RESUMO
Cellular senescence, a major driver of aging, can be stimulated by DNA damage, and is counteracted by the DNA repair machinery. Here we show that in p16INK4a-deficient cells, senescence induction by the environmental genotoxin B[a]P or ionizing radiation (IR) completely depends on p21CIP1. Immunoprecipitation-based mass spectrometry interactomics data revealed that during senescence induction and maintenance, p21CIP1 specifically inhibits CDK4 and thereby activates the DREAM complex. Genome-wide transcriptomics revealed striking similarities in the response induced by B[a]P and IR. Among the top 100 repressed genes 78 were identical between B[a]P and IR and 76 were DREAM targets. The DREAM complex transcriptionally silences the main proliferation-associated transcription factors E2F1, FOXM1 and B-Myb as well as multiple DNA repair factors. Knockdown of p21CIP1, E2F4 or E2F5 diminished both, repression of these factors and senescence. The transcriptional profiles evoked by B[a]P and IR largely overlapped with the profile induced by pharmacological CDK4 inhibition, further illustrating the role of CDK4 inhibition in genotoxic stress-induced senescence. Moreover, data obtained by live-cell time-lapse microscopy suggest the inhibition of CDK4 by p21CIP1 is especially important for arresting cells which slip through mitosis. Overall, we identified the p21CIP1/CDK4/DREAM axis as a master regulator of genotoxic stress-induced senescence.
Assuntos
Senescência Celular , Quinase 4 Dependente de Ciclina , Inibidor de Quinase Dependente de Ciclina p21 , Dano ao DNA , Proteínas Interatuantes com Canais de Kv , Senescência Celular/efeitos da radiação , Senescência Celular/genética , Inibidor de Quinase Dependente de Ciclina p21/metabolismo , Inibidor de Quinase Dependente de Ciclina p21/genética , Quinase 4 Dependente de Ciclina/metabolismo , Quinase 4 Dependente de Ciclina/genética , Humanos , Proteínas Interatuantes com Canais de Kv/metabolismo , Proteínas Interatuantes com Canais de Kv/genética , Radiação Ionizante , Reparo do DNA , Regulação da Expressão Gênica/efeitos da radiação , Proteínas RepressorasRESUMO
Histone deacetylases (HDACs) play a crucial role in transcriptional regulation and are implicated in various diseases, including cancer. They are involved in histone tail deacetylation and canonically linked to transcriptional repression. Previous studies suggested that HDAC recruitment to cell-cycle gene promoters via the retinoblastoma (RB) protein or the DREAM complex through SIN3B is essential for G1/S and G2/M gene repression during cell-cycle arrest and exit. Here we investigate the interplay among DREAM, RB, SIN3 proteins, and HDACs in the context of cell-cycle gene repression. Knockout of SIN3B does not globally derepress cell-cycle genes in non-proliferating HCT116 and C2C12 cells. Loss of SIN3A/B moderately upregulates several cell-cycle genes in HCT116 cells but does so independently of DREAM/RB. HDAC inhibition does not induce general upregulation of RB/DREAM target genes in arrested transformed or non-transformed cells. Our findings suggest that E2F:RB and DREAM complexes can repress cell-cycle genes without relying on HDAC activity.
Assuntos
Fatores de Transcrição E2F , Histona Desacetilases , Proteínas Repressoras , Proteína do Retinoblastoma , Humanos , Histona Desacetilases/metabolismo , Histona Desacetilases/genética , Células HCT116 , Proteínas Repressoras/metabolismo , Proteínas Repressoras/genética , Fatores de Transcrição E2F/metabolismo , Fatores de Transcrição E2F/genética , Proteína do Retinoblastoma/metabolismo , Proteína do Retinoblastoma/genética , Camundongos , Animais , Complexo Correpressor Histona Desacetilase e Sin3/metabolismo , Complexo Correpressor Histona Desacetilase e Sin3/genética , Proteínas Interatuantes com Canais de Kv/metabolismo , Proteínas Interatuantes com Canais de Kv/genética , Ciclo Celular/genética , Regiões Promotoras Genéticas/genética , Regulação da Expressão Gênica , Genes cdcRESUMO
KV channel-interacting proteins (KChIP1-4) belong to a family of Ca2+-binding EF-hand proteins that are able to bind to the N-terminus of the KV4 channel α-subunits. KChIPs are predominantly expressed in the brain and heart, where they contribute to the maintenance of the excitability of neurons and cardiomyocytes by modulating the fast inactivating-KV4 currents. As the auxiliary subunit, KChIPs are critically involved in regulating the surface protein expression and gating properties of KV4 channels. Mechanistically, KChIP1, KChIP2, and KChIP3 promote the translocation of KV4 channels to the cell membrane, accelerate voltage-dependent activation, and slow the recovery rate of inactivation, which increases KV4 currents. By contrast, KChIP4 suppresses KV4 trafficking and eliminates the fast inactivation of KV4 currents. In the heart, IKs, ICa,L, and INa can also be regulated by KChIPs. ICa,L and INa are positively regulated by KChIP2, whereas IKs is negatively regulated by KChIP2. Interestingly, KChIP3 is also known as downstream regulatory element antagonist modulator (DREAM) because it can bind directly to the downstream regulatory element (DRE) on the promoters of target genes that are implicated in the regulation of pain, memory, endocrine, immune, and inflammatory reactions. In addition, all the KChIPs can act as transcription factors to repress the expression of genes involved in circadian regulation. Altered expression of KChIPs has been implicated in the pathogenesis of several neurological and cardiovascular diseases. For example, KChIP2 is decreased in failing hearts, while loss of KChIP2 leads to increased susceptibility to arrhythmias. KChIP3 is increased in Alzheimer's disease and amyotrophic lateral sclerosis, but decreased in epilepsy and Huntington's disease. In the present review, we summarize the progress of recent studies regarding the structural properties, physiological functions, and pathological roles of KChIPs in both health and disease. We also summarize the small-molecule compounds that regulate the function of KChIPs. This review will provide an overview and update of the regulatory mechanism of the KChIP family and the progress of targeted drug research as a reference for researchers in related fields.
