIM2 in inducing the activated conformation of STIM1. By using conformational sensors of STIM2 and STIM1, together with protein interaction and functional studies, we show that STIM2 is constitutively localized within ER-PM junctions in ER-Ca2+ store replete cells. Importantly, STIM2 traps STIM1 and triggers remodeling of STIM1 C terminus, causing STIM1/Orai1 coupling and enhancement of Orai1 function in cells with relatively high ER-[Ca2+]. The increase in Ca2+ entry controls Ca2+-dependent transcription factor, NFAT, activation at low [agonist]. Our findings reveal that STIM2 modulates STIM1/Orai1 function to tune the fidelity of receptor-evoked Ca2+ signaling and the physiological response of cells.
Asunto(s)
Membrana Celular/metabolismo , Retículo Endoplásmico/metabolismo , Proteínas de Neoplasias/metabolismo , Proteína ORAI1/metabolismo , Molécula de Interacción Estromal 1/metabolismo , Molécula de Interacción Estromal 2/metabolismo , Calcio/metabolismo , Señalización del Calcio/efectos de los fármacos , Células HEK293 , Humanos , Indoles/farmacología , Microscopía Confocal , Factores de Transcripción NFATC/metabolismo , Proteínas de Neoplasias/antagonistas & inhibidores , Proteínas de Neoplasias/genética , Proteína ORAI1/antagonistas & inhibidores , Proteína ORAI1/genética , Técnicas de Placa-Clamp , Conformación Proteica , Interferencia de ARN , ARN Interferente Pequeño/metabolismo , Molécula de Interacción Estromal 1/antagonistas & inhibidores , Molécula de Interacción Estromal 1/genética , Molécula de Interacción Estromal 2/genéticaRESUMEN
Store-operated Ca2+ entry (SOCE) is critical for salivary gland fluid secretion. We report that radiation treatment caused persistent salivary gland dysfunction by activating a TRPM2-dependent mitochondrial pathway, leading to caspase-3-mediated cleavage of stromal interaction molecule 1 (STIM1) and loss of SOCE. After irradiation, acinar cells from the submandibular glands of TRPM2+/+ , but not those from TRPM2-/- mice, displayed an increase in the concentrations of mitochondrial Ca2+ and reactive oxygen species, a decrease in mitochondrial membrane potential, and activation of caspase-3, which was associated with a sustained decrease in STIM1 abundance and attenuation of SOCE. In a salivary gland cell line, silencing the mitochondrial Ca2+ uniporter or caspase-3 or treatment with inhibitors of TRPM2 or caspase-3 prevented irradiation-induced loss of STIM1 and SOCE. Expression of exogenous STIM1 in the salivary glands of irradiated mice increased SOCE and fluid secretion. We suggest that targeting the mechanisms underlying the loss of STIM1 would be a potentially useful approach for preserving salivary gland function after radiation therapy.
Asunto(s)
Canales de Calcio/metabolismo , Caspasa 3/metabolismo , Radioterapia/efectos adversos , Glándulas Salivales/patología , Glándulas Salivales/efectos de la radiación , Molécula de Interacción Estromal 1/metabolismo , Células Acinares/metabolismo , Células Acinares/patología , Células Acinares/efectos de la radiación , Animales , Calcio/metabolismo , Canales de Calcio/genética , Caspasa 3/genética , Células Cultivadas , Humanos , Potencial de la Membrana Mitocondrial/efectos de la radiación , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Mitocondrias/metabolismo , Mitocondrias/patología , Mitocondrias/efectos de la radiación , Proteína ORAI1/genética , Proteína ORAI1/metabolismo , Glándulas Salivales/metabolismo , Molécula de Interacción Estromal 1/genética , Canales Catiónicos TRPM/metabolismo , Rayos XRESUMEN
The signaling pathways involved in the generation and maintenance of exocrine gland acinar cells have not yet been established. Primary human salivary gland epithelial cells, derived from salivary gland biopsies, acquired an acinar-like phenotype when the [Ca(2+)] in the serum-free medium (keratinocyte growth medium, KGM) was increased from 0.05 mm (KGM-L) to 1.2 mm (KGM-H). Here we examined the mechanism underlying this Ca(2+)-dependent generation of the acinar cell phenotype. Compared with cells in KGM-L, those in KGM-H display enhancement of Orai1, STIM1, STIM2, and nuclear factor of activated T cells 1 (NFAT1) expression together with an increase in store-operated Ca(2+) entry (SOCE), SOCE-dependent nuclear translocation of pGFP-NFAT1, and NFAT-dependent but not NFκB-dependent gene expression. Importantly, AQP5, an acinar-specific protein critical for function, is up-regulated in KGM-H via SOCE/NFAT-dependent gene expression. We identified critical NFAT binding motifs in the AQP5 promoter that are involved in Ca(2+)-dependent up-regulation of AQP5. These important findings reveal that the Ca(2+)-induced switch of salivary epithelial cells to an acinar-like phenotype involves remodeling of SOCE and NFAT signaling, which together control the expression of proteins critically relevant for acinar cell function. Our data provide a novel strategy for generating and maintaining acinar cells in culture.
