RESUMEN
In T cells, stromal interaction molecule (STIM) and Orai are dispensable for conventional T cell development, but critical for activation and differentiation. This review focuses on novel STIM-dependent mechanisms for control of Ca2+ signals during T cell activation and its impact on mitochondrial function and transcriptional activation for control of T cell differentiation and function. We highlight areas that require further work including the roles of plasma membrane Ca2+ ATPase (PMCA) and partner of STIM1 (POST) in controlling Orai function. A major knowledge gap also exists regarding the independence of T cell development from STIM and Orai, despite compelling evidence that it requires Ca2+ signals. Resolving these and other outstanding questions ensures that the field will remain active for many years to come.
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Señalización del Calcio , Calcio , Proteína ORAI1/metabolismo , Molécula de Interacción Estromal 1/metabolismo , Membrana Celular/metabolismo , Diferenciación Celular , Calcio/metabolismo , Señalización del Calcio/fisiologíaRESUMEN
The role of store-operated Ca2+ entry (SOCE) in melanoma metastasis is highly controversial. To address this, we here examined UV-dependent metastasis, revealing a critical role for SOCE suppression in melanoma progression. UV-induced cholesterol biosynthesis was critical for UV-induced SOCE suppression and subsequent metastasis, although SOCE suppression alone was both necessary and sufficient for metastasis to occur. Further, SOCE suppression was responsible for UV-dependent differences in gene expression associated with both increased invasion and reduced glucose metabolism. Functional analyses further established that increased glucose uptake leads to a metabolic shift towards biosynthetic pathways critical for melanoma metastasis. Finally, examination of fresh surgically isolated human melanoma explants revealed cholesterol biosynthesis-dependent reduced SOCE. Invasiveness could be reversed with either cholesterol biosynthesis inhibitors or pharmacological SOCE potentiation. Collectively, we provide evidence that, contrary to current thinking, Ca2+ signals can block invasive behavior, and suppression of these signals promotes invasion and metastasis.
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Señalización del Calcio , Melanoma , Calcio/metabolismo , Canales de Calcio/metabolismo , Colesterol , Glucosa , Humanos , Melanoma/genética , Melanoma/metabolismo , Proteína ORAI1/metabolismo , Molécula de Interacción Estromal 1/metabolismoRESUMEN
The sense of taste starts with activation of receptor cells in taste buds by chemical stimuli which then communicate this signal via innervating oral sensory neurons to the CNS. The cell bodies of oral sensory neurons reside in the geniculate ganglion (GG) and nodose/petrosal/jugular ganglion. The geniculate ganglion contains two main neuronal populations: BRN3A+ somatosensory neurons that innervate the pinna and PHOX2B+ sensory neurons that innervate the oral cavity. While much is known about the different taste bud cell subtypes, considerably less is known about the molecular identities of PHOX2B+ sensory subpopulations. In the GG, as many as 12 different subpopulations have been predicted from electrophysiological studies, while transcriptional identities exist for only 3 to 6. Importantly, the cell fate pathways that diversify PHOX2B+ oral sensory neurons into these subpopulations are unknown. The transcription factor EGR4 was identified as being highly expressed in GG neurons. EGR4 deletion causes GG oral sensory neurons to lose their expression of PHOX2B and other oral sensory genes and up-regulate BRN3A. This is followed by a loss of chemosensory innervation of taste buds, a loss of type II taste cells responsive to bitter, sweet, and umami stimuli, and a concomitant increase in type I glial-like taste bud cells. These deficits culminate in a loss of nerve responses to sweet and umami taste qualities. Taken together, we identify a critical role of EGR4 in cell fate specification and maintenance of subpopulations of GG neurons, which in turn maintain the appropriate sweet and umami taste receptor cells.
