RESUMEN
Tacrolimus (Tac) is the cornerstone calcineurin inhibitor in transplantation. Extended-release Meltdose formulation (Tac-LCP) offers better bioavailability compared with immediate-release formulation (Tac-IR). We postulated that the less fluctuating pharmacokinetic (PK) profile of Tac-LCP might maintain a sustained inhibition of calcineurin activity (CNA) between dose intervals. Higher concentrations (peak plasma concentration (Cmax )) after Tac-IR may not result in a more potent CNA inhibition due to a capacity-limited effect. This study was aimed at evaluating the pharmacodynamic (PD)/PK profiles of Tac-IR compared with Tac-LCP. An open-label, prospective, nonrandomized, investigator-driven study was conducted. Twenty-five kidney transplant recipients receiving Tac-IR were switched to Tac-LCP. Before and 28 days after conversion, intensive CNA-PD and PK sampling were conducted using ultra-high-performance liquid chromatography-tandem accurate mass spectrometry. PD nonlinear mixed effects model was performed in Phoenix-WinNonlin. Statistically significant higher Cmax (P < 0.001) after Tac-IR did not result in lower CNA as compared with after Tac-LCP (P = 0.860). Tac-LCP showed a statistically more maintained CNA inhibition between dose intervals (area under the effect-time curve from 0 to 24 hours (AUE0-24h )) compared with Tac-IR, in which CNA returned to predose levels after 4 hours of drug intake (373.8 vs. 290.5 pmol RII·h/min·mg prot, Tac-LCP vs. Tac-IR; P = 0.039). No correlation was achieved between any PD and PK parameters in any formulations. Moreover, Tac concentration to elicit a 50% of the maximum response (half-maximal inhibitory concentration) was 9.24 ng/mL. The higher Cmax after Tac-IR does not result in an additional CNA inhibition compared with Tac-LCP attributable to a capacity-limited effect. Tac-LCP may represent an improvement of the PD of Tac due to the more sustained CNA inhibition during dose intervals.
Asunto(s)
Inhibidores de la Calcineurina/administración & dosificación , Inmunosupresores/administración & dosificación , Trasplante de Riñón , Modelos Biológicos , Tacrolimus/administración & dosificación , Anciano , Área Bajo la Curva , Calcineurina/efectos de los fármacos , Calcineurina/metabolismo , Inhibidores de la Calcineurina/farmacocinética , Inhibidores de la Calcineurina/farmacología , Cromatografía Líquida de Alta Presión , Preparaciones de Acción Retardada , Femenino , Humanos , Inmunosupresores/farmacocinética , Inmunosupresores/farmacología , Masculino , Persona de Mediana Edad , Estudios Prospectivos , Tacrolimus/farmacocinética , Tacrolimus/farmacología , Espectrometría de Masas en TándemRESUMEN
PURPOSE: Left ventricular hypertrophy (LVH) is a cardiovascular complication highly prevalent in patients with chronic kidney disease (CKD). Previous studies analyzing 1α-hydroxylase or vitamin D receptor (Vdr) knockout mice revealed active vitamin D as a promising agent inhibiting LVH progression. Paricalcitol, an active vitamin D analog, failed to suppress the progression of LV mass index (LVMI) in pre-dialysis patients with CKD. As target genes of activated VDR differ depending on its agonists, we examined the effects of maxacalcitol (22-oxacalcitriol: OCT), a less calcemic active vitamin D analog, on LVH in hemodialysis patients and animal LVH models with renal insufficiency. METHODS: In retrospective cohort study, patients treated with OCT who underwent hemodialysis were enrolled. Using cardiac echocardiography, LV mass was evaluated by the area-length method. In animal study, angiotensin II (Ang II)-infused Wister rats with heminephrectomy or Ang II-stimulated neonatal rat ventricular myocytes (NRVM) were treated with OCT. RESULTS: OCT significantly inhibited the progression of LVMI in hemodialysis patients. In Ang II-infused heminephrectomized rats, OCT suppressed the progression of LVH in a blood pressure-independent manner. OCT also suppressed the activity of calcineurin in the left ventricle of model rats. Specifically, OCT reduced the protein levels of calcineurin A, but not the mRNA levels of Ppp3ca (calcineurin Aα). Luciferase assays showed that OCT increased the promoter activity of Fbxo32 (atrogin1), an E3 ubiquitin ligase targeting calcineurin A. Finally, OCT promoted ubiquitination and degradation of calcineurin A. CONCLUSION: Our works indicated that OCT retards progression of LVH through calcineurin-NFAT pathway, which reveal a novel aspect of OCT in attenuating pathological LVH.
Asunto(s)
Calcitriol/análogos & derivados , Hipertrofia Ventricular Izquierda/tratamiento farmacológico , Hipertrofia Ventricular Izquierda/etiología , Hipertrofia Ventricular Izquierda/patología , Insuficiencia Renal/complicaciones , Anciano , Animales , Calcineurina/efectos de los fármacos , Calcitriol/farmacología , Técnicas de Cultivo de Célula , Modelos Animales de Enfermedad , Femenino , Humanos , Masculino , Persona de Mediana Edad , Miocitos Cardíacos/efectos de los fármacos , Factores de Transcripción NFATC/metabolismo , Embarazo , Ratas , Ratas Wistar , Estudios RetrospectivosRESUMEN
RCAN1 is an inhibitor of the phosphatase calcineurin, which is involved in the regulation of oxidative stress and apoptosis, among other important cell processes. Here we have used RCAN1 deficient mice (RCAN1-/-) to elucidate its role after an acute oxidative insult such as paraquat injection. We have observed that RCAN1-/- mice show less oxidative damage than wildtype (WT) mice after treatment. Under basal conditions, RCAN1-/- animals express more calcineurin, heme oxygenase-1, Nrf2, and catalase compared to WT mice (controls). This may explain the less severe effect of paraquat treatment on RCAN1-/- mice compared to WT. We showed that oxidative stress is involved in the early stages of apoptosis, thus we determined the apoptotic effector BAD and found that decreases in RCAN1-/- mice after treatment with paraquat compared with WT in similar experimental conditions. Our results suggest that RCAN1 may be involved in the balance between oxidant and antioxidant species production in vivo.
