RESUMO
TRPM2 is a Ca2+ permeable, non-selective cation channel in the plasma membrane that is involved in the innate immune response regulating, for example, chemotaxis in neutrophils and cytokine secretion in monocytes and macrophages. The intracellular adenine nucleotides ADP-ribose (ADPR) and 2'-deoxy-ADPR (2dADPR) activate the channel, in combination with their co-agonist Ca2+. Interestingly, activation of human TRPM2 (hsTRPM2) by 2dADPR is much more effective than activation by ADPR. However, the underlying mechanism of the nucleotides' differential effect on the channel is not yet fully understood. In this study, we performed whole-cell patch clamp experiments with HEK293 cells heterologously expressing hsTRPM2. We show that 2dADPR has an approx. 4-fold higher Ca2+ sensitivity than ADPR (EC50 = 190 and 690 nM). This allows 2dADPR to activate the channel at lower and thus physiological intracellular Ca2+ concentrations. Kinetic analysis of our data reveals that activation by 2dADPR is faster than activation by ADPR. Mutation in a calmodulin binding N-terminal IQ-like motif in hsTRPM2 completely abrogated channel activation by both agonists. However, mutation of a single amino acid residue (W1355A) in the C-terminus of hsTRPM2, at a site of extensive inter-domain interaction, resulted in slower activation by 2dADPR and neutralized the difference in rate of activation between the two agonists. Taken together, we propose a mechanism by which 2dADPR induces higher hsTRPM2 currents than ADPR by means of faster channel activation. The finding that 2dADPR has a higher Ca2+ sensitivity than ADPR may indicate that 2dADPR rather than ADPR activates hsTRPM2 in physiological contexts such as the innate immune response.
Assuntos
Adenosina Difosfato Ribose , Canais de Cátion TRPM , Humanos , Adenosina Difosfato Ribose/química , Adenosina Difosfato Ribose/farmacologia , Sinalização do Cálcio , Células HEK293 , Cinética , Canais de Cátion TRPM/genética , Canais de Cátion TRPM/metabolismoRESUMO
Ca2+ signaling is one of the essential signaling systems for T lymphocyte activation, the latter being an essential step in the pathogenesis of autoimmune diseases such as multiple sclerosis (MS). Store-operated Ca2+ entry (SOCE) ensures long lasting Ca2+ signaling and is of utmost importance for major downstream T lymphocyte activation steps, e.g. nuclear localization of the transcription factor 'nuclear factor of activated T cells' (NFAT). 2-Methoxyestradiol (2ME2), an endogenous metabolite of estradiol (E2), blocks nuclear translocation of NFAT. The likely underlying mechanism is inhibition of SOCE, as shown for its synthetic sulfamate ester analogue 2-ethyl-3-sulfamoyloxy-17ß-cyanomethylestra-1,3,5(10)-triene (STX564). Here, we demonstrate that another synthetic bis-sulfamoylated 2ME2 derivative, 2-methoxyestradiol-3,17-O,O-bis-sulfamate (2-MeOE2bisMATE, STX140), an orally bioavailable, multi-targeting anticancer agent and potent steroid sulfatase (STS) inhibitor, antagonized SOCE in T lymphocytes. Downstream events, e.g. secretion of the pro-inflammatory cytokines interferon-γ and interleukin-17, were decreased by STX140 in in vitro experiments. Remarkably, STX140 dosed in vivo completely blocked the clinical disease in both active and transfer experimental autoimmune encephalomyelitis (EAE) in Lewis rats, a T cell-mediated animal model for MS, at a dose of 10 mg/kg/day i.p., whereas neither 2ME2 nor Irosustat, a pure STS inhibitor, showed any effect. The STS inhibitory activity of STX140 is therefore not responsible for its activity in this model. Taken together, inhibition of SOCE by STX140 resulting in full antagonism of clinical symptoms in EAE in the Lewis rat, paired with the known excellent bioavailability and pharmaceutical profile of this drug, open potentially new therapeutic avenues for the treatment of MS.
