RESUMEN
The tumor suppressor gene F-box and WD repeat domain-containing (FBXW) 7 reduces cancer stemness properties by promoting the protein degradation of pluripotent stem cell markers. We recently demonstrated the transcriptional repression of FBXW7 by the three-dimensional (3D) spheroid formation of several cancer cells. In the present study, we found that the transcriptional activity of FBXW7 was promoted by the inhibition of the Ca2+-activated K+ channel, KCa1.1, in a 3D spheroid model of human prostate cancer LNCaP cells through the Akt-Nrf2 signaling pathway. The transcriptional activity of FBXW7 was reduced by the siRNA-mediated inhibition of the CCAAT-enhancer-binding protein C/EBP δ (CEBPD) after the transfection of miR223 mimics in the LNCaP spheroid model, suggesting the transcriptional regulation of FBXW7 through the Akt-Nrf2-CEBPD-miR223 transcriptional axis in the LNCaP spheroid model. Furthermore, the KCa1.1 inhibition-induced activation of FBXW7 reduced (1) KCa1.1 activity and protein levels in the plasma membrane and (2) the protein level of the cancer stem cell (CSC) markers, c-Myc, which is a molecule degraded by FBXW7, in the LNCaP spheroid model, indicating that KCa1.1 inhibition-induced FBXW7 activation suppressed CSC conversion in KCa1.1-positive cancer cells.
Asunto(s)
Proteína 7 que Contiene Repeticiones F-Box-WD , Regulación Neoplásica de la Expresión Génica , Factor 2 Relacionado con NF-E2 , Neoplasias de la Próstata , Transducción de Señal , Esferoides Celulares , Humanos , Proteína 7 que Contiene Repeticiones F-Box-WD/metabolismo , Proteína 7 que Contiene Repeticiones F-Box-WD/genética , Masculino , Factor 2 Relacionado con NF-E2/metabolismo , Factor 2 Relacionado con NF-E2/genética , Neoplasias de la Próstata/metabolismo , Neoplasias de la Próstata/genética , Neoplasias de la Próstata/patología , Esferoides Celulares/metabolismo , Línea Celular Tumoral , Regulación hacia Arriba , Canales de Potasio de Conductancia Intermedia Activados por el Calcio/metabolismo , Canales de Potasio de Conductancia Intermedia Activados por el Calcio/genética , Canales de Potasio de Conductancia Intermedia Activados por el Calcio/antagonistas & inhibidores , Subunidades alfa de los Canales de Potasio de Gran Conductancia Activados por Calcio/metabolismo , Subunidades alfa de los Canales de Potasio de Gran Conductancia Activados por Calcio/genética , MicroARNs/genética , MicroARNs/metabolismo , Proteínas Proto-Oncogénicas c-akt/metabolismoRESUMEN
Osteoblasts synthesize and deposit essential components of the extracellular bone matrix and collagen scaffolds, leading to mineralized bone formation. Therefore, the proliferation of preosteoblasts (precursors of mature osteoblasts) helps in regulating skeletal homeostasis. This study demonstrated that the functional expression of KCa3.1, an intermediate-conductance Ca2+-activated K+ channel, is markedly upregulated in murine preosteoblastic MC3T3-E1 cells in the G0/G1 phase. The enhancement of KCa3.1 is involved in the establishment of more negative membrane potentials in MC3T3-E1 cells. This hyperpolarization can promote intracellular Ca2+ signaling because store-operated Ca2+ channels are activated. Treatment with TRAM-34, a specific KCa3.1 inhibitor, attenuated the cell cycle progression from the G0/G1 phase to the S/G2/M phases. In MC3T3-E1 cells, KCa3.1 significantly promoted the transition from the G1 phase to the S phase. KCa3.1 inhibition also caused G0 phase cell accumulation. Furthermore, TRAM-34 decreased the expression of alkaline phosphatase, bone sialoprotein, and osteocalcin, osteoblast differentiation markers in MC3T3-E1 cells, and inhibited the endochondral ossification of murine metatarsals. These results reveal novel ways by which KCa3.1 activity can strongly modulate osteoblast maturation during bone formation.
RESUMEN
The large-conductance Ca2+-activated K+ channel, KCa1.1, plays a pivotal role in cancer progression, metastasis, and the acquisition of chemoresistance. Previous studies indicated that the pharmacological inhibition of KCa1.1 overcame resistance to doxorubicin (DOX) by down-regulating multidrug resistance-associated proteins in the three-dimensional spheroid models of human prostate cancer LNCaP, osteosarcoma MG-63, and chondrosarcoma SW-1353 cells. Investigations have recently focused on the critical roles of intratumoral, drug-metabolizing cytochrome P450 enzymes (CYPs) in chemoresistance. In the present study, we examined the involvement of CYPs in the acquisition of DOX resistance and its overcoming by inhibiting KCa1.1 in cancer spheroid models. Among the CYP isoforms involved in DOX metabolism, CYP3A4 was up-regulated by spheroid formation and significantly suppressed by the inhibition of KCa1.1 through the transcriptional repression of CCAAT/enhancer-binding protein, CEBPB, which is a downstream transcription factor of the Nrf2 signaling pathway. DOX resistance was overcome by the siRNA-mediated inhibition of CYP3A4 and treatment with the potent CYP3A4 inhibitor, ketoconazole, in cancer spheroid models. The phosphorylation levels of Akt were significantly reduced by inhibiting KCa1.1 in cancer spheroid models, and KCa1.1-induced down-regulation of CYP3A4 was reversed by the treatment with Akt and Nrf2 activators. Collectively, the present results indicate that the up-regulation of CYP3A4 is responsible for the acquisition of DOX resistance in cancer spheroid models, and the inhibition of KCa1.1 overcame DOX resistance by repressing CYP3A4 transcription mainly through the Akt-Nrf2-CEBPB axis.
