RESUMEN
The receptor-receptor interaction (RRI) of G protein-coupled receptors (GPCRs) leads to new functional entities that are conceptually distinct from the simple addition of signals mediated by the activation of the receptors that form the heteromers. Focusing on astrocytes, there is evidence for the existence of inhibitory and facilitatory RRIs, including the heteromers formed by the adenosine A2A and the dopamine D2 receptors, by A2A and the oxytocin receptor (OTR), and the D2-OTR heteromers. The possible involvement of these receptors in mosaicism has never been investigated in striatal astrocytes. By biophysical and functional approaches, we focused our attention on the existence of an A2A-D2-OTR high-order receptor complex and its role in modulating cytosolic calcium levels and endogenous glutamate release, when striatal astrocyte processes were stimulated with 4-aminopyridine. Functional data indicate a permissive role of OTR on dopamine signaling in the regulation of the glutamatergic transmission, and an inhibitory control mediated by A2A on both the D2-mediated signaling and on the OTR-facilitating effect on D2. Imaging biochemical and bioinformatic evidence confirmed the existence of the A2A-D2-OTR complex and its ternary structure in the membrane. In conclusion, the D2 receptor appears to be a hotspot in the control of the glutamate release from the astrocytic processes and may contribute to the regulation and integration of different neurotransmitter-mediated signaling in the striatum by the A2A-D2-OTR heterotrimers. Considering the possible selectivity of allosteric interventions on GPCRs organized as receptor mosaics, A2A-D2-OTR heterotrimers may offer selective pharmacological targets in neuropsychiatric disorders and neurodegenerative diseases.
Asunto(s)
Astrocitos , Cuerpo Estriado , Dopamina , Receptor de Adenosina A2A , Receptores de Dopamina D2 , Transducción de Señal , Astrocitos/metabolismo , Animales , Receptor de Adenosina A2A/metabolismo , Cuerpo Estriado/metabolismo , Cuerpo Estriado/citología , Receptores de Dopamina D2/metabolismo , Dopamina/metabolismo , Receptores de Oxitocina/metabolismo , Receptores de Oxitocina/genética , Humanos , Calcio/metabolismo , Ácido Glutámico/metabolismo , RatonesRESUMEN
Glioblastoma (GBM) is the most malignant brain tumor, characterized by cell heterogeneity comprising stem cells (GSCs) responsible for aggressiveness. The calpain/calpastatin (calp/cast) proteolytic system is involved in critical physiological processes and cancer progression. In this work we showed the expression profile of hcast 3-25 (a Type III calpastatin variant devoid of inhibitory units) and the members of the system in several patient-derived GSCs exploring the relationship between hcast 3-25 and activation/activity of calpains. Each GSC shows a peculiar calp/cast mRNA and protein expression pattern, and hcast 3-25 is the least expressed. Differentiation promotes upregulation of all the calp/cast system components except hcast 3-25 mRNA, which increased or decreased depending on individual GSC culture. Transfection of hcast 3-25-V5 into two selected GSCs indicated that hcast 3-25 effectively associates with calpains, supporting the digestion of selected calpain targets. Hcast 3-25 possibly affects the stem state promoting a differentiated, less aggressive phenotype.
RESUMEN
High-pressure and temperature extraction (HPTE) can effectively recover bioactive compounds from olive pomace (OP). HPTE extract obtained by extracting OP with ethanol and water (50:50 v/v) at 180 °C for 90 min demonstrated a pronounced ability to preserve intracellular calcium homeostasis, shielding neurons from the harmful effects induced by N-methyl-d-aspartate (NMDA) receptor (NMDAR) overactivation, such as aberrant calpain activation. In this study, the extraction temperature was changed from 37 to 180 °C, and the extracts were evaluated for their antioxidant potency and ability to preserve crucial intracellular Ca2+-homeostasis necessary for neuronal survival. Additionally, to verify the temperature-induced activity of the extract, further extractions on the exhausted olive pomace were conducted, aiming to identify variations in the quality and quantity of extracted phenolic molecules through HPLC analysis. The results revealed a significant increase in bioactive compounds as a function of temperature variation, reaching 6.31 ± 0.09 mgCAE/mL extract for the extraction performed at 180 °C. Subsequent extraction of the exhausted residues yielded extracts that remained active in preventing calcium-induced cell death. Moreover, despite increased antiradical power, extracts re-treated at 180 °C did not display cell protection activity. Our results indicate that the molecules able to maintain physiological Ca2+-homeostasis in murine cortical neurons in conditions of cytotoxic stimulation of NMDAR are wholly recovered from olive pomace only following extraction performed at 180 °C.
