RESUMEN
OBJECTIVE: In this study, we examine how advancements in novel antirheumatic drugs affect the clinicopathologic features of lymphoproliferative disorder (LPD) in patients with rheumatoid arthritis (RA). METHODS: In this multicenter study across 53 hospitals in Japan, we characterized patients with RA who developed LPDs and visited the hospitals between January 1999 and March 2021. The statistical tools used included Fisher's exact test, the Mann-Whitney U-test, the log-rank test, logistic regression analysis, and Cox proportional hazards models. RESULTS: Overall, 752 patients with RA-associated LPD (RA-LPD) and 770 with sporadic LPD were included in the study. We observed significant differences in the clinicopathologic features between patients with RA-LPD and those with sporadic LPD. Histopathological analysis revealed a high frequency of LPD-associated immunosuppressive conditions. Furthermore, patients with RA-LPD were evaluated based on the antirheumatic drugs administered. The methotrexate (MTX) plus tacrolimus and MTX plus tumor necrosis factor inhibitor (TNFi) groups had different affected site frequencies and histologic subtypes than the MTX-only group. Moreover, MTX and TNFi may synergistically affect susceptibility to Epstein-Barr virus infection. In case of antirheumatic drugs administered after LPD onset, tocilizumab (TCZ)-only therapy was associated with lower frequency of regrowth after spontaneous regression than other regimens. CONCLUSION: Antirheumatic drugs administered before LPD onset may influence the clinicopathologic features of RA-LPD, with patterns changing over time. Furthermore, TCZ-only regimens are recommended after LPD onset.
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Antirreumáticos , Artritis Reumatoide , Trastornos Linfoproliferativos , Metotrexato , Inhibidores del Factor de Necrosis Tumoral , Humanos , Artritis Reumatoide/tratamiento farmacológico , Artritis Reumatoide/complicaciones , Antirreumáticos/uso terapéutico , Antirreumáticos/efectos adversos , Trastornos Linfoproliferativos/inducido químicamente , Masculino , Femenino , Persona de Mediana Edad , Metotrexato/uso terapéutico , Anciano , Inhibidores del Factor de Necrosis Tumoral/uso terapéutico , Inhibidores del Factor de Necrosis Tumoral/efectos adversos , Japón , Tacrolimus/uso terapéutico , Tacrolimus/efectos adversos , Quimioterapia Combinada , Infecciones por Virus de Epstein-Barr/complicaciones , AdultoRESUMEN
Chronic lung diseases (CLD), including interstitial lung disease (ILD) and airway diseases (ADs), are common complications of rheumatoid arthritis (RA). Rheumatoid factor (RF) and anti-citrullinated peptide antibodies are reported to be associated with CLD in RA patients. The presence of anti-melanoma differentiation-associated gene 5 (MDA5) antibodies (Abs) is associated with clinically amyopathic dermatomyositis developing into rapidly progressive ILD. However, few studies on anti-MDA5 Abs in RA have been published. Here, we analyzed the association of anti-MDA5 Abs with CLD complications in RA. Anti-MDA5 Abs were quantified in sera from RA patients with or without CLD. Anti-MDA5 Ab levels were higher in RA patients with ADs than without (mean ± SDM, 4.4 ± 2.4 vs. 4.0 ± 4.2, p = 0.0001). AUC values of anti-MDA5 Ab and RF ROC curves were similar in RA patients with or without CLD (0.578, 95%CI 0.530-0.627 and 0.579, 95%CI 0.530-0.627, respectively, p = 0.9411). Multiple logistic regression analysis of anti-MDA5 Abs and clinical characteristics yielded an MDA5-index with a higher AUC value than anti-MDA5 Ab alone (0.694, 95%CI 0.648-0.740, p = 5.08 × 10-5). Anti-MDA5 Abs were associated with ADs in RA patients and could represent a biomarker for CLD, similar to RF. The involvement of anti-MDA5 Abs in the pathogenesis of ADs in RA is proposed.
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Artritis Reumatoide , Dermatomiositis , Enfermedades Pulmonares Intersticiales , Humanos , Helicasa Inducida por Interferón IFIH1 , Autoanticuerpos , Dermatomiositis/complicaciones , Enfermedades Pulmonares Intersticiales/complicaciones , Artritis Reumatoide/complicaciones , Estudios RetrospectivosRESUMEN
CONTEXT: Atypical femoral fractures (AFFs) are very rare atraumatic or mild trauma fractures in the subtrochanteric region or femoral shaft. Some unique genetic variants in Asian populations might confer susceptibility to AFF, since the incidence of AFFs is higher in Asian populations. OBJECTIVE: Because rare variants have been found to be causative in some diseases and the roles of osteomalacia causative genes have not been reported, we investigated rare variants in genes causing abnormal mineralization. METHODS: Exome sequencing was performed to detect variants in gene coding and boundary regions, and the frequencies of deleterious rare alleles were compared between Japanese patients with AFF (nâ =â 42) and controls of the 4.7KJPN panel of Tohoku Medical Megabank by whole genome sequencing (nâ =â 4773). RESULTS: The frequency of the deleterious rare allele of ENPP1 was significantly increased in AFF (Pâ =â .0012, corrected P [Pc]â =â .0155, OR 4.73, 95% CI 2.15-10.40). In multigene panel analysis, the frequencies of deleterious rare alleles of candidate genes were increased in AFF (Pâ =â .0025, OR 2.72, 95% CI 1.49-4.93). Principal component analysis of bone metabolism markers identified a subgroup of patients with AFF with higher frequencies of deleterious rare alleles in ENPP1 (Pâ =â 4.69 × 10-5, Pcâ =â .0006, OR 8.47, 95% CI 3.76-19.09) and the candidate genes (Pâ =â 1.08 × 10-5, OR 5.21, 95% CI 2.76-9.86). CONCLUSION: AFF is associated with genes including ENPP1 that cause abnormal mineralization, suggesting that osteomalacia is an underlying condition predisposing to AFF and that higher incident rates of AFFs in Asian populations might be explained by the genetic risk factors including ENPP1.
