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The pyroptosis of renal tubular epithelial cells leads to tubular loss and inflammation and then promotes renal fibrosis. The transcription factor Krüppel-like factor 4 (KLF4) can bidirectionally regulate the transcription of target genes. Our previous study revealed that sustained elevation of KLF4 is responsible for the transition of acute kidney injury (AKI) into chronic kidney disease (CKD) and renal fibrosis. In this study, we explored the upstream mechanisms of renal tubular epithelial cell pyroptosis from the perspective of posttranslational regulation and focused on the transcription factor KLF4. Mice were subjected to unilateral ureteral obstruction (UUO) surgery and euthanized on D7 or D14 for renal tissue harvesting. We showed that the pyroptosis of renal tubular epithelial cells mediated by both the Caspase-1/GSDMD and Caspase-3/GSDME pathways was time-dependently increased in UUO mouse kidneys. Furthermore, we found that the expression of the transcription factor KLF4 was also upregulated in a time-dependent manner in UUO mouse kidneys. Tubular epithelial cell-specific Klf4 knockout alleviated UUO-induced pyroptosis and renal fibrosis. In Ang II-treated mouse renal proximal tubular epithelial cells (MTECs), we demonstrated that KLF4 bound to the promoter regions of Caspase-3 and Caspase-1 and directly increased their transcription. In addition, we found that ubiquitin-specific protease 11 (USP11) was increased in UUO mouse kidneys. USP11 deubiquitinated KLF4. Knockout of Usp11 or pretreatment with the USP11 inhibitor mitoxantrone (3 mg/kg, i.p., twice a week for two weeks before UUO surgery) significantly alleviated the increases in KLF4 expression, pyroptosis and renal fibrosis. These results demonstrated that the increased expression of USP11 in renal tubular cells prevents the ubiquitin degradation of KLF4 and that elevated KLF4 promotes inflammation and renal fibrosis by initiating tubular cell pyroptosis.
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INTRODUCTION: Renal fibrosis (RF), being the most important pathological change in the progression of CKD, is currently assessed by the evaluation of a biopsy. This present study aimed to apply a novel functional MRI (fMRI) protocol named amide proton transfer (APT) weighting to evaluate RF noninvasively. METHODS: Male Sprague-Dawley (SD) rats were initially subjected to bilateral kidney ischemia/reperfusion injury (IRI), unilateral ureteral obstruction, and sham operation, respectively. All rats underwent APT mapping on the 7th and 14th days after operation. Besides, 26 patients underwent renal biopsy at the Nephrology Department of Shanghai Tongji Hospital between July 2022 and May 2023. Patients underwent APT and apparent diffusion coefficient (ADC) mappings within 1 week before biopsy. MRI results of both patients and rats were calculated by comparing with gold standard histology for fibrosis assessment. RESULTS: In animal models, the cortical APT (cAPT) and medullary APT (mAPT) values were positively correlated with the degree of RF. Compared to the sham group, IRI group showed significantly increased cAPT and mAPT values on the 7th and 14th days after surgery, but no group differences were found in ADC values. Similar results were found in human patients. Cortical/medullary APT values were significantly increased in patients with moderate-to-severe fibrosis than in patients with mild fibrosis. ROC curve analysis indicated that APT value displayed a better diagnostic value for RF. Furthermore, combination of cADC and cAPT improved fibrosis detection by imaging variables alone (p < 0.1). CONCLUSION: APT values had better diagnostic capability at early stage of RF compared to ADC values, and the addition of APT imaging to conventional ADC will significantly improve the diagnostic performance for predicting kidney fibrosis.
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Fibrose , Rim , Imageamento por Ressonância Magnética , Ratos Sprague-Dawley , Masculino , Animais , Fibrose/diagnóstico por imagem , Humanos , Ratos , Pessoa de Meia-Idade , Rim/diagnóstico por imagem , Rim/patologia , Imageamento por Ressonância Magnética/métodos , Traumatismo por Reperfusão/diagnóstico por imagem , Feminino , Adulto , Amidas , Prótons , Nefropatias/diagnóstico por imagem , Nefropatias/patologia , Nefropatias/diagnóstico , Idoso , Insuficiência Renal Crônica/diagnóstico por imagem , Insuficiência Renal Crônica/patologia , Obstrução Ureteral/diagnóstico por imagem , Modelos Animais de DoençasRESUMO
Acute kidney injury (AKI) is a worldwide public health problem characterized by the massive loss of tubular cells. However, the precise mechanism for initiating tubular cell death has not been fully elucidated. Here, we reported that phosphoglycerate mutase 5 (PGAM5) was upregulated in renal tubular epithelial cells during ischaemia/reperfusion or cisplatin-induced AKI in mice. PGAM5 knockout significantly alleviated the activation of the mitochondria-dependent apoptosis pathway and tubular apoptosis. Apoptosis inhibitors alleviated the activation of the mitochondria-dependent apoptosis pathway. Mechanistically, as a protein phosphatase, PGAM5 could dephosphorylate Bax and facilitate Bax translocation to the mitochondrial membrane. The translocation of Bax to mitochondria increased membrane permeability, decreased mitochondrial membrane potential and facilitated the release of mitochondrial cytochrome c (Cyt c) into the cytoplasm. Knockdown of Bax attenuated PGAM5 overexpression-induced Cyt c release and tubular cell apoptosis. Our results demonstrated that the increase in PGAM5-mediated Bax dephosphorylation and mitochondrial translocation was implicated in the development of AKI by initiating mitochondrial Cyt c release and activating the mitochondria-dependent apoptosis pathway. Targeting this axis might be beneficial for alleviating AKI.
