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1.
Proc Natl Acad Sci U S A ; 121(21): e2318874121, 2024 May 21.
Artigo em Inglês | MEDLINE | ID: mdl-38753510

RESUMO

The single-pass transmembrane protein Stromal Interaction Molecule 1 (STIM1), located in the endoplasmic reticulum (ER) membrane, possesses two main functions: It senses the ER-Ca2+ concentration and directly binds to the store-operated Ca2+ channel Orai1 for its activation when Ca2+ recedes. At high resting ER-Ca2+ concentration, the ER-luminal STIM1 domain is kept monomeric but undergoes di/multimerization once stores are depleted. Luminal STIM1 multimerization is essential to unleash the STIM C-terminal binding site for Orai1 channels. However, structural basis of the luminal association sites has so far been elusive. Here, we employed molecular dynamics (MD) simulations and identified two essential di/multimerization segments, the α7 and the adjacent region near the α9-helix in the sterile alpha motif (SAM) domain. Based on MD results, we targeted the two STIM1 SAM domains by engineering point mutations. These mutations interfered with higher-order multimerization of ER-luminal fragments in biochemical assays and puncta formation in live-cell experiments upon Ca2+ store depletion. The STIM1 multimerization impeded mutants significantly reduced Ca2+ entry via Orai1, decreasing the Ca2+ oscillation frequency as well as store-operated Ca2+ entry. Combination of the ER-luminal STIM1 multimerization mutations with gain of function mutations and coexpression of Orai1 partially ameliorated functional defects. Our data point to a hydrophobicity-driven binding within the ER-luminal STIM1 multimer that needs to switch between resting monomeric and activated multimeric state. Altogether, these data reveal that interactions between SAM domains of STIM1 monomers are critical for multimerization and activation of the protein.


Assuntos
Proteínas de Neoplasias , Multimerização Proteica , Molécula 1 de Interação Estromal , Humanos , Sítios de Ligação , Cálcio/metabolismo , Retículo Endoplasmático/metabolismo , Células HEK293 , Simulação de Dinâmica Molecular , Proteínas de Neoplasias/metabolismo , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/química , Proteína ORAI1/metabolismo , Proteína ORAI1/genética , Proteína ORAI1/química , Ligação Proteica , Domínios Proteicos , Molécula 1 de Interação Estromal/metabolismo , Molécula 1 de Interação Estromal/genética , Molécula 1 de Interação Estromal/química
2.
ACS Nano ; 17(20): 19667-19684, 2023 10 24.
Artigo em Inglês | MEDLINE | ID: mdl-37812740

RESUMO

The TWEAK receptor, Fn14, is a promising candidate for active targeting of cancer nanotherapeutics to many solid tumor types, including metastatic breast and primary brain cancers. Targeting of therapeutic nanoparticles (NPs) has been accomplished using a range of targeting moieties including monoclonal antibodies and related fragments, peptides, and small molecules. Here, we investigated a full-length Fn14-specific monoclonal antibody, ITEM4, or an ITEM4-Fab fragment as a targeting moiety to guide the development of a clinical formulation. We formulated NPs with varying densities of the targeting moieties while maintaining the decreased nonspecific adhesivity with receptor targeting (DART) characteristics. To model the conditions that NPs experience following intravenous infusion, we investigated the impact of serum exposure in relation to the targeting moiety type and surface density. To further evaluate performance at the cancer cell level, we performed experiments to assess differences in cellular uptake and trafficking in several cancer cell lines using confocal microscopy, imaging flow cytometry, and total internal reflection fluorescence microscopy. We observed that Fn14-targeted NPs exhibit enhanced cellular uptake in Fn14-high compared to Fn14-low cancer cells and that in both cell lines uptake levels were greater than observed with control, nontargeted NPs. We found that serum exposure increased Fn14-targeted NP specificity while simultaneously reducing the total NP uptake. Importantly, serum exposure caused a larger reduction in cancer cell uptake over time when the targeting moiety was an antibody fragment (Fab region of the monoclonal antibody) compared with the full-length monoclonal antibody targeting moiety. Lastly, we uncovered that full monoclonal antibody-targeted NPs enter cancer cells via clathrin-mediated endocytosis and traffic through the endolysosomal pathway. Taken together, these results support a pathway for developing a clinical formulation using a full-length Fn14 monoclonal antibody as the targeting moiety for a DART cancer nanotherapeutic agent.