Assuntos
Sistema Cardiovascular , Neurônios , Neurônios/metabolismo , Proteínas de Transporte/metabolismo , Proteínas Interatuantes com Canais de Kv/genética , Proteínas Interatuantes com Canais de Kv/metabolismo , Membrana Celular/metabolismo , Sistema Cardiovascular/metabolismoRESUMO
The transient outward potassium current (Itof) is generated by the activation of KV4 channels assembled with KChIP2 and other accessory subunits (DPP6 and KCNE2). To test the hypothesis that these subunits modify the channel pharmacology, we analyzed the electrophysiological effects of (3-(2-(3-phenoxyphenyl)acetamido)-2-naphthoic acid) (IQM-266), a new KChIP2 ligand, on the currents generated by KV4.3/KChIP2, KV4.3/KChIP2/DPP6 and KV4.3/KChIP2/KCNE2 channels. CHO cells were transiently transfected with cDNAs codifying for different proteins (KV4.3/KChIP2, KV4.3/KChIP2/DPP6 or KV4.3/KChIP2/KCNE2), and the potassium currents were recorded using the whole-cell patch-clamp technique. IQM-266 decreased the maximum peak of KV4.3/KChIP2, KV4.3/KChIP2/DPP6 and KV4.3/KChIP2/KCNE2 currents, slowing their time course of inactivation in a concentration-, voltage-, time- and use-dependent manner. IQM-266 produced an increase in the charge in KV4.3/KChIP2 channels that was intensified when DPP6 was present and abolished in the presence of KCNE2. IQM-266 induced an activation unblocking effect during the application of trains of pulses to cells expressing KV4.3/KChIP2 and KV4.3/KChIP2/KCNE2, but not in KV4.3/KChIP2/DPP6 channels. Overall, all these results are consistent with a preferential IQM-266 binding to an active closed state of Kv4.3/KChIP2 and Kv4.3/KChIP2/KCNE2 channels, whereas in the presence of DPP6, IQM-266 binds preferentially to an inactivated state. In conclusion, DPP6 and KCNE2 modify the pharmacological response of KV4.3/KChIP2 channels to IQM-266.
Assuntos
Proteínas Interatuantes com Canais de Kv , Canais de Potássio Shal , Animais , Cricetinae , Cricetulus , Proteínas Interatuantes com Canais de Kv/genética , Proteínas Interatuantes com Canais de Kv/metabolismo , Técnicas de Patch-Clamp , Potássio/metabolismo , Canais de Potássio Shal/genética , Canais de Potássio Shal/metabolismoRESUMO
Sympathetic regulation of the Kv4.2 transient outward potassium current (Ito) is critical for the acute electrical and contractile response of the myocardium under physiological and pathological conditions. Previous studies have suggested that KChIP2, the key auxiliary subunit of Kv4 channels, is required for the sympathetic regulation of Kv4.2 current densities. Of interest, Kv4.2 and KChIP2, and key components mediating acute sympathetic signaling transduction are present in lipid rafts, which are profoundly involved in regulation of Ito densities in rat ventricular myocytes. However, little is known about the mechanisms of Kv4.2-raft association and its connection with acute sympathetic regulation. With the aid of high-resolution fluorescent microscope, we demonstrated that KChIP2 assisted Kv4.2 localization in lipid rafts in HEK293 cells. Moreover, PKA-mediated Kv4.2 phosphorylation, the downstream signaling event of acute sympathetic stimulation, induced dissociation between Kv4.2 and KChIP2, resulting in Kv4.2 shifting out of lipid rafts in KChIP2-expressed HEK293. The mutation that mimics Kv4.2 phosphorylation by PKA (K4.2-S552D) similarly disrupted Kv4.2 interaction with KChIP2 and also decreased the surface stability of Kv4.2. The attenuated Kv4.2-KChIP2 interaction was also observed in native neonatal rat ventricular myocytes (NRVMs) upon acute adrenergic stimulation with phenylephrine (PE). Furthermore, PE stimulation decreased Kv4.2 location at lipid rafts and induced internalization of Kv4.2 as well as the effect of lipid rafts disruption. In conclusion, KChIP2 contributes to targeting Kv4.2 to lipid rafts. Acute adrenergic stimulation induces Kv4.2-KChIP2 dissociation, leading to Kv4.2 out of lipid rafts and internalization, reinforcing the critical role of Kv4.2-lipid raft association in the essential physiological response of Ito to acute sympathetic regulation.
Assuntos
Proteínas Interatuantes com Canais de Kv , Canais de Potássio Shal , Adrenérgicos , Animais , Células HEK293 , Humanos , Proteínas Interatuantes com Canais de Kv/genética , Proteínas Interatuantes com Canais de Kv/metabolismo , Microdomínios da Membrana/metabolismo , Fosforilação , Ratos , Canais de Potássio Shal/genética , Canais de Potássio Shal/metabolismoRESUMO
Fifteen percent of colorectal cancer (CRC) cells exhibit a mucin hypersecretory phenotype, which is suggested to provide resistance to immune surveillance and chemotherapy. We now formally show that CRC cells build a barrier to chemotherapeutics by increasing mucins' secretion. We show that low levels of KChIP3, a negative regulator of mucin secretion (Cantero-Recasens et al., 2018), is a risk factor for CRC patients' relapse in a subset of untreated tumours. Our results also reveal that cells depleted of KChIP3 are four times more resistant (measured as cell viability and DNA damage) to chemotherapeutics 5-fluorouracil + irinotecan (5-FU+iri.) compared to control cells, whereas KChIP3-overexpressing cells are 10 times more sensitive to killing by chemotherapeutics. A similar increase in tumour cell death is observed upon chemical inhibition of mucin secretion by the sodium/calcium exchanger (NCX) blockers (Mitrovic et al., 2013). Finally, sensitivity of CRC patient-derived organoids to 5-FU+iri. increases 40-fold upon mucin secretion inhibition. Reducing mucin secretion thus provides a means to control chemoresistance of mucinous CRC cells and other mucinous tumours.