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Olea , Animales , Ratones , Calcio , Temperatura , Neuronas , Receptores de N-Metil-D-Aspartato , Extractos Vegetales/farmacologíaRESUMEN
BACKGROUND: The most recent modulator combination, elexacaftor/tezacaftor/ivacaftor (Trikafta®), has been shown to improve clinical outcomes in most patients with cystic fibrosis (PwCF). Unfortunately, the clinical benefits are sometimes variable; thus, improving our knowledge of the possible causes of this variability can help reduce it. METHODS: Circulating mononuclear cells (CMCs) and plasma were collected from 16 PwCF (including those on Trikafta® therapy) and 4 non-CF subjects. Cystic fibrosis transmembrane conductance regulator (CFTR) activity and matrix metalloprotease 9 (MMP9) expression were monitored before and after therapy, together with some clinical parameters. The relationship between MMP9 expression and the modulation of the extracellular-regulated 1/2 (ERK1/2) and nuclear factor-kB (NF-kB) pathways was also analyzed. RESULTS: MMP9, markedly expressed in the CMCs and plasma of all the patients included in the study, was downregulated in the clinically responsive PwCF. In the non-responder, the MMP9 levels remained high. The modulation of MMP9 following treatment with Trikafta® may be controlled by the NF-kB pathway. CONCLUSIONS: These data strongly suggest that MMP9 downregulation is a potential biomarker of therapy efficacy and that it could be useful in understanding the molecular events underlying the variable clinical responses of patients to Trikafta®. This knowledge could be helpful for future studies of personalized medicine and thereby ensure improvements in individual responses to therapies.
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Regulador de Conductancia de Transmembrana de Fibrosis Quística , Fibrosis Quística , Humanos , Regulador de Conductancia de Transmembrana de Fibrosis Quística/genética , Fibrosis Quística/tratamiento farmacológico , Fibrosis Quística/genética , Metaloproteinasa 9 de la Matriz/genética , FN-kappa BRESUMEN
The ability of oxytocin (OT) to interact with the dopaminergic system through facilitatory D2-OT receptor (OTR) receptor-receptor interaction in the limbic system is increasingly considered to play roles in social or emotional behavior, and suggested to serve as a potential therapeutic target. Although roles of astrocytes in the modulatory effects of OT and dopamine in the central nervous system are well recognized, the possibility of D2-OTR receptor-receptor interaction in astrocytes has been neglected. In purified astrocyte processes from adult rat striatum, we assessed OTR and dopamine D2 receptor expression by confocal analysis. The effects of activation of these receptors were evaluated in the processes through a neurochemical study of glutamate release evoked by 4-aminopyridine; D2-OTR heteromerization was assessed by co-immunoprecipitation and proximity ligation assay (PLA). The structure of the possible D2-OTR heterodimer was estimated by a bioinformatic approach. We found that both D2 and OTR were expressed on the same astrocyte processes and controlled the release of glutamate, showing a facilitatory receptor-receptor interaction in the D2-OTR heteromers. Biochemical and biophysical evidence confirmed D2-OTR heterodimers on striatal astrocytes. The residues in the transmembrane domains four and five of both receptors are predicted to be mainly involved in the heteromerization. In conclusion, roles for astrocytic D2-OTR in the control of glutamatergic synapse functioning through modulation of astrocytic glutamate release should be taken into consideration when considering interactions between oxytocinergic and dopaminergic systems in striatum.