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Conservadores de la Densidad Ósea , Enfermedades Óseas , Raquitismo Hipofosfatémico Familiar , Fracturas del Fémur , Osteomalacia , Alelos , Conservadores de la Densidad Ósea/efectos adversos , Enfermedades Óseas/genética , Difosfonatos/efectos adversos , Raquitismo Hipofosfatémico Familiar/complicaciones , Femenino , Fracturas del Fémur/epidemiología , Fracturas del Fémur/genética , Humanos , Masculino , Osteomalacia/genéticaRESUMEN
OBJECTIVE: Rheumatoid arthritis (RA) is often complicated with chronic lung diseases (CLD), including interstitial lung disease (ILD) and airway disease, which occur as extra-articular manifestations. CLD in RA have been associated with the production of rheumatoid factor (RF), anti-citrullinated peptide antibody (ACPA), or anti-carbamylated protein (CarP) antibody. However, few validation studies have been performed thus far. In the present study, we investigated the association of RF, ACPA, and anti-CarP antibodies with RA complicated with CLD. METHODS: Sera from RA patients with or without CLD were collected. The levels of serum RF, RF immunoglobulin A (IgA), ACPA IgG, ACPA IgA, and ACPA secretory component (SC) were measured using enzyme-linked immunosorbent assay. RESULTS: The comparison of RA patients with and without CLD showed that RF IgA was associated with ILD (mean ± standard deviation: 206.6 ± 400.5 vs. 95.0 ± 523.1 U/ml, respectively, P = 1.13 × 10- 8), particularly usual interstitial pneumonia (UIP) (263.5 ± 502.0 U/ml, P = 1.00 × 10- 7). ACPA SC was associated with RA complicated with ILD (mean ± standard deviation: 8.6 ± 25.1 vs. 2.3 ± 3.4 U/ml, respectively, P = 0.0003), particularly nonspecific interstitial pneumonia (NSIP) (10.7 ± 31.5 U/ml, P = 0.0017). Anti-CarP antibodies were associated with RA complicated with ILD (0.042 ± 0.285 vs. 0.003 ± 0.011 U/ml, respectively, P = 1.04X10- 11). CONCLUSION: RF IgA and ACPA SC in RA were associated with UIP and NSIP, respectively, suggesting different specificities in patients with RA. Anti-CarP antibodies were associated with ILD in RA. These results may help elucidate the different pathogeneses of UIP and NSIP in RA.
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Artritis Reumatoide , Enfermedades Pulmonares Intersticiales , Anticuerpos Antiproteína Citrulinada , Artritis Reumatoide/diagnóstico , Autoanticuerpos , Humanos , Enfermedades Pulmonares Intersticiales/diagnóstico , Factor Reumatoide , Componente SecretorioRESUMEN
Objectives: Interstitial lung disease (ILD) is an extra-articular manifestation in rheumatoid arthritis (RA), detected in 10.7% of patients, and causing a poor prognosis. Hence, biomarkers for ILD are urgently required in RA. Low molecular weight metabolites can be assessed by metabolomic analyses, and although these have been conducted in RA and in idiopathic pulmonary fibrosis, few have been carried out for ILD in the context of RA. Therefore, we analyzed serum metabolomic profiles of ILD in RA to identify novel biomarkers. Methods: Serum samples from 100 RA patients with ILD and 100 matched RA patients without chronic lung disease (CLD) were collected. These samples were subjected to metabolomic analyses using capillary electrophoresis time-of-flight mass spectrometry. Results: A total of 299 metabolites were detected in the metabolomic analysis. By univariate analysis, serum levels of decanoic acid and morpholine were lower in RA with ILD (false discovery rate Q = 1.87 × 10-11 and 7.09 × 10-6, respectively), and glycerol was higher (Q = 1.20 × 10-6), relative to RA without CLD. Serum levels of these metabolites in RA with usual interstitial pneumonia or RA with non-specific interstitial pneumonia were also altered. The partial least squares-discriminant analysis model generated from these three metabolites could successfully discriminate ILD in RA (area under the curve: 0.919, 95% confidence interval: 0.867-0.968, sensitivity 0.880, specificity 0.780). Conclusions: Serum levels of some metabolites were significantly different in RA with ILD compared with RA without CLD. It is concluded that metabolomic profiling will be useful for discovering candidate screening biomarkers for ILD in RA.
RESUMEN
OBJECTIVES: To estimate the prevalence of chronic kidney disease in patients with rheumatoid arthritis (RA) and the administration of disease-modifying anti-rheumatic-drugs (DMARDs), using data from the National Database of Rheumatic Disease by iR-net in Japan (NinJa) 2012 study. METHODS: From a total of 11,940 RA patients, 7135 who underwent an estimated glomerular filtration rate (eGFR) test were studied. Renal dysfunction staging was assessed using Japanese eGFR equations and classified according to the Kidney Disease Improving Global Outcomes 2012 clinical practice guideline. RESULTS: The prevalence of GFR stages was as follows: stage G1, 25.4%; stage G2, 55.9%; stage G3, 17.5%; stage G4, 0.8%; and stage G5, 0.2%. Overall, 92.7% of patients received at least one DMARD. Sulfasalazine, tacrolimus, and biologics (except inflixmab) were administered in all GFR stages. Methotrexate was not prescribed in patients with stage G5, but methotrexate 3.5 mg/week (mean) was prescribed in four patients (6.8%) with stage G4. Non-steroidal anti-inflammatory drugs and glucocorticoids were prescribed in 40.5% and 43.7% of patients, respectively. CONCLUSION: The prevalence of kidney disease in this large sample of RA patients was higher than that in the general population, and the results suggest that RA patients with renal dysfunction require careful drug selection.