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Injúria Renal Aguda , Citocromos c , Camundongos , Animais , Citocromos c/metabolismo , Fosfoglicerato Mutase/metabolismo , Proteína X Associada a bcl-2/metabolismo , Apoptose/fisiologia , Mitocôndrias/metabolismo , Injúria Renal Aguda/induzido quimicamente , Injúria Renal Aguda/metabolismo , Proteínas de Transporte/metabolismo , Fosfoproteínas Fosfatases/metabolismoRESUMO
Transforming growth factor-ß1 (TGF-ß1) is regarded as a key factor in promoting renal fibrosis during chronic kidney disease (CKD). Signaling transduction of TGF-ß1 starts with binding to TGF-ß type II receptor (Tgfbr2), a constitutively activated kinase that phosphorylates TGF-ß type I receptor (Tgfbr1), and then activates downstream Smad2/3 or noncanonical pathways. Previous studies show that cellular senescence is associated with the progression of CKD, and accelerated tubular cell senescence is implicated in promoting renal fibrosis. In the present study we investigated the renal parenchymal cell senescence in fibrosis from the sight of posttranslational regulation and focused on Tgfbr2, the important gatekeeper for TGF-ß1 downstream signaling. In mice with unilateral ureteral obstruction (UUO) and folic acid (FA)-induced fibrotic kidneys, we found that Tgfbr2 was markedly elevated without obvious change in its mRNA levels. As an important member of deubiquitinating enzymes, ubiquitin-specific protease 11 (Usp11) was also significantly increased in fibrotic kidneys, and co-distributed with Tgfbr2 in tubular epithelial cells. Pretreatment with Usp11 inhibitor mitoxantrone (MTX, 30 mg · kg-1 · d-1, i.p.) twice a week, for 2 weeks significantly attenuated the elevation of Tgfbr2, activation in downstream senescence-related signaling pathway, as well as renal senescence and fibrosis. In cultured mouse tubular epithelial cells (MTECs), treatment with angiotensin II (Ang-II, 10-7, 10-6 M) dose-dependently elevated both Tgfbr2 and Usp11 levels. Inhibition or knockdown on Usp11 attenuated Ang-II-induced elevation in Tgfbr2 level, and attenuated the activation of downstream senescent-related signaling pathway and as well as cell senescence. We conducted Co-IP experiments, which revealed that Usp11 was able to interact with Tgfbr2, and inhibition of Usp11 increased the ubiquitination of Tgfbr2. Taken together, these results demonstrate that the elevation of Usp11 under pathological condition is implicated in promoting renal fibrosis. Usp11 promotes the development of renal fibrosis by deubiquitinating Tgfbr2, reducing Tgfbr2 ubiquitination degradation, and then facilitating the activation of downstream senescent signaling pathway.
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Senescência Celular , Enzimas Desubiquitinantes , Insuficiência Renal Crônica , Animais , Camundongos , Senescência Celular/fisiologia , Enzimas Desubiquitinantes/metabolismo , Células Epiteliais/metabolismo , Fibrose/metabolismo , Rim/patologia , Receptor do Fator de Crescimento Transformador beta Tipo II/metabolismo , Insuficiência Renal Crônica/patologia , Fator de Crescimento Transformador beta1/metabolismo , Ubiquitina/metabolismo , Obstrução Ureteral/complicaçõesRESUMO
Background: Disruption of the vascular immunological inflammatory microenvironment is linked to metabolic memory impairment. Even though it has been proven that the Shen-Qi compound (SQC) can efficiently halt metabolic memory and preserve vascular endothelial cells, extensive studies need to be done to investigate if it can also change the vascular immune-inflammatory microenvironment by regulating the immune system. This will help figure out the role of stopping metabolic memory. Methods: After 4 weeks on a high-fat diet (HFD), GK rats were used to create a model for diabetic thoracic aortic problems. The effect and mechanisms of SQC on diabetic thoracic aortic complications were assessed by hematoxylin-eosin (H&E) staining, enzyme-linked immunosorbent assay (ELISA), biochemical analysis, terminal deoxynucleotidyl transferase deoxyuridine triphosphate (dUTP) nick end labeling (TUNEL), reverse transcription, real-time polymerase chain reaction (RT-qPCR), immunofluorescence (IF), western blot, and luciferase reporter assays. Results: SQC treatment ameliorates the HFD-induced pathological symptoms as well as the HFD-induced increased concentrations of fasting blood glucose (FBG), fasting insulin (FINS), total cholesterol (TC), triglycerides (TGs), and low-density lipoprotein cholesterol (LDL-C) and decreased concentrations of high-density lipoprotein cholesterol (HDL-C). Besides, SQC counteracted the HFD-induced average fluorescence intensity of vascular cell adhesion molecule-1 (VCAM-1) and intercellular adhesion molecule-1 (ICAM-1), as well as the concentrations of endothelin-1 (ET-1) and monocyte chemoattractant protein-1 (MCP-1), while rescuing the HFD-induced concentrations of nitric oxide (NO) and nitric oxide synthetase (NOS). Also, SQC decreases apoptosis and oxidative stress in rats with diabetic thoracic aortic complications. In addition, SQC facilitated the polarization of macrophages, stimulated the activation of dendritic cells, and regulated the inflammatory milieu in rats with diabetic thoracic aortic complications. Furthermore, SQC also modulated the miR-223-3p/RBP-J/IRF8 axis in the macrophages of rats with diabetic thoracic aortic complications. Conclusion: SQC ameliorated diabetic thoracic aortic complications through the regulation of apoptosis, oxidative stress, and inflammatory microenvironment mediating by the miR-223-3p/RBP-J/IRF8 axis.
