RESUMO
In primary Sjögren's syndrome (pSS) patients, salivary gland (SG) epithelial cells (SGECs) could be exposed to chronic hyperosmotic stress (HOS), consecutive to their destruction and deregulation, that exacerbates an inflammatory response. The aims of this study were to assess the mechanism accounting for C-C motif chemokine ligand 2 (CCL2) expression in an immortalized human salivary gland epithelial acinar cell line (NS-SV-AC) subjected to HOS, as well as the involvement of CCL2 in pSS. CCL2 mRNA and protein levels were determined via RT-qPCR and ELISA. Reporter plasmids and a promoter pull-down assay were used to identify transcription factors associated with CCL2 mRNA increase. Our data showed that HOS-induced CCL2 mRNA increase was independent of the nuclear factor of activated T-cells 5 (NFAT5) and nuclear factor-kappa B (NFkB) but involved Kruppel-like factor 5 (KLF5). CCL2 protein levels, quantified by enzyme-linked immunosorbent assay (ELISA) in sera samples from pSS patients, correlated with the European Alliance of Associations for Rheumatology's Sjogren's syndrome disease activity index (ESSDAI) score for systemic activity. In addition, CCL2 protein levels were higher in patients with biological activity, cutaneous manifestations, and ESSDAI score superior or equal to five. Our data suggest that chronic HOS could exacerbate pSS disease by contributing to the inflammatory process induced by the expression and secretion of CCL2.
Assuntos
Síndrome de Sjogren , Humanos , Síndrome de Sjogren/genética , Ligantes , Glândulas Salivares , Quimiocinas , Fator V , RNA Mensageiro , Fatores de Transcrição , Quimiocina CCL2/genéticaRESUMO
Sjogren's syndrome (SS) is a chronic autoimmune disease characterized by the infiltration of exocrine glands including salivary and lachrymal glands responsible for the classical dry eyes and mouth symptoms (sicca syndrome). The spectrum of disease manifestations stretches beyond the classical sicca syndrome with systemic manifestations including arthritis, interstitial lung involvement, and neurological involvement. The pathophysiology underlying SS is not well deciphered, but several converging lines of evidence have supported the conjuncture of different factors interplaying together to foster the initiation and perpetuation of the disease. The innate and adaptive immune system play a cardinal role in this process. In this review, we discuss the inherent parts played by both the innate and adaptive immune system in the pathogenesis of SS.
Assuntos
Imunidade Adaptativa , Suscetibilidade a Doenças/imunologia , Imunidade Inata , Síndrome de Sjogren/imunologia , Animais , Comunicação Celular/imunologia , Células Dendríticas/imunologia , Células Dendríticas/metabolismo , Humanos , Sistema Imunitário/citologia , Sistema Imunitário/imunologia , Sistema Imunitário/metabolismo , Macrófagos/imunologia , Macrófagos/metabolismo , Especificidade de Órgãos/imunologia , Subpopulações de Linfócitos T/imunologia , Subpopulações de Linfócitos T/metabolismoRESUMO
Sjögren's syndrome (SS) is an exocrinopathy characterized by the hypofunction of salivary glands (SGs). Aquaporin-5 (AQP5); a water channel involved in saliva formation; is aberrantly distributed in SS SG acini and contributes to glandular dysfunction. We aimed to investigate the role of ezrin in AQP5 mislocalization in SS SGs. The AQP5-ezrin interaction was assessed by immunoprecipitation and proteome analysis and by proximity ligation assay in immortalized human SG cells. We demonstrated, for the first time, an interaction between ezrin and AQP5. A model of the complex was derived by computer modeling and in silico docking; suggesting that AQP5 interacts with the ezrin FERM-domain via its C-terminus. The interaction was also investigated in human minor salivary gland (hMSG) acini from SS patients (SICCA-SS); showing that AQP5-ezrin complexes were absent or mislocalized to the basolateral side of SG acini rather than the apical region compared to controls (SICCA-NS). Furthermore, in SICCA-SS hMSG acinar cells, ezrin immunoreactivity was decreased at the acinar apical region and higher at basal or lateral regions, accounting for altered AQP5-ezrin co-localization. Our data reveal that AQP5-ezrin interactions in human SGs could be involved in the regulation of AQP5 trafficking and may contribute to AQP5-altered localization in SS patients.