Assuntos
Neoplasias Colorretais/fisiopatologia , Resistencia a Medicamentos Antineoplásicos/fisiologia , Mucinas/fisiologia , Antimetabólitos Antineoplásicos/farmacologia , Fluoruracila/farmacologia , Regulação Neoplásica da Expressão Gênica , Células HT29 , Humanos , Irinotecano/farmacologia , Proteínas Interatuantes com Canais de Kv/genética , Mucina-5AC/genética , Mucina-5AC/metabolismo , Mucina-1 , Mucinas/biossíntese , Mucinas/genética , Recidiva Local de Neoplasia , Proteínas Repressoras/genética , Fatores de RiscoRESUMO
PURPOSE: Papillary thyroid carcinoma (PTC) is the common endocrine malignancy. Kv channel interacting protein 3 (KCNIP3) has been investigated in a variety of diseases, but its role and underlying mechanism in PTC are not fully delineated. Based on this, this study mainly explored the possible mechanism of KCNIP3 in PTC. METHODS: KCNIP3 expression in PTC tissues was analyzed by ENCORI and validated by quantitative real-time PCR (qRT-PCR). KCNIP3 overexpression (oe-KCNIP3) or KCNIP3 silence (si-KCNIP3) was transfected into IHH4 and FTC-133 cells, respectively. Then cell biological behaviors were detected by cell function assays. The expressions of epithelial-mesenchymal transition (EMT)- and Wnt/ß-catenin pathway-related proteins were quantified by qRT-PCR and western blot. Lastly, IHH4 cells were treated with LiCl and the above assays were performed again. RESULTS: The expression of KCNIP3 was decreased in PTC. After transfection, oe-KCNIP3 inhibited the PTC cell viability, cloning, migration and invasion but promoted apoptosis, and meanwhile, oe-KCNIP3 reduced the EMT and Wnt pathway activation. In contrast, si-KCNIP3 had the opposite effect. Moreover, LiCl, a Wnt signaling pathway activator, could reverse the above effects of oe-KCNIP3. CONCLUSION: KCNIP3 might play an anticarcinogenic role in PTC via inhibiting the activation of Wnt/ß-catenin signaling pathway.
Assuntos
Proteínas Interatuantes com Canais de Kv/genética , Proteínas Repressoras/genética , Neoplasias da Glândula Tireoide , Via de Sinalização Wnt , Linhagem Celular Tumoral , Movimento Celular , Proliferação de Células , Transição Epitelial-Mesenquimal/genética , Regulação Neoplásica da Expressão Gênica , Humanos , Proteínas Interatuantes com Canais de Kv/metabolismo , Proteínas Repressoras/metabolismo , Câncer Papilífero da Tireoide/genética , Câncer Papilífero da Tireoide/metabolismo , Câncer Papilífero da Tireoide/patologia , Neoplasias da Glândula Tireoide/genética , Neoplasias da Glândula Tireoide/metabolismo , Neoplasias da Glândula Tireoide/patologia , Via de Sinalização Wnt/genética , beta Catenina/genética , beta Catenina/metabolismoRESUMO
The interaction between neutrophils and endothelial cells is critical for the pathogenesis of vascular inflammation. However, the regulation of neutrophil adhesive function remains not fully understood. Intravital microscopy demonstrates that neutrophil DREAM promotes neutrophil recruitment to sites of inflammation induced by TNF-α but not MIP-2 or fMLP. We observe that neutrophil DREAM represses expression of A20, a negative regulator of NF-κB activity, and enhances expression of pro-inflammatory molecules and phosphorylation of IκB kinase (IKK) after TNF-α stimulation. Studies using genetic and pharmacologic approaches reveal that DREAM deficiency and IKKß inhibition significantly diminish the ligand-binding activity of ß2 integrins in TNF-α-stimulated neutrophils or neutrophil-like HL-60 cells. Neutrophil DREAM promotes degranulation through IKKß-mediated SNAP-23 phosphorylation. Using sickle cell disease mice lacking DREAM, we show that hematopoietic DREAM promotes vaso-occlusive events in microvessels following TNF-α challenge. Our study provides evidence that targeting DREAM might be a novel therapeutic strategy to reduce excessive neutrophil recruitment in inflammatory diseases.