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Astrocitos , Cuerpo Estriado , Receptores de Dopamina D2 , Receptores de Oxitocina , Animales , Ratas , Astrocitos/metabolismo , Cuerpo Estriado/metabolismo , Dopamina/metabolismo , Ácido Glutámico/metabolismo , Oxitocina/metabolismo , Receptores de Oxitocina/química , Receptores de Oxitocina/metabolismo , Receptores de Dopamina D2/química , Receptores de Dopamina D2/metabolismoRESUMEN
In this case report, we demonstrate how the correct positioning of implants, associated with optimal gingival conditioning, and the correct choice of biomaterial can yield very predictable and fantastic aesthetic results. OBJECTIVE: We aimed to use dental implants to rehabilitate the area of elements #11 and #21 in a satisfactory surgical and prosthetic manner, using guided surgery, connective tissue, nano-biomaterials, and a porcelain prosthesis. CASE REPORT: A 32-year-old male patient presented with bone loss of elements #11 and #21, which was proven radiographically and clinically. Thus, oral rehabilitation with the use of dental implants was required. It was decided to proceed via digital planning with the DSD program (Digital smile design) and with the software Exoplan, (Smart Dent-Germany) whenever it was possible to plan immediate provisional and accurate dental implant positioning through reverse diagnostics (Software Exoplan, Smart Dent-German). The dental elements were extracted atraumatically; then, a guide was established, the implants were positioned, the prosthetic components were placed, the conjunctive tissue was removed from the palate and redirected to the vestibular wall of the implants, the nano-graft (Blue Bone®) was conditioned in the gaps between the vestibular wall and the implants, and, finally, the cemented provision was installed. RESULTS: After a 5-month accompaniment, an excellent remodeling of the tissues had been achieved by the implants; consequently, the final prosthetic stage could begin, which also achieved a remarkable aesthetic result. CONCLUSIONS: This report demonstrates that the correct planning of dental implants, which is associated with appropriate soft tissue and bone manipulation, allows for the achievement of admirable clinical results.
RESUMEN
In mammalian cells, the content of polyamines is tightly regulated. Polyamines, including spermine, spermidine and putrescine, are involved in many cellular processes. Spermine oxidase specifically oxidizes spermine, and its deregulated activity has been reported to be linked to brain pathologies involving neuron damage. Spermine is a neuromodulator of a number of ionotropic glutamate receptors and types of ion channels. In this respect, the Dach-SMOX mouse model overexpressing spermine oxidase in the neocortex neurons was revealed to be a model of chronic oxidative stress, excitotoxicity and neuronal damage. Reactive astrocytosis, chronic oxidative and excitotoxic stress, neuron loss and the susceptibility to seizure in the Dach-SMOX are discussed here. This genetic model would help researchers understand the linkage between polyamine dysregulation and neurodegeneration and unveil the roles of polyamines in the crosstalk between astrocytes and neurons in neuroprotection or neurodegeneration.
RESUMEN
BACKGROUND: Roles of astrocytes in the modulatory effects of oxytocin (OT) in central nervous system are increasingly considered. Nevertheless, OT effects on gliotransmitter release have been neglected. METHODS: In purified astrocyte processes from adult rat striatum, we assessed OT receptor (OTR) and adenosine A2A receptor expression by confocal analysis. The effects of receptors activation on glutamate release from the processes were evaluated; A2A-OTR heteromerization was assessed by co-immunoprecipitation and PLA. Structure of the possible heterodimer of A2A and OT receptors was estimated by a bioinformatic approach. RESULTS: Both A2A and OT receptors were expressed on the same astrocyte processes. Evidence for A2A-OTR receptor-receptor interaction was obtained by measuring the release of glutamate: OT inhibited the evoked glutamate release, while activation of A2A receptors, per se ineffective, abolished the OT effect. Biochemical and biophysical evidence for A2A-OTR heterodimers on striatal astrocytes was also obtained. The residues in the transmembrane domains 4 and 5 of both receptors are predicted to be mainly involved in the heteromerization. CONCLUSIONS: When considering effects of OT in striatum, modulation of glutamate release from the astrocyte processes and of glutamatergic synapse functioning, and the interaction with A2A receptors on the astrocyte processes should be taken into consideration.