Assuntos
Aquaporina 5/metabolismo , Proteínas do Citoesqueleto/genética , Proteínas do Citoesqueleto/metabolismo , Regulação da Expressão Gênica , Glândulas Salivares/metabolismo , Síndrome de Sjogren/genética , Síndrome de Sjogren/metabolismo , Sequência de Aminoácidos , Aquaporina 5/química , Proteínas de Transporte , Proteínas do Citoesqueleto/química , Humanos , Modelos Moleculares , Ligação Proteica , Mapeamento de Interação de Proteínas , Mapas de Interação de Proteínas , Transporte Proteico , Síndrome de Sjogren/patologia , Relação Estrutura-AtividadeRESUMO
Aquaporins are a family of transmembrane proteins permeable to water. In mammals, they are subdivided into classical aquaporins that are permeable to water; aquaglyceroporins that are permeable to water, glycerol and urea; peroxiporins that facilitate the diffusion of H2O2 through cell membranes; and so called unorthodox aquaporins. Aquaporins ensure important physiological functions in both exocrine and endocrine pancreas. Indeed, they are involved in pancreatic fluid secretion and insulin secretion. Modification of aquaporin expression and/or subcellular localization may be involved in the pathogenesis of pancreatic insufficiencies, diabetes and pancreatic cancer. Aquaporins may represent useful drug targets for the treatment of pathophysiological conditions affecting pancreatic function, and/or diagnostic/predictive biomarker for pancreatic cancer. This review summarizes the current knowledge related to the involvement of aquaporins in the pancreas physiology and physiopathology.
Assuntos
Aquaporinas/metabolismo , Pâncreas/metabolismo , Pancreatopatias/metabolismo , Humanos , Insulina/metabolismo , Ilhotas Pancreáticas/metabolismo , Pâncreas Exócrino/metabolismoRESUMO
OBJECTIVES: The human salivary gland (HSG) cell line, labeled as a submandibular ductal cell line, is commonly used as in vitro models to study radiation therapy, Sjögren's syndrome, pleomorphic adenoma, mucocele, epithelial-to-mesenchymal transition, and epigenetics. However, the American Type Culture Collection (ATCC) has recently released a list of cross-contaminated cell lines that included HSG. Despite this notice, some research laboratories still use HSG as a salivary cell model. Therefore, this study examined the authenticity of HSG sampled from three different laboratories. METHODS: DNA was extracted from HSG and additional salivary cell lines (NS-SV-AC, NS-SV-DC, A253, HSY) and submitted for cell line authentication with short tandem repeat (STR) analysis. RESULTS: All HSG samples had STR profiles indicating >80% match with HeLa in both the ATCC and Deutsche Sammlung von Mikroorganismen und Zellkulturen (DSMZ) databases. This confirmed that HSG sampled from three different laboratories and HSY shared a common ancestry (host) with HeLa, whereas NS-SV-AC, NS-SV-DC, and A253 had unique STR profiles. CONCLUSION: Short tandem repeat analysis revealed that HSG was contaminated by the HeLa cell line. Furthermore, because genotyping of the original HSG cell line was not performed during its establishment, it will be difficult to authenticate an uncontaminated sample of HSG.
Assuntos
Contaminação por DNA , Repetições de Microssatélites , Glândulas Salivares/citologia , Células HeLa , Humanos , Análise de Sequência de DNARESUMO
Sjögren's syndrome (SS) is a chronic autoimmune disease characterized by lymphocytic infiltration of salivary and lacrimal glands resulting in diminished production of saliva and tears. The pathophysiology of SS has not yet been fully deciphered. Classically it has been postulated that sicca symptoms in SS patients are a double step process whereby lymphocytic infiltration of lacrimal and salivary glands (SG) is followed by epithelial cell destruction resulting in keratoconjunctivitis sicca and xerostomia. Recent advances in the field of the pathophysiology of SS have brought in new players, such as aquaporins (AQPs) and anti AQPs autoantibodies that could explain underlying mechanistic processes and unveil new pathophysiological pathways offering a deeper understanding of the disease. In this review, we delineate the link between the AQP and SS, focusing on salivary glands, and discuss the role of AQPs in the treatment of SS-induced xerostomia.