Asunto(s)
Neoplasias Óseas , Citocromo P-450 CYP3A , Humanos , Masculino , Línea Celular Tumoral , Citocromo P-450 CYP3A/genética , Citocromo P-450 CYP3A/metabolismo , Sistema Enzimático del Citocromo P-450/metabolismo , Regulación hacia Abajo , Doxorrubicina/farmacología , Resistencia a Antineoplásicos , Factor 2 Relacionado con NF-E2/metabolismo , Proteínas Proto-Oncogénicas c-akt/metabolismoRESUMEN
The KCa3.1 inhibition up-regulates IL-10 expression in regulatory T (Treg) cells in the recovery phase of inflammatory bowel disease (IBD) model mice; however, the underlying signaling pathway remains unclear. We investigated the involvement of AP-1 (Fos/Jun) and NF-κB in the expression of IL-10 and its transcription factors (TFs) in in vitro-induced mouse splenic Treg cells. The pharmacological inhibition of JNK reversed KCa3.1 inhibition-induced increases in the expression of IL-10 and its TFs. The inhibition of KCa3.1 increased phosphorylated JNK and c-Jun levels. Therefore, the JNK/c-Jun signaling pathway may contribute to the KCa3.1 inhibition-induced up-regulation of IL-10 in peripherally-induced Treg cells.
Asunto(s)
Expresión Génica/genética , Enfermedades Inflamatorias del Intestino/genética , Interleucina-10/genética , Interleucina-10/metabolismo , Canales de Potasio de Conductancia Intermedia Activados por el Calcio/antagonistas & inhibidores , Canales de Potasio de Conductancia Intermedia Activados por el Calcio/fisiología , Proteínas Quinasas JNK Activadas por Mitógenos/metabolismo , Sistema de Señalización de MAP Quinasas/genética , Sistema de Señalización de MAP Quinasas/fisiología , Proteínas Proto-Oncogénicas c-jun/metabolismo , Linfocitos T Reguladores/metabolismo , Animales , Modelos Animales de Enfermedad , Ratones , FN-kappa B/metabolismo , Fosforilación , Factor de Transcripción AP-1/metabolismo , Factores de Transcripción/genética , Factores de Transcripción/metabolismoRESUMEN
THP-1-differentiated macrophages are useful for investigating the physiological significance of tumor-associated macrophages (TAMs). In the tumor microenvironment (TME), TAMs with the M2-like phenotype play a critical role in promoting cancer progression and metastasis by inhibiting the immune surveillance system. We examined the involvement of Ca2+-activated K+ channel KCa3.1 in TAMs in expressing pro-tumorigenic cytokines and angiogenic growth factors. In THP-1-derived M2 macrophages, the expression levels of IL-8 and IL-10 were significantly decreased by treatment with the selective KCa3.1 activator, SKA-121, without changes in those of VEGF and TGF-ß1. Furthermore, under in vitro experimental conditions that mimic extracellular K+ levels in the TME, IL-8 and IL-10 levels were both significantly elevated, and these increases were reversed by combined treatment with SKA-121. Among several signaling pathways potentially involved in the transcriptional regulation of IL-8 and IL-10, respective treatments with ERK and JNK inhibitors significantly repressed their transcriptions, and treatment with SKA-121 significantly reduced the phosphorylated ERK, JNK, c-Jun, and CREB levels. These results strongly suggest that the KCa3.1 activator may suppress IL-10-induced tumor immune surveillance escape and IL-8-induced tumorigenicity and metastasis by inhibiting their production from TAMs through ERK-CREB and JNK-c-Jun cascades.
Asunto(s)
Interleucina-10 , Canales de Potasio de Conductancia Intermedia Activados por el Calcio/metabolismo , Neoplasias , Regulación hacia Abajo , Humanos , Interleucina-10/genética , Interleucina-10/metabolismo , Interleucina-8/genética , Interleucina-8/metabolismo , Macrófagos/metabolismo , Neoplasias/metabolismo , Microambiente TumoralRESUMEN
The large-conductance Ca2+ -activated K+ channel KCa 1.1 plays a pivotal role in tumor development and progression in several solid cancers. The three-dimensional (3D) in vitro cell culture system is a powerful tool for cancer spheroid formation, and mimics in vivo solid tumor resistance to chemotherapy in the tumor microenvironment (TME). KCa 1.1 is functionally expressed in osteosarcoma and chondrosarcoma cell lines. KCa 1.1 activator-induced hyperpolarizing responses were significantly larger in human osteosarcoma MG-63 cells isolated from 3D spheroid models compared with in those from adherent 2D monolayer cells. The present study investigated the mechanisms underlying the upregulation of KCa 1.1 and its role in chemoresistance using a 3D spheroid model. KCa 1.1 protein expression levels were significantly elevated in the lipid-raft-enriched compartments of MG-63 spheroids without changes in its transcriptional level. 3D spheroid formation downregulated the expression of the ubiquitin E3 ligase FBXW7, which is an essential contributor to KCa 1.1 protein degradation in breast cancer. The siRNA-mediated inhibition of FBXW7 in MG-63 cells from 2D monolayers upregulated KCa 1.1 protein expression. Furthermore, a treatment with a potent and selective KCa 1.1 inhibitor overcame the chemoresistance of the MG-63 and human chondrosarcoma SW-1353 spheroid models to paclitaxel, doxorubicin, and cisplatin. Among several multidrug resistance ATP-binding cassette transporters, the expression of the multidrug resistance-associated protein MRP1 was upregulated in both spheroids and restored by the inhibition of KCa 1.1. Therefore, the pharmacological inhibition of KCa 1.1 may be an attractive new strategy for acquiring resistance to chemotherapeutic drugs in the TME of KCa 1.1-positive sarcomas.