Assuntos
Inflamação/genética , Proteínas Interatuantes com Canais de Kv/genética , Microvasos/metabolismo , Infiltração de Neutrófilos/genética , Neutrófilos/metabolismo , Proteínas Repressoras/genética , Animais , Adesão Celular/efeitos dos fármacos , Regulação da Expressão Gênica , Células HL-60 , Humanos , Quinase I-kappa B/metabolismo , Inflamação/metabolismo , Proteínas Interatuantes com Canais de Kv/metabolismo , Camundongos Endogâmicos C57BL , Camundongos Knockout , Microvasos/patologia , NF-kappa B/metabolismo , Infiltração de Neutrófilos/efeitos dos fármacos , Neutrófilos/efeitos dos fármacos , Fosforilação/efeitos dos fármacos , Proteínas Repressoras/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Proteína 3 Induzida por Fator de Necrose Tumoral alfa/genética , Proteína 3 Induzida por Fator de Necrose Tumoral alfa/metabolismo , Fator de Necrose Tumoral alfa/farmacologiaRESUMO
Cellular senescence is a stable cell cycle arrest that normal cells undergo after a finite number of divisions, in response to a variety of intrinsic and extrinsic stimuli. Although senescence is largely established and maintained by the p53/p21WAF1/CIP1 and pRB/p16INK4A tumour suppressor pathways, the downstream targets responsible for the stability of the growth arrest are not known. We have employed a stable senescence bypass assay in conditionally immortalised human breast fibroblasts (CL3EcoR) to investigate the role of the DREAM complex and its associated components in senescence. DREAM is a multi-subunit complex comprised of the MuvB core, containing LIN9, LIN37, LIN52, LIN54, and RBBP4, that when bound to p130, an RB1 like protein, and E2F4 inhibits cell cycle-dependent gene expression thereby arresting cell division. Phosphorylation of LIN52 at Serine 28 is required for DREAM assembly. Re-entry into the cell cycle upon phosphorylation of p130 leads to disruption of the DREAM complex and the MuvB core, associating initially to B-MYB and later to FOXM1 to form MMB and MMB-FOXM1 complexes respectively. Here we report that simultaneous expression of MMB-FOXM1 complex components efficiently bypasses senescence with LIN52, B-MYB, and FOXM1 as the crucial components. Moreover, bypass of senescence requires non-phosphorylated LIN52 that disrupts the DREAM complex, thereby indicating a central role for assembly of the DREAM complex in senescence.
Assuntos
Mama/metabolismo , Proteínas de Ciclo Celular/metabolismo , Senescência Celular , Fibroblastos/metabolismo , Proteína Forkhead Box M1/metabolismo , Regulação da Expressão Gênica , Complexos Multiproteicos/metabolismo , Transativadores/metabolismo , Mama/citologia , Proteínas de Ciclo Celular/genética , Inibidor de Quinase Dependente de Ciclina p21/genética , Inibidor de Quinase Dependente de Ciclina p21/metabolismo , Fatores de Transcrição E2F/genética , Fatores de Transcrição E2F/metabolismo , Feminino , Fibroblastos/citologia , Proteína Forkhead Box M1/genética , Humanos , Proteínas Interatuantes com Canais de Kv/genética , Proteínas Interatuantes com Canais de Kv/metabolismo , Complexos Multiproteicos/genética , Fosforilação , Proteínas Repressoras/genética , Proteínas Repressoras/metabolismo , Proteínas de Ligação a Retinoblastoma/genética , Proteínas de Ligação a Retinoblastoma/metabolismo , Transativadores/genética , Proteína Supressora de Tumor p53/genética , Proteína Supressora de Tumor p53/metabolismo , Ubiquitina-Proteína Ligases/genética , Ubiquitina-Proteína Ligases/metabolismo , Proteínas de Sinalização YAP/genética , Proteínas de Sinalização YAP/metabolismoRESUMO
Ruxolitinib (RUX), a JAK1/2-inhibitor, is effective for myeloproliferative neoplasm (MPN) with both JAK2V617 F and calreticulin (CALR) mutations. However, many MPN patients develop resistance to RUX. Although mechanisms of RUX-resistance in cells with JAK2V617 F have already been characterized, those in cells with CALR mutations remain to be elucidated. In this study, we established RUX-resistant human cell lines with CALR mutations and characterized mechanisms of RUX-resistance. Here, we found that RUX-resistant cells had high levels of MPL transcripts, overexpression of both MPL and JAK2, and increased phosphorylation of JAK2 and STAT5. We also found that mature MPL proteins were more stable in RUX-resistant cells. Knockdown of MPL in RUX-resistant cells by shRNAs decreased JAK/STAT signaling. Immunoprecipitation assays showed that binding of mutant CALR to MPL was increased in RUX-resistant cells. Reduction of mutated CALR decreased proliferation of the resistant cells. When resistant cells were cultured in the absence of RUX, the RUX-resistance was reversed, with reduction of the mutant-CALR/MPL complex. In conclusion, MPL overexpression induces higher levels of a mutant-CALR/MPL complex, which may cause RUX-resistance in cells with CALR mutations. This mechanism may be a new therapeutic target to overcome RUX-resistance.