Assuntos
Aquaporinas , Autoanticorpos , Aparelho Lacrimal , Glândulas Salivares , Síndrome de Sjogren , Aquaporinas/imunologia , Aquaporinas/metabolismo , Autoanticorpos/imunologia , Autoanticorpos/metabolismo , Humanos , Aparelho Lacrimal/imunologia , Aparelho Lacrimal/metabolismo , Aparelho Lacrimal/patologia , Glândulas Salivares/imunologia , Glândulas Salivares/metabolismo , Glândulas Salivares/patologia , Síndrome de Sjogren/imunologia , Síndrome de Sjogren/metabolismo , Síndrome de Sjogren/patologia , Síndrome de Sjogren/terapiaRESUMO
Diabetic retinopathy is a frequent eyesight threatening complication of type 1 and type 2 diabetes. Under physiological conditions, the inner and the outer blood-retinal barriers protect the retina by regulating ion, protein, and water flux into and out of the retina. During diabetic retinopathy, many factors, including inflammation, contribute to the rupture of the inner and/or the outer blood-retinal barrier. This rupture leads the development of macular edema, a foremost cause of sight loss among diabetic patients. Under these conditions, it has been speculated that retinal pigmented epithelial cells, that constitute the outer blood-retinal barrier, may be subjected to hyperosmolar stress resulting from different mechanisms. Herein, we review the possible origins and consequences of hyperosmolar stress on retinal pigmented epithelial cells during diabetic retinopathy, with a special focus on the intimate interplay between inflammation and hyperosmolar stress, as well as the current and forthcoming new pharmacotherapies for the treatment of such condition.
Assuntos
Retinopatia Diabética/metabolismo , Epitélio Pigmentado da Retina/metabolismo , Barreira Hematorretiniana/metabolismo , Retinopatia Diabética/patologia , Humanos , Inflamação/metabolismo , Inflamação/patologia , Concentração Osmolar , Epitélio Pigmentado da Retina/patologiaRESUMO
Aquaglyceroporins-aquaporin membrane channels (AQP) that conduct glycerol and other small neutral solutes in addition to water-play major roles in obesity. In adipocytes, aquaglyceroporins mediate glycerol uptake and release across the plasma membrane, which are two key steps for triacylglycerols (TAGs) synthesis (lipogenesis) and hydrolysis (lipolysis). The aim of this study was to assess both glycerol permeability and metabolism in undifferentiated 3T3-L1 cells (UDCs) as well as in untreated (CTL-DCs) versus lipopolysaccharide (LPS-DCs)-treated differentiated 3T3-L1 adipocytes. Glycerol release, TAGs content and whole membrane glycerol permeability were significantly increased in DCs as compared to UDCs. Moreover, in DCs, LPS treatment significantly increased TAGs content and decreased glycerol permeability. In addition, a significant reduction in whole membrane glycerol permeability was observed in LPS-DCs as compared to CTL-DCs. The relative contributions of AQP3, AQP7 and AQP9 (facilitated diffusion), as well as that of the phospholipid bilayer (simple diffusion), to the whole membrane glycerol permeability, were estimated biophysically in UDCs, CTL-DCs and LPS-DCs, using selective AQP inhibitors. Further studies will be required to determine if modifications in either subcellular localization and/or activity of aquaglyceroporins could account for the data herein. Nevertheless, our findings provide novel insights in understanding the LPS-induced adipocyte hypertrophy that accompanies obesity.