Asunto(s)
Neoplasias Óseas/metabolismo , Resistencia a Antineoplásicos/efectos de los fármacos , Resistencia a Antineoplásicos/genética , Subunidades alfa de los Canales de Potasio de Gran Conductancia Activados por Calcio/metabolismo , Osteosarcoma/metabolismo , Esferoides Celulares/metabolismo , Regulación hacia Arriba/genética , Antineoplásicos/farmacología , Neoplasias Óseas/patología , Línea Celular Tumoral , Supervivencia Celular/efectos de los fármacos , Cisplatino/farmacología , Doxorrubicina/farmacología , Proteína 7 que Contiene Repeticiones F-Box-WD/genética , Proteína 7 que Contiene Repeticiones F-Box-WD/metabolismo , Humanos , Indoles/farmacología , Subunidades alfa de los Canales de Potasio de Gran Conductancia Activados por Calcio/antagonistas & inhibidores , Osteosarcoma/patología , Paclitaxel/farmacología , Bloqueadores de los Canales de Potasio/farmacología , ARN Interferente Pequeño/genética , Transfección , Microambiente Tumoral/efectos de los fármacos , Microambiente Tumoral/genéticaRESUMEN
Inflammatory bowel diseases (IBD) are chronic inflammatory diseases of the gastrointestinal tract arising from abnormal responses of the innate and adaptative immune systems. Interleukin (IL)-10-producing CD4+CD25+ regulatory T (Treg) cells play a protective role in the recovery phase of IBD. In the present study, the effects of the administration of the selective Ca2+-activated K+ channel KCa3.1 inhibitor TRAM-34 on disease activities were examined in chemically induced IBD model mice. IBD disease severity, as assessed by diarrhea, visible fecal blood, inflammation, and crypt damage in the colon, was significantly lower in mice administered 1 mg/kg TRAM-34 than in vehicle-administered mice. Quantitative real-time polymerase chain reaction examinations showed that IL-10 expression levels in the recovery phase were markedly increased by the inhibition of KCa3.1 in mesenteric lymph node (mLN) Treg cells of IBD model mice compared with vehicle-administered mice. Among several positive and negative transcriptional regulators (TRs) for IL-10, three positive TRs-E4BP4, KLF4, and Blimp1-were upregulated by the inhibition of KCa3.1 in the mLN Treg cells of IBD model mice. In mouse peripheral CD4+CD25+ Treg cells induced by lectin stimulation, IL-10 expression and secretion were enhanced by the treatment with TRAM-34, together with the upregulation of E4BP4, KLF4, and Blimp1. Collectively, the present results demonstrated that the pharmacological inhibition of KCa3.1 decreased IBD symptoms in the IBD model by increasing IL-10 production in peripheral Treg cells and that IL-10high Treg cells produced by the treatment with KCa3.1 inhibitor may contribute to efficient Treg therapy for chronic inflammatory disorders, including IBD. SIGNIFICANCE STATEMENT: Pharmacological inhibition of Ca2+-activated K+ channel KCa3.1 increased IL-10 expression in peripheral Treg cells, together with the upregulation of the transcriptional regulators of IL-10: Krüppel-like factor 4, E4 promoter-binding protein 4, and/or B lymphocyte-induced maturation protein 1. The manipulation of IL-10high-producing Treg cells by the pharmacological inhibition of KCa3.1 may be beneficial in the treatment of chronic inflammatory diseases such as inflammatory bowel disease.