Asunto(s)
Señalización del Calcio/fisiología , Calcio/metabolismo , Células Epiteliales/metabolismo , Factores de Transcripción NFATC/metabolismo , Glándulas Salivales/metabolismo , Regulación hacia Arriba/fisiología , Acuaporina 5/biosíntesis , Acuaporina 5/genética , Canales de Calcio/biosíntesis , Células Cultivadas , Células Epiteliales/citología , Humanos , Factores de Transcripción NFATC/genética , Glándulas Salivales/citologíaRESUMEN
Stromal interaction molecule 1 (STIM1) senses depletion of ER-Ca2+ store and clusters in ER-PM junctions where it associates with and gates Ca2+ influx channels, Orai1 and TRPC1. Clustering of TRPC1 with STIM1 and Orai1 in these junctions is critical since Orai1-mediated Ca2+ entry triggers surface expression of TRPC1 while STIM1 gates the channel. Thus, plasma membrane function of TRPC1 depends on the delivery of the channel to the sites where STIM1 puncta are formed. This study examines intracellular trafficking mechanism(s) that determine plasma membrane expression and function of TRPC1 in cells where Orai1 and TRPC1 are endogenously expressed and contribute to Ca2+ entry. We report that TRPC1 is internalized by Arf6-dependent pathway, sorted to Rab5-containing early endosomes, and trafficked to ER-PM junctions by Rab4-dependent fast recycling. Overexpression of Arf6, or Rab5, but not the respective dominant negative mutants, induced retention of TRPC1 in early endosomes and suppressed TRPC1 function. Notably, cells expressing Arf6 or Rab5 displayed an inwardly rectifying ICRAC current that is mediated by Orai1 instead of TRPC1-associated ISOC, demonstrating that Orai1 function was not altered. Importantly, expression of Rab4, but not STIM1, with Rab5 rescued surface expression and function of TRPC1, restoring generation of ISOC. Together, these data demonstrate that trafficking via fast recycling endosomes determines TRPC1-STIM1 clustering within ER-PM junctions following ER-Ca2+ store depletion which is critical for the surface expression and function of the channel. Ca2+ influx mediated by TRPC1 modifies Ca2+-dependent physiological response of cells.
Asunto(s)
Canales de Calcio/metabolismo , Membrana Celular/metabolismo , Endocitosis/fisiología , Retículo Endoplásmico/metabolismo , Proteínas de la Membrana/metabolismo , Proteínas de Neoplasias/metabolismo , Canales Catiónicos TRPC/metabolismo , Factor 6 de Ribosilación del ADP , Factores de Ribosilacion-ADP/genética , Factores de Ribosilacion-ADP/metabolismo , Calcio/metabolismo , Canales de Calcio/genética , Membrana Celular/genética , Retículo Endoplásmico/genética , Células HEK293 , Humanos , Proteínas de la Membrana/genética , Proteínas de Neoplasias/genética , Proteína ORAI1 , Molécula de Interacción Estromal 1 , Canales Catiónicos TRPC/genética , Proteínas de Unión al GTP rab4/genética , Proteínas de Unión al GTP rab4/metabolismo , Proteínas de Unión al GTP rab5/genética , Proteínas de Unión al GTP rab5/metabolismoRESUMEN
A central component of receptor-evoked Ca(2+) signaling is store-operated Ca(2+) entry (SOCE), which is activated by the assembly of STIM1-Orai1 channels in endoplasmic reticulum (ER) and plasma membrane (PM) (ER-PM) junctions in response to depletion of ER Ca(2+). We report that STIM2 enhances agonist-mediated activation of SOCE by promoting STIM1 clustering in ER-PM junctions at low stimulus intensities. Targeted deletion of STIM2 in mouse salivary glands diminished fluid secretion in vivo and SOCE activation in dispersed salivary acinar cells stimulated with low concentrations of muscarinic receptor agonists. STIM2 knockdown in human embryonic kidney (HEK) 293 cells diminished agonist-induced Ca(2+) signaling and nuclear translocation of NFAT (nuclear factor of activated T cells). STIM2 lacking five carboxyl-terminal amino acid residues did not promote formation of STIM1 puncta at low concentrations of agonist, whereas coexpression of STIM2 with STIM1 mutant lacking the polybasic region STIM1ΔK resulted in co-clustering of both proteins. Together, our findings suggest that STIM2 recruits STIM1 to ER-PM junctions at low stimulus intensities when ER Ca(2+) stores are mildly depleted, thus increasing the sensitivity of Ca(2+) signaling to agonists.