Assuntos
Nanopartículas , Neoplasias , Coroa de Proteína , Receptores do Fator de Necrose Tumoral/química , Receptores do Fator de Necrose Tumoral/metabolismo , Linhagem Celular Tumoral , Anticorpos Monoclonais , Nanopartículas/química
3.
Proc Natl Acad Sci U S A ; 119(3)2022 01 18.
Artigo em Inglês | MEDLINE | ID: mdl-35022238

RESUMO

Stromal interaction molecules, STIM1 and STIM2, sense decreases in the endoplasmic reticulum (ER) [Ca2+] ([Ca2+]ER) and cluster in ER-plasma membrane (ER-PM) junctions where they recruit and activate Orai1. While STIM1 responds when [Ca2+]ER is relatively low, STIM2 displays constitutive clustering in the junctions and is suggested to regulate basal Ca2+ entry. The cellular cues that determine STIM2 clustering under basal conditions is not known. By using gene editing to fluorescently tag endogenous STIM2, we report that endogenous STIM2 is constitutively localized in mobile and immobile clusters. The latter associate with ER-PM junctions and recruit Orai1 under basal conditions. Agonist stimulation increases immobile STIM2 clusters, which coordinate recruitment of Orai1 and STIM1 to the junctions. Extended synaptotagmin (E-Syt)2/3 are required for forming the ER-PM junctions, but are not sufficient for STIM2 clustering. Importantly, inositol 1,4,5-triphosphate receptor (IP3R) function and local [Ca2+]ER are the main drivers of immobile STIM2 clusters. Enhancing, or decreasing, IP3R function at ambient [IP3] causes corresponding increase, or attenuation, of immobile STIM2 clusters. We show that immobile STIM2 clusters denote decreases in local [Ca2+]ER mediated by IP3R that is sensed by the STIM2 N terminus. Finally, under basal conditions, ambient PIP2-PLC activity of the cell determines IP3R function, immobilization of STIM2, and basal Ca2+ entry while agonist stimulation augments these processes. Together, our findings reveal that immobilization of STIM2 clusters within ER-PM junctions, a first response to ER-Ca2+ store depletion, is facilitated by the juxtaposition of IP3R and marks a checkpoint for initiation of Ca2+ entry.


Assuntos
Receptores de Inositol 1,4,5-Trifosfato/química , Receptores de Inositol 1,4,5-Trifosfato/metabolismo , Molécula 2 de Interação Estromal/química , Molécula 2 de Interação Estromal/metabolismo , Cálcio/metabolismo , Sinalização do Cálcio/fisiologia , Membrana Celular/metabolismo , Análise por Conglomerados , Retículo Endoplasmático/metabolismo , Células HEK293 , Humanos , Receptores de Inositol 1,4,5-Trifosfato/genética , Proteínas de Neoplasias , Molécula 1 de Interação Estromal , Molécula 2 de Interação Estromal/genética
4.
J Cell Sci ; 134(9)2021 05 01.
Artigo em Inglês | MEDLINE | ID: mdl-34550354

RESUMO

Although RACK1 is known to act as a signaling hub in immune cells, its presence and role in mast cells (MCs) is undetermined. MC activation via antigen stimulation results in mediator release and is preceded by cytoskeleton reorganization and Ca2+ mobilization. In this study, we found that RACK1 was distributed throughout the MC cytoplasm both in vivo and in vitro. After RACK1 knockdown (KD), MCs were rounded, and the cortical F-actin was fragmented. Following antigen stimulation, in RACK1 KD MCs, there was a reduction in cortical F-actin, an increase in monomeric G-actin and a failure to organize F-actin. RACK1 KD also increased and accelerated degranulation. CD63+ secretory granules were localized in F-actin-free cortical regions in non-stimulated RACK1 KD MCs. Additionally, RACK1 KD increased antigen-stimulated Ca2+ mobilization, but attenuated antigen-stimulated depletion of ER Ca2+ stores and thapsigargin-induced Ca2+ entry. Following MC activation there was also an increase in interaction of RACK1 with Orai1 Ca2+-channels, ß-actin and the actin-binding proteins vinculin and MyoVa. These results show that RACK1 is a critical regulator of actin dynamics, affecting mediator secretion and Ca2+ signaling in MCs. This article has an associated First Person interview with the first author of the paper.