Asunto(s)
Enfermedades Inflamatorias del Intestino/metabolismo , Interleucina-10/metabolismo , Canales de Potasio de Conductancia Intermedia Activados por el Calcio/antagonistas & inhibidores , Moduladores del Transporte de Membrana/farmacología , Pirazoles/farmacología , Linfocitos T Reguladores/metabolismo , Animales , Factores de Transcripción con Cremalleras de Leucina de Carácter Básico/genética , Factores de Transcripción con Cremalleras de Leucina de Carácter Básico/metabolismo , Femenino , Enfermedades Inflamatorias del Intestino/tratamiento farmacológico , Enfermedades Inflamatorias del Intestino/patología , Interleucina-10/genética , Subunidad alfa del Receptor de Interleucina-2/genética , Subunidad alfa del Receptor de Interleucina-2/metabolismo , Canales de Potasio de Conductancia Intermedia Activados por el Calcio/metabolismo , Factor 4 Similar a Kruppel , Factores de Transcripción de Tipo Kruppel/genética , Factores de Transcripción de Tipo Kruppel/metabolismo , Moduladores del Transporte de Membrana/administración & dosificación , Moduladores del Transporte de Membrana/uso terapéutico , Ratones , Ratones Endogámicos C57BL , Factor 1 de Unión al Dominio 1 de Regulación Positiva/genética , Factor 1 de Unión al Dominio 1 de Regulación Positiva/metabolismo , Pirazoles/administración & dosificación , Pirazoles/uso terapéutico , Linfocitos T Reguladores/efectos de los fármacosRESUMEN
Bone-forming cells or osteoblasts play an important role in bone modeling and remodeling processes. Osteoblast differentiation or osteoblastogenesis is orchestrated by multiple intracellular signaling pathways (e.g., bone morphogenetic proteins (BMP) and Wnt signaling pathways) and is modulated by the extracellular environment (e.g., parathyroid hormone (PTH), vitamin D, transforming growth factor ß (TGF-ß), and integrins). The regulation of bone homeostasis depends on the proper differentiation and function of osteoblast lineage cells from osteogenic precursors to osteocytes. Intracellular Ca2+ signaling relies on the control of numerous processes in osteoblast lineage cells, including cell growth, differentiation, migration, and gene expression. In addition, hyperpolarization via the activation of K+ channels indirectly promotes Ca2+ signaling in osteoblast lineage cells. An improved understanding of the fundamental physiological and pathophysiological processes in bone homeostasis requires detailed investigations of osteoblast lineage cells. This review summarizes the current knowledge on the functional impacts of K+ channels and Ca2+-permeable channels, which critically regulate Ca2+ signaling in osteoblast lineage cells to maintain bone homeostasis.
Asunto(s)
Canales de Calcio/metabolismo , Osteoblastos/metabolismo , Osteogénesis , Canales de Potasio/metabolismo , Transducción de Señal , Animales , Calcio/química , Calcio/metabolismo , Cationes/metabolismo , Humanos , Osteoblastos/fisiología , Potasio/química , Potasio/metabolismoRESUMEN
Several types of K+ channels play crucial roles in tumorigenicity, stemness, invasiveness, and drug resistance in cancer. Spheroid formation of human prostate cancer (PC) LNCaP cells with ultra-low attachment surface cultureware induced the up-regulation of cancer stem cell markers, such as NANOG, and decreased the protein degradation of the Ca2+-activated K+ channel KCa1.1 by down-regulating the E3 ubiquitin ligase, FBXW7, compared with LNCaP monolayers. Accordingly, KCa1.1 activator-induced hyperpolarizing responses were larger in isolated cells from LNCaP spheroids. The pharmacological inhibition of KCa1.1 overcame the resistance of LNCaP spheroids to antiandrogens and doxorubicin (DOX). The protein expression of androgen receptors (AR) was significantly decreased by LNCaP spheroid formation and reversed by KCa1.1 inhibition. The pharmacological and genetic inhibition of MDM2, which may be related to AR protein degradation in PC stem cells, revealed that MDM2 was responsible for the acquisition of antiandrogen resistance in LNCaP spheroids, which was overcome by KCa1.1 inhibition. Furthermore, a member of the multidrug resistance-associated protein subfamily of ABC transporters, MRP5 was responsible for the acquisition of DOX resistance in LNCaP spheroids, which was also overcome by KCa1.1 inhibition. Collectively, the present results suggest the potential of KCa1.1 in LNCaP spheroids, which mimic PC stem cells, as a therapeutic target for overcoming antiandrogen- and DOX-resistance in PC cells.
Asunto(s)
Antineoplásicos/uso terapéutico , Resistencia a Antineoplásicos , Subunidades alfa de los Canales de Potasio de Gran Conductancia Activados por Calcio/antagonistas & inhibidores , Neoplasias de la Próstata/fisiopatología , Antagonistas de Andrógenos/uso terapéutico , Línea Celular Tumoral , Doxorrubicina/uso terapéutico , Regulación Neoplásica de la Expresión Génica , Humanos , Masculino , Proteínas Asociadas a Resistencia a Múltiples Medicamentos , Células Madre Neoplásicas , Neoplasias de la Próstata/tratamiento farmacológico , Neoplasias de la Próstata/genética , Neoplasias de la Próstata/metabolismo , Receptores Androgénicos/genética , Esferoides CelularesRESUMEN
The maturity of osteoblasts by proliferation and differentiation in preosteoblasts is essential for maintaining bone homeostasis. The beneficial effects of vitamin D on bone homeostasis in mammals have been demonstrated experimentally and clinically. However, the direct actions of vitamin D on preosteoblasts remain to be fully elucidated. In this study, we found that the functional activity of intermediate-conductance Ca2+-activated K+ channels (KCa3.1) positively regulated cell proliferation in MC3T3-E1 cells derived from mouse preosteoblasts by enhancing intracellular Ca2+ signaling. We examined the effects of treatment with vitamin D receptor (VDR) agonist on the expression and activity of KCa3.1 by real-time PCR examination, Western blotting, Ca2+ imaging, and patch clamp analyses in mouse MC3T3-E1 cells. Following the downregulation of KCa3.1 transcriptional modulators such as Fra-1 and HDAC2, KCa3.1 activity was suppressed in MC3T3-E1 cells treated with VDR agonists. Furthermore, application of the KCa3.1 activator DCEBIO attenuated the VDR agonist-evoked suppression of cell proliferation rate. These findings suggest that a decrease in KCa3.1 activity is involved in the suppression of cell proliferation rate in VDR agonist-treated preosteoblasts. Therefore, KCa3.1 plays an important role in bone formation by promoting osteoblastic proliferation under physiological conditions.