Asunto(s)
Señalización del Calcio/fisiología , Moléculas de Adhesión Celular/metabolismo , Membrana Celular/metabolismo , Retículo Endoplásmico/metabolismo , Proteínas de la Membrana/metabolismo , Proteínas de Neoplasias/metabolismo , Células Acinares/metabolismo , Análisis de Varianza , Animales , Proteínas Bacterianas , Western Blotting , Técnicas de Silenciamiento del Gen , Vectores Genéticos/genética , Células HEK293 , Humanos , Proteínas Luminiscentes , Ratones , Microscopía Confocal , Microscopía Fluorescente , Mutagénesis Sitio-Dirigida , ARN Interferente Pequeño/genética , Saliva/citología , Análisis de Secuencia de ADN , Molécula de Interacción Estromal 1 , Molécula de Interacción Estromal 2RESUMEN
Gaseous signaling molecules such as hydrogen sulfide (H2S) are produced endogenously and mediate effects through diverse mechanisms. H2S is one such gasotransmitters that regulates multiple signaling pathways in mammalian cells, and abnormal H2S metabolism has been linked to defects in bone homeostasis. Here, we demonstrate that bone marrow mesenchymal stem cells (BMMSCs) produce H2S in order to regulate their self-renewal and osteogenic differentiation, and H2S deficiency results in defects in BMMSC differentiation. H2S deficiency causes aberrant intracellular Ca(2+) influx because of reduced sulfhydration of cysteine residues on multiple Ca(2+) TRP channels. This decreased Ca(2+) flux downregulates PKC/Erk-mediated Wnt/ß-catenin signaling which controls osteogenic differentiation of BMMSCs. Consistently, H2S-deficient mice display an osteoporotic phenotype that can be rescued by small molecules that release H2S. These results demonstrate that H2S regulates BMMSCs and that restoring H2S levels via nontoxic donors may provide treatments for diseases such as osteoporosis that can arise from H2S deficiencies.
Asunto(s)
Células de la Médula Ósea/fisiología , Canales de Calcio Tipo T/metabolismo , Sulfuro de Hidrógeno/metabolismo , Células Madre Mesenquimatosas/fisiología , Osteoporosis/metabolismo , Animales , Canales de Calcio Tipo T/química , Señalización del Calcio/genética , Diferenciación Celular/genética , Células Cultivadas , Cistationina betasintasa/genética , Homeostasis , Humanos , Ratones , Ratones Noqueados , Osteogénesis/genética , Osteoporosis/patología , Proteína Quinasa C/metabolismo , ARN Interferente Pequeño/genética , Sulfurtransferasas/genética , Transaminasas/genética , beta Catenina/metabolismoRESUMEN
OBJECTIVE: The objective of this study was to determine the effect of epithelial barrier disruption, caused by deficiency of the membrane-anchored serine protease, matriptase, on salivary gland function and the induction of autoimmunity in an animal model. METHODS: Embryonic and acute ablation of matriptase expression in the salivary glands of mice was induced, leading to decreased epithelial barrier function. Mice were characterized for secretory epithelial function and the induction of autoimmunity including salivary and lacrimal gland dysfunction, lymphocytic infiltration, serum anti-Ro/SSA, anti-La/SSB and antinuclear antibodies. Salivary glands immune activation/regulation, barrier function as well as tight junction proteins expression also were determined. Expression of matriptase in minor salivary gland biopsies was compared among pSS patients and healthy volunteers. RESULTS: Embryonic ablation of matriptase expression in mice resulted in the loss of secretory epithelial cell function and the induction of autoimmunity similar to that observed in primary Sjögren's syndrome. Phenotypic changes included exocrine gland dysfunction, lymphocytic infiltrates, production of Sjögren's syndrome-specific autoantibodies, and overall activation of the immune system. Acute ablation of matriptase expression resulted in significant salivary gland dysfunction in the absence of overt immune activation. Analysis of the salivary glands indicates a loss of electrical potential across the epithelial layer as well as altered distribution of a tight junction protein. Moreover, a significant decrease in matriptase gene expression was detected in the minor salivary glands of pSS patients compared with healthy volunteers. CONCLUSIONS: Our findings demonstrate that local impairment of epithelial barrier function can lead to loss of exocrine gland function [corrected] in the absence of inflammation while systemic deletion can induce a primary Sjögren's syndrome like phenotype with autoimmunity and loss of gland function.