Asunto(s)
Antioxidantes/metabolismo , Calcineurina/efectos de los fármacos , Proteínas de Unión al ADN/metabolismo , Proteínas Musculares/metabolismo , Estrés Oxidativo/efectos de los fármacos , Paraquat/efectos adversos , Animales , RatonesRESUMEN
Cardiac hypertrophy is a common phenomenon observed in progressive heart disease associated with heart failure. Insulin-like growth factor receptor II (IGF-IIR) has been much implicated in myocardial hypertrophy. Our previous studies have found that increased activities of signaling mediators, such as calcium/calmodulin-dependent protein kinase II (CaMKII) and calcineurin induces pathological hypertrophy. Given the critical roles played by CaMKII and calcineurin signaling in the progression of maladaptive hypertrophy, we anticipated that inhibition of CaMKII and calcineurin signaling may attenuate IGF-IIR-induced cardiac hypertrophy. The current study, therefore, investigated the effects of IGF-IIR activation on the CaMKII and calcineurin signaling and whether the combinatorial inhibition of the CaMKIIδ and calcineurin signaling could ameliorate IGF-IIR-induced pathological hypertrophy. In the present study, we induced IGF-IIR through the cardiomyocyte-specific transduction of IGFIIY27L via adeno-associated virus 2 (AAV2) to evaluate its effects on cardiac hypertrophy. Interestingly, it was observed that the activation of IGF-IIR signaling through IGFIIY27L induces significant hypertrophy of the myocardium and increased cardiac apoptosis and fibrosis. Moreover, we found that Leu27 IGF-II significantly induced calcineurin and CaMKII expression. Furthermore and importantly, the combinatorial treatment with CaMKII and calcineurin inhibitors significantly alleviates IGF-IIR-induced hypertrophic responses. Thus, it could be envisaged that the inhibition of IGF-IIR may serve as a promising candidate for attenuating maladaptive hypertrophy. Both calcineurin and CaMKII could be valuable targets for developing treatment strategies against hypertension-induced cardiomyopathies.
Asunto(s)
Calcineurina/genética , Proteína Quinasa Tipo 2 Dependiente de Calcio Calmodulina/genética , Cardiomegalia/tratamiento farmacológico , Insuficiencia Cardíaca/tratamiento farmacológico , Receptor IGF Tipo 2/genética , Animales , Apoptosis/genética , Calcineurina/efectos de los fármacos , Inhibidores de la Calcineurina/farmacología , Proteína Quinasa Tipo 2 Dependiente de Calcio Calmodulina/antagonistas & inhibidores , Cardiomegalia/genética , Cardiomegalia/patología , Modelos Animales de Enfermedad , Insuficiencia Cardíaca/genética , Insuficiencia Cardíaca/patología , Humanos , Hipertensión/tratamiento farmacológico , Hipertensión/genética , Hipertensión/patología , Factor II del Crecimiento Similar a la Insulina/genética , Miocardio/metabolismo , Miocardio/patología , Miocitos Cardíacos/efectos de los fármacos , Miocitos Cardíacos/patología , Ratas , Transducción de Señal/efectos de los fármacosRESUMEN
The Phosphoprotein Phosphatase Calcineurin (CN, PP2B, PP3) recognizes and binds to two short linear motifs (SLiMs), PxIxIT and LxVP, in its regulators and substrates. These interactions enable CN function in many key biological processes. The identification of SLiMs is difficult because of their short, degenerate sequence and often low binding affinity. Here we combine Structure Based Shape Complementarity (SBSC) analysis and proteome-wide affinity purification-mass spectrometry to identify PxIxIT and LxVP containing CN interactors to expand and thereby redefine the LxVP motif. We find that the new πφ-LxVx primary sequence defines an ensemble of binding competent confirmations and thus the binding on-rate, making it difficult to predict the LxVP binding strength from its sequence. Our analysis confirms existing and, more importantly, identifies novel CN interactors, substrates, and thus biological functions of CN.
Asunto(s)
Calcineurina/efectos de los fármacos , Calcineurina/metabolismo , Humanos , Espectrometría de Masas/métodos , Transporte de Proteínas , Proteoma , Especificidad por SustratoRESUMEN
3-Nitropropionic acid (3-NP) induces a spectrum of Huntington's disease (HD)-like neuropathologies in the rat striatum. The present study aimed to demonstrate the neuroprotective effect of lercanidipine (LER) in rats with 3-NP-induced neurotoxicity, address the possible additional protective effect of combined treatment with bone marrow-derived mesenchymal stem cells (BM-MSCs) and LER, and investigate the possible involvement of the Ca2+/calcineurin (CaN)/nuclear factor of activated T cells c4 (NFATc4) and Wnt/ß-catenin signalling pathways. Rats were injected with 3-NP (10â¯mg/kg/day, i.p.) for two weeks and were divided into four subgroups; the first served as the control HD group, the second received a daily dose of LER (0.5â¯mg/kg, i.p.), the third received a single injection of BM-MSCs (1 x 106/rat, i.v.) and the last received a combination of both BM-MSCs and LER. The combined therapy improved motor and behaviour performance. Meanwhile, this treatment led to a marked reduction in striatal cytosolic Ca2+, CaN, tumour necrosis factor-alpha, and NFATc4 expression and the Bax/Bcl2 ratio. Combined therapy also increased striatal brain-derived neurotrophic factor, FOXP3, Wnt, and ß-catenin protein expression. Furthermore, haematoxylin-eosin and Nissl staining revealed an amelioration of striatum tissue injury with the combined treatment. In conclusion, the current study provides evidence for a neuroprotective effect of LER and/or BM-MSCs in 3-NP-induced neurotoxicity in rats. Interestingly, combined LER/BM-MSC therapy was superior to cell therapy alone in inhibiting 3-NP-induced neurological insults via modulation of the Ca2+/CaN/NFATc4 and Wnt/ß-catenin signalling pathways. LER/BM-MSC combined therapy may represent a feasible approach for improving the beneficial effects of stem cell therapy in HD.