Asunto(s)
Canales de Potasio de Conductancia Intermedia Activados por el Calcio/genética , Osteoblastos/metabolismo , Receptores de Calcitriol/genética , Vitamina D/genética , Células 3T3 , Animales , Bencimidazoles/farmacología , Calcio/metabolismo , Señalización del Calcio/genética , Diferenciación Celular/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Regulación de la Expresión Génica/genética , Histona Desacetilasa 2/genética , Humanos , Ratones , Osteoblastos/citología , Osteogénesis/efectos de los fármacos , Osteogénesis/genética , Técnicas de Placa-Clamp , Proteínas Proto-Oncogénicas c-fos/genética , Receptores de Calcitriol/agonistas , Transducción de Señal/efectos de los fármacosRESUMEN
The hyperpolarization induced by intermediate-conductance Ca2+-activated K+ channel (KCa3.1) activation increases the driving force for Ca2+ influx, which generally promotes cell proliferation, migration, and cytokine production in immunocompetent cells. Interleukin-10 (IL-10) from tumor-infiltrating lymphocytes and macrophages, lymphoma, and carcinoma cells facilitates escape from cancer immune surveillance; however, the role of KCa3.1 in IL-10 production remains unclear. The objective of the present study was to elucidate the involvement of KCa3.1 in IL-10 expression and production using the human T-cell lymphoma HuT-78 cells. In HuT-78 cells, IL-10 gene expression and production were reduced by treatment with the KCa3.1 activator, as 6-hour Western blotting showed that the protein expression ratio of phosphorylated Smad2 (P-Smad2)/Smad2, but not P-Smad3/Smad3, was decreased by the treatment with KCa3.1 activator in HuT-78 cells. Concomitant with this, the nuclear translocation of P-Smad2 was inhibited by KCa3.1 activator. Furthermore, the KCa3.1 activator-induced transcriptional repression of IL-10 disappeared with pretreatment with the calmodulin kinase II (CaMKII) inhibitor KN-62 for 1 hour, and KCa3.1 activator-induced decreases in the nuclear translocation of P-Smad2 were also prevented by pretreatment with KN-62. Taken together, the KCa3.1 activator-induced transcriptional repression of IL-10 is due to the inhibition of the nuclear translocation of P-Smad2 in HuT-78 cells, resulting in the prevention of P-Smad2/3 complex formation in nuclei, and the activation of CaMKII induced by KCa3.1 activators suppresses the constitutive activation of P-Smad2/3 in HuT-78 cells. Therefore, KCa3.1 activators have potential as a therapeutic option to suppress the tumor-promoting activities of IL-10.
Asunto(s)
Interleucina-10/metabolismo , Canales de Potasio de Conductancia Intermedia Activados por el Calcio/metabolismo , Linfoma de Células T/metabolismo , Transducción de Señal/fisiología , Proteína Smad2/metabolismo , Proteína smad3/metabolismo , Transcripción Genética/fisiología , Proteína Quinasa Tipo 2 Dependiente de Calcio Calmodulina/metabolismo , Línea Celular Tumoral , Núcleo Celular/metabolismo , Proliferación Celular/fisiología , Humanos , Células K562 , Fosforilación/fisiología , Células THP-1RESUMEN
Previous studies have reported the up-regulation of the two-pore domain K+ channel K2P5.1 in the CD4+ T cells of patients with multiple sclerosis (MS) and rheumatoid arthritis (RA), as well as in a mouse model of inflammatory bowel disease (IBD). However, the mechanisms underlying this up-regulation remain unclear. Inflammation-associated hypoxia is involved in the pathogenesis of autoimmune diseases, such as IBD, MS, and RA, and T cells are exposed to a hypoxic environment during their recruitment from inflamed tissues to secondary lymphoid tissues. We herein investigated whether inflammation-associated hypoxia is attributable to the increased expression and activity of K2P5.1 in the splenic CD4+ T cells of chemically-induced IBD model mice. Significant increases in hypoxia-inducible factor (HIF)-1α transcripts and proteins were found in the splenic CD4+ T cells of the IBD model. In the activated splenic CD4+ T cells, hypoxia (1.5% O2) increased K2P5.1 expression and activity, whereas a treatment with the HIF inhibitor FM19G11 but not the selective HIF-2 inhibitor exerted the opposite effect. Hypoxia-exposed K2P5.1 up-regulation was also detected in stimulated thymocytes and the mouse T-cell line. The class III histone deacetylase sirtuin-1 (SIRT1) is a downstream molecule of HIF-1α signaling. We examined the effects of the SIRT1 inhibitor NCO-01 on K2P5.1 transcription in activated CD4+ T cells, and we found no significant effects on the K2P5.1 transcription. No acute compensatory responses of K2P3.1-K2P5.1 up-regulation were found in the CD4+ T cells of the IBD model and the hypoxia-exposed T cells. Collectively, these results suggest a mechanism for K2P5.1 up-regulation via HIF-1 in the CD4+ T cells of the IBD model.