Asunto(s)
Astrocitos/metabolismo , Ácido Glutámico/metabolismo , Receptor de Adenosina A2A/metabolismo , Receptores de Oxitocina/metabolismo , Animales , Cuerpo Estriado/metabolismo , Masculino , Neostriado/metabolismo , Oxitocina/metabolismo , Ratas , Ratas Sprague-DawleyRESUMEN
BACKGROUND: In the brain, polyamines are mainly synthesized in neurons, but preferentially accumulated in astrocytes, and are proposed to be involved in neurodegenerative/neuroinflammatory disorders and neuron injury. A transgenic mouse overexpressing spermine oxidase (SMOX, which specifically oxidizes spermine) in the neocortex neurons (Dach-SMOX mouse) was proved to be a model of increased susceptibility to excitotoxic injury. METHODS: To investigate possible alterations in synapse functioning in Dach-SMOX mouse, both cerebrocortical nerve terminals (synaptosomes) and astrocytic processes (gliosomes) were analysed by assessing polyamine levels, ezrin and vimentin content, glutamate AMPA receptor activation, calcium influx, and catalase activity. RESULTS: The main findings are as follows: (i) the presence of functional calcium-permeable AMPA receptors in synaptosomes from both control and Dach-SMOX mice, and in gliosomes from Dach-SMOX mice only; (ii) reduced content of spermine in gliosomes from Dach-SMOX mice; and (iii) down-regulation and up-regulation of catalase activity in synaptosomes and gliosomes, respectively, from Dach-SMOX mice. CONCLUSIONS: Chronic activation of SMOX in neurons leads to major changes in the astrocyte processes including reduced spermine levels, increased calcium influx through calcium-permeable AMPA receptors, and stimulation of catalase activity. Astrocytosis and the astrocyte process alterations, depending on chronic activation of polyamine catabolism, result in synapse dysregulation and neuronal suffering.
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Gliosis/metabolismo , Gliosis/patología , Poliaminas/metabolismo , Animales , Astrocitos/efectos de los fármacos , Astrocitos/metabolismo , Astrocitos/patología , Calcio/metabolismo , Catalasa/metabolismo , Corteza Cerebral/metabolismo , Corteza Cerebral/patología , Proteínas del Citoesqueleto/metabolismo , Modelos Animales de Enfermedad , Ratones , Ratones Transgénicos , Terminaciones Nerviosas/efectos de los fármacos , Terminaciones Nerviosas/metabolismo , Neuroglía/efectos de los fármacos , Neuroglía/metabolismo , Neuronas/efectos de los fármacos , Neuronas/metabolismo , Estrés Oxidativo/efectos de los fármacos , Oxidorreductasas actuantes sobre Donantes de Grupo CH-NH/metabolismo , Receptores AMPA/metabolismo , Espermina/análogos & derivados , Espermina/metabolismo , Espermina/farmacología , Sinaptosomas/efectos de los fármacos , Sinaptosomas/metabolismo , Vimentina/metabolismo , Poliamino OxidasaRESUMEN
The aim of this study was the identification of specific proteomic profiles, related to a restored cystic fibrosis transmembrane conductance regulator (CFTR) activity in cystic fibrosis (CF) leukocytes before and after ex vivo treatment with the potentiator VX770. We used leukocytes, isolated from CF patients carrying residual function mutations and eligible for Ivacaftor therapy, and performed CFTR activity together with proteomic analyses through micro-LC-MS. Bioinformatic analyses of the results obtained revealed the downregulation of proteins belonging to the leukocyte transendothelial migration and regulation of actin cytoskeleton pathways when CFTR activity was rescued by VX770 treatment. In particular, we focused our attention on matrix metalloproteinase 9 (MMP9), because the high expression of this protease potentially contributes to parenchyma lung destruction and dysfunction in CF. Thus, the downregulation of MMP9 could represent one of the possible positive effects of VX770 in decreasing the disease progression, and a potential biomarker for the prediction of the efficacy of therapies targeting the defect of Cl- transport in CF.