Assuntos
Adipócitos/metabolismo , Glicerol/metabolismo , Lipopolissacarídeos/farmacologia , Células 3T3-L1 , Animais , Aquagliceroporinas/metabolismo , Transporte Biológico/efeitos dos fármacos , Camundongos , Triglicerídeos/metabolismoRESUMO
PURPOSE: Macular edema, a frequently encountered complication of diabetic retinopathy (DR), results from alterations of the blood retinal barrier (BRB) and leads to modifications of the retinal pigmented epithelium (RPE) functions. Osmolar changes of the surrounding medium could be responsible for modifications of the RPE functions leading to disturbance of retinal homeostasis. The expression, activation and function of the key hyperosmolar response factor Tonicity Enhancer Binding Protein (TonEBP also called nuclear factor of activated T-cell 5 - NFTA5) was investigated in ARPE-19 cells, derived from human RPE, in response to hyperosmolar stimulation. METHODS: ARPE-19 cells were exposed to hyperosmolar medium. TonEBP mRNA and protein levels were quantified by qRT-PCR and semi-quantitative Western blot. TonEBP nuclear translocation was investigated by immunofluorescence. TonEBP transactivation activity was measured using a reported plasmid containing TonEBP binding sites. RESULTS: In response to hyperosmolar stimulation of ARPE-19 cells, a dose-dependent increase in TonEBP mRNA and protein levels, as well as TonEBP nuclear translocation were observed. TonEBP transactivation activity was further demonstrated using a reporter plasmid containing TonEBP binding sites. A dominant negative form of TonEBP abolished NaCl-induced increase in TonEBP transactivation activity, and inhibited the increase of the target genes aldose reductase and sodium-dependent taurine transporter mRNA levels. SB203580, an inhibitor of two of the p38 protein kinase's isoforms (p38α and p38ß) inhibited the TonEBP nuclear translocation and transactivation activity in ARPE-19 cells exposed to hyperosmolar stimulation. CONCLUSIONS: Our data demonstrates the involvement of TonEBP in the mechanisms responsible for osmoadaptation to hyperosmolar stress in RPE cells. Given the emerging role of TonEBP in different pathological pathways, these data open new perspectives for the analysis of the mechanisms involved in the modification of functions of the RPE during macular edema.
Assuntos
Pressão Osmótica , RNA Mensageiro/genética , Epitélio Pigmentado da Retina/metabolismo , Fatores de Transcrição/genética , Western Blotting , Linhagem Celular , Primers do DNA , Inibidores Enzimáticos/farmacologia , Técnica Indireta de Fluorescência para Anticorpo , Regulação da Expressão Gênica/fisiologia , Humanos , Imidazóis/farmacologia , Concentração Osmolar , Piridinas/farmacologia , Reação em Cadeia da Polimerase em Tempo Real , Cloreto de Sódio/farmacologia , Fatores de Transcrição/metabolismo , TransfecçãoRESUMO
In an obese state, Toll-like receptor-4 (TLR-4) upregulates proinflammatory adipokines secretion including monocyte chemotactic protein-1 (MCP-1) in adipose tissue. In contrast, G-protein coupled receptor 120 (GPR120) mediates antiobesity effects. The aim of this study was to determine the signaling pathway by which Forskolin (FK), a cyclic adenosine monophosphate- (cAMP-) promoting agent causing positive changes in body composition in overweight and obese adult men, affects MCP-1 and GPR120 expression during an inflammatory response induced by lipopolysaccharide (LPS) in adipocytes, such as in an obese state. 3T3-L1 cells differentiated into adipocytes (DC) were stimulated with LPS in the absence or presence of FK and inhibitors of TLR-4 and inhibitor of kappa B (IκBα). In DC, LPS increased MCP-1, TLR-4, and nuclear factor-κB1 (NFκB1) mRNA levels, whereas it decreased GPR120 mRNA levels. In DC, FK inhibited the LPS-induced increase in MCP-1, TLR-4, and NFκB1 mRNA levels and the LPS-induced decrease in GPR120 mRNA. BAY11-7082 and CLI-095 abolished these LPS-induced effects. In conclusion, FK inhibits LPS-induced increase in MCP-1 mRNA levels and decrease in GPR120 mRNA levels in adipocytes and may be a potential treatment for inflammation in obesity. Furthermore, TLR-4-induced activation of NFκB may be involved in the LPS-induced regulation of these genes.