Assuntos
Regulação da Expressão Gênica , Proteínas Interatuantes com Canais de Kv/genética , Proteínas Interatuantes com Canais de Kv/metabolismo , Mutação , Transtornos Mieloproliferativos/genética , Receptores de Trombopoetina/genética , Receptores de Trombopoetina/metabolismo , Animais , Calreticulina , Linhagem Celular Tumoral , Análise Mutacional de DNA , Gerenciamento Clínico , Modelos Animais de Doenças , Suscetibilidade a Doenças , Resistencia a Medicamentos Antineoplásicos , Humanos , Imuno-Histoquímica , Janus Quinase 2/genética , Megacariócitos/metabolismo , Camundongos , Transtornos Mieloproliferativos/diagnóstico , Transtornos Mieloproliferativos/tratamento farmacológico , Nitrilas , Mielofibrose Primária/genética , Mielofibrose Primária/metabolismo , Ligação Proteica , Pirazóis/farmacologia , Pirazóis/uso terapêutico , PirimidinasRESUMO
OBJECTIVE: To determine if long interspersed element-1 (L1) retrotransposons convey risk for idiopathic temporal lobe epilepsy (TLE). METHODS: Surgically resected temporal cortex from individuals with TLE (N = 33) and postmortem temporal cortex from individuals with no known neurological disease (N = 33) were analyzed for L1 content by Restriction Enzyme Based Enriched L1Hs sequencing (REBELseq). Expression of three KCNIP4 splice variants was assessed by droplet digital PCR (ddPCR). Protein ANalysis THrough Evolutionary Relationships (PANTHER) was used to determine ontologies and pathways for lists of genes harboring L1 insertions. RESULTS: We identified novel L1 insertions specific to individuals with TLE, and others specific to controls. Although there were no statistically significant differences between cases and controls in the numbers of known and novel L1 insertions, PANTHER analyses of intragenic L1 insertions showed statistically significant enrichments for epilepsy-relevant gene ontologies in both cases and controls. Gene ontologies "neuron projection development" and "calcium ion transmembrane transport" were among those found only in individuals with TLE. We confirmed novel L1 insertions in several genes associated with seizures/epilepsy, including a de novo somatic L1 retrotransposition in KCNIP4 that occurred after neural crest formation in one patient. However, ddPCR results suggest this de novo L1 did not alter KCNIP4 mRNA expression. SIGNIFICANCE: Given current data from this small cohort, we conclude that L1 elements, either rare heritable germline insertions or de novo somatic retrotranspositions, may contribute only minimally to overall genetic risk for idiopathic TLE. We suggest that further studies in additional patients and additional brain regions are warranted.
Assuntos
Elementos de DNA Transponíveis/genética , Epilepsia do Lobo Temporal/genética , Elementos Nucleotídeos Longos e Dispersos/genética , Adulto , Cálcio/metabolismo , Biologia Computacional , Eletroencefalografia , Epilepsia do Lobo Temporal/epidemiologia , Feminino , Humanos , Proteínas Interatuantes com Canais de Kv/genética , Imageamento por Ressonância Magnética , Masculino , Pessoa de Meia-Idade , Neurônios/patologia , Valores de Referência , Fatores de Risco , Lobo Temporal/químicaRESUMO
Animals respond to visual threats, such as a looming object, with innate defensive behaviors. Here, we report that a specific type of retinal ganglion cell (RGC), the OFF-transient alpha RGC, is critical for the detection of looming objects. We identified Kcnip2 as its molecular marker. The activity of the Kcnip2-expressing RGCs encodes the size of the looming object. Ablation or suppression of these RGCs abolished or severely impaired the escape and freezing behaviors of mice in response to a looming object, while activation of their somas in the retina, or their axon terminals in the superior colliculus, triggered immediate escape behavior. Our results link the activity of a single type of RGC to visually triggered innate defensive behaviors and underscore that ethologically significant visual information is encoded by a labeled line strategy as early as in the retina.
Assuntos
Reação de Fuga/fisiologia , Medo/fisiologia , Reação de Congelamento Cataléptica/fisiologia , Células Ganglionares da Retina/fisiologia , Percepção de Tamanho/fisiologia , Percepção Visual/fisiologia , Animais , Feminino , Proteínas Interatuantes com Canais de Kv/genética , Proteínas Interatuantes com Canais de Kv/metabolismo , Masculino , Camundongos , Optogenética , Células Ganglionares da Retina/metabolismo , Colículos Superiores/citologia , Colículos Superiores/fisiologiaRESUMO
Ion channels are macromolecular complexes present in the plasma membrane and intracellular organelles of cells. Dysfunction of ion channels results in a group of disorders named channelopathies, which represent an extraordinary challenge for study and treatment. In this review, we will focus on voltage-gated potassium channels (KV), specifically on the KV4-family. The activation of these channels generates outward currents operating at subthreshold membrane potentials as recorded from myocardial cells (ITO, transient outward current) and from the somata of hippocampal neurons (ISA). In the heart, KV4 dysfunctions are related to Brugada syndrome, atrial fibrillation, hypertrophy, and heart failure. In hippocampus, KV4.x channelopathies are linked to schizophrenia, epilepsy, and Alzheimer's disease. KV4.x channels need to assemble with other accessory subunits (ß) to fully reproduce the ITO and ISA currents. ß Subunits affect channel gating and/or the traffic to the plasma membrane, and their dysfunctions may influence channel pharmacology. Among KV4 regulatory subunits, this review aims to analyze the KV4/KChIPs interaction and the effect of small molecule KChIP ligands in the A-type currents generated by the modulation of the KV4/KChIP channel complex. Knowledge gained from structural and functional studies using activators or inhibitors of the potassium current mediated by KV4/KChIPs will better help understand the underlying mechanism involving KV4-mediated-channelopathies, establishing the foundations for drug discovery, and hence their treatments.