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BACKGROUND: Xia-Gibbs syndrome (XGS, OMIM: 615829), caused by mutations within the AT-Hook DNA-binding motif-containing protein 1 (AHDC1) gene (OMIM: 615790), located on the short arm of chromosome 1 within the cytogenetic band 1p36.11, contains five noncoding 5 exons, a single 4.9-kb coding exon, and a noncoding 3 exon. CASE SUMMARY: In this case report, we diagnosed and treated a 6-mo-old girl with XGS. The primary clinical symptoms included global developmental delay, hypotonia, and mild dysmorphic features. Using high-throughput whole-exosome sequencing to sequence the patient and her parents, and the results showed a novel frameshift mutation of c.1155dupG (p.Arg386Alafs*3) in the AHDC1 gene. The paternal gene was wild type. CONCLUSION: This report extends the mutation spectrum of the AHDC1 gene to provide the diagnostic basis for genetic counseling in families with XGS.
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Ischemia/reperfusion (I/R) injury is a major cause of acute kidney injury (AKI) in clinic. The activation of NLRP3 inflammasome is associated with inflammation and renal injury in I/R-induced AKI. In the current study we explored the molecular and cellular mechanisms for NLRP3 inflammasome activation following renal I/R. Mice were subjected to I/R renal injury by clamping bilateral renal pedicles. We showed that I/R injury markedly increased caspase-11 expression and the cleavage of pannexin 1 (panx1) in the kidneys accompanied by NLRP3 inflammasome activation evidenced by the activation of caspase-1 and interlukin-1ß (IL-1ß) maturation. In Casp-11-/- mice, I/R-induced panx1 cleavage, NLRP3 inflammasome activation as well as renal functional deterioration and tubular morphological changes were significantly attenuated. In cultured primary tubular cells (PTCs) and NRK-52E cells, hypoxia/reoxygenation (H/R) markedly increased caspase-11 expression, NLRP3 inflammasome activation, IL-1ß maturation and panx1 cleavage. Knockdown of caspase-11 attenuated all those changes; similar effects were observed in PTCs isolated from Casp-11-/- mice. In NRK-52E cells, overexpression of caspase-11 promoted panx1 cleavage; pretreatment with panx1 inhibitor carbenoxolone or knockdown of panx1 significantly attenuated H/R-induced intracellular ATP reduction, extracellular ATP elevation and NLRP3 inflammasome activation without apparent influence on H/R-induced caspase-11 increase; pretreatment with P2X7 receptor inhibitor AZD9056 also attenuated NLRP3 inflammasome activation. The above results demonstrate that the cleavage of panx1 by upregulated caspase-11 is involved in facilitating ATP release and then NLRP3 inflammasome activation in I/R-induced AKI. This study provides new insight into the molecular mechanism of NLRP3 inflammasome activation in AKI.
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Injúria Renal Aguda/metabolismo , Caspases Iniciadoras/metabolismo , Conexinas/metabolismo , Inflamassomos/metabolismo , Proteína 3 que Contém Domínio de Pirina da Família NLR/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Traumatismo por Reperfusão/metabolismo , Injúria Renal Aguda/patologia , Animais , Caspases Iniciadoras/deficiência , Células Cultivadas , Relação Dose-Resposta a Droga , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Estrutura Molecular , Traumatismo por Reperfusão/patologia , Relação Estrutura-AtividadeRESUMO
Acute renal injury (AKI) causes a long-term risk for progressing into chronic kidney disease (CKD) and interstitial fibrosis. Yes-associated protein (YAP), a key transcriptional cofactor in Hippo signaling pathway, shuttles between the cytoplasm and nucleus, which is required for the renal tubular epithelial cells repair in the acute phase of AKI. In this study we investigated the role of YAP during ischemia-reperfusion (IR)-induced AKI to CKD. Mice were subjected to left kidney IR followed by removal of the right kidney on the day before tissue harvests. Mouse shRNA expression adenovirus (Ad-shYAP or Ad-shKLF4) and mouse KLF4 expression adenovirus (Ad-KLF4) were delivered to mice by intrarenal injection on D7 after IR. We showed that the expression and nucleus distribution of YAP were persistently increased until the end of experiment (D21 after IR). The sustained activation of YAP in post-acute phase of AKI was accompanied by renal dysfunction and interstitial fibrosis. Knockdown of YAP significantly attenuated IR-induced renal dysfunction and decreased the expression of fibrogenic factors TGF-ß and CTGF in the kidney. We showed that the expression of the transcription factor KLF4, lined on the upstream of YAP, was also persistently increased. Knockdown on KLF4 attenuated YAP increase and nuclear translocation as well as renal functional deterioration and interstitial fibrosis in IR mice, whereas KLF4 overexpression caused opposite effects. KLF4 increased the expression of ITCH, and ITCH facilitated YAP nuclear translocation via degrading LATS1. Furthermore, we demonstrated in primary cultured renal tubular cells that KLF4 bound to the promoter region of YAP and positively regulates YAP expression. In biopsy sample from CKD patients, we also observed increased expression and nuclear distribution of YAP. In conclusion, the activation of YAP in the post-acute phase of AKI is implicated in renal functional deterioration and fibrosis although it exhibits beneficial effect in acute phase. Reprogramming factor KLF4 is responsible for the persistent activation of YAP. Blocking the activation of KLF4-YAP pathway might be a way to prevent the transition of AKI into CKD.