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Papilas Gustativas , Gusto , Gusto/fisiología , Ganglio Geniculado/metabolismo , Lengua/inervación , Papilas Gustativas/metabolismo , Factores de Transcripción/metabolismo , Células Receptoras Sensoriales/metabolismoRESUMEN
Ca2+ dynamics and oxidative signaling are fundamental mechanisms for mitochondrial bioenergetics and cell function. The MCU complex is the major pathway by which these signals are integrated in mitochondria. Whether and how these coactive elements interact with MCU have not been established. As an approach toward understanding the regulation of MCU channel by oxidative milieu, we adapted inflammatory and hypoxia models. We identified the conserved cysteine 97 (Cys-97) to be the only reactive thiol in human MCU that undergoes S-glutathionylation. Furthermore, biochemical, structural, and superresolution imaging analysis revealed that MCU oxidation promotes MCU higher order oligomer formation. Both oxidation and mutation of MCU Cys-97 exhibited persistent MCU channel activity with higher [Ca2+]m uptake rate, elevated mROS, and enhanced [Ca2+]m overload-induced cell death. In contrast, these effects were largely independent of MCU interaction with its regulators. These findings reveal a distinct functional role for Cys-97 in ROS sensing and regulation of MCU activity.
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Canales de Calcio/metabolismo , Señalización del Calcio , Calcio/metabolismo , Células Endoteliales/metabolismo , Activación del Canal Iónico , Mitocondrias/metabolismo , Membranas Mitocondriales/metabolismo , Especies Reactivas de Oxígeno/metabolismo , Animales , Células COS , Canales de Calcio/química , Canales de Calcio/genética , Señalización del Calcio/efectos de los fármacos , Muerte Celular , Hipoxia de la Célula , Chlorocebus aethiops , Cisteína , Células Endoteliales/efectos de los fármacos , Células Endoteliales/patología , Metabolismo Energético , Glutatión/metabolismo , Células HEK293 , Células HeLa , Humanos , Activación del Canal Iónico/efectos de los fármacos , Lipopolisacáridos/farmacología , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Mitocondrias/efectos de los fármacos , Mitocondrias/patología , Membranas Mitocondriales/efectos de los fármacos , Membranas Mitocondriales/patología , Mutación , Oxidación-Reducción , Multimerización de Proteína , Procesamiento Proteico-Postraduccional , Estructura Cuaternaria de Proteína , Relación Estructura-Actividad , Trombina/farmacología , Factores de Tiempo , TransfecciónRESUMEN
Although Ras/mitogen-activated protein kinase (MAPK) signaling is activated in most human cancers, attempts to target this pathway using kinase-active site inhibitors have not typically led to durable clinical benefit. To address this shortcoming, we sought to test the feasibility of an alternative targeting strategy, focused on the ERK2 substrate binding domains, D and DEF binding pocket (DBP). Disabling the ERK2-DBP domain in mice caused baseline erythrocytosis. Consequently, we investigated the role of the ERK2-D and -DBP domains in disease, using a JAK2-dependent model of polycythemia vera (PV). Of note, inactivation of the ERK2-DBP domain promoted the progression of disease from PV to myelofibrosis, suggesting that the ERK2-DBP domain normally opposes progression. ERK2-DBP inactivation also prevented oncogenic JAK2 kinase (JAK2V617F) from promoting oncogene-induced senescence in vitro. The ERK2-DBP mutation attenuated JAK2-mediated oncogene-induced senescence by preventing the physical interaction of ERK2 with the transcription factor Egr1. Because inactivation of the ERK2-DBP created a functional ERK2 kinase limited to binding substrates through its D domain, these data suggested that the D domain substrates were responsible for promoting oncogene-induced progenitor growth and tumor progression and that pharmacologic targeting of the ERK2-D domain may attenuate cancer cell growth. Indeed, pharmacologic agents targeting the ERK2-D domain were effective in attenuating the growth of JAK2-dependent myeloproliferative neoplasm cell lines. Taken together, these data indicate that the ERK-D and -DBP domains can play distinct roles in the progression of neoplasms and that the D domain has the potential to be a potent therapeutic target in Ras/MAPK-dependent cancers.