Asunto(s)
Linfocitos T CD4-Positivos/metabolismo , Subunidad alfa del Factor 1 Inducible por Hipoxia/genética , Enfermedades Inflamatorias del Intestino/genética , Canales de Potasio de Dominio Poro en Tándem/genética , Animales , Benzamidas/farmacología , Hipoxia de la Célula , Línea Celular , Sulfato de Dextran/efectos adversos , Modelos Animales de Enfermedad , Regulación de la Expresión Génica , Humanos , Subunidad alfa del Factor 1 Inducible por Hipoxia/metabolismo , Enfermedades Inflamatorias del Intestino/inducido químicamente , Enfermedades Inflamatorias del Intestino/metabolismo , Ratones , Canales de Potasio de Dominio Poro en Tándem/metabolismo , Sirtuina 1/genética , Sirtuina 1/metabolismo , Timocitos/citología , Timocitos/metabolismoRESUMEN
Recent studies have indicated that the intracellular concentration of chloride ions (Cl- ) regulates gene expression in several types of cells and that Cl- modulators positively or negatively regulate the PI3K/AKT/mammalian target of rapamycin (mTOR) and signal transducer and activator of transcription (STAT)3 signaling pathways. We previously reported that the Ca2+ -activated Cl- channel anoctamine (ANO)1 regulated human epidermal growth factor receptor 2 (HER2) transcription in breast cancer YMB-1 cells. However, the mechanisms underlying ANO1-regulated HER2 gene expression have not yet been elucidated. In the present study, we showed the involvement of intracellular organelle ClC-3 Cl- /H+ transporter in HER2 transcription in breast cancer MDA-MB-453 cells. The siRNA-mediated inhibition of ClC-3, but not ANO1, markedly repressed HER2 transcription in MDA-MB-453 cells. Subsequently, treatments with the AKT inhibitor AZD 5363 and mTOR inhibitor everolimus significantly enhanced HER2 transcription in MDA-MB-453 cells, whereas that with the STAT3 inhibitor 5,15-diphenylporphyrin (5,15-DPP) inhibited it. AKT and mTOR inhibitors also significantly enhanced HER2 transcription in YMB-1 cells. The siRNA-mediated inhibition of ClC-3 and ANO1 resulted in increased AKT phosphorylation and decreased STAT3 phosphorylation in MDA-MB-453 and YMB-1 cells, respectively. The intracellular Cl- channel protein CLIC1 was expressed in both cells; however, its siRNA-mediated inhibition did not elicit the transcriptional repression of HER2. Collectively, our results demonstrate that intracellular Cl- regulation by ANO1/ClC-3 participates in HER2 transcription, mediating the PI3K/AKT/mTOR and/or STAT3 signaling pathway(s) in HER2-positive breast cancer cells, and support the potential of ANO1/ClC-3 blockers as therapeutic options for patients with resistance to anti-HER2 therapies.
Asunto(s)
Neoplasias de la Mama/metabolismo , Canales de Cloruro/fisiología , Regulación Neoplásica de la Expresión Génica , Receptor ErbB-2/genética , Anoctamina-1/fisiología , Neoplasias de la Mama/patología , Línea Celular Tumoral , Cloruros/metabolismo , Femenino , Histonas/metabolismo , Humanos , Proteínas de Neoplasias/fisiología , Fosfatidilinositol 3-Quinasas/fisiología , Proteínas Proto-Oncogénicas c-akt/fisiología , Factor de Transcripción STAT3/fisiología , Transducción de Señal/fisiología , Serina-Treonina Quinasas TOR/fisiologíaRESUMEN
In lymphoid and myeloid cells, membrane hyperpolarization by the opening of K+ channels increases the activity of Ca2+ release-activated Ca2+ (CRAC) channels and transient receptor potential (TRP) Ca2+ channels. The intermediate-conductance Ca2+-activated K+ channel KCa3.1 plays an important role in cell proliferation, differentiation, migration, and cytokine production in innate and adaptive immune systems. KCa3.1 is therefore an attractive therapeutic target for allergic, inflammatory, and autoimmune disorders. In the past several years, studies have provided new insights into 1) KCa3.1 pharmacology and its auxiliary regulators; 2) post-transcriptional and proteasomal regulation of KCa3.1; 3) KCa3.1 as a regulator of immune cell migration, cytokine production, and phenotypic polarization; 4) the role of KCa3.1 in the phosphorylation and nuclear translocation of Smad2/3; and 5) KCa3.1 as a therapeutic target for cancer immunotherapy. In this review, we have assembled a comprehensive overview of current research on the physiological and pathophysiological significance of KCa3.1 in the immune system.
Asunto(s)
Enfermedades del Sistema Inmune/inmunología , Canales de Potasio de Conductancia Intermedia Activados por el Calcio/inmunología , Animales , Movimiento Celular , Citocinas/inmunología , Humanos , Enfermedades del Sistema Inmune/tratamiento farmacológico , Enfermedades del Sistema Inmune/metabolismo , Canales de Potasio de Conductancia Intermedia Activados por el Calcio/metabolismo , Fenotipo , Fosforilación , Proteína Smad2/metabolismo , Proteína smad3/metabolismoRESUMEN
The up-regulated expression of the Ca2+-activated K⺠channel KCa3.1 in inflammatory CD4⺠T cells has been implicated in the pathogenesis of inflammatory bowel disease (IBD) through the enhanced production of inflammatory cytokines, such as interferon-γ (IFN-γ). However, the underlying mechanisms have not yet been elucidated. The objective of the present study is to clarify the involvement of histone deacetylases (HDACs) in the up-regulation of KCa3.1 in the CD4⺠T cells of IBD model mice. The expression levels of KCa3.1 and its regulators, such as function-modifying molecules and transcription factors, were quantitated using a real-time polymerase chain reaction (PCR) assay, Western blotting, and depolarization responses, which were induced by the selective KCa3.1 blocker TRAM-34 (1 µM) and were measured using a voltage-sensitive fluorescent dye imaging system. The treatment with 1 µM vorinostat, a pan-HDAC inhibitor, for 24 h repressed the transcriptional expression of KCa3.1 in the splenic CD4⺠T cells of IBD model mice. Accordingly, TRAM-34-induced depolarization responses were significantly reduced. HDAC2 and HDAC3 were significantly up-regulated in the CD4⺠T cells of IBD model mice. The down-regulated expression of KCa3.1 was observed following treatments with the selective inhibitors of HDAC2 and HDAC3. The KCa3.1 K⺠channel regulates inflammatory cytokine production in CD4⺠T cells, mediating epigenetic modifications by HDAC2 and HDAC3.