Assuntos
Actinas , Cálcio , Citoesqueleto de Actina , Actinas/genética , Humanos , Mastócitos , Proteínas de Neoplasias/genética , Receptores de Quinase C Ativada/genética , Tapsigargina
5.
Proc Natl Acad Sci U S A ; 117(28): 16638-16648, 2020 07 14.
Artigo em Inglês | MEDLINE | ID: mdl-32601188

RESUMO

The Orai1 channel is regulated by stromal interaction molecules STIM1 and STIM2 within endoplasmic reticulum (ER)-plasma membrane (PM) contact sites. Ca2+ signals generated by Orai1 activate Ca2+-dependent gene expression. When compared with STIM1, STIM2 is a weak activator of Orai1, but it has been suggested to have a unique role in nuclear factor of activated T cells 1 (NFAT1) activation triggered by Orai1-mediated Ca2+ entry. In this study, we examined the contribution of STIM2 in NFAT1 activation. We report that STIM2 recruitment of Orai1/STIM1 to ER-PM junctions in response to depletion of ER-Ca2+ promotes assembly of the channel with AKAP79 to form a signaling complex that couples Orai1 channel function to the activation of NFAT1. Knockdown of STIM2 expression had relatively little effect on Orai1/STIM1 clustering or local and global [Ca2+]i increases but significantly attenuated NFAT1 activation and assembly of Orai1 with AKAP79. STIM1ΔK, which lacks the PIP2-binding polybasic domain, was recruited to ER-PM junctions following ER-Ca2+ depletion by binding to Orai1 and caused local and global [Ca2+]i increases comparable to those induced by STIM1 activation of Orai1. However, in contrast to STIM1, STIM1ΔK induced less NFAT1 activation and attenuated the association of Orai1 with STIM2 and AKAP79. Orai1-AKAP79 interaction and NFAT1 activation were recovered by coexpressing STIM2 with STIM1ΔK. Replacing the PIP2-binding domain of STIM1 with that of STIM2 eliminated the requirement of STIM2 for NFAT1 activation. Together, these data demonstrate an important role for STIM2 in coupling Orai1-mediated Ca2+ influx to NFAT1 activation.


Assuntos
Proteínas de Ancoragem à Quinase A/metabolismo , Cálcio/metabolismo , Fatores de Transcrição NFATC/metabolismo , Proteínas de Neoplasias/metabolismo , Proteína ORAI1/metabolismo , Molécula 1 de Interação Estromal/metabolismo , Molécula 2 de Interação Estromal/metabolismo , Proteínas de Ancoragem à Quinase A/genética , Membrana Celular/genética , Membrana Celular/metabolismo , Retículo Endoplasmático/genética , Retículo Endoplasmático/metabolismo , Células HEK293 , Humanos , Fatores de Transcrição NFATC/genética , Proteínas de Neoplasias/genética , Proteína ORAI1/genética , Ligação Proteica , Transdução de Sinais , Molécula 1 de Interação Estromal/genética , Molécula 2 de Interação Estromal/genética
6.
PLoS Biol ; 18(4): e3000700, 2020 04.
Artigo em Inglês | MEDLINE | ID: mdl-32330125

RESUMO

Trimeric intracellular cation (TRIC) channels have been proposed to modulate Ca2+ release from the endoplasmic reticulum (ER) and determine oscillatory Ca2+ signals. Here, we report that TRIC-A-mediated amplitude and frequency modulation of ryanodine receptor 2 (RyR2)-mediated Ca2+ oscillations and inositol 1,4,5-triphosphate receptor (IP3R)-induced cytosolic signals is based on attenuating store-operated Ca2+ entry (SOCE). Further, TRIC-A-dependent delay in ER Ca2+ store refilling contributes to shaping the pattern of Ca2+ oscillations. Upon ER Ca2+ depletion, TRIC-A clusters with stromal interaction molecule 1 (STIM1) and Ca2+-release-activated Ca2+ channel 1 (Orai1) within ER-plasma membrane (PM) junctions and impairs assembly of the STIM1/Orai1 complex, causing a decrease in Orai1-mediated Ca2+ current and SOCE. Together, our findings demonstrate that TRIC-A is a negative regulator of STIM1/Orai1 function. Thus, aberrant SOCE could contribute to muscle disorders associated with loss of TRIC-A.