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Janus Quinasa 2 , Policitemia Vera , Animales , Línea Celular , Humanos , Janus Quinasa 2/genética , Sistema de Señalización de MAP Quinasas , Ratones , Proteínas Quinasas Activadas por Mitógenos , Fosforilación , Transducción de SeñalRESUMEN
Stromal interaction molecule (STIM) proteins function in cells as dynamic coordinators of cellular calcium (Ca(2+)) signals. Spanning the endoplasmic reticulum (ER) membrane, they sense tiny changes in the levels of Ca(2+) stored within the ER lumen. As ER Ca(2+) is released to generate primary Ca(2+) signals, STIM proteins undergo an intricate activation reaction and rapidly translocate into junctions formed between the ER and the plasma membrane. There, STIM proteins tether and activate the highly Ca(2+)-selective Orai channels to mediate finely controlled Ca(2+) signals and to homeostatically balance cellular Ca(2+). Details are emerging on the remarkable organization within these STIM-induced junctional microdomains and the identification of new regulators and alternative target proteins for STIM.
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Señalización del Calcio , Calcio/metabolismo , Moléculas de Adhesión Celular/metabolismo , Retículo Endoplásmico/metabolismo , Proteínas de la Membrana/metabolismo , Proteínas de Neoplasias/metabolismo , Animales , Membrana Celular/metabolismo , Humanos , Uniones Intercelulares/metabolismo , Modelos Biológicos , Molécula de Interacción Estromal 1 , Molécula de Interacción Estromal 2RESUMEN
To determine the intrinsic role of Orai1 in osteoclast development, Orai1-floxed mice were bred with LysMcre mice to delete Orai1 from the myeloid lineage. PCR, in situ labelling and Western analysis showed Orai1 deletion in myeloid-lineage cells, including osteoclasts, as expected. Surprisingly, bone resorption was maintained in vivo, despite loss of multinucleated osteoclasts; instead, a large number of mononuclear cells bearing tartrate resistant acid phosphatase were observed on cell surfaces. An in vitro resorption assay confirmed that RANKL-treated Orai1 null cells, also TRAP-positive but mononuclear, degraded matrix, albeit at a reduced rate compared to wild type osteoclasts. This shows that mononuclear osteoclasts can degrade bone, albeit less efficiently. Further unexpected findings included that Orai1fl/fl -LysMcre vertebrae showed slightly reduced bone density in 16-week-old mice, despite Orai1 deletion only in myeloid cells; however, this mild difference resolved with age. In summary, in vitro analysis showed a severe defect in osteoclast multinucleation in Orai1 negative mononuclear cells, consistent with prior studies using less targeted strategies, but with evidence of resorption in vivo and unexpected secondary effects on bone formation leaving bone mass largely unaffected.
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Desarrollo Óseo , Calcio/metabolismo , Diferenciación Celular , Proteína ORAI1/fisiología , Osteoclastos/citología , Fosfatasa Ácida Tartratorresistente/metabolismo , Animales , Femenino , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Osteoclastos/metabolismoRESUMEN
While the zinc finger transcription factors EGR1, EGR2, and EGR3 are recognized as critical for T-cell function, the role of EGR4 remains unstudied. Here, we show that EGR4 is rapidly upregulated upon TCR engagement, serving as a critical "brake" on T-cell activation. Hence, TCR engagement of EGR4-/- T cells leads to enhanced Ca2+ responses, driving sustained NFAT activation and hyperproliferation. This causes profound increases in IFNγ production under resting and diverse polarizing conditions that could be reversed by pharmacological attenuation of Ca2+ entry. Finally, an in vivo melanoma lung colonization assay reveals enhanced anti-tumor immunity in EGR4-/- mice, attributable to Th1 bias, Treg loss, and increased CTL generation in the tumor microenvironment. Overall, these observations reveal for the first time that EGR4 is a key regulator of T-cell differentiation and function.