Asunto(s)
Aminofenoles/farmacología , Regulador de Conductancia de Transmembrana de Fibrosis Quística/genética , Fibrosis Quística/tratamiento farmacológico , Metaloproteinasa 9 de la Matriz/genética , Quinolonas/farmacología , Citoesqueleto de Actina/genética , Adulto , Biomarcadores/sangre , Movimiento Celular/efectos de los fármacos , Fibrosis Quística/sangre , Fibrosis Quística/genética , Fibrosis Quística/patología , Femenino , Regulación de la Expresión Génica/efectos de los fármacos , Humanos , Leucocitos Mononucleares/efectos de los fármacos , Masculino , Proteoma/genéticaRESUMEN
We have recently demonstrated that bioactive molecules, extracted by high pressure and temperature from olive pomace, counteract calcium-induced cell damage to different cell lines. Here, our aim was to study the effect of the same extract on murine cortical neurons, since the preservation of the intracellular Ca2+-homeostasis is essential for neuronal function and survival. Accordingly, we treated neurons with different stimuli in order to evoke cytotoxic glutamatergic activation. In these conditions, the high-pressure and temperature extract from olive pomace (HPTOPE) only abolished the effects of N-methyl-d-aspartate (NMDA). Particularly, we observed that HPTOPE was able to promote the neuron rescue from NMDA-induced cell death. Moreover, we demonstrated that HPTOPE is endowed with the ability to maintain the intracellular Ca2+-homeostasis following NMDA receptor overactivation, protecting neurons from Ca2+-induced adverse effects, including aberrant calpain proteolytic activity. Moreover, we highlight the importance of the extraction conditions used that, without producing toxic molecules, allow us to obtain protecting molecules belonging to proanthocyanidin derivatives like procyanidin B2. In conclusion, we can hypothesize that HPTOPE, due to its functional and nontoxic properties on neuronal primary culture, can be utilized for future therapeutic interventions for neurodegeneration.
Asunto(s)
Biflavonoides/farmacología , Señalización del Calcio/efectos de los fármacos , Catequina/farmacología , N-Metilaspartato/efectos adversos , Neuronas/metabolismo , Olea/química , Extractos Vegetales/farmacología , Proantocianidinas/farmacología , Receptores de N-Metil-D-Aspartato/metabolismo , Animales , Biflavonoides/química , Catequina/química , Muerte Celular/efectos de los fármacos , Células Cultivadas , Ratones , N-Metilaspartato/farmacología , Neuronas/patología , Extractos Vegetales/química , Proantocianidinas/químicaRESUMEN
Several human acute and chronic diseases involve calpain over-activation. However, the mechanistic linkages between the etiology and the progression of cell damages are not yet completely understood. Here we show that different human cells and tissues, including brain tumor specimens, cell lines of nerve origin, breast tumor samples and peripheral blood mononuclear cells from healthy donors, express a calpastatin form that lacks all the exons coding for the domains responsible of calpain inhibition. The open reading frame of this new form of calpastatin, named hcast 3-25, starts inside the L-domain (exons 2 and 3) and continues with the exons from 25 to 29 that code for the conserved C-terminal tail shared by all the full-length calpastatins. We have here observed that unlike the other calpastatins forms, that are predominantly Δ3 splice variants, hcast 3-25 is endowed with exon 3. At a functional level, recombinant hcast 3-25 operates as a positive modulator of calpain-1 in vitro by preventing 1) calpain-1-mediated proteolytic degradation of the activated enzyme and 2) binding to calpain-1 of inhibitory calpastatins that contain the L-domain. Thus hcast 3-25 can be considered as a novel member and possible modulator of the calpain/calpastatin system acting by a mechanism alternative to inhibition.