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Injúria Renal Aguda/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Fibrose/metabolismo , Fatores de Transcrição Kruppel-Like/metabolismo , Traumatismo por Reperfusão/metabolismo , Injúria Renal Aguda/etiologia , Animais , Núcleo Celular/metabolismo , Células Cultivadas , Fibrose/etiologia , Fator 4 Semelhante a Kruppel , Masculino , Camundongos Endogâmicos C57BL , Insuficiência Renal Crônica/etiologia , Insuficiência Renal Crônica/metabolismo , Traumatismo por Reperfusão/complicações , Ubiquitina-Proteína Ligases/metabolismo , Regulação para Cima/fisiologia , Proteínas de Sinalização YAPRESUMO
Diabetic nephropathy (DN) is characterized by sterile inflammation with continuous injury and loss of renal inherent parenchyma cells. Podocyte is an essential early injury target in DN. The injury and loss of podocytes are closely associated with proteinuria, the early symptom of renal injury in DN. However, the exact mechanism for podocyte injury and death in DN remains ambiguous. In this study we investigated whether pyroptosis, a newly discovered cell death pathway was involved in DN. Diabetic mice were generated by high-fat diet/STZ injections. We showed that the expression levels of caspase-11 and cleavage of gasdermin D (GSDMD-N) in podocytes were significantly elevated, accompanied by reduced expression of podocyte makers nephrin and podocin, loss and fusion in podocyte foot processes, increased inflammatory cytokines NF-κB, IL-1ß, and IL-18, macrophage infiltration, glomerular matrix expansion and increased urinary albumin to creatinine ratio (UACR). All these changes in diabetic mice were blunted by knockout of caspase-11 or GSDMD. Cultured human and mouse podocytes were treated with high glucose (30 mM), which significantly increased the expression levels of caspase-11 or caspase-4 (the homolog of caspase-11 in human), GSDMD-N, NF-κB, IL-1ß, and IL-18, and decreased the expression of nephrin and podocin. Either caspase-4 or GSDMD knockdown by siRNA significantly blunted these changes. In summary, our results demonstrate that caspase-11/4 and GSDMD-mediated pyroptosis is activated and involved in podocyte loss under hyperglycemia condition and the development of DN.
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Caspases Iniciadoras/metabolismo , Nefropatias Diabéticas/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Proteínas de Ligação a Fosfato/metabolismo , Podócitos/metabolismo , Piroptose/fisiologia , Animais , Caspases Iniciadoras/genética , Células Cultivadas , Diabetes Mellitus Experimental/induzido quimicamente , Diabetes Mellitus Experimental/metabolismo , Diabetes Mellitus Experimental/patologia , Nefropatias Diabéticas/complicações , Nefropatias Diabéticas/patologia , Dieta Hiperlipídica , Técnicas de Inativação de Genes , Glucose/farmacologia , Humanos , Inflamação/etiologia , Inflamação/metabolismo , Inflamação/patologia , Peptídeos e Proteínas de Sinalização Intracelular/genética , Glomérulos Renais/patologia , Macrófagos/metabolismo , Masculino , Camundongos Endogâmicos C57BL , Proteínas de Ligação a Fosfato/genética , Podócitos/efeitos dos fármacos , EstreptozocinaRESUMO
To scientifically evaluate the intervention effect of Chinese medicine preventive administration(combined use of Huo-xiang Zhengqi Oral Liquid and Jinhao Jiere Granules) on community population in the case of coronavirus disease 2019(COVID-19), a large cohort, prospective, randomized, and parallel-controlled clinical study was conducted. Total 22 065 subjects were included and randomly divided into 2 groups. The non-intervention group was given health guidance only, while the traditional Chinese medicine(TCM) intervention group was given two coordinated TCM in addition to health guidance. The medical instructions were as follows. Huoxiang Zhengqi Oral Liquid: oral before meals, 10 mL/time, 2 times/day, a course of 5 days. Jinhao Jiere Granules: dissolve in boiling water and take after meals, 8 g/time, 2 times/day, a course of 5 days, followed up for 14 days, respectively. The study found that with the intake of medication, the incidence rate of TCM intervention group was basically maintained at a low and continuous stable level(0.01%-0.02%), while the non-intervention group showed an overall trend of continuous growth(0.02%-0.18%) from 3 to 14 days. No suspected or confirmed COVID-19 case occurred in either group. There were 2 cases of colds in the TCM intervention group and 26 cases in the non-intervention group. The incidence of colds in the TCM intervention group was significantly lower(P<0.05) than that in the non-intervention group. In the population of 16-60 years old, the incidence rate of non-intervention and intervention groups were 0.01% and 0.25%, respectively. The difference of colds incidence between the two groups was statistically significant(P<0.05). In the population older than 60 years old, they were 0.04% and 0.21%, respectively. The incidence of colds in the non-intervention group was higher than that in the intervention group, but not reaching statistical difference. The protection rate of TCM for the whole population was 91.8%, especially for the population of age 16-60(95.0%). It was suggested that TCM intervention(combined use of Huoxiang Zhengqi Oral Liquid and Jinhao Jiere Granules) could effectively protect community residents against respiratory diseases, such as colds, which was worthy of promotion in the community. In addition, in terms of safety, the incidence of adverse events and adverse reactions in the TCM intervention group was relatively low, which was basically consistent with the drug instructions.