Assuntos
Doença de Alzheimer/fisiopatologia , Canalopatias/fisiopatologia , Epilepsia/fisiopatologia , Proteínas Interatuantes com Canais de Kv/farmacologia , Canais de Potássio de Abertura Dependente da Tensão da Membrana/farmacologia , Esquizofrenia/fisiopatologia , Canais de Potássio Shal/farmacologia , Doença de Alzheimer/etiologia , Sequência de Aminoácidos , Canalopatias/complicações , Epilepsia/etiologia , Coração/fisiopatologia , Hipocampo/metabolismo , Hipocampo/fisiopatologia , Humanos , Proteínas Interatuantes com Canais de Kv/genética , Proteínas Interatuantes com Canais de Kv/metabolismo , Potenciais da Membrana , Modelos Moleculares , Neurônios/metabolismo , Canais de Potássio de Abertura Dependente da Tensão da Membrana/genética , Canais de Potássio de Abertura Dependente da Tensão da Membrana/metabolismo , Esquizofrenia/etiologia , Alinhamento de Sequência , Canais de Potássio Shal/genética , Canais de Potássio Shal/metabolismoRESUMO
The DREAM complex orchestrates cell quiescence and the cell cycle. However, how the DREAM complex is deregulated in cancer remains elusive. Here, we report that PAF (PCLAF/KIAA0101) drives cell quiescence exit to promote lung tumorigenesis by remodeling the DREAM complex. PAF is highly expressed in lung adenocarcinoma (LUAD) and is associated with poor prognosis. Importantly, Paf knockout markedly suppressed LUAD development in mouse models. PAF depletion induced LUAD cell quiescence and growth arrest. PAF is required for the global expression of cell-cycle genes controlled by the repressive DREAM complex. Mechanistically, PAF inhibits DREAM complex formation by binding to RBBP4, a core DREAM subunit, leading to transactivation of DREAM target genes. Furthermore, pharmacological mimicking of PAF-depleted transcriptomes inhibited LUAD tumor growth. Our results unveil how the PAF-remodeled DREAM complex bypasses cell quiescence to promote lung tumorigenesis and suggest that the PAF-DREAM axis may be a therapeutic vulnerability in lung cancer.
Assuntos
Carcinogênese/genética , Proteínas de Ligação a DNA/genética , Proteínas Interatuantes com Canais de Kv/genética , Neoplasias Pulmonares/genética , Pulmão/patologia , Proteínas Repressoras/genética , Células A549 , Adenocarcinoma de Pulmão/genética , Adenocarcinoma de Pulmão/patologia , Animais , Carcinogênese/patologia , Divisão Celular/genética , Linhagem Celular , Linhagem Celular Tumoral , Proliferação de Células/genética , Feminino , Humanos , Neoplasias Pulmonares/patologia , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Knockout , Camundongos Nus , Células NIH 3T3 , Ativação Transcricional/genética , Transcriptoma/genéticaRESUMO
The pharmacological inhibition of glial activation is one of the new approaches for combating neuropathic pain in which the role of glia in the modulation of neuropathic pain has attracted significant interest and attention. Neuron-glial crosstalk is achieved with N-methyl-D-aspartate-2B receptor (NMDAR-2B) activation. This study aims to determine the effect of ifenprodil, a potent noncompetitive NMDAR-2B antagonist, on activated microglia, brain-derived neurotrophic factors (BDNF) and downstream regulatory element antagonist modulator (DREAM) protein expression in the spinal cord of streptozotocin-induced painful diabetic neuropathy (PDN) rats following formalin injection. In this experimentation, 48 Sprague-Dawley male rats were randomly selected and divided into four groups: (n = 12): control, PDN, and ifenprodil-treated PDN rats at 0.5 µg or 1.0 µg for 7 days. Type I diabetes mellitus was then induced by injecting streptozotocin (60 mg/kg, i.p.) into the rats which were then over a 2-week period allowed to progress into the early phase of PDN. Ifenprodil was administered in PDN rats while saline was administered intrathecally in the control group. A formalin test was conducted during the fourth week to induce inflammatory nerve injury, in which the rats were sacrificed at 72 h post-formalin injection. The lumbar enlargement region (L4-L5) of the spinal cord was dissected for immunohistochemistry and western blot analyses. The results demonstrated a significant increase in formalin-induced flinching and licking behavior with an increased spinal expression of activated microglia, BDNF and DREAM proteins. It was also shown that the ifenprodil-treated rats following both doses reduced the extent of their flinching and duration of licking in PDN in a dose-dependent manner. As such, ifenprodil successfully demonstrated inhibition against microglia activation and suppressed the expression of BDNF and DREAM proteins in the spinal cord of PDN rats. In conclusion, ifenprodil may alleviate PDN by suppressing spinal microglia activation, BDNF and DREAM proteins.
Assuntos
Fator Neurotrófico Derivado do Encéfalo/biossíntese , Neuropatias Diabéticas/tratamento farmacológico , Proteínas Interatuantes com Canais de Kv/biossíntese , Microglia/efeitos dos fármacos , Proteínas do Tecido Nervoso/biossíntese , Neuralgia/tratamento farmacológico , Piperidinas/uso terapêutico , Proteínas Repressoras/biossíntese , Medula Espinal/efeitos dos fármacos , Animais , Fator Neurotrófico Derivado do Encéfalo/genética , Neuropatias Diabéticas/metabolismo , Formaldeído/toxicidade , Proteínas Interatuantes com Canais de Kv/genética , Masculino , Proteínas do Tecido Nervoso/genética , Neuralgia/metabolismo , Piperidinas/farmacologia , Distribuição Aleatória , Ratos , Ratos Sprague-Dawley , Receptores de N-Metil-D-Aspartato/antagonistas & inibidores , Proteínas Repressoras/genética , Medula Espinal/metabolismoRESUMO
Clinical reports suggest a high incidence of ICU mortality with the use of hyperoxia during mechanical ventilation in patients. Our laboratory is pioneer in studying effect of hyperoxia on cardiac pathophysiology. In this study for the first time, we are reporting the sequence of cardiac pathophysiological events in mice under hyperoxic conditions in time-dependent manner. C57BL/6J male mice, aged 8-10 weeks, were treated with either normal air or >90% oxygen for 24, 48, and 72 h. Following normal air or hyperoxia treatment, physical, biochemical, functional, electrical, and molecular parameters were analyzed. Our data showed that significant reduction of body weight observed as early as 24 h hyperoxia treatment, whereas, no significant changes in heart weight until 72 h. Although we do not see any fibrosis in these hearts, but observed significant increase in cardiomyocyte size with hyperoxia treatment in time-dependent manner. Our data also demonstrated that arrhythmias were present in mice at 24 h hyperoxia, and worsened comparatively after 48 and 72 h. Echocardiogram data confirmed cardiac dysfunction in time-dependent manner. Dysregulation of ion channels such as Kv4.2 and KChIP2; and serum cardiac markers confirmed that hyperoxia-induced effects worsen with each time point. From these observations, it is evident that electrical remodeling precedes structural remodeling, both of which gets worse with length of hyperoxia exposure, therefore shorter periods of hyperoxia exposure is always beneficial for better outcome in ICU/critical care units.