Asunto(s)
Antirreumáticos/uso terapéutico , Artritis Reumatoide/tratamiento farmacológico , Tasa de Filtración Glomerular/fisiología , Insuficiencia Renal Crónica/epidemiología , Anciano , Artritis Reumatoide/complicaciones , Femenino , Humanos , Japón/epidemiología , Masculino , Persona de Mediana Edad , Prevalencia , Insuficiencia Renal Crónica/complicaciones , Insuficiencia Renal Crónica/fisiopatologíaRESUMEN
Insulin-like growth factor-1 (IGF-1) plays important roles in the regulation of neuronal development. The electrical activity of Na(+) channels is crucial for the regulation of synaptic formation and maintenance/repair of neuronal circuits. Here, we examined the effects of chronic IGF-1 treatment on cell surface expression and function of Na(+) channels. In cultured bovine adrenal chromaffin cells expressing Na(V)1.7 isoform of voltage-dependent Na(+) channels, chronic IGF-1 treatment increased cell surface [(3)H]saxitoxin binding by 31%, without altering the Kd value. In cells treated with IGF-1, veratridine-induced (22)Na(+) influx, and subsequent (45)Ca(2+) influx and catecholamine secretion were augmented by 35%, 33%, 31%, respectively. Pharmacological properties of Na(+) channels characterized by neurotoxins were similar between nontreated and IGF-1-treated cells. IGF-1-induced up-regulation of [(3)H]saxitoxin binding was prevented by phosphatydil inositol-3 kinase inhibitors (LY204002 or wortmannin), or Akt inhibitor (Akt inhibitor IV). Glycogen synthase kinase-3 (GSK-3) inhibitors (LiCl, valproic acid, SB216763 or SB415286) also increased cell surface [(3)H]saxitoxin binding by â¼ 33%, whereas simultaneous treatment of IGF-1 with GSK-3 inhibitors did not produce additive increasing effect on [(3)H]saxitoxin binding. IGF-1 (100 nM) increased Ser(437)-phosphorylated Akt and Ser(9)-phosphorylated GSK-3ß, and inhibited GSK-3ß activity. Treatment with IGF-1, LiCl or SB216763 increased protein level of Na(+) channel α-subunit; it was prevented by cycloheximide. Either treatment increased α-subunit mRNA level by â¼ 48% and accelerated α-subunit gene transcription by â¼ 30% without altering α-subunit mRNA stability. Thus, inhibition of GSK-3ß caused by IGF-1 up-regulates cell surface expression of functional Na(+) channels via acceleration of α-subunit gene transcription.
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Catecolaminas/metabolismo , Células Cromafines/efectos de los fármacos , Glucógeno Sintasa Quinasa 3/metabolismo , Factor I del Crecimiento Similar a la Insulina/farmacología , Canales de Sodio/metabolismo , Regulación hacia Arriba/efectos de los fármacos , Glándulas Suprarrenales/citología , Animales , Calcio/metabolismo , Bovinos , Células Cultivadas , Células Cromafines/metabolismo , Relación Dosis-Respuesta a Droga , Inhibidores Enzimáticos/farmacología , Glucógeno Sintasa Quinasa 3 beta , Inmunoprecipitación , Fosforilación/efectos de los fármacos , Unión Proteica/efectos de los fármacos , ARN Mensajero/metabolismo , Radioisótopos/farmacocinética , Receptor IGF Tipo 1/metabolismo , Receptor de Insulina/metabolismo , Saxitoxina/farmacocinética , Sodio/metabolismo , Canales de Sodio/genética , Factores de TiempoRESUMEN
In cultured bovine adrenal chromaffin cells expressing Nav1.7 sodium channel isoform, we previously showed that veratridine-induced Na+ influx via Nav1.7 and the subsequent Ca2+ influx via voltage-dependent calcium channels activated protein kinase C-alpha and Akt, which converged on increasing inhibitory Ser9-phosphorylation of glycogen synthase kinase-3beta, decreasing constitutive Ser396-phosphorylation of tau. Here, veratridine increased constitutive Tyr204-phosphorylation of extracellular signal-regulated kinase-1/-2 (ERK1/ERK2) and constitutive Thr180/Tyr182-dual phosphorylation of p38 by approximately 118% (EC50=2.8 microM). Veratridine-induced increased phosphorylation levels of ERK1/ERK2 and p38 were abolished by tetrodotoxin, extracellular Ca2+ removal, or Gö6976 [12-(2-cyanoethyl)-6,7,12,13-tetrahydro-13-methyl-5-oxo-5H-indolo(2,3-a)pyrrolo(3,4-c)-carbazole;Go6976] (protein kinase C-alpha inhibitor). PD98059 (2'-amino-3'-methoxyflavone) (ERK1/ERK2 inhibitor) or SB203580 [4-(4-fluorophenyl)-2-(4-methylsulfinylphenyl)-5-(4-pyridyl)1H-imidazole] (p38 inhibitor) attenuated veratridine-induced increased phosphorylation of glycogen synthase kinase-3beta and decreased phosphorylation of tau by approximately 54% and approximately 56%, as partial blockade by Gö6976. Additionally, basal constitutive phosphorylation levels of ERK1/ERK2 and p38 were decreased by PD98059 or SB203580, but not by SB216763 [3-(2,4-dichlorophenyl)-4-(1-methyl-1H-indolo-3-yl)-1H-pyrrole-2,5-dione] (glycogen synthase kinase-3beta inhibitor) or extracellular Ca2+ removal. In this condition, PD98059 or SB203580 (but not SB216763 or extracellular Ca2+ removal) inhibited veratridine-induced 22Na+ influx and 45Ca2+ influx, without changing nicotine-induced 22Na+ influx via nicotinic receptor-associated cation channels and nicotine-induced 45Ca2+ influx via voltage-dependent calcium channels. These results suggest that Nav1.7-Ca2+ influx-protein kinase C-alpha pathway activated ERK1/ERK2 and p38, which increased phosphorylation of glycogen synthase kinase-3beta, decreasing tau phosphorylation. In veratridine-nontreated cells, basal constitutive activities of ERK1/ERK2 and p38 primed Nav1.7 to increase 22Na+ influx.