Assuntos
Encefalomielite Autoimune Experimental , Linfócitos T , Ratos , Animais , 2-Metoxiestradiol , Encefalomielite Autoimune Experimental/tratamento farmacológico , Ratos Endogâmicos Lew , Preparações FarmacêuticasRESUMO
The formation of Ca2+ microdomains during T cell activation is initiated by the production of nicotinic acid adenine dinucleotide phosphate (NAADP) from its reduced form NAADPH. The reverse reactionNAADP to NAADPHis catalyzed by glucose 6-phosphate dehydrogenase (G6PD). Here, we identified NADPH oxidases NOX and DUOX as NAADP-forming enzymes that convert NAADPH to NAADP under physiological conditions in vitro. T cells express NOX1, NOX2, and, to a minor extent, DUOX1 and DUOX2. Local and global Ca2+ signaling were decreased in mouse T cells with double knockout of Duoxa1 and Duoxa2 but not with knockout of Nox1 or Nox2. Ca2+ microdomains in the first 15 s upon T cell activation were significantly decreased in Duox2−/− but not in Duox1−/− T cells, whereas both DUOX1 and DUOX2 were required for global Ca2+ signaling between 4 and 12 min after stimulation. Our findings suggest that a DUOX2- and G6PD-catalyzed redox cycle rapidly produces and degrades NAADP through NAADPH as an inactive intermediate.
Assuntos
Sinalização do Cálcio , Oxidases Duais , Ativação Linfocitária , NADPH Oxidases , NADP/biossíntese , Linfócitos T , Animais , Oxidases Duais/genética , Células HEK293 , Humanos , Células Jurkat , Camundongos Knockout , NADP/análogos & derivados , NADPH Oxidases/genética , Linfócitos T/enzimologiaRESUMO
NAADP-evoked Ca2+ release through type 1 ryanodine receptors (RYR1) is a major mechanism underlying the earliest signals in T cell activation, which are the formation of Ca2+ microdomains. In our characterization of the molecular machinery underlying NAADP action, we identified an NAADP-binding protein, called hematological and neurological expressed 1-like protein (HN1L) [also known as Jupiter microtubule-associated homolog 2 (JPT2)]. Gene deletion of Hn1l/Jpt2 in human Jurkat and primary rat T cells resulted in decreased numbers of initial Ca2+ microdomains and delayed the onset and decreased the amplitude of global Ca2+ signaling. Photoaffinity labeling demonstrated direct binding of NAADP to recombinant HN1L/JPT2. T cell receptor/CD3-dependent coprecipitation of HN1L/JPT2 with RYRs and colocalization of these proteins suggest that HN1L/JPT2 connects NAADP formation with the activation of RYR channels within the first seconds of T cell activation. Thus, HN1L/JPT2 enables NAADP to activate Ca2+ release from the endoplasmic reticulum through RYR.
Assuntos
Cálcio/metabolismo , Microdomínios da Membrana/metabolismo , Proteínas Associadas aos Microtúbulos/metabolismo , NADP/análogos & derivados , Animais , Complexo CD3/metabolismo , Sinalização do Cálcio , Retículo Endoplasmático/metabolismo , Humanos , Células Jurkat , Ativação Linfocitária , Proteínas Associadas aos Microtúbulos/genética , NADP/metabolismo , Ligação Proteica , Ratos , Receptores de Antígenos de Linfócitos T/metabolismo , Canal de Liberação de Cálcio do Receptor de Rianodina/metabolismo , Linfócitos T/metabolismoRESUMO
T cell activation starts with formation of second messengers that release Ca2+ from the endoplasmic reticulum (ER) and thereby activate store-operated Ca2+ entry (SOCE), one of the essential signals for T cell activation. Recently, the steroidal 2-methoxyestradiol was shown to inhibit nuclear translocation of the nuclear factor of activated T cells (NFAT). We therefore investigated 2-methoxyestradiol for inhibition of Ca2+ entry in T cells, screened a library of 2-methoxyestradiol analogues, and characterized the derivative 2-ethyl-3-sulfamoyloxy-17ß-cyanomethylestra-1,3,5(10)-triene (STX564) as a novel, potent and specific SOCE inhibitor. STX564 inhibits Ca2+ entry via SOCE without affecting other ion channels and pumps involved in Ca2+ signaling in T cells. Downstream effects such as cytokine expression and cell proliferation were also inhibited by both 2-methoxyestradiol and STX564, which has potential as a new chemical biology tool.