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Betacoronavirus , Infecções por Coronavirus , Medicamentos de Ervas Chinesas , Pandemias , Pneumonia Viral , Adolescente , Adulto , COVID-19 , Infecções por Coronavirus/tratamento farmacológico , Humanos , Medicina Tradicional Chinesa , Pessoa de Meia-Idade , Pneumonia Viral/tratamento farmacológico , Estudos Prospectivos , SARS-CoV-2 , Adulto Jovem , Tratamento Farmacológico da COVID-19RESUMO
Aberrant activation of estrogen signaling through three ESR (estrogen receptor) subtypes, termed ESR1/ERα, ESR2/ERß, and GPER1 (G protein-coupled estrogen receptor 1), is implicated in breast cancer pathogenesis and progression. Antiestrogens tamoxifen (TAM) and fulvestrant (FUL) are effective for treatment of ESR1-positive breast tumors, but development of resistance represents a major clinical challenge. However, the molecular mechanisms behind these events remain largely unknown. Here, we report that 17ß-estradiol (E2), TAM, and FUL stabilize MORC2 (MORC family CW-type zinc finger 2), an emerging oncoprotein in human cancer, in a GPER1-dependent manner. Mechanistically, GPER1 activates PRKACA (protein kinase cAMP-activated catalytic subunit alpha), which in turn phosphorylates MORC2 at threonine 582 (T582). Phosphorylated MORC2 decreases its interaction with HSPA8 (heat shock protein family A [Hsp70] member 8) and LAMP2A (lysosomal associated membrane protein 2A), two core components of the chaperone-mediated autophagy (CMA) machinery, thus protecting MORC2 from lysosomal degradation by CMA. Functionally, knockdown of MORC2 attenuates E2-induced cell proliferation and enhances cellular sensitivity to TAM and FUL. Moreover, introduction of wild-type MORC2, but not its phosphorylation-lacking mutant (T582A), in MORC2-depleted cells restores resistance to antiestrogens. Clinically, the phosphorylation levels of MORC2 at T582 are elevated in breast tumors from patients undergoing recurrence after TAM treatment. Together, these findings delineate a phosphorylation-dependent mechanism for MORC2 stabilization in response to estrogen and antiestrogens via blocking CMA-mediated lysosomal degradation and uncover a dual role for MORC2 in both estrogen-induced proliferation and resistance to antiestrogen therapies of breast cancer cells. ABBREVIATIONS: 4-OHT: 4-hydroxytamoxifen; Baf A1: bafilomycin A1; CMA: chaperone-mediated autophagy; E2: 17ß-estradiol; ESR: estrogen receptor; FUL: fulvestrant; GPER1: G protein-coupled estrogen receptor 1; HSPA8: heat shock protein family A (Hsp70) member 8; LAMP2A: lysosomal associated membrane protein 2A; MORC2: MORC family CW-type zinc finger 2; PRKACA: protein kinase cAMP-activated catalytic subunit alpha; TAM: tamoxifen; VCL: vinculin.
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Autofagia/efeitos dos fármacos , Neoplasias da Mama/metabolismo , Quimases/metabolismo , Subunidades Catalíticas da Proteína Quinase Dependente de AMP Cíclico/metabolismo , Moduladores de Receptor Estrogênico/farmacologia , Estrogênios/farmacologia , Receptores de Estrogênio/metabolismo , Receptores Acoplados a Proteínas G/metabolismo , Fatores de Transcrição/metabolismo , Neoplasias da Mama/tratamento farmacológico , Neoplasias da Mama/genética , Neoplasias da Mama/patologia , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Resistencia a Medicamentos Antineoplásicos , Estradiol/farmacologia , Feminino , Fulvestranto/farmacologia , Proteínas de Choque Térmico HSC70/metabolismo , Humanos , Imuno-Histoquímica , Proteína 2 de Membrana Associada ao Lisossomo/metabolismo , Lisossomos/efeitos dos fármacos , Lisossomos/metabolismo , Fosforilação , Estabilidade Proteica , RNA Interferente Pequeno , Transdução de Sinais/efeitos dos fármacos , Tamoxifeno/farmacologia , Fatores de Transcrição/genéticaRESUMO
Deregulation of E3 ubiquitin ligases is intimately implicated in breast cancer pathogenesis and progression, but the underlying mechanisms still remain elusive. Here we report that RING finger protein 144A (RNF144A), a poorly characterized member of the RING-in-between-RING family of E3 ubiquitin ligases, functions as a tumor suppressor in breast cancer. RNF144A was downregulated in a subset of primary breast tumors and restoration of RNF144A suppressed breast cancer cell proliferation, colony formation, migration, invasion in vitro, tumor growth, and lung metastasis in vivo. In contrast, knockdown of RNF144A promoted malignant phenotypes of breast cancer cells. Quantitative proteomics and biochemical analysis revealed that RNF144A interacted with and targeted heat-shock protein family A member 2 (HSPA2), a putative oncoprotein that is frequently upregulated in human cancer and promotes tumor growth and progression, for ubiquitination and degradation. Notably, the ligase activity-defective mutants of RNF144A impaired its ability to induce ubiquitination and degradation of HSPA2, and to suppress breast cancer cell proliferation, migration, and invasion as compared with its wild-type counterpart. Moreover, RNF144A-mediated suppression of breast cancer cell proliferation, migration, and invasion was rescued by ectopic HSPA2 expression. Clinically, low RNF144A and high HSPA2 expression in breast cancer patients was correlated with aggressive clinicopathological characteristics and decreased overall and disease-free survival. Collectively, these findings reveal a previously unappreciated role for RNF144A in suppression of breast cancer growth and metastasis, and identify RNF144A as the first, to our knowledge, E3 ubiquitin ligase for HSPA2 in human cancer.