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Calcio/metabolismo , Quinasas MAP Reguladas por Señal Extracelular/metabolismo , Glucógeno Sintasa Quinasa 3/metabolismo , Activación del Canal Iónico , Canales de Sodio/metabolismo , Proteínas Quinasas p38 Activadas por Mitógenos/metabolismo , Proteínas tau/metabolismo , Animales , Carbazoles/farmacología , Bovinos , Relación Dosis-Respuesta a Droga , Quinasas MAP Reguladas por Señal Extracelular/química , Flavonoides/farmacología , Glucógeno Sintasa Quinasa 3 beta , Conos de Crecimiento/efectos de los fármacos , Conos de Crecimiento/metabolismo , Humanos , Imidazoles/farmacología , Activación del Canal Iónico/efectos de los fármacos , Proteína Quinasa 1 Activada por Mitógenos/química , Proteína Quinasa 1 Activada por Mitógenos/metabolismo , Proteína Quinasa 3 Activada por Mitógenos/química , Proteína Quinasa 3 Activada por Mitógenos/metabolismo , Nicotina/farmacología , Fosforilación/efectos de los fármacos , Proteína Quinasa C-alfa/metabolismo , Piridinas/farmacología , Receptores Nicotínicos/metabolismo , Canales de Sodio/genética , Tetrodotoxina/farmacología , Factores de Tiempo , Veratridina/farmacologíaRESUMEN
In cultured bovine adrenal chromaffin cells, approximately 24 h-treatment with insulin-like growth factor-I (IGF-I) decreased cell surface (125)I-IGF-I binding capacity and IGF-I receptor protein level by approximately 64% (EC(50) = 5.0 nM; t(1/2) = approximately 7 h). IGF-I-induced IGF-I receptor decrease was abolished by LY294002 (phosphoinositide 3-kinase inhibitor) and partially attenuated by rapamycin (an inhibitor of mammalian target of rapamycin [mTOR]). SB216763 (an inhibitor of glycogen synthase kinase-3 [GSK-3]) down-regulated IGF-I receptor, which was further decreased by IGF-I. IGF-I increased inhibitory Ser(9)-phosphorylation of GSK-3beta and stimulatory Ser(2448)-phosphorylation of mTOR. l-leucine increased phosphorylation of mTOR (but not GSK-3beta), and down-regulated IGF-I receptor, both events being abolished by rapamycin. IGF-I-induced IGF-I receptor decrease was not prevented by proteolysis inhibitors. Pulse-label with [(35)S]methionine/cysteine followed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed that SB216763 or L-leucine retarded synthesis of IGF-I receptor and its precursor molecule. SB216763 (but not l-leucine) destabilized IGF-I receptor mRNA and decreased its level, without changing IGF-I receptor gene transcription. In SB216763-treated cells, IGF-I-induced Tyr-autophosphorylation of IGF-I receptor was decreased by 36%, compared to nontreated cells. IGF-I attenuated constitutive Ser(396)-phosphorylation of tau by 30% in nontreated cells, but not in SB216763-treated cells. IGF-I-induced down-regulations of (125)I-IGF-I binding and IGF-I receptor, as well as IGF-I-induced phosphorylations of GSK-3beta and mTOR were restored to the control levels of nontreated cells after washout of IGF-I (10 nM for 12 h)-treated cells. Thus, IGF-I down-regulated functional IGF-I receptor via GSK-3beta inhibition and mTOR activation; constitutive activity of GSK-3beta maintained IGF-I receptor level in nonstimulated cells.
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Glándulas Suprarrenales/metabolismo , Células Cromafines/metabolismo , Glucógeno Sintasa Quinasa 3/metabolismo , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Proteínas Serina-Treonina Quinasas/metabolismo , Receptor IGF Tipo 1/metabolismo , Proteínas tau/metabolismo , Glándulas Suprarrenales/efectos de los fármacos , Animales , Bovinos , Células Cultivadas , Células Cromafines/efectos de los fármacos , Regulación hacia Abajo/efectos de los fármacos , Glucógeno Sintasa Quinasa 3/antagonistas & inhibidores , Glucógeno Sintasa Quinasa 3 beta , Factor I del Crecimiento Similar a la Insulina/metabolismo , Leucina/metabolismo , Fosfatidilinositol 3-Quinasas/metabolismo , Fosforilación , ARN Mensajero/metabolismo , Receptor IGF Tipo 1/genética , Transducción de Señal/efectos de los fármacos , Serina-Treonina Quinasas TORRESUMEN
Multiple signaling pathways via insulin receptor substrate-1 and -2 play crucial roles in health, diseases, and therapeutics (i.e., longevity, tumorigenesis, and neuroprotection). The 90-kDa heat-shock protein (Hsp90) is an emerging target molecule of therapeutics, Hsp90 inhibitors being promising against various diseases (e.g., cancer, brain and cardiac ischemia, and neurodegenerative diseases). Much remains, however, unknown whether Hsp90 could regulate insulin receptor substrate-1 and -2 signaling pathways. In cultured bovine adrenal chromaffin cells, we observed that 24-h treatment with 1 microM geldanamycin (an inhibitor of Hsp90) decreased insulin receptor substrate-1 level, while increasing insulin receptor substrate-2 level; besides, geldanamycin lowered phosphoinositide 3-kinase, phosphoinositide-dependent kinase-1, Akt, glycogen synthase kinase-3beta, and Raf-1 levels, without changing extracellular signal-regulated kinase and its upstream kinase levels. Chronic (>or=12h) treatment with 0.1-10 microM Hsp90 inhibitor (geldanamycin, 17-allylamino-17-demethoxy-geldanamycin, herbimycin A, and radicicol) decreased insulin receptor substrate-1 level by approximately 66%, while increasing insulin receptor substrate-2 level by approximately 160%. These effects of geldanamycin (IC(50) 155 nM, EC(50) 177 nM) and 17-allylamino-17-demethoxy-geldanamycin (IC(50) 310 nM, EC(50) 260 nM) were time- and concentration-dependent. Geldanamycin-induced decrease of insulin receptor substrate-1 was attenuated by lactacystin, beta-lactone or MG132 (proteasome inhibitor), but not by calpastatin (calpain inhibitor) or leupeptin (lysosome inhibitor); geldanamycin did not affect heteroprotein complex formation between insulin receptor substrate-1 or -2 and Hsp90. Geldanamycin-induced increase of insulin receptor substrate-2 was prevented by cycloheximide or actinomycin D. Geldanamycin lowered insulin receptor substrate-1 mRNA level by approximately 39%, while raising insulin receptor substrate-2 mRNA level by approximately 109% between 3 and 24h, without changing the stability of insulin receptor substrate-1 and -2 mRNAs. Nuclear run-on assay revealed that geldanamycin retarded insulin receptor substrate-1 gene transcription by 42%, while accelerating insulin receptor substrate-2 gene transcription by 41%. Hsp90 inhibitors oppositely altered insulin receptor substrate-1 and -2 levels via proteasomal degradation and gene transcription.