Asunto(s)
Linfocitos T CD4-Positivos/metabolismo , Histona Desacetilasas/metabolismo , Enfermedades Inflamatorias del Intestino/metabolismo , Canales de Potasio de Conductancia Intermedia Activados por el Calcio/genética , Animales , Linfocitos T CD4-Positivos/efectos de los fármacos , Células Cultivadas , Inhibidores de Histona Desacetilasas/farmacología , Canales de Potasio de Conductancia Intermedia Activados por el Calcio/antagonistas & inhibidores , Canales de Potasio de Conductancia Intermedia Activados por el Calcio/metabolismo , Masculino , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Bloqueadores de los Canales de Potasio/farmacología , Pirazoles/farmacologíaRESUMEN
The Ca2+-activated Cl- channel ANO1 contributes to tumorigenesis and metastasis in several carcinomas including breast cancer (BCA). Cl- channels have recently been attracting attention as 'transcriptional modulators'. Human epidermal growth factor receptor 2 (HER2) is overexpressed in approximately 30% of patients with BCA, and anti-HER2 monoclonal antibodies such as trastuzumab have emerged as a treatment for metastatic BCA. Among the seven human BCA cell lines examined in the present study, MDA-MB-453 and YMB-1 cells were HER2-positive; however, YMB-1 cell viability showed resistance to trastuzumab. Whole-cell patch-clamp configurations indicated that ANO1 was the main Cl- conductance in YMB-1 cells, and the pharmacological and siRNA-mediated inhibition of ANO1 significantly prevented HER2 transcription in YMB-1 cells. The expression levels of insulin-like growth factor-binding protein 5 (IGFBP5), which is a risk factor for BCA recurrence and metastasis, was not affected by the inhibition of ANO1 in YMB-1 cells. These results suggest that ANO1 Cl- channels may function as a transcriptional regulator of HER2, and ANO1 inhibitors have potential in the treatment of BCA patients with resistance to HER2-targeted therapy.
Asunto(s)
Antineoplásicos/farmacología , Neoplasias de la Mama/tratamiento farmacológico , Neoplasias de la Mama/metabolismo , Canales de Cloruro/metabolismo , Proteínas de Neoplasias/metabolismo , Receptor ErbB-2/genética , Activación Transcripcional/efectos de los fármacos , Trastuzumab/farmacología , Anoctamina-1 , Neoplasias de la Mama/patología , Línea Celular Tumoral , Supervivencia Celular/efectos de los fármacos , Canales de Cloruro/genética , Relación Dosis-Respuesta a Droga , Resistencia a Antineoplásicos/efectos de los fármacos , Humanos , Proteínas de Neoplasias/genética , Receptor ErbB-2/metabolismo , Trastuzumab/administración & dosificaciónRESUMEN
The two-pore domain K+ channel K2P5.1 has been implicated in the pathogenesis of autoimmune diseases. We investigated the changes in K2P5.1 activity caused by a defect in normal pre-mRNA splicing in concanavalin A-activated mouse splenic CD4+ T cells. The pre-mRNA splicing inhibitor, pladienolide B (1 µM) markedly decreased full-length K2P5.1 transcription in activated CD4+ T cells, resulting in the disappearance of K2P5.1 activity and an imbalance in Th17 and Treg cytokines. These results suggest that the defect in K2P5.1 splicing by the pre-mRNA splicing inhibitor regulates pro- and/or anti-inflammatory cytokine production in K2P5.1-associated autoimmune diseases.
Asunto(s)
Compuestos Epoxi/farmacología , Macrólidos/farmacología , Canales de Potasio de Dominio Poro en Tándem/genética , Precursores del ARN/genética , Empalme del ARN/efectos de los fármacos , Animales , Linfocitos T CD4-Positivos , Masculino , Ratones , Ratones Endogámicos C57BL , Canales de Potasio de Dominio Poro en Tándem/metabolismo , Empalme del ARN/genéticaRESUMEN
Vitamin D (VD) reduces the risk of breast cancer and improves disease prognoses. Potential VD analogs are being developed as therapeutic agents for breast cancer treatments. The large-conductance Ca2+-activated K⺠channel KCa1.1 regulates intracellular Ca2+ signaling pathways and is associated with high grade tumors and poor prognoses. In the present study, we examined the effects of treatments with VD receptor (VDR) agonists on the expression and activity of KCa1.1 in human breast cancer MDA-MB-453 cells using real-time PCR, Western blotting, flow cytometry, and voltage-sensitive dye imaging. Treatments with VDR agonists for 72 h markedly decreased the expression levels of KCa1.1 transcripts and proteins in MDA-MB-453 cells, resulting in the significant inhibition of depolarization responses induced by paxilline, a specific KCa1.1 blocker. The specific proteasome inhibitor MG132 suppressed VDR agonist-induced decreases in KCa1.1 protein expression. These results suggest that KCa1.1 is a new downstream target of VDR signaling and the down-regulation of KCa1.1 through the transcriptional repression of KCa1.1 and enhancement of KCa1.1 protein degradation contribute, at least partly, to the antiproliferative effects of VDR agonists in breast cancer cells.