Assuntos
Canais Iônicos/metabolismo , Proteínas de Neoplasias/metabolismo , Proteína ORAI1/metabolismo , Molécula 1 de Interação Estromal/metabolismo , Animais , Sinalização do Cálcio/fisiologia , Membrana Celular/metabolismo , Retículo Endoplasmático/metabolismo , Células HEK293 , Humanos , Masculino , Camundongos Endogâmicos C57BL , Complexos Multiproteicos/metabolismo , Proteínas de Neoplasias/genética , Proteína ORAI1/genética , Técnicas de Patch-Clamp , Canal de Liberação de Cálcio do Receptor de Rianodina/metabolismo , Molécula 1 de Interação Estromal/genética
7.
Biochim Biophys Acta Mol Cell Res ; 1866(7): 1037-1045, 2019 07.
Artigo em Inglês | MEDLINE | ID: mdl-30521873

RESUMO

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.


Assuntos
Sinalização do Cálcio/fisiologia , Cálcio/metabolismo , Membrana Celular/metabolismo , Retículo Endoplasmático/metabolismo , Proteína ORAI1/metabolismo , Canais de Cátion TRPC/metabolismo , Animais , Humanos , Proteínas de Neoplasias/metabolismo , Molécula 1 de Interação Estromal/metabolismo , Molécula 2 de Interação Estromal/metabolismo
8.
Cells ; 7(7)2018 Jul 11.
Artigo em Inglês | MEDLINE | ID: mdl-29997338

RESUMO

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.

9.
Cell Rep ; 23(2): 522-534, 2018 Apr 10.
Artigo em Inglês | MEDLINE | ID: mdl-29642009

RESUMO

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.


Assuntos
Membrana Celular/metabolismo , Retículo Endoplasmático/metabolismo , Proteínas de Neoplasias/metabolismo , Proteína ORAI1/metabolismo , Molécula 1 de Interação Estromal/metabolismo , Molécula 2 de Interação Estromal/metabolismo , Cálcio/metabolismo , Sinalização do Cálcio/efeitos dos fármacos , Células HEK293 , Humanos , Indóis/farmacologia , Microscopia Confocal , Fatores de Transcrição NFATC/metabolismo , Proteínas de Neoplasias/antagonistas & inibidores , Proteínas de Neoplasias/genética , Proteína ORAI1/antagonistas & inibidores , Proteína ORAI1/genética , Técnicas de Patch-Clamp , Conformação Proteica , Interferência de RNA , RNA Interferente Pequeno/metabolismo , Molécula 1 de Interação Estromal/antagonistas & inibidores , Molécula 1 de Interação Estromal/genética , Molécula 2 de Interação Estromal/genética
10.
Sci Signal ; 10(482)2017 Jun 06.
Artigo em Inglês | MEDLINE | ID: mdl-28588080

RESUMO

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.


Assuntos
Canais de Cálcio/metabolismo , Caspase 3/metabolismo , Radioterapia/efeitos adversos , Glândulas Salivares/patologia , Glândulas Salivares/efeitos da radiação , Molécula 1 de Interação Estromal/metabolismo , Células Acinares/metabolismo , Células Acinares/patologia , Células Acinares/efeitos da radiação , Animais , Cálcio/metabolismo , Canais de Cálcio/genética , Caspase 3/genética , Células Cultivadas , Humanos , Potencial da Membrana Mitocondrial/efeitos da radiação , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Mitocôndrias/metabolismo , Mitocôndrias/patologia , Mitocôndrias/efeitos da radiação , Proteína ORAI1/genética , Proteína ORAI1/metabolismo , Glândulas Salivares/metabolismo , Molécula 1 de Interação Estromal/genética , Canais de Cátion TRPM/metabolismo , Raios X
11.
Adv Exp Med Biol ; 981: 253-276, 2017.
Artigo em Inglês | MEDLINE | ID: mdl-29594865