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Señalización del Calcio , Factores de Transcripción de la Respuesta de Crecimiento Precoz , Neoplasias , Animales , Diferenciación Celular , Activación de Linfocitos , Ratones , Microambiente Tumoral , Dedos de ZincRESUMEN
T cell-mediated adaptive immunity requires naïve, unstimulated T cells to transition from a quiescent metabolic state into a highly proliferative state upon T cell receptor engagement. This complex process depends on transcriptional changes mediated by Ca2+-dependent NFAT signaling, mTOR-mediated signaling and increased activity of the guanine nucleotide biosynthetic inosine-5'-monophosphate (IMP) dehydrogenase 1 and 2 enzymes (IMPDH1 and IMPDH2, hereafter IMPDH). Inhibitors of these pathways serve as potent immunosuppressants. Unexpectedly, we discovered that all three pathways converge to promote the assembly of IMPDH protein into micron-scale macromolecular filamentous structures in response to T cell activation. Assembly is post-transcriptionally controlled by mTOR and the Ca2+ influx regulator STIM1. Furthermore, IMPDH assembly and catalytic activity were negatively regulated by guanine nucleotide levels, suggesting a negative feedback loop that limits biosynthesis of guanine nucleotides. Filamentous IMPDH may be more resistant to this inhibition, facilitating accumulation of the higher GTP levels required for T cell proliferation.
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IMP Deshidrogenasa/metabolismo , Linfocitos T/enzimología , Animales , Células Cultivadas , Nucleótidos de Guanina/metabolismo , IMP Deshidrogenasa/genética , Activación de Linfocitos , Masculino , Ratones , Ratones Endogámicos C57BL , Receptores de Antígenos de Linfocitos T/genética , Receptores de Antígenos de Linfocitos T/inmunología , Bazo/enzimología , Bazo/inmunología , Molécula de Interacción Estromal 1/genética , Molécula de Interacción Estromal 1/metabolismo , Linfocitos T/inmunología , Serina-Treonina Quinasas TOR/genética , Serina-Treonina Quinasas TOR/metabolismoRESUMEN
RATIONALE: Pathological increases in cardiac afterload result in myocyte hypertrophy with changes in myocyte electrical and mechanical phenotype. Remodeling of contractile and signaling Ca2+ occurs in pathological hypertrophy and is central to myocyte remodeling. STIM1 (stromal interaction molecule 1) regulates Ca2+ signaling in many cell types by sensing low endoplasmic reticular Ca2+ levels and then coupling to plasma membrane Orai channels to induce a Ca2+ influx pathway. Previous reports suggest that STIM1 may play a role in cardiac hypertrophy, but its role in electrical and mechanical phenotypic alterations is not well understood. OBJECTIVE: To define the contributions of STIM1-mediated Ca2+ influx on electrical and mechanical properties of normal and diseased myocytes, and to determine whether Orai channels are obligatory partners for STIM1 in these processes using a clinically relevant large animal model of hypertrophy. METHODS AND RESULTS: Cardiac hypertrophy was induced by slow progressive pressure overload in adult cats. Hypertrophied myocytes had increased STIM1 expression and activity, which correlated with altered Ca2+-handling and action potential (AP) prolongation. Exposure of hypertrophied myocytes to the Orai channel blocker BTP2 caused a reduction of AP duration and reduced diastolic Ca2+ spark rate. BTP2 had no effect on normal myocytes. Forced expression of STIM1 in cultured adult feline ventricular myocytes increased diastolic spark rate and prolonged AP duration. STIM1 expression produced an increase in the amount of Ca2+ stored within the sarcoplasmic reticulum and activated Ca2+/calmodulin-dependent protein kinase II. STIM1 expression also increased spark rates and induced spontaneous APs. STIM1 effects were eliminated by either BTP2 or by coexpression of a dominant negative Orai construct. CONCLUSIONS: STIM1 can associate with Orai in cardiac myocytes to produce a Ca2+ influx pathway that can prolong the AP duration and load the sarcoplasmic reticulum and likely contributes to the altered electromechanical properties of the hypertrophied heart.