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Neoplasias Encefálicas/metabolismo , Proteínas de Unión al Calcio/metabolismo , Calpaína/metabolismo , Proteínas de Neoplasias/metabolismo , Proteolisis , Neoplasias Encefálicas/genética , Proteínas de Unión al Calcio/genética , Calpaína/genética , Línea Celular Tumoral , Humanos , Proteínas de Neoplasias/genética , Dominios Proteicos , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismoRESUMEN
We are reporting in the present study that molecules extracted from olive pomace prevent cell death induced by Ca2+-overloading in different cell types. Exposure of cells to these molecules counteracts the Ca2+-induced cell damages by reducing the activation of the Ca2+-dependent protease calpain, acting possibly through the modification of the permeability to Ca2+ of the plasma membrane. The purification step by RP-HPLC suggests that effective compound(s), differing from the main biophenols known to be present in the olive pomace extract, could be responsible for this effect. Our observations suggest that bioactive molecules present in the olive pomace could be potential candidates for therapeutic applications in pathologies characterised by alterations of intracellular Ca2+ homeostasis.
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Calcio/metabolismo , Citoprotección/efectos de los fármacos , Aceite de Oliva/análisis , Extractos Vegetales/farmacología , Línea Celular Tumoral , Supervivencia Celular/efectos de los fármacos , Células Cultivadas , Cromatografía Líquida de Alta Presión , Cromatografía de Fase Inversa , Homeostasis , Humanos , Transporte IónicoRESUMEN
Autoproteolysis of human erythrocyte calpain-1 proceeds in vitro at high [Ca2+], through the conversion of the 80-kDa catalytic subunit into a 75-kDa activated enzyme that requires lower [Ca2+] for catalysis. Importantly, here we detect a similar 75 kDa calpain-1 form also in vivo, in human meningiomas. Although calpastatin is so far considered the specific inhibitor of calpains, we have previously identified in rat brain a calpastatin transcript truncated at the end of the L-domain (cast110, L-DOM), coding for a protein lacking the inhibitory units. Aim of the present study was to characterize the possible biochemical role of the L-DOM during calpain-1 autoproteolysis in vitro, at high (100 µM) and low (5 µM) [Ca2+]. Here we demonstrate that the L-DOM binds the 80 kDa proenzyme in the absence of Ca2+ Consequently, we have explored the ability of the 75 kDa activated protease to catalyze at 5 µM Ca2+ the intermolecular activation of native calpain-1 associated with the L-DOM. Notably, this [Ca2+] is too low to promote the autoproteolytic activation of calpain-1 but enough to support the catalysis of the 75 kDa calpain. We show for the first time that the L-DOM preserves native calpain-1 from the degradation mediated by the 75 kDa form. Taken together, our data suggest that the free L-domain of calpastatin is a novel member of the calpain/calpastatin system endowed with a function alternative to calpain inhibition. For this reason, it will be crucial to define the intracellular relevance of the L-domain in controlling calpain activation/activity in physiopathological conditions having altered Ca2+ homeostasis.
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Proteínas de Unión al Calcio/química , Calcio/metabolismo , Calpaína/química , Eritrocitos/química , Proteolisis , Calcio/química , Proteínas de Unión al Calcio/metabolismo , Calpaína/metabolismo , Eritrocitos/metabolismo , Humanos , Dominios ProteicosRESUMEN
Matrix metalloproteinase 9 (MMP9) is physiologically involved in remodeling the extracellular matrix components but its abnormal release has been observed in several human pathologies. We here report that peripheral blood mononuclear cells (PBMCs), isolated from cystic fibrosis (CF) patients homozygous for F508del-cystic fibrosis transmembrane conductance regulator (CFTR), express constitutively and release at high rate MMP9 due to the alteration in their intracellular Ca(2+) homeostasis. This spontaneous and sustained MMP9 secretion may contribute to the accumulation of this protease in fluids of CF patients. Conversely, in PBMCs isolated from healthy donors, expression and secretion of MMP9 are undetectable but can be evoked, after 12 h of culture, by paracrine stimulation which also promotes an increase in [Ca(2+)]i. We also demonstrate that in both CF and control PBMCs the Ca(2+)-dependent MMP9 secretion is mediated by the concomitant activation of calpain and protein kinase Cα (PKCα), and that MMP9 expression involves extracellular signal-regulated protein kinases 1 and 2 (ERK1/2) phosphorylation. Our results are supported by the fact that either the inhibition of Ca(2+) entry or chelation of [Ca(2+)]i as well as the inhibition of single components of the signaling pathway or the restoration of CFTR activity all promote the reduction of MMP9 secretion.