RESUMO

Store-operated calcium entry (SOCE), a unique plasma membrane Ca2+ entry mechanism, is activated when ER-[Ca2+] is decreased. SOCE is mediated via the primary channel, Orai1, as well as others such as TRPC1. STIM1 and STIM2 are ER-Ca2+ sensor proteins that regulate Orai1 and TRPC1. SOCE requires assembly of STIM proteins with the plasma membrane channels which occurs within distinct regions in the cell that have been termed as endoplasmic reticulum (ER)-plasma membrane (PM) junctions. The PM and ER are in close proximity to each other within this region, which allows STIM1 in the ER to interact with and activate either Orai1 or TRPC1 in the plasma membrane. Activation and regulation of SOCE involves dynamic assembly of various components that are involved in mediating Ca2+ entry as well as those that determine the formation and stabilization of the junctions. These components include proteins in the cytosol, ER and PM, as well as lipids in the PM. Recent studies have also suggested that SOCE and its components are compartmentalized within ER-PM junctions and that this process might require remodeling of the plasma membrane lipids and reorganization of structural and scaffolding proteins. Such compartmentalization leads to the generation of spatially- and temporally-controlled Ca2+signals that are critical for regulating many downstream cellular functions.


Assuntos
Sinalização do Cálcio/fisiologia , Cálcio/metabolismo , Membrana Celular/metabolismo , Retículo Endoplasmático/metabolismo , Proteína ORAI1/metabolismo , Canais de Cátion TRPC/metabolismo , Animais , Membrana Celular/genética , Retículo Endoplasmático/genética , Humanos , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/metabolismo , Proteína ORAI1/genética , Molécula 1 de Interação Estromal/genética , Molécula 1 de Interação Estromal/metabolismo , Molécula 2 de Interação Estromal/genética , Molécula 2 de Interação Estromal/metabolismo , Canais de Cátion TRPC/genética
12.
EBioMedicine ; 10: 216-26, 2016 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-27381477

RESUMO

Primary Sjögren's syndrome (pSS) is a systemic autoimmune disease that is associated with inflammation and dysfunction of salivary and lacrimal glands. The molecular mechanism(s) underlying this exocrinopathy is not known, although the syndrome has been associated with viruses, such as the Epstein Barr Virus (EBV). We report herein that an EBV-specific microRNA (ebv-miR-BART13-3p) is significantly elevated in salivary glands (SGs) of pSS patients and we show that it targets stromal interacting molecule 1 (STIM1), a primary regulator of the store-operated Ca(2+) entry (SOCE) pathway that is essential for SG function, leading to loss of SOCE and Ca(2+)-dependent activation of NFAT. Although EBV typically infects B cells and not salivary epithelial cells, ebv-miR-BART13-3p is present in both cell types in pSS SGs. Importantly, we further demonstrate that ebv-miR-BART13-3p can be transferred from B cells to salivary epithelial cells through exosomes and it recapitulates its functional effects on calcium signaling in a model system.


Assuntos
Exossomos/metabolismo , MicroRNAs/genética , MicroRNAs/metabolismo , Síndrome de Sjogren/etiologia , Síndrome de Sjogren/metabolismo , Molécula 1 de Interação Estromal/genética , Molécula 1 de Interação Estromal/metabolismo , Aquaporina 5/genética , Linfócitos B/imunologia , Linfócitos B/metabolismo , Transporte Biológico , Regulação para Baixo , Células Epiteliais/metabolismo , Humanos , Fatores de Transcrição NFATC/metabolismo , Interferência de RNA , Glândulas Salivares/metabolismo
13.
Adv Exp Med Biol ; 898: 87-109, 2016.
Artigo em Inglês | MEDLINE | ID: mdl-27161226