Asunto(s)
Eliminación de Gen , Aparato Lagrimal/patología , Glándulas Salivales/patología , Serina Endopeptidasas/genética , Síndrome de Sjögren/genética , Síndrome de Sjögren/patología , Adulto , Animales , Anticuerpos Antinucleares/biosíntesis , Autoinmunidad , Permeabilidad de la Membrana Celular , Movimiento Celular , Modelos Animales de Enfermedad , Células Epiteliales/inmunología , Células Epiteliales/patología , Femenino , Regulación de la Expresión Génica , Humanos , Aparato Lagrimal/inmunología , Linfocitos/inmunología , Linfocitos/patología , Ratones , Persona de Mediana Edad , Glándulas Salivales/inmunología , Serina Endopeptidasas/deficiencia , Síndrome de Sjögren/inmunología , Proteínas de Uniones Estrechas/genética , Proteínas de Uniones Estrechas/metabolismo , Uniones Estrechas/inmunología , Uniones Estrechas/patologíaRESUMEN
BACKGROUND: Transplantation of islets of Langerhans (islets) was used to treat insulin-dependent diabetes mellitus. However, islet grafts must be maintained by administration of immunosuppressive drugs, which can lead to complications in the long term. An approach that avoids immunosuppressive drug use is desirable. METHODS: Co-aggregates of Sertoli cells and islet cells from BALB/c mice that were prepared by the hanging drop method were transplanted into C57BL/6 mouse liver through the portal vein as in human clinical islet transplantation. RESULTS: The core part of the aggregates contained mainly Sertoli cells, and these cells were surrounded by islet cells. The co-aggregates retained the functions of both Sertoli and islet cells. When 800 co-aggregates were transplanted into seven C57BL/6 mice via the portal vein, six of seven recipient mice demonstrated quasi-normoglycemia for more than 100 days. CONCLUSIONS: The hanging drop method is suitable for preparing aggregates of Sertoli and islet cells for transplantation. Notably, transplantation of these allogeneic co-aggregates into mice with chemically induced diabetes via the portal vein resulted in long-term graft survival without systemic immunosuppression.
Asunto(s)
Diabetes Mellitus Tipo 1/terapia , Terapia de Inmunosupresión/métodos , Trasplante de Islotes Pancreáticos/métodos , Islotes Pancreáticos/citología , Trasplante de Hígado/métodos , Células de Sertoli/citología , Activinas/química , Animales , Diabetes Mellitus Experimental/terapia , Supervivencia de Injerto , Tolerancia Inmunológica , Inmunosupresores/efectos adversos , Insulina/sangre , Insulina/metabolismo , Secreción de Insulina , Islotes Pancreáticos/inmunología , Hígado/patología , Masculino , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Ratones Transgénicos , Vena Porta/patologíaRESUMEN
OBJECTIVE: Primary Sjögren's syndrome (SS) is characterized by autoimmune activation and loss of function in secretory epithelia. The present study was undertaken to investigate and characterize changes in the epithelia associated with the loss of gland function in primary SS. METHODS: To identify changes in epithelial gene expression, custom microarrays were probed with complementary RNA (cRNA) isolated from minor salivary glands (MSGs) of female patients with primary SS who had low focus scores and low salivary flow rates, and the results were compared with those obtained using cRNA from the MSGs of sex-matched healthy volunteers. The effect of bone morphogenetic protein 6 (BMP-6) on salivary gland function was tested using adeno-associated virus-mediated gene transfer to the salivary glands of C57BL/6 mice. RESULTS: A significant increase in expression of BMP-6 was observed in RNA isolated from SS patients compared with healthy volunteers. Overexpression of BMP-6 locally in the salivary or lacrimal glands of mice resulted in the loss of fluid secretion as well as changes in the connective tissue of the salivary gland. Assessment of the fluid movement in either isolated acinar cells from mice overexpressing BMP-6 or a human salivary gland cell line cultured with BMP-6 revealed a loss in volume regulation in these cells. Lymphocytic infiltration in the submandibular gland of BMP-6 vector-treated mice was increased. No significant changes in the production of proinflammatory cytokines or autoantibodies associated with SS (anti-Ro/SSA and anti-La/SSB) were found after BMP-6 overexpression. CONCLUSION: In addition to identifying BMP-6 expression in association with xerostomia and xerophthalmia in primary SS, the present results suggest that BMP-6-induced salivary and lacrimal gland dysfunction in primary SS is independent of the autoantibodies and immune activation associated with the disease.