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Médula Suprarrenal/metabolismo , Células Cromafines/metabolismo , Proteínas HSP90 de Choque Térmico/metabolismo , Proteínas Sustrato del Receptor de Insulina/metabolismo , Transducción de Señal/fisiología , Médula Suprarrenal/citología , Animales , Benzoquinonas/farmacología , Bovinos , Células Cultivadas , Células Cromafines/citología , Células Cromafines/efectos de los fármacos , Regulación hacia Abajo/efectos de los fármacos , Regulación hacia Abajo/genética , Inhibidores Enzimáticos/farmacología , Regulación de la Expresión Génica/efectos de los fármacos , Regulación de la Expresión Génica/fisiología , Proteínas HSP90 de Choque Térmico/antagonistas & inhibidores , Proteínas Sustrato del Receptor de Insulina/efectos de los fármacos , Proteínas Sustrato del Receptor de Insulina/genética , Lactamas Macrocíclicas/farmacología , Fosfotransferasas/efectos de los fármacos , Fosfotransferasas/metabolismo , Complejo de la Endopetidasa Proteasomal/efectos de los fármacos , Complejo de la Endopetidasa Proteasomal/metabolismo , Inhibidores de la Síntesis de la Proteína/farmacología , ARN Mensajero/efectos de los fármacos , ARN Mensajero/metabolismo , Transducción de Señal/efectos de los fármacos , Transcripción Genética/efectos de los fármacos , Transcripción Genética/genética , Activación Transcripcional/efectos de los fármacos , Activación Transcripcional/genética , Regulación hacia Arriba/efectos de los fármacos , Regulación hacia Arriba/genéticaRESUMEN
In cultured bovine adrenal chromaffin cells expressing Na(V)1.7 isoform of voltage-dependent Na(+) channels, we have previously reported that lithium chloride (LiCl) inhibits function of Na(+) channels independent of glycogen synthase kinase-3 (GSK-3) (Yanagita et al., 2007). Here, we further examined the effects of chronic lithium treatment on Na(+) channels. LiCl treatment (1-30 mM, > or = 12 h) increased cell surface [(3)H]saxitoxin ([(3)H]STX) binding by approximately 32% without altering the affinity of [(3)H]STX binding. This increase was prevented by cycloheximide and actinomycin D. SB216763 and SB415286 (GSK-3 inhibitors) also increased cell surface [(3)H]STX binding by approximately 31%. Simultaneous treatment with LiCl and SB216763 or SB415286 did not produce an increased effect on [(3)H]STX binding compared with either treatment alone. LiCl increased Na(+) channel alpha-subunit mRNA level by 32% at 24 h. LiCl accelerated alpha-subunit gene transcription by 35% without altering alpha-subunit mRNA stability. In LiCl-treated cells, LiCl inhibited veratridine-induced (22)Na(+) influx as in untreated cells. However, washout of LiCl after chronic treatment enhanced veratridine-induced (22)Na(+) influx, (45)Ca(2+) influx and catecholamine secretion by approximately 30%. Washout of LiCl after 24 h treatment shifted concentration-response curve of veratridine upon (22)Na(+) influx upward, without altering its EC(50) value. Ptychodiscus brevis toxin-3 allosterically enhanced veratridine-induced (22)Na(+) influx by two-fold in untreated and LiCl-treated cells. Whole-cell patch-clamp analysis indicated that I-V curve and steady-state inactivation/activation curves were comparable between untreated and LiCl-treated cells. Thus, GSK-3 inhibition by LiCl up-regulated cell surface Na(V)1.7 via acceleration of alpha-subunit gene transcription, enhancing veratridine-induced Na(+) influx, Ca(2+) influx and catecholamine secretion.
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Antimaníacos/farmacología , Células Cromafines/efectos de los fármacos , Glucógeno Sintasa Quinasa 3/antagonistas & inhibidores , Cloruro de Litio/farmacología , Canales de Sodio/metabolismo , Animales , Calcio/metabolismo , Canales de Calcio/metabolismo , Catecolaminas/metabolismo , Bovinos , Membrana Celular/efectos de los fármacos , Membrana Celular/metabolismo , Células Cultivadas , Células Cromafines/metabolismo , Glucógeno Sintasa Quinasa 3/metabolismo , Potenciales de la Membrana/efectos de los fármacos , Potenciales de la Membrana/fisiología , Estabilidad Proteica/efectos de los fármacos , ARN Mensajero/metabolismo , Sodio/metabolismo , Canales de Sodio/genética , Transcripción Genética/efectos de los fármacosRESUMEN
In cultured bovine adrenal chromaffin cells expressing Na(v)1.7 sodium channel isoform, veratridine increased Ser(473)-phosphorylation of Akt and Ser(9)-phosphorylation of glycogen synthase kinase-3beta by approximately 217 and approximately 195%, while decreasing Ser(396)-phosphorylation of tau by approximately 36% in a concentration (EC(50)=2.1 microM)- and time (t(1/2)=2.7 min)-dependent manner. These effects of veratridine were abolished by tetrodotoxin or extracellular Ca(2+) removal. Veratridine (10 microM for 5 min) increased translocation of Ca(2+)-dependent conventional protein kinase C-alpha from cytoplasm to membranes by 47%; it was abolished by tetrodotoxin, extracellular Ca(2+) removal, or Gö6976 (an inhibitor of protein kinase C-alpha), and partially attenuated by LY294002 (an inhibitor of phosphatidylinositol 3-kinase). LY294002 (but not Gö6976) abrogated veratridine-induced Akt phosphorylation. In contrast, either LY294002 or Gö6976 alone attenuated veratridine-induced glycogen synthase kinase-3beta phosphorylation by 65 or 42%; however, LY294002 plus Gö6976 completely blocked it. Veratridine (10 microM for 5 min)-induced decrease of tau phosphorylation was partially attenuated by LY294002 or Gö6976, but completely blocked by LY294002 plus Gö6976; okadaic acid or cyclosporin A (inhibitors of protein phosphatases 1, 2A, and 2B) failed to alter tau phosphorylation. These results suggest that Na(+) influx via Na(v)1.7 sodium channel and the subsequent Ca(2+) influx via voltage-dependent calcium channel activated (1) Ca(2+)/protein kinase C-alpha pathway, as well as (2) Ca(2+)/phosphatidylinositol 3-kinase/Akt and (3) Ca(2+)/phosphatidylinositol 3-kinase/protein kinase C-alpha pathways; these parallel pathways converged on inhibitory phosphorylation of glycogen synthase kinase-3beta, decreasing tau phosphorylation.