Assuntos
Adipócitos/efeitos dos fármacos , Adipócitos/metabolismo , Quimiocina CCL2/metabolismo , Colforsina/farmacologia , Lipopolissacarídeos/farmacologia , Receptores Acoplados a Proteínas G/metabolismo , Células 3T3-L1 , Animais , Western Blotting , Quimiocina CCL2/genética , Ensaio de Imunoadsorção Enzimática , Camundongos , NF-kappa B/metabolismo , RNA Mensageiro/genética , Receptores Acoplados a Proteínas G/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Receptor 4 Toll-Like/genética , Receptor 4 Toll-Like/metabolismo , beta-Arrestina 2/genética , beta-Arrestina 2/metabolismoRESUMO
Salivary glands are involved in saliva secretion that ensures proper oral health. Aquaporins are expressed in salivary glands and play a major role in saliva secretion. This review will provide an overview of the salivary gland morphology and physiology of saliva secretion, and focus on the expression, subcellular localization and role of aquaporins under physiological and pathophysiological conditions, as well as clinical applications involving aquaporins. This review is highlighting expression and localization of aquaporins in human, rat and mouse, the most studied species and is pointing out possible difference between major salivary glands, i.e., parotid, submandibular and sublingual glands.
Assuntos
Aquaporinas/metabolismo , Doenças da Boca/metabolismo , Glândulas Salivares/metabolismo , Animais , Aquaporinas/genética , Terapia Genética , Humanos , Doenças da Boca/terapia , Saúde Bucal , Saliva/metabolismo , Glândulas Salivares/citologiaRESUMO
Aquaglyceroporins, belonging to the family of aquaporins (AQPs), are integral plasma membrane proteins permeable to water and glycerol that have emerged as key players in obesity. The aim of this study was to investigate the expression profile of AQPs in undifferentiated and differentiated 3T3-L1 cells and to investigate the changes in expression of aquaglyceroporins in 3T3-L1 cells differentiated into adipocytes and subjected to lipopolysaccharide (LPS) mimicking inflammation occurring during obesity. Furthermore, the study aimed at identifying the signaling cascade involved in the regulation of aquaglyceroporins expression upon LPS stimulation. 3T3-L1 cells were grown as undifferentiated cells (UDC; preadipocytes) or cells differentiated into adipocytes (DC, adipocytes). DC were incubated in the presence or absence of LPS with or without inhibitors of various protein kinases. AQPs mRNA expression levels were measured by real-time quantitative polymerase chain reaction (RT-qPCR). AQP1, AQP2, AQP3, AQP9 and AQP11 mRNA were expressed in both UDC and DC, whereas AQP4, AQP7 and AQP8 mRNA were expressed only in DC. In DC, LPS up-regulated AQP3 mRNA levels (p < 0.05) compared to control; these effects were inhibited by CLI095, SP600125 and BAY11-7082 (p < 0.05). LPS decreased both AQP7 and AQP11 mRNA levels (p < 0.01) in DC as compared to control; this decrease was inhibited by CLI095 and BAY11-7082 (p < 0.05) and additionally by SP00125 for AQP7 (p < 0.05). SB203580 had no effect on LPS-induced AQP3, AQP7 and AQP11 mRNA levels modulations. In conclusion, our results clearly show that many AQPs are expressed in murine 3T3-L1 adipocytes. Moreover, in DCs, LPS led to decreased AQP7 and AQP11 mRNA levels but to increased AQP3 mRNA levels, resulting from the Toll-like receptor 4 (TLR4)-induced activation of JNK and/or NFκB pathway.
Assuntos
Adipócitos/imunologia , Adipogenia , Aquagliceroporinas/genética , Regulação da Expressão Gênica , Lipopolissacarídeos/imunologia , Sistema de Sinalização das MAP Quinases , NF-kappa B/imunologia , Células 3T3-L1 , Adipócitos/citologia , Adipócitos/metabolismo , Animais , Aquagliceroporinas/imunologia , Camundongos , Obesidade/genética , Obesidade/imunologia , RNA Mensageiro/genética , Transdução de Sinais , Receptor 4 Toll-Like/imunologiaRESUMO
A deeper understanding of aquaporins (AQPs) expression and transcriptional regulation will provide useful information for liver pathophysiology. We established a complete AQPs mRNA expression profile in human and mouse liver, as well as protein localization of expressed AQPs. Additionally, the modulation of AQPs mRNA levels in response to various agents was determined in human HuH7 cells and in primary culture of mouse hepatocytes. AQP1, AQP3, AQP7, AQP8, and AQP9 mRNA and protein expressions were detected in human liver, while only AQP6 and AQP11 mRNAs were detected. We reported for the first time the localization of AQP3 in Kupffer cells, AQP7 in hepatocytes and endothelial cells, and AQP9 in cholangiocytes. In addition, we confirmed the localization of AQP1 in endothelial cells, and of AQP8 and AQP9 in hepatocytes. On HuH7 cells, we reported the presence of AQP4 mRNA, confirmed the presence of AQP3, AQP7, and AQP11 mRNAs, but not of AQP8 mRNA. On primary culture of murine hepatocytes, AQP1 and AQP7 mRNAs were identified, while the presence of AQP3, AQP8, AQP9, and AQP11 mRNAs was confirmed. At the protein level, murine endothelial liver cells expressed AQP1 and AQP9, while hepatocytes expressed AQP3, AQP7, AQP8, and AQP9, and macrophages expressed AQP3. Dexamethasone, forskolin, AICAR, rosiglitazone, octanoylated, and non-octanoylated ghrelin regulated some AQP expression in primary culture of murine hepatocytes and human HuH7 cells. Additional studies will be required to further assess the role of AQPs expression in human and murine liver and understand the transcriptional regulation of AQPs in hepatocytes under pathophysiological conditions.