Asunto(s)
Bloqueadores de los Canales de Calcio/uso terapéutico , Dihidropiridinas/uso terapéutico , Enfermedad de Huntington/terapia , Trasplante de Células Madre Mesenquimatosas/métodos , Transducción de Señal/efectos de los fármacos , Animales , Conducta Animal/efectos de los fármacos , Calcineurina/efectos de los fármacos , Señalización del Calcio/efectos de los fármacos , Citosol/efectos de los fármacos , Citosol/metabolismo , Enfermedad de Huntington/inducido químicamente , Enfermedad de Huntington/tratamiento farmacológico , Masculino , Factores de Transcripción NFATC/efectos de los fármacos , Proteínas del Tejido Nervioso/efectos de los fármacos , Nitrocompuestos , Propionatos , Proteínas Proto-Oncogénicas c-bcl-2/metabolismo , Ratas , Ratas Wistar , Factor de Necrosis Tumoral alfa/metabolismo , Vía de Señalización Wnt/efectos de los fármacos , Proteína X Asociada a bcl-2/metabolismo , beta Catenina/efectos de los fármacosRESUMEN
BACKGROUND The inhibitory effect of arsenic trioxide (As2O3) on lung cancer has been reported in some preclinical studies. However, its effect on small cell lung cancer (SCLC) has been poorly explored. Calcineurin and its substrate, nuclear factor of activated T cells (NFAT), mediate the downstream signaling of VEGF, and is critical in the process endothelium activation and tumor metastasis. In this study, we aimed to evaluate whether As2O3 had inhibitory effects on endothelial cells activation and the metastasis of SCLC, and to explore the possible mechanisms. MATERIAL AND METHODS In vitro, human umbilical vein endothelial cells (HUVECs) were used. Cell Counting Kit-8 assay and cell migration assay were performed to determine the effect of As2O3 on HUVECs proliferation and migration. The level of calcineurin, NFAT, downstream factors for Down syndrome candidate region 1 (DSCR1), and the endogenous inhibitor of calcineurin, were evaluated by quantitative PCR and western blotting. In vivo, SCLC metastasis models were established by injecting NCI-H446 cells into tail veins of nude mice. Tumor-bearing mice were treated with As2O3 or calcineurin inhibitor for 10 days, after which tumor metastasis in target organs was evaluated. RESULTS As2O3 significantly inhibited the proliferation and migration of endothelial cells. Also, As2O3 inhibited the expression levels of calcineurin, NFAT, and the downstream target genes CXCR7 and RND1, while it upregulated the level of DSCR1. Both As2O3 and calcineurin inhibitor exhibited notable inhibitory effect on the metastasis of SCLC, without obvious side effects. CONCLUSIONS These findings suggested that As2O3 had remarkable inhibitory effects on the endothelial cell activation and SCLC metastasis, and the mechanism might be related to the blocking of calcineurin-NFAT signaling by upregulating DSCR1.
Asunto(s)
Trióxido de Arsénico/farmacología , Factores de Transcripción NFATC/efectos de los fármacos , Carcinoma Pulmonar de Células Pequeñas/tratamiento farmacológico , Animales , Trióxido de Arsénico/metabolismo , Calcineurina/efectos de los fármacos , Movimiento Celular/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , China , Proteínas de Unión al ADN , Endotelio Vascular/metabolismo , Células Endoteliales de la Vena Umbilical Humana/efectos de los fármacos , Humanos , Péptidos y Proteínas de Señalización Intracelular/efectos de los fármacos , Masculino , Ratones , Ratones Desnudos , Proteínas Musculares/efectos de los fármacos , Factores de Transcripción NFATC/metabolismo , Metástasis de la Neoplasia/tratamiento farmacológico , Neovascularización Patológica/metabolismo , Receptores CXCR/efectos de los fármacos , Transducción de Señal , Regulación hacia Arriba/efectos de los fármacos , Factor A de Crecimiento Endotelial Vascular/efectos de los fármacos , Proteínas de Unión al GTP rho/efectos de los fármacosRESUMEN
Long-term treatment with the potent immunosuppressive drug cyclosporine A (CsA) results in chronic nephrotoxicity. Its immunosuppressive properties are due to the inhibition of the calcium- and calmodulin-dependent phosphatase protein calcineurin A (CnA) which has three catalytic isoforms. Of those, the CnAα and ß isoforms are ubiquitously expressed, particularly in the kidney. Additionally, chronic nephrotoxicity has been associated with an imbalance of extracellular matrix (ECM) synthesis and degradation resulting in an accumulation of ECM molecules. This study evaluates whether the expressions of matrix metalloproteinases (MMP-2 and MMP-9) induced by CsA are calcineurin isoform specific. Wild-type (WT), CnAα knockout (CnAα-/-) and CnAß knockout (CnAß-/-) kidney fibroblast cell lines (an in vitro innovative tool that was previously created in our lab) were treated with CsA at 10â¯ng/ml for 48â¯h. ELISA analysis demonstrated that the CsA-induced secretion profile of MMP-9 was highest in CnAα-/- cells and lowest in CnAß-/- cells vs. WT cells. In contrast, CsA did not induce an increase in MMP-2 protein levels in WT, CnAα-/- nor CnAß-/- renal fibroblasts. These results indicate that MMP-9 secretion is CnA-isoform specific, i.e. the CnAß isoform contributes to the CsA-induced upregulation of MMP-9 while the CnAα does not. As such, understanding the role of calcineurin A isoforms in the regulation of the homeostasis of ECM degradation in the kidney after long-term CsA treatment needs to be further investigated.