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Cardiomegalia/metabolismo , Cardiomegalia/fisiopatología , Contracción Miocárdica/fisiología , Proteínas de Neoplasias/biosíntesis , Molécula de Interacción Estromal 1/biosíntesis , Potenciales de Acción/fisiología , Animales , Gatos , Células Cultivadas , MasculinoRESUMEN
Antigen presentation to the T-cell receptor leads to sustained cytosolic Ca2+ elevation, which is critical for T-cell activation. We previously showed that in activated T cells, Ca2+ clearance is inhibited by the endoplasmic reticulum Ca2+ sensor stromal interacting molecule 1 (STIM1) via association with the plasma membrane Ca2+/ATPase 4 (PMCA4) Ca2+ pump. Having further observed that expression of both proteins is increased in activated T cells, the current study focused on mechanisms regulating both up-regulation of STIM1 and PMCA4 and assessing how this up-regulation contributes to control of Ca2+ clearance. Using a STIM1 promoter luciferase vector, we found that the zinc finger transcription factors early growth response (EGR) 1 and EGR4, but not EGR2 or EGR3, drive luciferase activity. We further found that neither STIM1 nor PMCA4 is up-regulated when both EGR1 and EGR4 are knocked down using RNA interference. Further, under these conditions, activation-induced Ca2+ clearance inhibition was eliminated with little effect on Ca2+ entry. Finally, we found that nuclear factor of activated T-cell (NFAT) activity is profoundly attenuated if Ca2+ clearance is not inhibited by STIM1. These findings reveal a critical role for STIM1-mediated control of Ca2+ clearance in NFAT induction during T-cell activation.-Samakai, E., Hooper, R., Martin, K. A., Shmurak, M., Zhang, Y., Kappes, D. J., Tempera, I., Soboloff, J. Novel STIM1-dependent control of Ca2+ clearance regulates NFAT activity during T-cell activation.
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Canales de Calcio/metabolismo , Calcio/metabolismo , Activación de Linfocitos/fisiología , Proteínas de la Membrana/metabolismo , Factores de Transcripción NFATC/metabolismo , Proteínas de Neoplasias/metabolismo , Molécula de Interacción Estromal 1/metabolismo , Linfocitos T/metabolismo , Señalización del Calcio/fisiología , Retículo Endoplásmico/metabolismo , Humanos , Receptores de Antígenos de Linfocitos T/metabolismo , Regulación hacia ArribaRESUMEN
Sigma-1 receptor (Sig-1R) is an intracellular chaperone protein with many ligands, located at the endoplasmic reticulum (ER). Binding of cocaine to Sig-1R has previously been found to modulate endothelial functions. In the present study, we show that cocaine dramatically inhibits store-operated Ca(2+) entry (SOCE), a Ca(2+) influx mechanism promoted by depletion of intracellular Ca(2+) stores, in rat brain microvascular endothelial cells (RBMVEC). Using either Sig-1R shRNA or pharmacological inhibition with the unrelated Sig-1R antagonists BD-1063 and NE-100, we show that cocaine-induced SOCE inhibition is dependent on Sig-1R. In addition to revealing new insight into fundamental mechanisms of cocaine-induced changes in endothelial function, these studies indicate an unprecedented role for Sig-1R as a SOCE inhibitor.
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Calcio/metabolismo , Cocaína/farmacología , Células Endoteliales/metabolismo , Microvasos/metabolismo , Receptores sigma/fisiología , Animales , Canales de Calcio/metabolismo , Señalización del Calcio/efectos de los fármacos , Señalización del Calcio/fisiología , Células Cultivadas , Células Endoteliales/efectos de los fármacos , Microvasos/efectos de los fármacos , Ratas , Receptores sigma/agonistas , Receptor Sigma-1RESUMEN
T-cell activation involves a complex signalling cascade uniquely dependent on elevated cytosolic Ca(2+) levels. Further, the spatiotemporal characteristics of this Ca(2+) signal play a critical role in this process via selective activation of transcription factors. In T cells, store-operated Ca(2+) entry (SOCe) is the primary Ca(2+) influx pathway; however, cytosolic Ca(2+) concentration depends upon the balance between Ca(2+) influx and extrusion. The plasma membrane Ca(2+) ATPase (PMCA) has previously been identified as a critical player in Ca(2+) clearance in T cells. Here, we provide data revealing both functional and physical links between the activation of stromal interacting molecule 1 (STIM1) and PMCA-mediated Ca(2+) clearance. Due to the ubiquitous expression of both STIM1 and PMCA, these findings have wide-ranging implications for Ca(2+) signalling in multiple cell types.