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Glándulas Suprarrenales/metabolismo , Calcio/metabolismo , Células Cromafines/metabolismo , Glucógeno Sintasa Quinasa 3/metabolismo , Canales de Sodio/metabolismo , Proteínas tau/metabolismo , Glándulas Suprarrenales/citología , Animales , Western Blotting , Bloqueadores de los Canales de Calcio/farmacología , Señalización del Calcio/efectos de los fármacos , Bovinos , Inhibidores Enzimáticos/farmacología , Glucógeno Sintasa Quinasa 3 beta , Proteína Oncogénica v-akt/metabolismo , Fosfatidilinositol 3-Quinasas/metabolismo , Inhibidores de las Quinasa Fosfoinosítidos-3 , Fosforilación , Tetrodotoxina/farmacología , Veratridina/farmacologíaRESUMEN
The ability of calcineurin to regulate IRS-1 and IRS-2 levels has not been examined in any given cells, although calcineurin inhibition by therapeutic immunosuppressants produced cytoprotective and cytotoxic effects (e.g., new-onset of diabetes mellitus, seizure). Chronic (>or=3h) treatment of cultured bovine adrenal chromaffin cells with cyclosporin A or FK506 decreased IRS-2 protein level by approximately 50% (IC(50)=200 or 10nM), without changing IRS-2 mRNA level, and insulin receptor, insulin-like growth factor-I (IGF-I) receptor, IRS-1, PI3K/PDK-1/Akt/GSK-3beta and ERK1/ERK2 protein levels. When the cells were washed to remove the test drug, the decreased IRS-2 level restored to the control level. Cyclosporin A or FK506 treatment inhibited calcineurin activity (IC(50)=500 or 40 nM, in vitro assay). Rapamycin, an FK506-binding protein ligand unable to inhibit calcineurin, failed to decrease IRS-2, but reversed FK506-induced decreases of calcineurin activity and IRS-2 level. Pulse-label followed by polyacrylamide gel electrophoresis revealed that cyclosporin A or FK506 accelerated IRS-2 degradation rate (t(1/2)) from >24 to approximately 4.2h, without altering IRS-2 synthesis. IRS-2 reduction by cyclosporin A or FK506 was prevented by lactacystin (proteasome inhibitor), but not by calpeptin (calpain inhibitor) or leupeptin (lysosome inhibitor). Cyclosporin A or FK506 increased serine-phosphorylation and ubiquitination of IRS-2. Cell surface (125)I-IGF-I binding capacity was not changed in cyclosporin A- or FK506-treated cells; however, IGF-I-induced phosphorylations of GSK-3beta and ERK1/ERK2 were attenuated by approximately 50%, which were prevented by rapamycin or lactacystin. Thus, calcineurin inhibition decreased IRS-2 level via proteasomal IRS-2 degradation, attenuating IGF-I-induced GSK-3beta and ERK pathways.
Asunto(s)
Glándulas Suprarrenales/citología , Células Cromafines/efectos de los fármacos , Inhibidores Enzimáticos/farmacología , Factor I del Crecimiento Similar a la Insulina/metabolismo , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Fosfoproteínas/metabolismo , Transducción de Señal/fisiología , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Animales , Bovinos , Ciclosporina/farmacología , Relación Dosis-Respuesta a Droga , Quinasas MAP Reguladas por Señal Extracelular/metabolismo , Regulación de la Expresión Génica/efectos de los fármacos , Glucógeno Sintasa Quinasa 3/metabolismo , Glucógeno Sintasa Quinasa 3 beta , Inmunoprecipitación , Proteínas Sustrato del Receptor de Insulina , Péptidos y Proteínas de Señalización Intracelular/genética , Fosfoproteínas/genética , Complejo de la Endopetidasa Proteasomal/metabolismo , Transducción de Señal/efectos de los fármacos , Tacrolimus/farmacología , Factores de TiempoRESUMEN
The inwardly rectifying K+ channels, Kir1.1, Kir2.3 and Kir4.1-Kir5.1, are the candidate chemosensory molecules for CO2/H+. We determined the mRNA expression and immunohistochemical localization of these channels in the medulla oblongata of the rat. RT-PCR analysis revealed mRNAs of Kir1.1, Kir2.3, Kir4.1 and Kir5.1 were detected in the medulla. The immunoreactivities for Kir1.1, Kir2.3, Kir4.1, and Kir5.1 were observed in the medulla, and immunolabeling pattern was varied by the subunit. Immunoreactivities for Kir1.1 and Kir2.3 were observed in the nerve cell bodies and glial cells both in the chemosensory areas [nucleus tractus solitarius (NTS), nucleus raphe obscurus (RO), pre-Bötzinger complex (PreBötC)] and non-chemosensory area [hypoglossal nucleus (XII), inferior olive nucleus (IO)]. Kir4.1 immunoreactivity was observed in the glial cells and neuropil, especially in XII and IO. Kir5.1 immunoreactivity was observed in the nerve cell bodies in the XII, RO, and PreBötC, but not in the NTS or IO. In the NTS, a dense network of varicose nerve fibers showed immunoreactivity for Kir5.1. Our findings suggest that Kir channels may not act specific to the central chemoreception, but regulate the ionic properties of cellular membranes in various neurons and glial cells.