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Calpaína/sangre , Regulador de Conductancia de Transmembrana de Fibrosis Quística/genética , Fibrosis Quística/sangre , Leucocitos Mononucleares/metabolismo , Metaloproteinasa 9 de la Matriz/sangre , Proteína Quinasa C-alfa/sangre , Adolescente , Adulto , Anciano , Calcio/metabolismo , Activación Enzimática , Células Epiteliales/citología , Quinasas MAP Reguladas por Señal Extracelular/metabolismo , Eliminación de Gen , Regulación Enzimológica de la Expresión Génica , Homeostasis , Homocigoto , Humanos , Persona de Mediana Edad , Fosforilación , Valor Predictivo de las Pruebas , Adulto JovenRESUMEN
Several evidences suggest that NK cells can patrol the body and eliminate tumors in their initial phases but may hardly control established solid tumors. Multiple factors, including the transition of tumor cells towards a proinvasive/prometastatic phenotype, the immunosuppressive effect of the tumor microenvironment, and the tumor structure complexity, may account for limited NK cell efficacy. Several putative mechanisms of NK cell suppression have been defined in these last years; conversely, the cross talk between NK cells and tumor cells undergoing different transitional phases remains poorly explored. Nevertheless, recent in vitro studies and immunohistochemical analyses on tumor biopsies suggest that NK cells could not only kill tumor cells but also influence their evolution. Indeed, NK cells may induce tumor cells to change the expression of HLA-I, PD-L1, or NKG2D-L and modulate their susceptibility to the immune response. Moreover, NK cells may be preferentially located in the borders of tumor masses, where, indeed, tumor cells can undergo Epithelial-to-Mesenchymal Transition (EMT) acquiring prometastatic phenotype. Finally, the recently highlighted role of HMGB1 both in EMT and in amplifying the recruitment of NK cells provides further hints on a possible effect of NK cells on tumor progression and fosters new studies on this issue.
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Inmunoterapia , Células Asesinas Naturales/inmunología , Neoplasias/inmunología , Neoplasias/terapia , Animales , Antígeno B7-H1/metabolismo , Citotoxicidad Inmunológica , Progresión de la Enfermedad , Transición Epitelial-Mesenquimal/inmunología , Antígenos HLA/metabolismo , Proteína HMGB1/fisiología , Humanos , Tolerancia Inmunológica , Células Asesinas Naturales/química , Subfamilia K de Receptores Similares a Lectina de Células NK/metabolismo , Metástasis de la Neoplasia/prevención & control , Neoplasias/fisiopatología , Microambiente Tumoral/inmunologíaRESUMEN
High mobility group box protein-1 (HMGB1) is released from cells under various pathological conditions and it plays a pivotal role as an alarmin signaling tissue damage. Little is known about the impact of HMGB1 in bone repair and remodeling. To this aim, we focused on HMGB1-induced effects on the in vitro osteoblast model SaOS-2. Cell proliferation was stimulated with a maximum at concentration of 2.5 nM, and such a dose also stimulated cell migration and scratch wound healing. We then characterized the modulatory effect of HMGB1 on bone biology, by using osteogenesis/mineralization assays, a PCR array, and the analysis of a series of osteogenic markers. We performed also a proteomic screening using SWATH-MS on SaOS-2 cell exposed to HMGB1 and we provide evidence for proteins modulated in HMGB1 exposed cells. Taken together, our data demonstrate that SaOS-2 cell proliferation, migration, and osteogenic differentiation were increased by HMGB1. We, therefore, propose that HMGB1 could be a potent bone-remodeling signal but the physiological meaning of this property remains to be more ascertained. J. Cell. Biochem. 117: 2559-2569, 2016. © 2016 Wiley Periodicals, Inc.