Asunto(s)
Fibroblastos/metabolismo , Riñón/citología , Metaloproteinasa 9 de la Matriz/metabolismo , Animales , Calcineurina/efectos de los fármacos , Calcineurina/metabolismo , Línea Celular , Ciclosporina/farmacología , Expresión Génica , Humanos , Riñón/enzimología , Metaloproteinasas de la Matriz/metabolismo , Isoformas de Proteínas/efectos de los fármacosRESUMEN
BACKGROUND: Calcineurin (CaN) having a high expression in hippocampal neurons is closely related to apoptosis. Pretreatment with nimodipine can lower the apoptosis rate of hippocampal neuron to reduce the incidence of postoperative cognitive dysfunction (POCD). However, the relationship between cerebral protective effect of pretreatment with nimodipine and CaN is controversial in the literature. The aim of this study is to evaluate the relationship between neuroprotective effect of nimodipine and CaN on POCD in aged rats. METHODS: Ninety-six 18-month-old male Sprague-Dawley rats were randomly assigned into 4 groups (n = 24 each): control group (Group C), nimodipine group (Group N), surgery group (Group S) and nimodipine + surgery group (Group N + S). In Group N and Group N + S, nimodipine 1 mg/kg was intraperitoneally injected, while the equal volume of normal saline was given instead in Group S. 30 min later, Group N and Group C inhaled pure oxygen for 2 h, and Group S and N + S inhaled 3% sevoflurane for 2 h when exploratory laparotomy was performed. Morris water maze test was performed on 1 day before operation and 1, 3 and 7 days after operation. After the end of Morris water maze test at 1 day before operation and 1 and 7 days after operation, 8 rats were sacrificed, brains were removed and hippocampal tissues were obtained for detection of apoptosis in hippocampal neurons, cytoplasmic calcium ([Ca2+]i), and hippocampal CaN and caspase-3 expression. RESULTS: Compared with the 1st day before operation, the escape latency, apoptosis rate, [Ca2+]i, expression of CaN and caspase-3 increased significantly, but the frequency of crossing the original platform decreased dramatically in Group S and N + S(P<0.05). In addition, the escape latency, apoptosis rate, [Ca2+]i, and expression of CaN and caspase-3 decreased markedly, but the frequency of crossing the original platform increased significantly in Group N + S as compared with Group S (P<0.05). CONCLUSIONS: Pretreatment with nimodipine reduces the incidence of POCD by decreasing CaN mediated hippocampal neuroapoptosis in aged rats.
Asunto(s)
Calcineurina/efectos de los fármacos , Bloqueadores de los Canales de Calcio/farmacología , Trastornos del Conocimiento/prevención & control , Hipocampo/efectos de los fármacos , Nimodipina/farmacología , Complicaciones Posoperatorias/prevención & control , Envejecimiento , Animales , Apoptosis/efectos de los fármacos , Calcineurina/metabolismo , Modelos Animales de Enfermedad , Hipocampo/patología , Masculino , Neuronas/efectos de los fármacos , Fármacos Neuroprotectores/farmacología , Ratas , Ratas Sprague-DawleyRESUMEN
Ciguatoxins (CTXs) are lipid-soluble polyether compounds produced by dinoflagellates from the genus Gambierdiscus spp. typically found in tropical and subtropical zones. This endemic area is however rapidly expanding due to environmental perturbations, and both toxic Gambierdiscus spp. and ciguatoxic fishes have been recently identified in the North Atlantic Ocean (Madeira and Canary islands) and Mediterranean Sea. Ciguatoxins bind to Voltage Gated Sodium Channels on the membranes of sensory neurons, causing Ciguatera Fish Poisoning (CFP) in humans, a disease characterized by a complex array of gastrointestinal, neurological, neuropsychological, and cardiovascular symptoms. Although CFP is the most frequently reported non bacterial food-borne poisoning worldwide, there is still no simple and quick way of detecting CTXs in contaminated samples. In the prospect to engineer rapid and easy-to-use CTXs live cells-based tests, we have studied the effects of CTXs on the yeast Saccharomyces cerevisiae, a unicellular model which displays a remarkable conservation of cellular signalling pathways with higher eukaryotes. Taking advantage of this high level of conservation, yeast strains have been genetically modified to encode specific transcriptional reporters responding to CTXs exposure. These yeast strains were further exposed to different concentrations of either purified CTX or micro-algal extracts containing CTXs. Our data establish that CTXs are not cytotoxic to yeast cells even at concentrations as high as 1µM, and cause an increase in the level of free intracellular calcium in yeast cells. Concomitantly, a dose-dependent activation of the calcineurin signalling pathway is observed, as assessed by measuring the activity of specific transcriptional reporters in the engineered yeast strains. These findings offer promising prospects regarding the potential development of a yeast cells-based test that could supplement or, in some instances, replace current methods for the routine detection of CTXs in seafood products.