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Proteína Morfogenética Ósea 6/metabolismo , Aparato Lagrimal/metabolismo , Glándulas Salivales/metabolismo , Síndrome de Sjögren/metabolismo , Animales , Autoanticuerpos/metabolismo , Proteína Morfogenética Ósea 6/genética , Femenino , Técnicas de Transferencia de Gen , Humanos , Aparato Lagrimal/inmunología , Aparato Lagrimal/fisiopatología , Ratones , Ratones Endogámicos C57BL , Glándulas Salivales/inmunología , Glándulas Salivales/fisiopatología , Síndrome de Sjögren/inmunología , Síndrome de Sjögren/fisiopatología , Xerostomía/inmunología , Xerostomía/metabolismo , Xerostomía/fisiopatologíaRESUMEN
Store-operated calcium entry (SOCE) is activated in response to depletion of the endoplasmic reticulum-Ca(2+) stores following stimulation of plasma membrane receptors that couple to PIP2 hydrolysis and IP3 generation. Search for the molecular components of SOCE channels led to the identification of mammalian transient receptor potential canonical (TRPC) family of calcium-permeable channels (TRPC1-TRPC7), which are all activated in response to stimuli that result in PIP2 hydrolysis. While several TRPCs, including TRPC1, TRPC3, and TRPC4, have been implicated in SOCE, the data are most consistent for TRPC1. Extensive studies in cell lines and knockout mouse models have established the contribution of TRPC1 to SOCE. Furthermore, there is a critical functional interaction between TRPC1 and the key components of SOCE, STIM1, and Orai1, which determines the activation of TRPC1. Orai1-mediated Ca(2+) entry is required for recruitment of TRPC1 and its insertion into surface membranes while STIM1 gates the channel. Notably, TRPC1 and Orai1 generate distinct patterns of Ca(2+) signals in cells that are decoded for the regulation of specific cellular functions. Thus, SOCE appears to be a complex process that depends on temporal and spatial coordination of several distinct steps mediated by proteins in different cellular compartments. Emerging data suggest that, in many cell types, the net Ca(2+) entry measured in response to store depletion is the result of the coordinated regulation of different calcium-permeable ion channels. Orai1 and STIM1 are central players in this process, and by mediating recruitment or activation of other Ca(2+) channels, Orai1-CRAC function can elicit rapid changes in global and local [Ca(2+)]i signals in cells. It is most likely that the type of channels and the [Ca(2+)]i signature that are generated by this process reflect the physiological function of the cell that is regulated by Ca(2+).