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Neoplasias Óseas/metabolismo , Movimiento Celular , Proteína HMGB1/metabolismo , Osteosarcoma/metabolismo , Proteoma/análisis , Proteómica/métodos , Western Blotting , Neoplasias Óseas/genética , Neoplasias Óseas/patología , Remodelación Ósea , Diferenciación Celular , Proliferación Celular , Proteína HMGB1/genética , Humanos , Osteosarcoma/genética , Osteosarcoma/patología , ARN Mensajero/genética , Reacción en Cadena en Tiempo Real de la Polimerasa , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Transducción de Señal , Células Tumorales Cultivadas , Cicatrización de HeridasRESUMEN
In this study we characterize a new mechanism by which Natural Killer (NK) cells may amplify their recruitment to tumors. We show that NK cells, upon interaction with melanoma cells, can release a chemotactic form of High Mobility Group Box-1 (HMGB1) protein capable of attracting additional activated NK cells. We first demonstrate that the engagement of different activating NK cell receptors, including those mainly involved in tumor cell recognition can induce the active release of HMGB1. Then we show that during NK-mediated tumor cell killing two HMGB1 forms are released, each displaying a specific electrophoretic mobility possibly corresponding to a different redox status. By the comparison of normal and perforin-defective NK cells (which are unable to kill target cells) we demonstrate that, in NK/melanoma cell co-cultures, NK cells specifically release an HMGB1 form that acts as chemoattractant, while dying tumor cells passively release a non-chemotactic HMGB1. Finally, we show that Receptor for Advanced Glycation End products is expressed by NK cells and mediates HMGB1-induced NK cell chemotaxis. Proteomic analysis of NK cells exposed to recombinant HMGB1 revealed that this molecule, besides inducing immediate chemotaxis, also promotes changes in the expression of proteins involved in the regulation of the cytoskeletal network. Importantly, these modifications could be associated with an increased motility of NK cells. Thus, our findings allow the definition of a previously unidentified mechanism used by NK cells to amplify their response to tumors, and provide additional clues for the emerging role of HMGB1 in immunomodulation and tumor immunity.
RESUMEN
We have recently demonstrated that in resting conditions calpain 1, but not calpain 2, is specifically associated to the N-Methyl-D-Aspartate receptor (NMDAR) multiprotein complex. We are here reporting that in SKNBE neuroblastoma cells or in freshly isolated nerve terminals from adult rat hippocampus, the proteolytic activity of calpain 1 resident at the NMDAR is very low under basal conditions and greatly increases following NMDAR stimulation. Since the protease resides at the NMDAR in saturating amounts, variations in Ca2+ influx promote an increase in calpain 1 activity without affecting the amount of the protease originally associated to NMDAR. In all the conditions examined, resident calpain 1 specifically cleaves NR2B at the C-terminal region, leading to its internalization together with NR1 subunit. While in basal conditions intracellular membranes include small amounts of NMDAR containing the calpain-digested NR2B, upon NMDAR stimulation nearly all the receptor molecules are internalized. We here propose that resident calpain 1 is involved in NMDAR turnover, and following an increase in Ca2+ influx, the activated protease, by promoting the removal of NMDAR from the plasma membranes, can decrease Ca2+ entrance through this channel. Due to the absence of calpastatin in such cluster, the activity of resident calpain 1 may be under the control of HSP90, whose levels are directly related to the activation of this protease. Observations of different HSP90/calpain 1 ratios in different ultrasynaptic compartments support this conclusion.