RESUMO

Store-operated calcium entry (SOCE) is a ubiquitous Ca(2+) entry pathway that is activated in response to depletion of Ca(2+) stores within the endoplasmic reticulum (ER) and contributes to the control of various physiological functions in a wide variety of cell types. The transient receptor potential canonical (TRPC) channels (TRPCs 1-7), that are activated by stimuli leading to PIP2 hydrolysis, were first identified as molecular components of SOCE channels. TRPC channels show a miscellany of tissue expression, physiological functions and channel properties. However, none of the TRPC members display currents that resemble I CRAC. Intensive search for the CRAC channel component led to identification of Orai1 and STIM1, now established as being the primary constituents of the CRAC channel. There is now considerable evidence that STIM1 activates both Orai1 and TRPC1 via distinct domains in its C-terminus. Intriguingly, TRPC1 function is not only dependent on STIM1 but also requires Orai1. The critical functional interaction between TRPC1 and Orai1, which determines the activation of TRPC1, has also been identified. In this review, we will discuss current concepts regarding the role of TRPC channels in SOCE, the physiological functions regulated by TRPC-mediated SOCE, and the complex mechanisms underlying the regulation of TRPCs, including the functional interactions with Orai1 and STIM1.


Assuntos
Cálcio/metabolismo , Canais de Cátion TRPC/fisiologia , Moléculas de Adesão Celular/metabolismo , Humanos , Transporte de Íons , Proteínas de Membrana/metabolismo , Proteínas de Neoplasias/metabolismo , Molécula 1 de Interação Estromal , Molécula 2 de Interação Estromal , Canais de Cátion TRPC/metabolismo
14.
J Biol Chem ; 291(16): 8709-20, 2016 Apr 15.
Artigo em Inglês | MEDLINE | ID: mdl-26903518

RESUMO

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.


Assuntos
Sinalização do Cálcio/fisiologia , Cálcio/metabolismo , Células Epiteliais/metabolismo , Fatores de Transcrição NFATC/metabolismo , Glândulas Salivares/metabolismo , Regulação para Cima/fisiologia , Aquaporina 5/biossíntese , Aquaporina 5/genética , Canais de Cálcio/biossíntese , Células Cultivadas , Células Epiteliais/citologia , Humanos , Fatores de Transcrição NFATC/genética , Glândulas Salivares/citologia
15.
Biochim Biophys Acta ; 1853(10 Pt A): 2709-21, 2015 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-26232624

RESUMO

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.


Assuntos
Canais de Cálcio/metabolismo , Membrana Celular/metabolismo , Endocitose/fisiologia , Retículo Endoplasmático/metabolismo , Proteínas de Membrana/metabolismo , Proteínas de Neoplasias/metabolismo , Canais de Cátion TRPC/metabolismo , Fator 6 de Ribosilação do ADP , Fatores de Ribosilação do ADP/genética , Fatores de Ribosilação do ADP/metabolismo , Cálcio/metabolismo , Canais de Cálcio/genética , Membrana Celular/genética , Retículo Endoplasmático/genética , Células HEK293 , Humanos , Proteínas de Membrana/genética , Proteínas de Neoplasias/genética , Proteína ORAI1 , Molécula 1 de Interação Estromal , Canais de Cátion TRPC/genética , Proteínas rab4 de Ligação ao GTP/genética , Proteínas rab4 de Ligação ao GTP/metabolismo , Proteínas rab5 de Ligação ao GTP/genética , Proteínas rab5 de Ligação ao GTP/metabolismo
16.
Sci Signal ; 8(359): ra3, 2015 Jan 13.
Artigo em Inglês | MEDLINE | ID: mdl-25587190

RESUMO

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.


Assuntos
Sinalização do Cálcio/fisiologia , Moléculas de Adesão Celular/metabolismo , Membrana Celular/metabolismo , Retículo Endoplasmático/metabolismo , Proteínas de Membrana/metabolismo , Proteínas de Neoplasias/metabolismo , Células Acinares/metabolismo , Análise de Variância , Animais , Proteínas de Bactérias , Western Blotting , Técnicas de Silenciamento de Genes , Vetores Genéticos/genética , Células HEK293 , Humanos , Proteínas Luminescentes , Camundongos , Microscopia Confocal , Microscopia de Fluorescência , Mutagênese Sítio-Dirigida , RNA Interferente Pequeno/genética , Saliva/citologia , Análise de Sequência de DNA , Molécula 1 de Interação Estromal , Molécula 2 de Interação Estromal
17.
Handb Exp Pharmacol ; 223: 1005-34, 2014.
Artigo em Inglês | MEDLINE | ID: mdl-24961978