Assuntos
Arritmias Cardíacas/etiologia , Cardiomegalia/etiologia , Sistema de Condução Cardíaco/fisiopatologia , Frequência Cardíaca , Hiperóxia/complicações , Miócitos Cardíacos/patologia , Função Ventricular Esquerda , Remodelação Ventricular , Potenciais de Ação , Animais , Arritmias Cardíacas/metabolismo , Arritmias Cardíacas/patologia , Arritmias Cardíacas/fisiopatologia , Cardiomegalia/metabolismo , Cardiomegalia/patologia , Cardiomegalia/fisiopatologia , Cardiotoxicidade , Tamanho Celular , Modelos Animais de Doenças , Regulação da Expressão Gênica , Sistema de Condução Cardíaco/metabolismo , Proteínas Interatuantes com Canais de Kv/genética , Proteínas Interatuantes com Canais de Kv/metabolismo , L-Lactato Desidrogenase/sangue , Masculino , Camundongos Endogâmicos C57BL , Miócitos Cardíacos/metabolismo , Canais de Potássio Shal/genética , Canais de Potássio Shal/metabolismo , Fatores de Tempo , Troponina I/sangueRESUMO
Transcriptional regulation of DNA repair is of outmost importance for the restoration of DNA integrity upon genotoxic stress. Here we report that the potent environmental carcinogen benzo[a]pyrene (B[a]P) activates a cellular DNA damage response resulting in transcriptional repression of mismatch repair (MMR) genes (MSH2, MSH6, EXO1) and of RAD51, the central homologous recombination repair (HR) component, ultimately leading to downregulation of MMR and HR. B[a]P-induced gene repression is caused by abrogated E2F1 signalling. This occurs through proteasomal degradation of E2F1 in G2-arrested cells and downregulation of E2F1 mRNA expression in G1-arrested cells. Repression of E2F1-mediated transcription and silencing of repair genes is further mediated by the p21-dependent E2F4/DREAM complex. Notably, repression of DNA repair is also observed following exposure to the active B[a]P metabolite BPDE and upon ionizing radiation and occurs in response to a p53/p21-triggered, irreversible cell cycle arrest marking the onset of cellular senescence. Overall, our results suggest that repression of MMR and HR is an early event during genotoxic-stress induced senescence. We propose that persistent downregulation of DNA repair might play a role in the maintenance of the senescence phenotype, which is associated with an accumulation of unrepairable DNA lesions.
Assuntos
Benzo(a)pireno/toxicidade , Carcinógenos/toxicidade , Senescência Celular/genética , DNA/genética , Fator de Transcrição E2F1/genética , Fator de Transcrição E2F4/genética , Pontos de Checagem do Ciclo Celular , Linhagem Celular Transformada , Linhagem Celular Tumoral , Inibidor de Quinase Dependente de Ciclina p21/genética , Inibidor de Quinase Dependente de Ciclina p21/metabolismo , DNA/metabolismo , Dano ao DNA , Reparo de Erro de Pareamento de DNA/efeitos dos fármacos , Reparo de Erro de Pareamento de DNA/efeitos da radiação , Enzimas Reparadoras do DNA/genética , Enzimas Reparadoras do DNA/metabolismo , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Fator de Transcrição E2F1/metabolismo , Fator de Transcrição E2F4/metabolismo , Células Epiteliais/citologia , Células Epiteliais/efeitos dos fármacos , Células Epiteliais/metabolismo , Células Epiteliais/efeitos da radiação , Exodesoxirribonucleases/genética , Exodesoxirribonucleases/metabolismo , Fibroblastos/citologia , Fibroblastos/efeitos dos fármacos , Fibroblastos/metabolismo , Fibroblastos/efeitos da radiação , Raios gama , Humanos , Proteínas Interatuantes com Canais de Kv/genética , Proteínas Interatuantes com Canais de Kv/metabolismo , Células MCF-7 , Proteína 2 Homóloga a MutS/genética , Proteína 2 Homóloga a MutS/metabolismo , Rad51 Recombinase/genética , Rad51 Recombinase/metabolismo , Reparo de DNA por Recombinação/efeitos dos fármacos , Reparo de DNA por Recombinação/efeitos da radiação , Proteínas Repressoras/genética , Proteínas Repressoras/metabolismo , Transdução de SinaisRESUMO
BACKGROUND: Soluble epoxide hydrolase inhibitors (sEHi) have anti-arrhythmic effects, and we previously found that the novel sEHi t-AUCB (trans-4[-4-(3-adamantan-1-yl-ureido)-cyclohexyloxy]-benzoic acid) significantly inhibited ventricular arrhythmias after myocardial infarction (MI). However, the mechanism is unknown. It's known that microRNA-29 (miR-29) participates in the occurrence of arrhythmias. In this study, we investigated whether sEHi t-AUCB was protective against ischemic arrhythmias by modulating miR-29 and its target genes KCNJ12 and KCNIP2. METHODS: Male 8-week-old C57BL/6 mice were divided into five groups and fed distilled water only or distilled water with t-AUCB of different dosages for seven days. Then, the mice underwent MI or sham surgery. The ischemic region of the myocardium was obtained 24 hours after MI to detect miR-29, KCNJ12, and KCNIP2 mRNA expression levels via real-time PCR and KCNJ12 and KCNIP2 protein expression levels via western blotting. RESULTS: MiR-29 expression levels were significantly increased in the ischemic region of MI mouse hearts and the mRNA and protein expression levels of its target genes KCNJ12 and KCNIP2 were significantly decreased. T-AUCB prevented these changes dose-dependently. CONCLUSION: The sEHi t-AUCB regulates the expression levels of miR-29 and its target genes KCNJ12 and KCNIP2, suggesting a possible mechanism for its potential therapeutic application in ischemic arrhythmia.