Asunto(s)
Calcineurina , Ciguatoxinas , Saccharomyces cerevisiae/metabolismo , Animales , Calcineurina/efectos de los fármacos , Calcineurina/metabolismo , Intoxicación por Ciguatera , Ciguatoxinas/análisis , Ciguatoxinas/toxicidad , Humanos , Mar Mediterráneo , Saccharomyces cerevisiae/efectos de los fármacos , EspañaRESUMEN
Antitubulin chemotherapeutics agents, such as paclitaxel, are effective chemotherapy drugs for cancer treatment. However, painful neuropathy is a major adverse effect limiting the wider application of chemotherapeutics. In this study, we found that A-kinase anchor protein 150 (AKAP150) was significantly upregulated after paclitaxel injection. Inhibition of AKAP150 via siRNA or AKAP150flox/flox in rodents alleviated the pain behavior induced by paclitaxel, and partly restored the decreased calcineurin (CN) phosphatase activity after paclitaxel treatment. Paclitaxel decreased the expression of anti-inflammatory cytokine interleukin-4 (IL-4), and intrathecal injections of IL-4 effectively alleviated paclitaxel-induced hypersensitivity and the frequency of dorsal root ganglion (DRG) neurons action potential. The decreased CN enzyme activity, resulted in reduced protein expression of nuclear factor of activated T cells 2 (NFAT2) in cell nuclei. Chromatin immunoprecipitation showed that, NFAT2 binds to the IL-4 gene promoter regulating the protein expression of IL-4. Overexpression of NFAT2 by intrathecal injection of the AAV5-NFAT2-GFP virus alleviated the pain behavior induced by paclitaxel via increasing the expression of IL-4. Knocked down AKAP150 by siRNA or AAV5-Cre-GFP partly restored the expression of IL-4 in DRG. Our results indicated that regulation of IL-4 via the CN/NFAT2 pathway mediated by AKAP150 could be a pivotal treatment target for paclitaxel-induced neuropathic pain and or other neuropsychiatric disorders.
Asunto(s)
Proteínas de Anclaje a la Quinasa A/metabolismo , Neuralgia/metabolismo , Proteínas de Anclaje a la Quinasa A/fisiología , Animales , Calcineurina/efectos de los fármacos , Calcineurina/metabolismo , Citocinas/metabolismo , Regulación hacia Abajo , Ganglios Espinales/metabolismo , Hiperalgesia/metabolismo , Inyecciones Espinales , Interleucina-4/metabolismo , Masculino , Factores de Transcripción NFATC/efectos de los fármacos , Factores de Transcripción NFATC/metabolismo , Neuralgia/fisiopatología , Paclitaxel/efectos adversos , Ratas , Ratas Sprague-Dawley , Médula Espinal/metabolismo , Regulación hacia ArribaRESUMEN
Psoriasis is an immune-mediated chronic skin disorder where T cells play a main role, and numerous inflammatory cytokines are implicated in its pathogenesis by initiating keratinocyte proliferation. Interleukin-22 (IL-22), an IL-10 family cytokines, is critical in the pathogenesis and development of psoriasis. To determine the target of microRNA (miR) -548a-3p and investigate its role in keratinocyte proliferation after treating human keratinocytes (HaCaT) with IL-22, we used quantitative reverse transcriptase PCR to measure the expression of miR-548a-3p in both HaCaT cells stimulated with IL-22 and psoriatic lesions, and then detected the biological function of miR-548a-3p in HaCaT cells by performing Counting Kit-8 (CCK-8) assays. Luciferase reporter assay was conducted to determine the target gene of miR-548a-3p. Immunohistochemistry and Western blot were performed to verify the target gene. Results showed that miR-548a-3p was significantly upregulated both in HaCaT cells treated with IL-22 and psoriatic lesions. The over expression of miR-548a-3p could promote the proliferation of HaCaT cells. Luciferase was mutated in the 3'UTR of PPP3R1, a gene coding Calcineurin. Immunohistochemistry and Western blot demonstrated that the expression of PPP3R1 decreased respectively in psoriatic lesions and HaCaT cells. In conclusion, the expression of miR-548a-3p is upregulated in IL-22 mediated keratinocyte proliferative disorder like psoriasis. The impact of miR-548a-3p on keratinocyte proliferation may be implemented by targeting PPP3R1 and T regulatory cells may be involved in the pathogenesis of psoriasis.
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Calcineurina/efectos de los fármacos , Proliferación Celular , Interleucinas/farmacología , Queratinocitos/citología , MicroARNs/fisiología , Línea Celular , Humanos , MicroARNs/genética , Reacción en Cadena de la Polimerasa , Psoriasis/tratamiento farmacológico , Activación Transcripcional , Regulación hacia Arriba , Interleucina-22RESUMEN
BACKGROUND: We sought to evaluate, in patients on a liver transplantation waiting list, potential biomarkers of the base calcineurin pathway activity with use of a new model of nonstimulated peripheral blood mononuclear cells (PBMC) and ex vivo response to tacrolimus (TAC). METHODS: The calcineurin pathway activity was explored ex vivo in stimulated and nonstimulated PBMC from 19 patients. The inhibition of NFAT1 translocation to PBMC nuclei, expression of intracellular IL-2, and membrane CD25 in different T-cell subsets were measured by multiparametric flow cytometry before and after exposure to TAC. We also studied the influence on the individual response of polymorphisms in 3 key genes of the calcineurin pathway: PPIA, PPP3CA, and IL2RA. RESULTS: All pharmacodynamics profiles closely fitted an I/Imax sigmoid model. Interindividual variability was higher in nonstimulated than in stimulated conditions, as well as in the presence of TAC. IL-2+CD8+ cells at TAC Imax showed the highest interindividual variability, suggesting its usefulness as a biomarker of individual TAC effects integrating many different sources of regulation and variability. Moreover, in the absence of TAC, patients with end-stage liver disease exhibited lower NFAT1 translocation and T-cell activation than healthy volunteers from a previous study under similar conditions. Multivariate statistical analysis showed strong and significant associations between TAC pharmacodynamic parameters and 2 polymorphisms in the gene-coding cyclophilin A (rs8177826 and rs6850). CONCLUSIONS: We show the feasibility of using nonstimulated PBMCs to explore the calcineurin pathway under more physiologic conditions and point toward potential biomarkers for TAC pharmacodynamic monitoring. ClinicalTrials.gov Identifier: NCT01760356.