RESUMO

The TRP-canonical (TRPC) subfamily, which consists of seven members (TRPC1-TRPC7), are Ca(2+)-permeable cation channels that are activated in response to receptor-mediated PIP2 hydrolysis via store-dependent and store-independent mechanisms. These channels are involved in a variety of physiological functions in different cell types and tissues. Of these, TRPC6 has been linked to a channelopathy resulting in human disease. Two key players of the store-dependent regulatory pathway, STIM1 and Orai1, interact with some TRPC channels to gate and regulate channel activity. The Ca(2+) influx mediated by TRPC channels generates distinct intracellular Ca(2+) signals that regulate downstream signaling events and consequent cell functions. This requires localization of TRPC channels in specific plasma membrane microdomains and precise regulation of channel function which is coordinated by various scaffolding, trafficking, and regulatory proteins.


Assuntos
Canais de Cátion TRPC/fisiologia , Cálcio/metabolismo , Canais de Cálcio/fisiologia , Humanos , Microdomínios da Membrana/metabolismo , Proteínas de Membrana/fisiologia , Proteínas de Neoplasias/fisiologia , Proteína ORAI1 , Molécula 1 de Interação Estromal
18.
Curr Top Membr ; 71: 149-79, 2013.
Artigo em Inglês | MEDLINE | ID: mdl-23890115

RESUMO

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+).


Assuntos
Sinalização do Cálcio , Canais de Cátion TRPC/fisiologia , Animais , Canais de Cálcio/metabolismo , Retículo Endoplasmático/metabolismo , Humanos , Microdomínios da Membrana/metabolismo , Proteínas de Membrana/metabolismo , Proteínas de Neoplasias/metabolismo , Proteína ORAI1 , Multimerização Proteica , Molécula 1 de Interação Estromal
19.
PLoS One ; 7(9): e42541, 2012.
Artigo em Inglês | MEDLINE | ID: mdl-23049731

RESUMO

BACKGROUND: Endothelial progenitor cells (EPCs) may be recruited from bone marrow to sustain tumor vascularisation and promote the metastatic switch. Understanding the molecular mechanisms driving EPC proliferation and tubulogenesis could outline novel targets for alternative anti-angiogenic treatments. Store-operated Ca(2+) entry (SOCE), which is activated by a depletion of the intracellular Ca(2+) pool, regulates the growth of human EPCs, where is mediated by the interaction between the endoplasmic reticulum Ca(2+)-sensor, Stim1, and the plasmalemmal Ca(2+) channel, Orai1. As oncogenesis may be associated to the capability of tumor cells to grow independently on Ca(2+) influx, it is important to assess whether SOCE regulates EPC-dependent angiogenesis also in tumor patients. METHODOLOGY/PRINCIPAL FINDINGS: The present study employed Ca(2+) imaging, recombinant sub-membranal and mitochondrial aequorin, real-time polymerase chain reaction, gene silencing techniques and western blot analysis to investigate the expression and the role of SOCE in EPCs isolated from peripheral blood of patients affected by renal cellular carcinoma (RCC; RCC-EPCs) as compared to control EPCs (N-EPCs). SOCE, activated by either pharmacological (i.e. cyclopiazonic acid) or physiological (i.e. ATP) stimulation, was significantly higher in RCC-EPCs and was selectively sensitive to BTP-2, and to the trivalent cations, La(3+) and Gd(3+). Furthermore, 2-APB enhanced thapsigargin-evoked SOCE at low concentrations, whereas higher doses caused SOCE inhibition. Conversely, the anti-angiogenic drug, carboxyamidotriazole (CAI), blocked both SOCE and the intracellular Ca(2+) release. SOCE was associated to the over-expression of Orai1, Stim1, and transient receptor potential channel 1 (TRPC1) at both mRNA and protein level The intracellular Ca(2+) buffer, BAPTA, BTP-2, and CAI inhibited RCC-EPC proliferation and tubulogenesis. The genetic suppression of Stim1, Orai1, and TRPC1 blocked CPA-evoked SOCE in RCC-EPCs. CONCLUSIONS: SOCE is remodelled in EPCs from RCC patients and stands out as a novel molecular target to interfere with RCC vascularisation due to its ability to control proliferation and tubulogenesis.