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Neoplasias da Mama/metabolismo , Proteínas de Transporte/metabolismo , Proteínas de Choque Térmico HSP70/metabolismo , Oncogenes , Proteínas Supressoras de Tumor/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , Animais , Neoplasias da Mama/patologia , Linhagem Celular Tumoral , Movimento Celular , Proliferação de Células , Regulação para Baixo/genética , Feminino , Humanos , Neoplasias Pulmonares/secundário , Camundongos Endogâmicos BALB C , Camundongos Nus , Modelos Biológicos , Invasividade Neoplásica , Prognóstico , Complexo de Endopeptidases do Proteassoma/metabolismo , Estabilidade Proteica , Proteólise , Ubiquitinação , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Decondesation of the highly compacted chromatin architecture is essential for efficient DNA repair, but how this is achieved remains largely unknown. Here, we report that microrchidia family CW-type zinc finger protein 2 (MORC2), a newly identified ATPase-dependent chromatin remodeling enzyme, is required for nucleosome destabilization after DNA damage through loosening the histone-DNA interaction. Depletion of MORC2 attenuates phosphorylated histone H2AX (γH2AX) focal formation, compromises the recruitment of DNA repair proteins, BRCA1, 53BP1, and Rad51, to sites of DNA damage, and consequently reduces cell survival following treatment with DNA-damaging chemotherapeutic drug camptothecin (CPT). Furthermore, we demonstrate that MORC2 can form a homodimer through its C-terminal coiled-coil (CC) domain, a process that is enhanced in response to CPT-induced DNA damage. Deletion of the C-terminal CC domain in MORC2 disrupts its homodimer formation and impairs its ability to destabilize histone-DNA interaction after DNA damage. Consistently, expression of dimerization-defective MORC2 mutant results in impaired the recruitment of DNA repair proteins to damaged chromatin and decreased cell survival after CPT treatment. Together, these findings uncover a new mechanism for MORC2 in modulating chromatin dynamics and DDR signaling through its c-terminal dimerization.
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Cromatina/metabolismo , Fatores de Transcrição/metabolismo , Antineoplásicos Fitogênicos/farmacologia , Camptotecina/farmacologia , Sobrevivência Celular/efeitos dos fármacos , Células Cultivadas , Dano ao DNA , Reparo do DNA , Dimerização , Células HEK293 , Células HeLa , Humanos , Células MCF-7 , Domínios Proteicos , Transdução de Sinais/efeitos dos fármacos , Fatores de Transcrição/deficiência , Fatores de Transcrição/genéticaRESUMO
c-Myc plays an important role in cell proliferation, differentiation, and cell apoptosis. FasL/Fas pathway is a key regulator of cell apoptosis. This study was aimed to investigate the effects of c-Myc on the FasL/Fas pathway in ischemia-reperfusion (I/R)-induced renal injury. Rats were objected to bilateral renal ischemia for 60 min and reperfused for 24 or 48 h. NRK-52E cells were treated with hypoxia-reoxygenation (H/R) or FasL. Immunohistochemistry was used to identify the distribution of c-Myc. Cell apoptosis was assessed by TUNEL staining. Ad-c-Myc and recombinant pcDAN 3.0 were used to overexpress c-Myc and c-FLIP, respectively. ChIP assay and luciferase assay were used to detect the binding of c-Myc to c-FLIP promoter. In I/R rats, c-Myc was increased significantly and mainly located in renal tubular epithelial cells; meanwhile, c-FLIP was decreased, cleaved caspase-8, cleaved caspase-3 and TUNEL-positive staining cells were increased. Treatment of I/R rats with c-Myc inhibitor 10058-F4 significantly attenuated the decrease in c-FLIP, the increase in cleaved caspase-8, cleaved caspase-3, TUNEL-positive cells, Scr and BUN in I/R rats. In NRK-52E cells, hypoxia and reoxygen induced the increase in c-Myc and decrease in c-FLIP. ChIP and luciferase assay results indicated that c-Myc binds to the promoter region of c-FLIP gene. Overexpression of c-Myc markedly decreased c-FLIP. Overexpression of c-FLIP inhibited the increase in cleaved caspase-8 and caspase-3 induced by FasL. Data indicated that c-Myc is increased in kidneys of I/R rats and negatively regulates the expression of c-FLIP, then enhanced FasL-induced cell apoptosis in I/R stress.
Assuntos
Apoptose/fisiologia , Proteína Reguladora de Apoptosis Semelhante a CASP8 e FADD/metabolismo , Nefropatias/fisiopatologia , Proteínas Proto-Oncogênicas c-myc/metabolismo , Traumatismo por Reperfusão/fisiopatologia , Animais , Proteína Reguladora de Apoptosis Semelhante a CASP8 e FADD/genética , Caspase 8/metabolismo , Linhagem Celular , Proteína Ligante Fas/metabolismo , Rim/metabolismo , Rim/patologia , Túbulos Renais/citologia , Regiões Promotoras Genéticas , Proteínas Proto-Oncogênicas c-myc/antagonistas & inibidores , Ratos Sprague-Dawley , Tiazóis/farmacologia , Receptor fas/metabolismoRESUMO
Caspase-11 is a key upstream modulator for activation of inflammatory response under pathological conditions. In this study, we investigated the roles of caspase-11 in the maturation of interleukin-1ß (IL-1ß) and development of renal interstitial fibrosis in vivo and in vitro. Mice were subjected to unilateral ureteral obstruction (UUO). The mice were treated with either caspase-11 inhibitor wedelolactone (Wed, 30 mg/kg/day, ig) for 7 days or caspase-11 siRNA (10 nmol/20 g body weight per day, iv) for 14 days. The mice were euthanized on day 14, their renal tissue and blood sample were collected. We found that the obstructed kidney had significantly higher caspase-11 levels and obvious tubular injury and interstitial fibrosis. Treatment with Wed or caspase-11 siRNA significantly mitigated renal fibrosis in UUO mice, evidenced by the improved histological changes. Furthermore, caspase-11 inhibition significantly blunted caspase-1 activation, IL-1ß maturation, transforming growth factor-ß (TGF-ß), fibronectin, and collagen I expressions in the obstructed kidney. Renal tubular epithelial NRK-52E cells were treated in vitro with angiotensin (Ang, 1 µmol/L), which stimulated caspase-11 activation and IL-1ß maturation. Treatment with IL-1ß (20 ng/ml) significantly increased the expression of TGF-ß, fibronectin, and collagen I in the cells. Ang II-induced expression of TGF-ß, fibronectin, and collagen I were suppressed by caspase-11 siRNA or Wed. Finally, we revealed using co-immunoprecipitation that caspase-11 was able to interact with caspase-1 in NRK-52E cells. These results suggest that caspase-11 is involved in UUO-induced renal fibrosis. Elevation of caspase-11 in the obstructed kidney promotes renal fibrosis by stimulating caspase-1 activation and IL-1ß maturation.