Assuntos
Aquaporinas/metabolismo , Hepatócitos/metabolismo , Fígado/metabolismo , Animais , Aquaporinas/genética , Linhagem Celular , Células Endoteliais/metabolismo , Feminino , Perfilação da Expressão Gênica/métodos , Regulação da Expressão Gênica , Hepatócitos/efeitos dos fármacos , Humanos , Imuno-Histoquímica , Células de Kupffer/metabolismo , Fígado/efeitos dos fármacos , Masculino , Camundongos , Cultura Primária de Células , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Reação em Cadeia da Polimerase em Tempo Real , Transcrição GênicaRESUMO
PURPOSE OF REVIEW: To understand the current trend of ghrelin genetic variations on the control of satiety, eating behaviours, obesity, and metabolic alterations, and its development over the last 18 months. RECENT FINDINGS: Several polymorphisms of the ghrelin gene, its receptor gene and ghrelin's acylating enzyme, ghrelin O-acyl transferase, have been identified and studied over the last decade in relation to control of satiety, obesity, eating behaviours, metabolic syndrome, glucose homeostasis, and type 2 diabetes. However, the effects described are either small or nonsignificant and often subjected to contradictory conclusions between studies. In the last 18 months, several of these areas of investigations have been revisited under more controlled conditions or have been subjected to meta-analysis. SUMMARY: The effects of ghrelin gene polymorphism, is a complex area of investigation, due to ghrelin's interplay with a host of various factors part of an integrative network. However, taken together, results suggest that there are no or nonsignificant effects of the common genetic variants. A better understanding of the network, probably by a systems biology type approach, will be necessary to assign the exact role played by gene polymorphism of the component of the ghrelin axis.
Assuntos
Grelina/genética , Polimorfismo Genético , Saciação/fisiologia , Animais , Diabetes Mellitus Tipo 2/sangue , Diabetes Mellitus Tipo 2/genética , Modelos Animais de Doenças , Comportamento Alimentar , Grelina/sangue , Homeostase , Humanos , Síndrome Metabólica/sangue , Síndrome Metabólica/genética , Obesidade/sangue , Obesidade/genética , Receptores de Grelina/genética , Receptores de Grelina/metabolismoRESUMO
PURPOSE: Aquaporin-1 (AQP1) is involved in cell migration and proliferation; therefore, the purpose of the study was to investigate its expression in proliferative vitreoretinopathy (PVR) and epiretinal membranes (ERM). METHODS: 19 membranes from PVR and ERM were collected following eye surgery. AQP1 mRNA and protein expressions were determined by RT-qPCR and immunofluorescence in the membranes from PVR and ERM. RESULTS: AQP1 mRNA and protein were expressed in both PVR and ERM as shown by RT-qPCR and immunofluorescence. AQP1 protein expression was heterogeneous among and between PVR and ERM and colocalized with alpha-smooth muscle actin ( α SMA) and with glial fibrillary acidic protein (GFAP). There were a higher percentage of cells coexpressing AQP1 and α SMA than AQP1 and GFAP. GFAP and α SMA did not colocalize. CONCLUSION: Our data show for the first time AQP1 expression in both PVR and ERM. AQP1 is expressed mostly by the α SMA-positive cells, presumably myofibroblasts, but also by GFAP-positive cells, assumed to be glial cells. These original findings warrant further functional investigations aiming at studying the potential role of AQP1 in cell migration and proliferation occurring during the development of PVR and ERM.