Asunto(s)
Neoplasias de la Mama/genética , Regulación hacia Abajo , Subunidades alfa de los Canales de Potasio de Gran Conductancia Activados por Calcio/genética , Receptores de Calcitriol/agonistas , Neoplasias de la Mama/patología , Calcitriol/análogos & derivados , Calcitriol/farmacología , Línea Celular Tumoral , Supervivencia Celular/efectos de los fármacos , Regulación hacia Abajo/efectos de los fármacos , Femenino , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Humanos , Subunidades alfa de los Canales de Potasio de Gran Conductancia Activados por Calcio/antagonistas & inhibidores , Subunidades alfa de los Canales de Potasio de Gran Conductancia Activados por Calcio/metabolismo , Leupeptinas/farmacología , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/metabolismo , Inhibidores de Proteasoma/farmacología , Proteolisis/efectos de los fármacos , ARN Mensajero/genética , ARN Mensajero/metabolismo , ARN Interferente Pequeño/metabolismo , Receptores de Calcitriol/metabolismoRESUMEN
Store-operated Ca(2+) entry (SOCE) via Orai1 and STIM1 complex is supposed to have obligatory roles in the regulation of cellular functions of vascular endothelial cells, while little is known about the contribution of Orai2. Quantitative PCR and Western blot analyses indicated the expression of Orai2 and STIM2, in addition to Orai1 and STIM1 in bovine brain capillary endothelial cell line, t-BBEC117. During the exponential growth of t-BBEC117, the knockdown of Orai1 and STIM1 significantly reduced the SOCE activity, whereas Orai2 and STIM2 siRNAs had no effect. To examine whether endogenous SOCE activity contributes to the regulation of cell cycle progression, t-BBEC117 were synchronized using double thymidine blockage. At the G2/M phase, Ca(2+) influx via SOCE was decreased and Orai2 expression was increased compared to the G0/G1 phase. When Orai2 was knocked down at the G2/M phase, the decrease in SOCE was removed, and cell proliferation was partly attenuated. Taken together, Orai1 significantly contributes to cell proliferation via the functional expression, which is presumably independent of the cell cycle phases. In construct, Orai2 is specifically up-regulated during the G2/M phase, negatively modulates the SOCE activity, and may contribute to the regulation of cell cycle progression in brain capillary endothelial cells.
Asunto(s)
Encéfalo/irrigación sanguínea , Canales de Calcio/metabolismo , Señalización del Calcio , Animales , Barrera Hematoencefálica/citología , Barrera Hematoencefálica/metabolismo , Encéfalo/citología , Encéfalo/metabolismo , Canales de Calcio/genética , Bovinos , Puntos de Control del Ciclo Celular/fisiología , Línea Celular , Proliferación Celular , Células Endoteliales/citología , Células Endoteliales/metabolismo , Técnicas de Silenciamiento del Gen , Glicoproteínas de Membrana/antagonistas & inhibidores , Glicoproteínas de Membrana/genética , Glicoproteínas de Membrana/metabolismo , ARN Interferente Pequeño/genética , Regulación hacia ArribaRESUMEN
The intermediate-conductance Ca(2+)-activated K(+) channel KCa3.1/KCNN4 plays an important role in the modulation of Ca(2+) signaling through the control of the membrane potential in T lymphocytes. Here, we study the involvement of KCa3.1 in the enlargement of the mesenteric lymph nodes (MLNs) in a mouse model of inflammatory bowel disease (IBD). The mouse model of IBD was prepared by exposing male C57BL/6J mice to 5% dextran sulfate sodium for 7 days. Inflammation-induced changes in KCa3.1 activity and the expressions of KCa3.1 and its regulators in MLN CD4(+) T lymphocytes were monitored by real-time PCR, Western blot, voltage-sensitive dye imaging, patch-clamp, and flow cytometric analyses. Concomitant with an upregulation of KCa3.1a and nucleoside diphosphate kinase B (NDPK-B), a positive KCa3.1 regulator, an increase in KCa3.1 activity was observed in MLN CD4(+) T lymphocytes in the IBD model. Pharmacological blockade of KCa3.1 elicited the following results: 1) a significant decrease in IBD disease severity, as assessed by diarrhea, visible fecal blood, inflammation, and crypt damage of the colon and MLN enlargement compared with control mice, and 2) the restoration of the expression levels of KCa3.1a, NDPK-B, and Th1 cytokines in IBD model MLN CD4(+) T lymphocytes. These findings suggest that the increase in KCa3.1 activity induced by the upregulation of KCa3.1a and NDPK-B may be involved in the pathogenesis of IBD by mediating the enhancement of the proliferative response in MLN CD4(+) T lymphocyte and, therefore, that the pharmacological blockade of KCa3.1 may decrease the risk of IBD.