Assuntos
Carcinoma de Células Renais/irrigação sanguínea , Células Endoteliais/metabolismo , Regulação Neoplásica da Expressão Gênica , Neoplasias Renais/irrigação sanguínea , Proteínas de Membrana/genética , Células-Tronco Neoplásicas/metabolismo , Trifosfato de Adenosina/farmacologia , Adulto , Idoso , Idoso de 80 Anos ou mais , Compostos de Boro/farmacologia , Cádmio/farmacologia , Canais de Cálcio/genética , Canais de Cálcio/metabolismo , Carcinoma de Células Renais/genética , Carcinoma de Células Renais/metabolismo , Células Endoteliais/efeitos dos fármacos , Células Endoteliais/patologia , Feminino , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Humanos , Indóis/farmacologia , Proteínas Sensoras de Cálcio Intracelular , Neoplasias Renais/genética , Neoplasias Renais/metabolismo , Lantânio/farmacologia , Masculino , Proteínas de Membrana/metabolismo , Pessoa de Meia-Idade , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/metabolismo , Células-Tronco Neoplásicas/efeitos dos fármacos , Células-Tronco Neoplásicas/patologia , Neovascularização Patológica , Proteína ORAI1 , Cultura Primária de Células , Transdução de Sinais/efeitos dos fármacos , Molécula 1 de Interação Estromal , Canais de Cátion TRPC/genética , Canais de Cátion TRPC/metabolismo
20.
Proc Natl Acad Sci U S A ; 109(33): 13434-9, 2012 Aug 14.
Artigo em Inglês | MEDLINE | ID: mdl-22778404

RESUMO

In vivo recycling of nitrate (NO(3)(-)) and nitrite (NO(2)(-)) is an important alternative pathway for the generation of nitric oxide (NO) and maintenance of systemic nitrate-nitrite-NO balance. More than 25% of the circulating NO(3)(-) is actively removed and secreted by salivary glands. Oral commensal bacteria convert salivary NO(3)(-) to NO(2)(-), which enters circulation and leads to NO generation. The transporters for NO(3)(-) in salivary glands have not yet been identified. Here we report that sialin (SLC17A5), mutations in which cause Salla disease and infantile sialic acid storage disorder (ISSD), functions as an electrogenic 2NO(3)(-)/H(+) cotransporter in the plasma membrane of salivary gland acinar cells. We have identified an extracellular pH-dependent anion current that is carried by NO(3)(-) or sialic acid (SA), but not by Br(-), and is accompanied by intracellular acidification. Both responses were reduced by knockdown of sialin expression and increased by the plasma membrane-targeted sialin mutant (L22A-L23A). Fibroblasts from patients with ISSD displayed reduced SA- and NO(3)(-)-induced currents compared with healthy controls. Furthermore, expression of disease-associated sialin mutants in fibroblasts and salivary gland cells suppressed the H(+)-dependent NO(3)(-) conductance. Importantly, adenovirus-dependent expression of the sialinH183R mutant in vivo in pig salivary glands decreased NO(3)(-) secretion in saliva after intake of a NO(3)(-)-rich diet. Taken together, these data demonstrate that sialin mediates nitrate influx into salivary gland and other cell types. We suggest that the 2NO(3)(-)/H(+) transport function of sialin in salivary glands can contribute significantly to clearance of serum nitrate, as well as nitrate recycling and physiological nitrite-NO homeostasis.


Assuntos
Proteínas de Transporte de Ânions/metabolismo , Membrana Celular/metabolismo , Transportadores de Ânions Orgânicos/metabolismo , Simportadores/metabolismo , Ácidos/metabolismo , Adenoviridae/metabolismo , Animais , Ânions , Transporte Biológico , Fibroblastos/metabolismo , Fibroblastos/patologia , Espaço Intracelular/metabolismo , Mutação/genética , Ácido N-Acetilneuramínico/metabolismo , Transportadores de Nitrato , Nitratos/metabolismo , Transportadores de Ânions Orgânicos/genética , Prótons , Doença do Armazenamento de Ácido Siálico/metabolismo , Glândula Submandibular/citologia , Glândula Submandibular/metabolismo , Sus scrofa , Simportadores/genética
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