Asunto(s)
Señalización del Calcio , Canales Catiónicos TRPC/fisiología , Animales , Canales de Calcio/metabolismo , Retículo Endoplásmico/metabolismo , Humanos , Microdominios de Membrana/metabolismo , Proteínas de la Membrana/metabolismo , Proteínas de Neoplasias/metabolismo , Proteína ORAI1 , Multimerización de Proteína , Molécula de Interacción Estromal 1RESUMEN
Xerostomia as a result of salivary gland damage is a permanent and debilitating side effect of radiotherapy for head and neck cancers. Effective treatments for protecting, or restoring, salivary gland function are not available. Here we report that irradiation treatment leads to activation of the calcium-permeable channel, transient potential melastatin-like 2 (TRPM2), via stimulation of poly-ADP-ribose polymerase. Importantly, irradiation induced an irreversible loss of salivary gland fluid secretion in TRPM2+/+ mice while a transient loss was seen in TRPM2-/- mice with >60% recovery by 30 days after irradiation. Treatment of TRPM2+/+ mice with the free radical scavenger Tempol or the PARP1 inhibitor 3-aminobenzamide attenuated irradiation-induced activation of TRPM2 and induced significant recovery of salivary fluid secretion. Furthermore, TPL (4-hydroxy-2,2,6,6-tetramethylpiperidine-N-oxyl) induced complete recovery of function in irradiated TRPM2-/- mice. These novel data demonstrate that TRPM2 is activated by irradiation, via PARP1 activation, and contributes to irreversible loss of salivary gland function.
Asunto(s)
Traumatismos por Radiación/prevención & control , Traumatismos por Radiación/fisiopatología , Glándulas Salivales/fisiopatología , Glándulas Salivales/efectos de la radiación , Canales Catiónicos TRPM/deficiencia , Células Acinares/efectos de los fármacos , Células Acinares/metabolismo , Células Acinares/patología , Células Acinares/efectos de la radiación , Animales , Benzamidas/farmacología , Calcio/metabolismo , Carbacol/farmacología , Línea Celular , Supervivencia Celular/efectos de los fármacos , Supervivencia Celular/efectos de la radiación , Inhibidores Enzimáticos/farmacología , Humanos , Peróxido de Hidrógeno/farmacología , Activación del Canal Iónico/efectos de los fármacos , Activación del Canal Iónico/efectos de la radiación , Ratones , Ratones Endogámicos C57BL , Piperidinas/farmacología , Inhibidores de Poli(ADP-Ribosa) Polimerasas , Poli(ADP-Ribosa) Polimerasas/metabolismo , Traumatismos por Radiación/patología , Saliva/efectos de los fármacos , Saliva/metabolismo , Saliva/efectos de la radiación , Glándulas Salivales/metabolismo , Glándulas Salivales/patología , Salivación/efectos de los fármacos , Salivación/efectos de la radiación , Glándula Submandibular/metabolismo , Glándula Submandibular/patología , Glándula Submandibular/fisiopatología , Glándula Submandibular/efectos de la radiación , Canales Catiónicos TRPM/metabolismo , Rayos XRESUMEN
Up to 25% of the circulating nitrate in blood is actively taken up, concentrated, and secreted into saliva by the salivary glands. Salivary nitrate can be reduced to nitrite by the commensal bacteria in the oral cavity or stomach and then further converted to nitric oxide (NO) in vivo, which may play a role in gastric protection. However, whether salivary nitrate is actively secreted in human beings has not yet been determined. This study was designed to determine whether salivary nitrate is actively secreted in human beings as an acute stress response and what role salivary nitrate plays in stress-induced gastric injury. To observe salivary nitrate function under stress conditions, alteration of salivary nitrate and nitrite was analyzed among 22 healthy volunteers before and after a strong stress activity, jumping down from a platform at the height of 68 m. A series of stress indexes was analyzed to monitor the stress situation. We found that both the concentration and the total amount of nitrate in mixed saliva were significantly increased in the human volunteers immediately after the jump, with an additional increase 1h later (p<0.01). Saliva nitrite reached a maximum immediately after the jump and was maintained 1h later. To study the biological functions of salivary nitrate and nitrite in stress protection, we further carried out a water-immersion-restraint stress (WIRS) assay in male adult rats with bilateral parotid and submandibular duct ligature (BPSDL). Intragastric nitrate, nitrite, and NO; gastric mucosal blood flow; and gastric ulcer index (UI) were monitored and nitrate was administrated in drinking water to compensate for nitrate secretion in BPSDL animals. Significantly decreased levels of intragastric nitrate, nitrite, and NO and gastric mucosal blood flow were measured in BPSDL rats during the WIRS assay compared to sham control rats (p<0.05). Recovery was observed in the BPSDL rats upon nitrate administration. The WIRS-induced UI was significantly higher in the BPSDL animals compared to controls, and nitrate administration rescued the WIRS-induced gastric injury in BPSDL rats. In conclusion, this study suggests that stress promotes salivary nitrate secretion and nitrite formation, which may play important roles in gastric protection against stress-induced injury via the nitrate-dependent NO pathway.