Assuntos
2-Metoxiestradiol/farmacologia , Sinalização do Cálcio/efeitos dos fármacos , Estrenos/farmacologia , Fatores de Transcrição NFATC/metabolismo , Linfócitos T/citologia , 2-Metoxiestradiol/análogos & derivados , Animais , Cálcio/metabolismo , Linhagem Celular , Núcleo Celular/efeitos dos fármacos , Núcleo Celular/metabolismo , Estrenos/síntese química , Estrenos/química , Regulação da Expressão Gênica/efeitos dos fármacos , Humanos , Células Jurkat , Ativação Linfocitária/efeitos dos fármacos , Transporte Proteico/efeitos dos fármacos , Ratos , Linfócitos T/efeitos dos fármacos , Linfócitos T/metabolismoRESUMO
The NAD-glycohydrolase/ADP-ribosyl cyclase CD38 catalyzes the metabolism of nicotinamide adenine dinucleotide (NAD) to the Ca2+ mobilizing second messengers ADP-ribose (ADPR), 2'-deoxy-ADPR, and cyclic ADP-ribose (cADPR). In the present study, we investigated binding and metabolism of NAD by a soluble fragment of human CD38, sCD38, and its catalytically inactive mutant by monitoring changes in endogenous tryptophan (Trp) fluorescence. Addition of NAD resulted in a concentration-dependent decrease in sCD38 fluorescence that is mainly caused by the Trp residue W189. Amplitude of the fluorescence decrease was fitted as one-site binding curve revealing a dissociation constant for NAD of 29⯵M. A comparable dissociation constant was found with the catalytically inactive sCD38 mutant (KD 37⯵M NAD) indicating that binding of NAD is not significantly affected by the mutation. The NAD-induced decrease in Trp fluorescence completely recovered in case of sCD38. Kinetics of recovery was slowed down with decreasing temperature and sCD38 concentration and increasing NAD concentration demonstrating that recovery in fluorescence is proportional to the enzymatic activity of sCD38. Accordingly, recovery in fluorescence was not observed with the catalytically inactive mutant. This article is part of a Special Issue entitled: ECS Meeting edited by Claus Heizmann, Joachim Krebs and Jacques Haiech.
Assuntos
ADP-Ribosil Ciclase 1/química , Glicoproteínas de Membrana/química , NAD/química , ADP-Ribosil Ciclase 1/genética , ADP-Ribosil Ciclase 1/metabolismo , Sítios de Ligação , Células HEK293 , Humanos , Cinética , Glicoproteínas de Membrana/genética , Glicoproteínas de Membrana/metabolismo , NAD/metabolismo , Triptofano/química , Triptofano/genética , Triptofano/metabolismoRESUMO
The effect of erythropoietin (Epo) on the expression of mitogen-activated protein kinase (MAPK) target genes egr-1 and c-fos was investigated in Epo-responsive murine erythroblastic cell line ELM-I-1. Epo induced a transient rise in egr-1 mRNA without a similar effect on c-fos expression. The induction of egr-1 correlated with a rapid ERK1/2 phosphorylation and was prevented with MEK1/2 inhibitors PD 98059 and UO126. The p38 inhibitor SB 203580 enhanced ERK1/2 phosphorylation and egr-1 mRNA levels. Longer incubations of ELM-I-1 cells with Epo revealed a second later phase of increase in egr-1 expression which was also prevented by MEK1/2 inhibitors, whereas SB 203580 had a stimulatory effect. In contrast, the beta-globin mRNA production was enhanced in the presence of PD 98059 and UO126 and reduced by SB 203580. The results suggest a regulatory role of egr-1 expression in Epo signal transduction and provide pharmacological evidence for the negative modulation of differentiation-specific gene expression by the ERK1/2 pathway in murine erythroleukemia cells.