Assuntos
Aquaporina 1/genética , Membrana Epirretiniana/genética , Expressão Gênica , Vitreorretinopatia Proliferativa/genética , Actinas/metabolismo , Adulto , Idoso , Idoso de 80 Anos ou mais , Aquaporina 1/metabolismo , Membrana Epirretiniana/metabolismo , Feminino , Proteína Glial Fibrilar Ácida/metabolismo , Humanos , Masculino , Pessoa de Meia-Idade , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Vitreorretinopatia Proliferativa/metabolismoRESUMO
UNLABELLED: BACGROUNS/AIMS: Several insulinotropic agents were recently reported to cause ß-cell swelling. The possible participation of AQP7 to water transport was investigated in AQP7(+/+) or AQP7(-/-) mice. METHODS: Aquaporin expression, insulin secretion, cell swelling and electrical activity were investigated in pancreatic islets. RESULTS: RT-PCR revealed the expression of AQP5 and AQP8 mRNA. Double immunofluorescent labeling indicated their presence in ß-cells. Whilst basal insulin release from isolated pancreatic islets incubated at 2.8 mM D-glucose did not differ between AQP7(+/+) or AQP7(-/-) mice, the secretion of insulin evoked by the omission of 50 mM NaCl, the substitution of 50 mM NaCl by 100 mM glycerol or a rise in D-glucose concentration to 8.3 mM and 16.7 mM was severely impaired in the islets from AQP7(-/-) mice. Yet, exposure of ß-cells to either the hypotonic medium or a rise in D-glucose concentration caused a similar degree of swelling and comparable pattern of electrical activity in cells from AQP7(+/+) and AQP7(-/-) mice. Both the cell swelling and change in membrane potential were only impaired in AQP7(-/-) cells when exposed to 50 mM glycerol. CONCLUSION: It is proposed, therefore, that AQP7 may, directly or indirectly, play a role at a distal site in the exocytotic pathway.
Assuntos
Aquaporinas/metabolismo , Aquaporinas/fisiologia , Insulina/metabolismo , Animais , Aquaporina 5/genética , Aquaporina 5/metabolismo , Aquaporinas/genética , Tamanho Celular/efeitos dos fármacos , Feminino , Glucose/farmacologia , Glicerol/farmacologia , Secreção de Insulina , Células Secretoras de Insulina/efeitos dos fármacos , Células Secretoras de Insulina/fisiologia , Masculino , Potenciais da Membrana/efeitos dos fármacos , Camundongos , Camundongos Knockout , Cloreto de Sódio/químicaRESUMO
The glucagon receptor belongs to the B family of G-protein coupled receptors. Little structural information is available about this receptor and its association with glucagon. We used the substituted cysteine accessibility method and three-dimensional molecular modeling based on the gastrointestinal insulinotropic peptide and glucagon-like peptide 1 receptor structures to study the N-terminal domain of this receptor, a central element for ligand binding and specificity. Our results showed that Asp(63), Arg(116), and Lys(98) are essential for the receptor structure and/or ligand binding because mutations of these three residues completely disrupted or markedly impaired the receptor function. In agreement with these data, our models revealed that Asp(63) and Arg(116) form a salt bridge, whereas Lys(98) is engaged in cation-π interactions with the conserved tryptophans 68 and 106. The native receptor could not be labeled by hydrophilic cysteine biotinylation reagents, but treatment of intact cells with [2-(trimethylammonium)ethyl]methanethiosulfonate increased the glucagon binding site density. This result suggested that an unidentified protein with at least one free cysteine associated with the receptor prevented glucagon recognition and that [2-(trimethylammonium)ethyl]methanethiosulfonate treatment relieved this inhibition. The substituted cysteine accessibility method was also performed on 15 residues selected using the three-dimensional models. Several receptor mutants, despite a relatively high predicted cysteine accessibility, could not be labeled by specific reagents. The three-dimensional models show that these mutated residues are located on one face of the protein. This could be part of the interface between the receptor and the unidentified inhibitory protein, making these residues inaccessible to biotinylation compounds.