Asunto(s)
Bulbo Raquídeo/citología , Canales de Potasio de Rectificación Interna/genética , ARN Mensajero/metabolismo , Animales , Cartilla de ADN/genética , Inmunohistoquímica , Bulbo Raquídeo/metabolismo , Microscopía Fluorescente , Canales de Potasio de Rectificación Interna/metabolismo , Ratas , Reacción en Cadena de la Polimerasa de Transcriptasa InversaRESUMEN
In cultured bovine adrenal chromaffin cells, where Akt1 is the predominant isoform over Akt2 and Akt3, chronic (> or =12 h) treatment with 1-20 mM LiCl, an inhibitor of glycogen synthase kinase-3, decreased Akt1 level by approximately 52% (EC50=3.7 mM; t1/2=l2 h); it was associated with LiCl-induced increased levels of Ser9-phosphorylated glycogen synthase kinase-3beta (approximately 37%) and beta-catenin (approximately 59%), two hallmarks of glycogen synthase kinase-3beta inhibition. The same LiCl treatment did not change phosphoinositide 3-kinase, phosphoinositide-dependent kinase 1, and extracellular signal-regulated kinase-1/2 levels. Treatment with SB216763 [3-(2,4-dichlorophenyl)-4-(1-methyl-1H-indol-3-yl)-1H-pyrrole-2,5-dione], a selective inhibitor of glycogen synthase kinase-3, lowered Akt1 level by approximately 67% (EC50=2 microM; t1/2=l2 h), when SB216763 caused concentration- and time-dependent increase of beta-catenin level by approximately 76%. LiCl- or SB216763-induced Akt1 decrease, as well as increases of Ser9-phosphorylated glycogen synthase kinase-3beta and beta-catenin were restored to the control levels of nontreated cells after the washout of LiCl (20 mM for 24 h)- or SB216763 (30 microM for 24 h)-treated cells. LiCl-induced Akt1 reduction was not prevented by beta-lactone, lactacystin (two inhibitors of proteasome), calpastatin (an inhibitor of calpain), or leupeptin (an inhibitor of lysosome). LiCl decreased Akt1 mRNA level by 20% at 6 h, with no effect on Akt1 mRNA stability. These results suggest that glycogen synthase kinase-3beta inhibition caused down-regulation of Akt1 mRNA and Akt1 protein levels; conversely, constitutive activity of glycogen synthase kinase-3beta maintains steady-state level of Akt1 in quiescent adrenal chromaffin cells.
Asunto(s)
Células Cromafines/metabolismo , Glucógeno Sintasa Quinasa 3/fisiología , Indoles/farmacología , Cloruro de Litio/farmacología , Maleimidas/farmacología , Proteína Oncogénica v-akt/biosíntesis , ARN Mensajero/biosíntesis , Proteínas Quinasas Dependientes de 3-Fosfoinosítido , Animales , Northern Blotting , Western Blotting , Bovinos , Células Cultivadas , Células Cromafines/efectos de los fármacos , Glucógeno Sintasa Quinasa 3 beta , Proteína Quinasa 1 Activada por Mitógenos/metabolismo , Proteína Quinasa 3 Activada por Mitógenos/metabolismo , Fosfatidilinositol 3-Quinasas/metabolismo , Fosforilación , Inhibidores de Proteasas/farmacología , Proteínas Serina-Treonina Quinasas/metabolismo , beta Catenina/metabolismoRESUMEN
In cultured bovine adrenal chromaffin cells, chronic (> or = 24 h) treatment with lysophosphatidic acid (LPA) augmented veratridine-induced 22Na+ influx via Na(v)1.7 by approximately 22% (EC(50) = 1 nmol/L), without changing nicotine-induced 22Na+ influx via nicotinic receptor-associated channel. LPA enhanced veratridine (but not nicotine)-induced 45Ca2+ influx via voltage-dependent calcium channel and catecholamine secretion. LPA shifted concentration-response curve of veratridine for 22Na+ influx upward, without altering the EC(50) of veratridine. Ptychodiscus brevis toxin-3 allosterically enhanced veratridine-induced 22Na+ influx by twofold in non-treated and LPA-treated cells. Whole-cell patch-clamp analysis showed that peak Na+ current amplitude was greater by 39% in LPA (100 nmol/L for 36 h)-treated cells; however, I-V curve and steady-state inactivation/activation curves were comparable between non-treated and LPA-treated cells. LPA treatment (> or = 24 h) increased cell surface [3H]saxitoxin binding by approximately 28%, without altering the K(d) value; the increase was prevented by cycloheximide, actinomycin D, or Ki16425, dioctylglycerol pyrophosphate 8:0 (two inhibitors of LPA(1) and LPA3 receptors), or botulinum toxin C3 (Rho inhibitor), Y27632 (Rho kinase inhibitor), consistent with LPA(1) receptor expression in adrenal chromaffin cells. LPA raised Nav1.7 mRNA level by approximately 37%. Thus, LPA-LPA(1) receptor-Rho/Rho kinase pathway up-regulated cell surface Nav1.7 and Nav1.7 mRNA levels, enhancing veratridine-induced Ca2+ influx and catecholamine secretion.
Asunto(s)
Glándulas Suprarrenales/citología , Calcio/metabolismo , Células Cromafines/efectos de los fármacos , Transducción de Señal/fisiología , Canales de Sodio/genética , Sodio/metabolismo , Regulación hacia Arriba/efectos de los fármacos , Animales , Catecolaminas/metabolismo , Bovinos , Células Cultivadas , Inhibidores Enzimáticos/farmacología , Lisofosfolípidos/farmacología , Potenciales de la Membrana/efectos de los fármacos , Potenciales de la Membrana/fisiología , Potenciales de la Membrana/efectos de la radiación , Técnicas de Placa-Clamp/métodos , ARN Mensajero/metabolismo , Radioisótopos/metabolismo , Receptores del Ácido Lisofosfatídico/fisiología , Transducción de Señal/efectos de los fármacos , Canales de Sodio/metabolismo , Veratridina/farmacología , Proteínas de Unión al GTP rho/fisiología , Quinasas Asociadas a rho/fisiologíaRESUMEN
Lithium has been proven to be effective in the therapy of bipolar disorder, but its mechanism of pharmacological action is not clearly defined. We examined the effects of lithium on voltage-dependent Na(+) channels, nicotinic acetylcholine receptors, and voltage-dependent Ca(2+) channels, as well as catecholamine secretion in cultured bovine adrenal chromaffin cells. Lithium chloride (LiCl) reduced veratridine-induced (22)Na(+) influx in a concentration-dependent manner, even in the presence of ouabain, an inhibitor of Na(+), K(+)-ATPase. Glycogen synthase kinase-3 (GSK-3) inhibitors (SB216763, SB415286 or the GSK-3 inhibitor IX) did not affect veratridine-induced (22)Na(+) influx, as well as inhibitory effect of LiCl on veratridine-induced (22)Na(+) influx. Enhancement of veratridine (site 2 toxin)-induced (22)Na(+) influx caused by alpha-scorpion venom (site 3 toxin), beta-scorpion venom (site 4 toxin), or Ptychodiscus brevis toxin-3 (site 5 toxin), still occurred in the presence of LiCl in the same manner as in the control cells. LiCl also reduced veratridine-induced (45)Ca(2+) influx and catecholamine secretion. In contrast, LiCl (< or = 30 mM) had no effect on nicotine-induced (22)Na(+) influx, (45)Ca(2+) influx and catecholamine secretion, as well as on high K(+)-induced (45)Ca(2+) influx and catecholamine secretion. Chronic treatment with LiCl at 100mM (but not at < or = 30 mM) significantly reduced cell viability in a time-dependent manner. These results suggest that lithium selectively inhibits Na(+) influx thorough Na(+) channels and subsequent Ca(2+) influx and catecholamine secretion, independent of GSK-3 inhibition.