Assuntos
Caspase 1/metabolismo , Caspases/metabolismo , Interleucina-1beta/metabolismo , Nefropatias/etiologia , Angiotensina II/metabolismo , Animais , Inibidores de Caspase/farmacologia , Caspases/genética , Caspases Iniciadoras , Cumarínicos/farmacologia , Ativação Enzimática , Matriz Extracelular/metabolismo , Fibrose , Inativação Gênica , Rim/patologia , Nefropatias/tratamento farmacológico , Nefropatias/patologia , Masculino , Camundongos Endogâmicos C57BL , RNA Interferente Pequeno/genética , Ratos , Obstrução Ureteral/complicaçõesRESUMO
Triple-negative breast cancer (TNBC) is the most lethal subtype of breast cancer, with a high propensity for distant metastasis and limited treatment options, yet its molecular underpinnings remain largely unknown. Microrchidia family CW-type zinc finger 2 (MORC2) is a newly identified chromatin remodeling protein whose mutations have been causally implicated in several neurologic disorders. Here, we report that a cancer-associated substitution of methionine to isoleucine at residue 276 (M276I) of MORC2 confers gain-of-function properties in the metastatic progression of TNBC. Expression of mutant MORC2 in TNBC cells increased cell migration, invasion, and lung metastasis without affecting cell proliferation and primary tumor growth compared with its wild-type counterpart. The M276I mutation enhanced binding of MORC2 to heterogeneous nuclear ribonucleoprotein M (hnRNPM), a component of the spliceosome machinery. This interaction promoted an hnRNPM-mediated splicing switch of CD44 from the epithelial isoform (CD44v) to the mesenchymal isoform (CD44s), ultimately driving epithelial-mesenchymal transition (EMT). Knockdown of hnRNPM reduced the binding of mutant MORC2 to CD44 pre-mRNA and reversed the mutant MORC2-induced CD44 splicing switch and EMT, consequently impairing the migratory, invasive, and lung metastatic potential of mutant MORC2-expressing cells. Collectively, these findings provide the first functional evidence for the M276I mutation in promoting TNBC progression. They also establish the first mechanistic connection between MORC2 and RNA splicing and highlight the importance of deciphering unique patient-derived mutations for optimizing clinical outcomes of this highly heterogeneous disease.Significance: A gain-of-function effect of a single mutation on MORC2 promotes metastasis of triple-negative breast cancer by regulating CD44 splicing. Cancer Res; 78(20); 5780-92. ©2018 AACR.
Assuntos
Processamento Alternativo , Ribonucleoproteínas Nucleares Heterogêneas Grupo M/metabolismo , Receptores de Hialuronatos/metabolismo , Fatores de Transcrição/genética , Neoplasias de Mama Triplo Negativas/genética , Animais , Linhagem Celular Tumoral , Movimento Celular , Sobrevivência Celular , Progressão da Doença , Transição Epitelial-Mesenquimal , Éxons , Feminino , Humanos , Isoleucina/química , Metionina/química , Camundongos , Camundongos Endogâmicos NOD , Camundongos SCID , Mutação , Invasividade Neoplásica , Metástase Neoplásica , Isoformas de Proteínas , Fatores de Transcrição/metabolismo , Neoplasias de Mama Triplo Negativas/metabolismo , Neoplasias de Mama Triplo Negativas/patologiaRESUMO
The molecular underpinnings behind malignant progression of breast cancer from a localized lesion to an invasive and ultimately metastatic disease are incompletely understood. Here, we report that F-box only protein 22 (FBXO22) plays a dual role in mammary tumorigenesis and metastasis. FBXO22 was upregulated in primary breast tumors and promoted cell proliferation and colony formation in vitro and xenograft tumorigenicity in vivo Surprisingly, FBXO22 suppressed epithelial-mesenchymal transition (EMT), cell motility, and invasiveness in vitro and metastatic lung colonization in vivo Clinical data showed that expression levels of FBXO22 were associated with favorable clinical outcomes, supporting the notion that metastasis, rather than primary cancer, is the major determinant of the mortality of patients with breast cancer. Mechanistic investigations further revealed that FBXO22 elicits its antimetastatic effects by targeting SNAIL, a master regulator of EMT and breast cancer metastasis, for ubiquitin-mediated proteasomal degradation in a glycogen synthase kinase 3ß phosphorylation-dependent manner. Importantly, expression of SNAIL rescued FBXO22-mediated suppression of EMT, cell migration, and invasion. A patient-derived tryptophan-to-arginine mutation at residue 52 (W52R) within the F-box domain impaired FBXO22 binding to the SKP1-Cullin1 complex and blocked FBXO22-mediated SNAIL degradation, thus abrogating the ability of FBXO22 to suppress cell migration, invasion, and metastasis. Collectively, these findings uncover an unexpected dual role for FBXO22 in mammary tumorigenesis and metastatic progression and delineate the mechanism of an oncogenic mutation of FBXO22 in breast cancer progression.Significance: These findings highlight the paradoxical roles of FBXO22 in breast cancer, as it promotes breast tumor cell proliferation but prevents EMT and metastasis. Cancer Res; 78(18); 5274-86. ©2018 AACR.