Asunto(s)
Calcineurina/sangre , Inmunosupresores/farmacología , Trasplante de Hígado , Tacrolimus/farmacología , Listas de Espera , Calcineurina/efectos de los fármacos , Calcineurina/genética , Humanos , Leucocitos Mononucleares/metabolismo , FarmacogenéticaRESUMEN
Diffuse axonal injury (DAI) is the most common and significant pathological features of traumatic brain injury (TBI). However, there are still no effective drugs to combat the formation and progression of DAI in affected individuals. FK506, also known as tacrolimus, is an immunosuppressive drug, which is widely used in transplantation medicine for the reduction of allograft rejection. Previous studies have identified that FK506 may play an important role in the nerve protective effect of the central nervous system. In the present study, apoptosis of neuronal cells was observed following the induction of experimental DAI. The results demonstrated that it was closely related with the upregulation of deathassociated protein kinase 1 (DAPK1). It was hypothesized that FK506 may inhibit the activity of DAPK1 by inhibiting calcineurin activity, which may be primarily involved in antiapoptosis following DAI induction. Through researching the expression of nerve regeneration associated proteins (NFH and GAP43) following DAI, the present study provides novel data to suggest that FK506 promotes axon formation and nerve regeneration following experimental DAI. Therefore, FK506 may be a potent therapeutic for inhibiting nerve injury, as well as promoting the nerve regeneration following DAI.
Asunto(s)
Apoptosis/efectos de los fármacos , Axones/efectos de los fármacos , Lesión Axonal Difusa/tratamiento farmacológico , Tacrolimus/farmacología , Animales , Axones/metabolismo , Axones/patología , Lesiones Traumáticas del Encéfalo/tratamiento farmacológico , Lesiones Traumáticas del Encéfalo/metabolismo , Lesiones Traumáticas del Encéfalo/patología , Tronco Encefálico/efectos de los fármacos , Tronco Encefálico/patología , Región CA1 Hipocampal/efectos de los fármacos , Región CA1 Hipocampal/patología , Calcineurina/efectos de los fármacos , Proteínas Quinasas Asociadas a Muerte Celular/antagonistas & inhibidores , Proteínas Quinasas Asociadas a Muerte Celular/metabolismo , Lesión Axonal Difusa/metabolismo , Lesión Axonal Difusa/patología , Proteína GAP-43/metabolismo , Masculino , Regeneración Nerviosa/efectos de los fármacos , Proteínas de Neurofilamentos/metabolismo , Ratas , Ratas Sprague-Dawley , Regulación hacia ArribaRESUMEN
The accumulation of iron in neurons has been proposed to contribute to the pathology of numerous neurodegenerative diseases, such as Alzheimer's disease and Parkinson's disease. However, insufficient research has been conducted on the precise mechanism underlying iron toxicity in neurons. In this study, we investigated mitochondrial dynamics in hippocampal HT-22 neurons exposed to ferric ammonium citrate (FAC) as a model of iron overload and neurodegeneration. Incubation with 150 µM FAC for 48 h resulted in decreased cell viability and apoptotic death in HT-22 cells. The FAC-induced iron overload triggered mitochondrial fragmentation, which was accompanied by Drp1(Ser637) dephosphorylation. Iron chelation with deferoxamine prevented the FAC-induced mitochondrial fragmentation and apoptotic cell death by inhibiting Drp1(Ser637) dephosphorylation. In addition, a S637D mutation of Drp1, which resulted in a phosphorylation-mimetic form of Drp1 at Ser637, protected against the FAC-induced mitochondrial fragmentation and neuronal apoptosis. FK506 and cyclosporine A, inhibitors of calcineurin activation, determined that calcineurin was associated with the iron-induced changes in mitochondrial morphology and the phosphorylation levels of Drp1. These results indicate that the FAC-induced dephosphorylation of Drp1-dependent mitochondrial fragmentation was rescued by the inhibition of calcineurin activation. Therefore, these findings suggest that calcineurin-mediated phosphorylation of Drp1(Ser637) acts as a key regulator of neuronal cell loss by modulating mitochondrial dynamics in iron-induced toxicity. These results may contribute to the development of novel therapies for treatment of neurodegenerative disorders related to iron toxicity.