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Calcio/metabolismo , Activación de Linfocitos , Proteínas de la Membrana/metabolismo , Proteínas de Neoplasias/metabolismo , ATPasas Transportadoras de Calcio de la Membrana Plasmática/metabolismo , Linfocitos T/inmunología , Cationes Bivalentes/metabolismo , Línea Celular , Citosol/química , Humanos , ATPasas Transportadoras de Calcio de la Membrana Plasmática/antagonistas & inhibidores , Unión Proteica , Mapeo de Interacción de Proteínas , Transducción de Señal , Molécula de Interacción Estromal 1 , Linfocitos T/metabolismoRESUMEN
INTRODUCTION: Parkinson's disease (PD) is the most prevalent disorder of the basal ganglia, propagated by the degeneration of axon terminals within the striatum and subsequent loss of dopaminergic neurons in the substantia nigra (SN). Exposure of environmental neurotoxins and mutations of several mitochondrial and proteasomal genes are primarily responsible. METHODS: To determine whether signal transducer and activator of transcription 3 (STAT3) could protect dopaminergic neurons against degeneration, we first screened it in the in vitro capacity using immortalized rat dopaminergic N27 cells under 6-OHDA neurotoxicity. We then evaluated the effectiveness of constitutively active (ca) STAT3 as a neuroprotective agent on N27 cells in a 6-hydroxydopamine (6-OHDA) induced rat model of PD and compared it to control animals or animals where AAV/caRheb was expressed in SN. Behavioral outcomes were assessed using rotational and cylinder assays and mitochondrial function using reactive oxygen species (ROS) levels. RESULTS: Using flow cytometry, the in vitro analysis determined caSTAT3 significantly decreased dopaminergic neuronal death under 6-OHDA treatment conditions. Importantly, in vivo overexpression of caSTAT3 in SN dopaminergic neurons using AAV-mediated expression demonstrated significant neuroprotection of dopaminergic neurons following 6-OHDA. Both caSTAT3 and caRheb + caSTAT3 co-injection into substantia nigra reduced D-amphetamine-induced rotational behavior and increased ipsilateral forelimb function when compared to control animals. In addition, caSTAT3 decreased mitochondrial ROS production following 6-OHDA induced neurotoxicity. CONCLUSION: caSTAT3 confers resistance against ROS production in mitochondria of susceptible SN dopaminergic neurons potentially offering a new avenue for treatment against PD.
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Fármacos Neuroprotectores , Enfermedad de Parkinson , Ratas , Animales , Enfermedad de Parkinson/metabolismo , Neuronas Dopaminérgicas/metabolismo , Oxidopamina/toxicidad , Oxidopamina/metabolismo , Ratas Sprague-Dawley , Especies Reactivas de Oxígeno/metabolismo , Factor de Transcripción STAT3/metabolismo , Modelos Animales de Enfermedad , Sustancia Negra/metabolismo , Fármacos Neuroprotectores/farmacología , Fármacos Neuroprotectores/metabolismoRESUMEN
We review the abnormal bone turnover that is the basis of idiopathic inflammatory or rheumatoid arthritis and bone loss, with emphasis on Tumor Necrosis Factor-alpha (TNFα)-related mechanisms. We review selected data on idiopathic arthritis in juvenile human disease, and discuss mouse models focusing on induction of bone resorbing cells by TNFα and Receptor Activator of Nuclear Factor kappa B Ligand (RANKL). In both humans and animal models, macrophage-derived cells in the joint, particularly in the synovium and periosteum, degrade bone and cartilage. Mouse models of rheumatoid arthritis share with human disease bone resorbing cells and strong relation to TNFα expression. In humans, differences in therapy and prognosis of arthritis vary with age, and results from early intervention for inflammatory cytokines in juvenile patients are particularly interesting. Mechanisms that contribute to inflammatory arthritis reflect, in large part, inflammatory cytokines that play minor roles in normal bone turnover. Changes in inflammatory cytokines, particularly TNFα, are many times larger, and presented in different locations, than cytokines that regulate normal bone turnover. Recent data from in vitro and mouse models include novel mechanisms described in differentiation of bone resorbing cells in inflammatory arthritis dependent on the Transient Receptor Potential Channel (TRPC) family of calcium channels. Low-molecular weight (MW) inhibitors of TRPC channels add to their potential importance. Associations with inflammatory arthritis unrelated to TNFα are briefly summarized as pointing to alternative mechanisms. We suggest that early detection and monoclonal antibodies targeting cytokines mediating disease progression deserves emphasis.