Assuntos
Substituição de Aminoácidos , Modelos Moleculares , Receptores de Glucagon/química , Receptores de Glucagon/metabolismo , Linhagem Celular , Cisteína/química , Cisteína/genética , Cisteína/metabolismo , Humanos , Mutagênese , Mutação de Sentido Incorreto , Estrutura Terciária de Proteína , Receptores de Glucagon/genéticaRESUMO
Saliva secretion requires effective translocation of aquaporin 5 (AQP5) water channel to the salivary glands (SGs) acinar apical membrane. Patients with Sjögren's syndrome (SS) display abnormal AQP5 localization within acinar cells from SGs that correlate with sicca manifestation and glands hypofunction. Several proteins such as Prolactin-inducible protein (PIP) may regulate AQP5 trafficking as observed in lacrimal glands from mice. However, the role of the AQP5-PIP complex remains poorly understood. In the present study, we show that PIP interacts with AQP5 in vitro and in mice as well as in human SGs and that PIP misexpression correlates with an altered AQP5 distribution at the acinar apical membrane in PIP knockout mice and SS hMSG. Furthermore, our data show that the protein-protein interaction involves the AQP5 C-terminus and the N-terminal of PIP (one molecule of PIP per AQP5 tetramer). In conclusion, our findings highlight for the first time the role of PIP as a protein controlling AQP5 localization in human salivary glands but extend beyond due to the PIP-AQP5 interaction described in lung and breast cancers.
Assuntos
Aquaporina 5/metabolismo , Proteínas de Membrana Transportadoras/metabolismo , Glândulas Salivares/metabolismo , Síndrome de Sjogren/metabolismo , Células Acinares/metabolismo , Animais , Aquaporina 5/química , Aquaporina 5/genética , Sítios de Ligação , Linhagem Celular , Humanos , Proteínas de Membrana Transportadoras/química , Proteínas de Membrana Transportadoras/genética , Camundongos , Camundongos Knockout , Ligação Proteica , Síndrome de Sjogren/genéticaRESUMO
Primary Sjögren's syndrome (pSS) is a chronic systemic autoimmune rheumatic disease characterized by lymphoplasmacytic infiltration of the salivary and lacrimal glands, whereby sicca syndrome and/or systemic manifestations are the clinical hallmarks, associated with a particular autoantibody profile. pSS is the most frequent connective tissue disease after rheumatoid arthritis, affecting 0.3-3% of the population. Women are more prone to develop pSS than men, with a sex ratio of 9:1. Considered in the past as innocent collateral passive victims of autoimmunity, the epithelial cells of the salivary glands are now known to play an active role in the pathogenesis of the disease. The aetiology of the "autoimmune epithelitis" still remains unknown, but certainly involves genetic, environmental and hormonal factors. Later during the disease evolution, the subsequent chronic activation of B cells can lead to the development of systemic manifestations or non-Hodgkin's lymphoma. The aim of the present comprehensive review is to provide the current state of knowledge on pSS. The review addresses the clinical manifestations and complications of the disease, the diagnostic workup, the pathogenic mechanisms and the therapeutic approaches.
RESUMO
The main role of salivary glands (SG) is the production and secretion of saliva, in which aquaporins (AQPs) play a key role by ensuring water flow. The AQPs are transmembrane channel proteins permeable to water to allow water transport across cell membranes according to osmotic gradient. This review gives an insight into SG AQPs. Indeed, it gives a summary of the expression and localization of AQPs in adult human, rat and mouse SG, as well as of their physiological role in SG function. Furthermore, the review provides a comprehensive view of the involvement of AQPs in pathological conditions affecting SG, including Sjögren's syndrome, diabetes, agedness, head and neck cancer radiotherapy and SG cancer. These conditions are characterized by salivary hypofunction resulting in xerostomia. A specific focus is given on current and future therapeutic strategies aiming at AQPs to treat xerostomia. A deeper understanding of the AQPs involvement in molecular mechanisms of saliva secretion and diseases offered new avenues for therapeutic approaches, including drugs, gene therapy and tissue engineering. As such, AQP5 represents a potential therapeutic target in different strategies for the treatment of xerostomia.