Asunto(s)
Glándulas Suprarrenales/citología , Antimaníacos/farmacología , Catecolaminas/metabolismo , Células Cromafines/efectos de los fármacos , Cloruro de Litio/farmacología , Canales de Sodio/fisiología , Análisis de Varianza , Animales , Calcio/metabolismo , Bovinos , Supervivencia Celular/efectos de los fármacos , Células Cultivadas , Relación Dosis-Respuesta a Droga , Interacciones Farmacológicas , Inhibidores Enzimáticos/farmacología , Glucógeno Sintasa Quinasa 3/metabolismo , Activación del Canal Iónico/efectos de los fármacos , Sodio/metabolismo , Bloqueadores de los Canales de Sodio/farmacología , Factores de Tiempo , Veratridina/farmacologíaRESUMEN
In cultured bovine adrenal chromaffin cells, 48 h-treatment with 20 mmol/L LiCl, 1 mmol/L valproic acid, 30 micromol/L SB216763, 30 micromol/L SB415286, or 100 nmol/L insulin, a condition that inhibits constitutive active glycogen synthase kinase-3 (GSK-3), decreased cell surface (125)I-insulin binding capacity by approximately 39%, without altering the K(d) value; LiCl, SB216763 or insulin decreased insulin receptor (IR) and IR precursor levels, attenuating insulin-induced Tyr-autophosphorylation of IR. LiCl increased inhibitory Ser9-phosphorylation of GSK-3beta at 6 h, decreasing (125)I-insulin binding at 24 h. SB216763-induced (125)I-insulin binding reduction (IC(50) = 3 micromol/L) was preceded by beta-catenin level increase by SB216763 (EC(50) = 11 micromol/L), a hallmark of GSK-3 inhibition. Insulin-induced rapid (> 1 min) Ser9-phosphorylation of GSK-3beta (Nemoto et al. 2006) was followed by approximately 48% decrease of IR level. LiCl did not stimulate endocytosis, nor proteolysis of IR. LiCl destabilized IR mRNA (t(1/2) = 9.3 vs. 6.5 h), decreasing IR mRNA level by approximately 47%, without altering IR gene transcription. Decreases of (125)I-insulin binding and IR level, as well as increased Ser9-phosphorylation of GSK-3beta were restored to the control levels by washing the test compound-treated cells. Thus, GSK-3beta regulates IR level via controlling IR mRNA stability.
Asunto(s)
Glándulas Suprarrenales/citología , Células Cromafines/metabolismo , Glucógeno Sintasa Quinasa 3/metabolismo , Estabilidad del ARN/fisiología , Receptor de Insulina/fisiología , Análisis de Varianza , Animales , Bovinos , Células Cultivadas , Células Cromafines/efectos de los fármacos , Inhibidores Enzimáticos/farmacología , Glucógeno Sintasa Quinasa 3 beta , Insulina/farmacología , Fosforilación/efectos de los fármacos , Unión Proteica/efectos de los fármacos , Estabilidad del ARN/efectos de los fármacos , Ensayo de Unión Radioligante/métodos , Tirosina/metabolismo , beta Catenina/metabolismoRESUMEN
In cultured bovine adrenal chromaffin cells, 12-h treatment with 1-20 mM LiCl, an inhibitor of glycogen synthase kinase-3 (GSK-3), increased Ser(9) phosphorylation of GSK-3beta by approximately 44%, while decreasing insulin receptor substrate-1 (IRS-1) and IRS-2 protein levels by approximately 38 and approximately 62% in a concentration-dependent manner. Treatment with SB216763 (0.1-30 microM for 12 h), a selective inhibitor of GSK-3, lowered IRS-1 and IRS-2 levels by approximately 38 and approximately 48%, while increasing beta-catenin protein level by approximately 47%, due to the prevention of GSK-3-induced degradation of beta-catenin by SB216763. Insulin (100 nM for 24 h) increased Ser(9) phosphorylation of GSK-3beta by approximately 104%, while decreasing IRS-1 and IRS-2 levels by approximately 41 and approximately 72%; the insulin-induced Ser(9) phosphorylation of GSK-3beta, as well as down-regulations of IRS-1 and IRS-2 levels were restored to the control levels of nontreated cells at 24 h after the washout of the insulin (100 nM for 12 h)-treated cells. Either clasto-lactacystin beta-lactone or lactacystin (an inhibitor of proteasome) prevented LiCl- or SB216763-induced decreases of IRS-1 and IRS-2 levels by approximately 100 and approximately 69%, respectively. In contrast, calpastatin (an inhibitor of calpain) and leupeptin (an inhibitor of lysosome) failed to prevent the decreases of IRS-1 and IRS-2 levels caused by LiCl or SB216763. LiCl or SB216763 lowered IRS-2 mRNA level, with no effect on IRS-1 mRNA level. These results suggest that constitutive activity of GSK-3beta in quiescent cells positively maintains steady-state levels of IRS-1 and IRS-2 via regulating proteasomal degradation and/or synthesis of IRS-1 and IRS-2 proteins.