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Artritis Juvenil , Modelos Animales de Enfermedad , Factor de Necrosis Tumoral alfa , Animales , Artritis Juvenil/metabolismo , Artritis Juvenil/inmunología , Humanos , Factor de Necrosis Tumoral alfa/metabolismo , Ratones , Remodelación Ósea , Ligando RANK/metabolismo , Osteoclastos/metabolismoRESUMEN
This study provides a detailed understanding of the preclinical pharmacokinetics and metabolism of ELP-004, an osteoclast inhibitor in development for the treatment of bone erosion. Current treatments for arthritis, including biological disease-modifying antirheumatic drugs, are not well-tolerated in a substantial subset of arthritis patients and are expensive; therefore, new treatments are needed. Pharmacokinetic parameters of ELP-004 were tested with intravenous, oral, and subcutaneous administration and found to be rapidly absorbed and distributed. We found that ELP-004 was non-mutagenic, did not induce chromosome aberrations, non-cardiotoxic, and had minimal off-target effects. Using in vitro hepatic systems, we found that ELP-004 is primarily metabolized by CYP1A2 and CYP2B6 and predicted metabolic pathways were identified. Finally, we show that ELP-004 inhibits osteoclast differentiation without suppressing overall T-cell function. These preclinical data will inform future development of an oral compound as well as in vivo efficacy studies in mice.
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Osteoclastos , Animales , Ratones , Osteoclastos/efectos de los fármacos , Masculino , Evaluación Preclínica de Medicamentos , Femenino , Ratones Endogámicos C57BL , Administración Oral , Humanos , Diferenciación Celular/efectos de los fármacos , Linfocitos T/efectos de los fármacos , Linfocitos T/metabolismo , Antirreumáticos/farmacología , Antirreumáticos/farmacocinética , Antirreumáticos/administración & dosificaciónRESUMEN
Store-operated Ca(2+) entry (SOCE) is an essential process in T cell activation. SOCE is controlled by the Ca(2+) release-activated Ca(2+) (CRAC) channel encoded by the gene Orai1 that is expressed on the plasma membrane and activated by STIM1 when ER Ca(2+) stores are depleted. Our earlier work showed that a somatic T-cell line Jurkat mutant H123 bearing a defect in Ca(2+) signaling was susceptible to the apoptotic effects of type I interferons (IFN-α/ß). The nature of the mutation and whether this mutation was linked to IFN-α/ß apoptotic susceptibility was unknown. Here we show that H123 cells lacked Orai1 and exhibit reduced STIM1 protein. Reconstitution of both Orai1 and STIM1 in H123 cells rescued SOCE in response to thapsigargin and ionomycin and abrogated IFN-α/ß-induced apoptosis. Reciprocally, overexpression of the dominant negative Orai1-E106A in either parental Jurkat cells or an unrelated human T cell line (CEM391) inhibited SOCE and led to sensitization to IFN-α/ß-induced apoptosis. Furthermore, we showed that the Ca(2+) response pathway antagonized the IFN-α/ß -induced transcriptional responses; in the absence of SOCE, this negative regulatory effect was lost. However, the inhibitory effect of Ca(2+) on type I IFN-induced gene transcription was diminished by pharmacological inhibition of NF-κB in cells with intact SOCE. Our findings reveal an unexpected and novel regulatory crosstalk mechanism between type I IFNs and store-operated Ca(2+) signaling pathways mediated at least in part by NF-κB activity with significant clinical implications to both viral and tumor immunology.