Assuntos
Regulação da Expressão Gênica , Proteínas Interatuantes com Canais de Kv/genética , MicroRNAs/genética , Infarto do Miocárdio/genética , Miocárdio/metabolismo , Canais de Potássio Corretores do Fluxo de Internalização/genética , Animais , Western Blotting , Modelos Animais de Doenças , Regulação para Baixo , Proteínas Interatuantes com Canais de Kv/biossíntese , Masculino , Camundongos , Camundongos Endogâmicos C57BL , MicroRNAs/biossíntese , Infarto do Miocárdio/metabolismo , Infarto do Miocárdio/patologia , Miocárdio/patologia , Canais de Potássio Corretores do Fluxo de Internalização/biossíntese , RNA/genética , RNA/metabolismoRESUMO
Voltage-gated potassium (Kv) channels of the Kv4 subfamily associate with Kv channel-interacting proteins (KChIPs), which leads to enhanced surface expression and shapes the inactivation gating of these channels. KChIP3 has been reported to also interact with the late endosomal/lysosomal membrane glycoprotein CLN3 (ceroid lipofuscinosis neuronal 3), which is modified because of gene mutation in juvenile neuronal ceroid lipofuscinosis (JNCL). The present study was undertaken to find out whether and how CLN3, by its interaction with KChIP3, may indirectly modulate Kv4.2 channel expression and function. To this end, we expressed KChIP3 and CLN3, either individually or simultaneously, together with Kv4.2 in HEK 293 cells. We performed co-immunoprecipitation experiments and found a lower amount of KChIP3 bound to Kv4.2 in the presence of CLN3. In whole-cell patch-clamp experiments, we examined the effects of CLN3 co-expression on the KChIP3-mediated modulation of Kv4.2 channels. Simultaneous co-expression of CLN3 and KChIP3 with Kv4.2 resulted in a suppression of the typical KChIP3-mediated modulation; i.e. we observed less increase in current density, less slowing of macroscopic current decay, less acceleration of recovery from inactivation, and a less positively shifted voltage dependence of steady-state inactivation. The suppression of the KChIP3-mediated modulation of Kv4.2 channels was weaker for the JNCL-related missense mutant CLN3R334C and for a JNCL-related C-terminal deletion mutant (CLN3ΔC). Our data support the notion that CLN3 is involved in Kv4.2/KChIP3 somatodendritic A-type channel formation, trafficking, and function, a feature that may be lost in JNCL.
Assuntos
Regulação da Expressão Gênica/genética , Proteínas Interatuantes com Canais de Kv , Glicoproteínas de Membrana , Chaperonas Moleculares , Mutação de Sentido Incorreto , Lipofuscinoses Ceroides Neuronais , Proteínas Repressoras , Canais de Potássio Shal , Substituição de Aminoácidos , Células HEK293 , Humanos , Proteínas Interatuantes com Canais de Kv/genética , Proteínas Interatuantes com Canais de Kv/metabolismo , Lipofuscinoses Ceroides Neuronais/genética , Lipofuscinoses Ceroides Neuronais/metabolismo , Lipofuscinoses Ceroides Neuronais/patologia , Ligação Proteica , Proteínas Repressoras/genética , Proteínas Repressoras/metabolismo , Canais de Potássio Shal/biossíntese , Canais de Potássio Shal/genéticaRESUMO
A form of hereditary cerebellar ataxia has recently been described in the Norwegian Buhund dog breed. This study aimed to identify the genetic cause of the disease. Whole-genome sequencing of two Norwegian Buhund siblings diagnosed with progressive cerebellar ataxia was carried out, and sequences compared with 405 whole genome sequences of dogs of other breeds to filter benign common variants. Nine variants predicted to be deleterious segregated among the genomes in concordance with an autosomal recessive mode of inheritance, only one of which segregated within the breed when genotyped in additional Norwegian Buhunds. In total this variant was assessed in 802 whole genome sequences, and genotyped in an additional 505 unaffected dogs (including 146 Buhunds), and only four affected Norwegian Buhunds were homozygous for the variant. The variant identified, a T to C single nucleotide polymorphism (SNP) (NC_006585.3:g.88890674T>C), is predicted to cause a tryptophan to arginine substitution in a highly conserved region of the potassium voltage-gated channel interacting protein KCNIP4. This gene has not been implicated previously in hereditary ataxia in any species. Evaluation of KCNIP4 protein expression through western blot and immunohistochemical analysis using cerebellum tissue of affected and control dogs demonstrated that the mutation causes a dramatic reduction of KCNIP4 protein expression. The expression of alternative KCNIP4 transcripts within the canine cerebellum, and regional differences in KCNIP4 protein expression, were characterised through RT-PCR and immunohistochemistry respectively. The voltage-gated potassium channel protein KCND3 has previously been implicated in spinocerebellar ataxia, and our findings suggest that the Kv4 channel complex KCNIP accessory subunits also have an essential role in voltage-gated potassium channel function in the cerebellum and should be investigated as potential candidate genes for cerebellar ataxia in future studies in other species.