Asunto(s)
Calcineurina/farmacología , Hipocampo/efectos de los fármacos , Hipocampo/patología , Sobrecarga de Hierro/patología , Mitocondrias/efectos de los fármacos , Mitocondrias/patología , Neuronas/efectos de los fármacos , Neuronas/patología , Animales , Apoptosis/efectos de los fármacos , Calcineurina/efectos de los fármacos , Línea Celular , Supervivencia Celular/efectos de los fármacos , Dinaminas/efectos de los fármacos , Ratones , Mitocondrias/ultraestructura , Interferencia de ARN , Transducción de Señal/efectos de los fármacosRESUMEN
Chronic immunosuppressive therapy is often complicated by the development of both arterial hypertension and renal dysfunction. The principal aim of this study was to assess the effects of dual inhibition of renin-angiotensin system (RAS) and other antihypertensive treatment on blood pressure and renal function in normotensive and hypertensive Fawn-Hooded (FH) strains during chronic calcineurin inhibitor (CNI) administration. Combinations of perindopril (5 mg kg(-1) per day) and losartan (50 mg kg(-1) per day) or amlodipine (6 mg kg(-1) per day) and metoprolol (80 mg kg(-1) per day) were administered to normotensive (FHL) and hypertensive (FHH) rats, fed with diet containing tacrolimus (Tac; 12 mg kg(-1) per day). Tac-induced arterial hypertension in both animal strains (FHL: 151±4; FHH: 198±6 mm Hg) was prevented by dual RAS inhibition (FHL: 132±3 mm Hg, P<0.05; FHH: 153±3 mm Hg, P<0.05) as well as by a combination of amlodipine and metoprolol (FHL: 136±3 mm Hg, P<0.05; FHH: 166±4 mm Hg, P<0.05). However, significant nephroprotection was observed only in animals on dual RAS inhibition where albuminuria was reduced in both FHL (51.1±3.9 vs. 68.3±4.5 µg per day; P<0.05) and FHH rats (13.1±0.3 vs. 18.8±0.7 mg per day; P<0.05). We also found Tac-induced enhancement in renal angiotensin II activity that was significantly reduced by dual RAS inhibition in both FHL (63.5±3.2 vs. 23.1±3.0 fmol g(-1)) and FHH (79.8±8.5 vs. 32.2±5.8 fmol g(-1)). In addition, histological analysis revealed that RAS inhibition noticeably diminished glomerulosclerosis and tubulo-interstitial injury. This study indicates that dual blockade of RAS significantly attenuates Tac-induced arterial hypertension and nephrotoxicity in FH rats and further supports the notion that RAS inhibitors display efficient renoprotective properties during CNI treatment.
Asunto(s)
Lesión Renal Aguda/inducido químicamente , Lesión Renal Aguda/prevención & control , Inmunosupresores/antagonistas & inhibidores , Inmunosupresores/toxicidad , Sistema Renina-Angiotensina/efectos de los fármacos , Tacrolimus/antagonistas & inhibidores , Tacrolimus/toxicidad , Antagonistas Adrenérgicos beta/farmacología , Bloqueadores del Receptor Tipo 1 de Angiotensina II/farmacología , Inhibidores de la Enzima Convertidora de Angiotensina/farmacología , Animales , Calcineurina/efectos de los fármacos , Bloqueadores de los Canales de Calcio/farmacología , Dieta , Masculino , RatasRESUMEN
We aimed to determine the mechanisms of the anabolic actions of androgens in skeletal muscle by investigating potential androgen receptor (AR)-regulated genes in in vitro and in vivo models. The expression of the myogenic regulatory factor myogenin was significantly decreased in skeletal muscle from testosterone-treated orchidectomized male mice compared to control orchidectomized males, and was increased in muscle from male AR knockout mice that lacked DNA binding activity (AR(ΔZF2)) versus wildtype mice, demonstrating that myogenin is repressed by the androgen/AR pathway. The ubiquitin ligase Fbxo32 was repressed by 12 h dihydrotestosterone treatment in human skeletal muscle cell myoblasts, and c-Myc expression was decreased in testosterone-treated orchidectomized male muscle compared to control orchidectomized male muscle, and increased in AR(∆ZF2) muscle. The expression of a group of genes that regulate the transition from myoblast proliferation to differentiation, Tceal7 , p57(Kip2), Igf2 and calcineurin Aa, was increased in AR(∆ZF2) muscle, and the expression of all but p57(Kip2) was also decreased in testosterone-treated orchidectomized male muscle compared to control orchidectomized male muscle. We conclude that in males, androgens act via the AR in part to promote peak muscle mass by maintaining myoblasts in the proliferative state and delaying the transition to differentiation during muscle growth and development, and by suppressing ubiquitin ligase-mediated atrophy pathways to preserve muscle mass in adult muscle.
Asunto(s)
Proteínas Musculares/genética , Mioblastos Esqueléticos/metabolismo , Miogenina/genética , Proteínas Proto-Oncogénicas c-myc/genética , ARN Mensajero/metabolismo , Receptores Androgénicos/genética , Proteínas Ligasas SKP Cullina F-box/genética , Animales , Calcineurina/efectos de los fármacos , Calcineurina/genética , Diferenciación Celular/efectos de los fármacos , Diferenciación Celular/genética , Línea Celular , Inhibidor p57 de las Quinasas Dependientes de la Ciclina/efectos de los fármacos , Inhibidor p57 de las Quinasas Dependientes de la Ciclina/genética , Dihidrotestosterona/farmacología , Expresión Génica/efectos de los fármacos , Humanos , Técnicas In Vitro , Factor II del Crecimiento Similar a la Insulina/efectos de los fármacos , Factor II del Crecimiento Similar a la Insulina/genética , Masculino , Ratones , Ratones Noqueados , Proteínas Musculares/efectos de los fármacos , Músculo Esquelético , Mioblastos Esqueléticos/efectos de los fármacos , Proteínas Nucleares/efectos de los fármacos , Proteínas Nucleares/genética , Orquiectomía , Proteínas Proto-Oncogénicas c-myc/efectos de los fármacos , ARN Mensajero/efectos de los fármacos , Proteínas Ligasas SKP Cullina F-box/efectos de los fármacos , Testosterona/farmacologíaRESUMEN
BACKGROUND: Alzheimer's disease (AD) is a neurodegenerative disorder featured by deposition of beta-amyloid (Aß) plaques in the hippocampus and associated cortices and progressive cognitive decline. Tropisetron, a selective 5-HT3 receptor antagonist, is conventionally used to counteract chemotherapy-induced emesis. Recent investigations describe antiphlogistic properties for tropisetron. It has been shown that tropisetron protects against rat embolic stroke. We investigated protective properties of tropisetron in a beta-amyloid (Aß) rat model of AD and possible involvement of 5-HT3 receptors. MATERIAL AND METHODS: Aß (1-42) was injected into the hippocampus of male rats. Animals were treated intracerebroventricularly with tropisetron, mCPBG (selective 5-HT3 receptor agonist) or mCPBG plus tropisetron on days 1, 3, 5 and 7. Seven days following Aß administration, inflammatory markers (TNF-α, COX-2, iNOS and NF-κB), apoptotic markers (caspase 3 cytochrome c release) and calcineurin phosphatase activity were assessed in hippocampus. RESULTS: Seven days following Aß inoculation, control animals displayed dramatic increase in TNF-α, COX-2, iNOS, NF-κB, active caspase 3, cytochrome c release and calcineurin phosphatase activity in the hippocampus. Tropisetron significantly diminished the elevated levels of these markers and reversed the cognitive deficit. Interestingly, tropisetron was also found to be a potent inhibitor of calcineurin phosphatase activity. The selective 5-HT3 receptor agonist mCPBG, when co-administered with tropisetron, completely reversed the procognitive and anti-apoptotic properties of tropisetron while it could only partially counteract the anti-inflammatory effects. mCPBG alone significantly aggravated Aß-induced injury. CONCLUSION: Our findings indicate that tropisetron protects against Aß-induced neurotoxicity in vivo through both 5-HT3 receptor-dependent and independent pathways.
Asunto(s)
Enfermedad de Alzheimer/tratamiento farmacológico , Apoptosis/efectos de los fármacos , Indoles/farmacología , Antagonistas del Receptor de Serotonina 5-HT3/farmacología , Péptidos beta-Amiloides/toxicidad , Animales , Calcineurina/efectos de los fármacos , Ciclooxigenasa 2/metabolismo , Citocromos c/metabolismo , Encefalitis/tratamiento farmacológico , Hipocampo/efectos de los fármacos , Hipocampo/metabolismo , Masculino , Aprendizaje por Laberinto/efectos de los fármacos , FN-kappa B/metabolismo , Óxido Nítrico Sintasa de Tipo II/metabolismo , Nitritos/metabolismo , Ratas , Ratas Wistar , Tropisetrón , Factor de Necrosis Tumoral alfa/metabolismoRESUMEN
INTRODUCTION: Pimecrolimus , a calcineurin inhibitor, is a non-steroidal treatment option in patients aged ≥ 2 years with mild-to-moderate atopic eczema (AE). It was approved as a viable therapeutic option by the FDA in 2001 and in the European Union a year later in 2002. Calcineurin inhibitors inhibit the synthesis of inflammatory cytokines released from T cells and mast cells. In contrast to corticosteroids, calcineurin inhibitors act specifically on proinflammatory cells. Pimecrolimus shows comparative efficacy to mild topical corticosteroids and a special antipruritic effect. Furthermore, examinations of the systemic absorption of pimecrolimus implicated no systemic immunosuppression. In 2006, the FDA set a black box warning in the packaging materials of pimecrolimus alluding to the risk of skin malignancy or lymphomas due to theoretical consideration. AREAS COVERED: The authors provide a review of pimecrolimus as a treatment for AE. Specifically, the authors present the pharmacokinetic and pharmacodynamic information on pimecrolimus and also review its efficacy. The authors also discuss pimecrolimus' safety and tolerability profile. EXPERT OPINION: Pimecrolimus represents a valuable part of active and proactive therapy in AE. That being said, the long-term safety of topical calcineurin inhibitors remains to be investigated. Given the results from experimental photocarcinogenicity studies, effective sun protection should be employed during the therapy, although an increased risk for skin malignancies and lymphomas was not found in recent studies. Pimecrolimus should be considered as an alternative therapeutic approach in AE treatment management going along with a corticoid-sparing effect.
Asunto(s)
Calcineurina/efectos de los fármacos , Dermatitis Atópica/tratamiento farmacológico , Fármacos Dermatológicos/administración & dosificación , Inhibidores Enzimáticos/administración & dosificación , Tacrolimus/análogos & derivados , Calcineurina/metabolismo , Fármacos Dermatológicos/efectos adversos , Fármacos Dermatológicos/farmacocinética , Relación Dosis-Respuesta a Droga , Evaluación Preclínica de Medicamentos , Inhibidores Enzimáticos/efectos adversos , Inhibidores Enzimáticos/farmacocinética , Humanos , Ensayos Clínicos Controlados Aleatorios como Asunto , Piel/efectos de los fármacos , Piel/patología , Tacrolimus/administración & dosificación , Tacrolimus/efectos adversos , Tacrolimus/farmacocinéticaRESUMEN
Disruption of fast axonal transport (FAT) is an early pathological event in Alzheimer's disease (AD). Soluble amyloid-ß oligomers (AßOs), increasingly recognized as proximal neurotoxins in AD, impair organelle transport in cultured neurons and transgenic mouse models. AßOs also stimulate hyperphosphorylation of the axonal microtubule-associated protein, tau. However, the role of tau in FAT disruption is controversial. Here we show that AßOs reduce vesicular transport of brain-derived neurotrophic factor (BDNF) in hippocampal neurons from both wild-type and tau-knockout mice, indicating that tau is not required for transport disruption. FAT inhibition is not accompanied by microtubule destabilization or neuronal death. Significantly, inhibition of calcineurin (CaN), a calcium-dependent phosphatase implicated in AD pathogenesis, rescues BDNF transport. Moreover, inhibition of protein phosphatase 1 and glycogen synthase kinase 3ß, downstream targets of CaN, prevents BDNF transport defects induced by AßOs. We further show that AßOs induce CaN activation through nonexcitotoxic calcium signaling. Results implicate CaN in FAT regulation and demonstrate that tau is not required for AßO-induced BDNF transport disruption.