Assuntos
Neoplasias da Mama/patologia , Carcinogênese , Proteínas F-Box/fisiologia , Metástase Neoplásica , Receptores Citoplasmáticos e Nucleares/fisiologia , Motivos de Aminoácidos , Animais , Neoplasias da Mama/metabolismo , Movimento Celular , Proliferação de Células , Progressão da Doença , Transição Epitelial-Mesenquimal , Feminino , Glicogênio Sintase Quinase 3 beta/metabolismo , Humanos , Neoplasias Pulmonares/patologia , Neoplasias Pulmonares/secundário , Células MCF-7 , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Invasividade Neoplásica , Transplante de Neoplasias , Fosforilação , Resultado do TratamentoRESUMO
Alternative splicing (AS) and its regulation play critical roles in cancer, yet the dysregulation of AS and its molecular bases in breast cancer development have not yet been elucidated. Using an in vivo CRISPR screen targeting RNA-binding proteins, we identified PHD finger protein 5A (PHF5A) as a key splicing factor involved in tumor progression. PHF5A expression was frequently upregulated in breast cancer and correlated with poor survival, and knockdown of PHF5A significantly suppressed cell proliferation, migration, and tumor formation. PHF5A was required for SF3b spliceosome stability and linked the complex to histones, and the PHF5A-SF3b complex modulated AS changes in apoptotic signaling. In addition, expression of a short truncated FAS-activated serine/threonine kinase (FASTK) protein was increased after PHF5A ablation and facilitated Fas-mediated apoptosis. This PHF5A-modulated FASTK-AS axis was widely present in breast cancer specimens, particularly those of the triple-negative subtype. Taken together, our findings reveal that PHF5A serves as an epigenetic suppressor of apoptosis and thus provides a mechanistic basis for breast cancer progression and may be a valuable therapeutic target.Significance: This study provides an epigenetic mechanistic basis for the aggressive biology of breast cancer and identifies a translatable therapeutic target. Cancer Res; 78(12); 3190-206. ©2018 AACR.
Assuntos
Apoptose/genética , Neoplasias da Mama/genética , Proteínas de Transporte/metabolismo , Epigênese Genética , Regulação Neoplásica da Expressão Gênica , Adulto , Idoso , Idoso de 80 Anos ou mais , Processamento Alternativo/genética , Animais , Mama/patologia , Neoplasias da Mama/mortalidade , Neoplasias da Mama/patologia , Proteínas de Transporte/genética , Linhagem Celular Tumoral , Proliferação de Células/genética , Progressão da Doença , Feminino , Técnicas de Silenciamento de Genes , Células HEK293 , Histonas/genética , Histonas/metabolismo , Humanos , Camundongos , Pessoa de Meia-Idade , Proteínas Serina-Treonina Quinases/metabolismo , Fatores de Processamento de RNA/metabolismo , Proteínas de Ligação a RNA , Transdução de Sinais/genética , Spliceossomos/metabolismo , Análise de Sobrevida , Transativadores , Regulação para Cima , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Poly(ADP-ribose) polymerase 1 (PARP1), a critical DNA repair protein, is frequently upregulated in breast tumors with a key role in breast cancer progression. Consequently, PARP inhibitors have emerged as promising therapeutics for breast cancers with DNA repair deficiencies. However, relatively little is known about the regulatory mechanism of PARP1 expression and the determinants of PARP inhibitor sensitivity in breast cancer cells. Here, we report that ring finger protein 144A (RNF144A), a RING-between-RING (RBR)-type E3 ubiquitin ligase with an unexplored functional role in human cancers, interacts with PARP1 through its carboxy-terminal region containing the transmembrane domain, and targets PARP1 for ubiquitination and subsequent proteasomal degradation. Moreover, induced expression of RNF144A decreases PARP1 protein levels and renders breast cancer cells resistant to the clinical-grade PARP inhibitor olaparib. Conversely, knockdown of endogenous RNF144A increases PARP1 protein levels and enhances cellular sensitivity to olaparib. Together, these findings define RNF144A as a novel regulator of PARP1 protein abundance and a potential determinant of PARP inhibitor sensitivity in breast cancer cells, which may eventually guide the optimal use of PARP inhibitors in the clinic.
RESUMO
BACKGROUND: Up to now, numerous case-control studies have reported the associations between fat mass and obesity associated (FTO) gene rs9939609 A/T polymorphism and polycystic ovary syndrome (PCOS), however, without a consistent result. Hence we performed current systematic review and meta-analysis to clarify the controversial results. METHODS: Case-control studies reporting the relationship of rs9939609 A/T polymorphism and PCOS published before April 2015 were searched in Pubmed database without language restriction. Data was analyzed by Review Manager 5.2. RESULTS: A total of five studies involving 5010 PCOS patients and 5300 controls were included for further meta-analysis. The results of meta-analysis showed that the FTO gene rs9939609 A/T polymorphism was significantly different between PCOS group and control group in different gene models (For AA + AT vs. TT: OR = 1.41, 95% CI = 1.28-1.55, P < 0.00001. For AA vs. AT + TT: OR = 1.54, 95% CI = 1.25-1.89, P < 0.0001. For AA vs. TT: OR = 1.74, 95% CI = 1.38-2.18, P < 0.00001. For A vs. T: OR = 1.36, 95% CI = 1.25-1.47, P < 0.00001, respectively) suggesting that A allele was a risk factor for PCOS susceptibility. Furthermore, subgroup analysis in Asian and Caucasian ethnicities also found significant association between rs9939609 A/T polymorphism and PCOS (In Asian subgroup: OR = 1.43, 95% CI = 1.29-1.59, P < 0.0001. In Caucasian subgroup: OR = 1.33, 95% CI = 1.08-1.64, P = 0.008) CONCLUSION: This meta-analysis suggests that rs9939609 A/T polymorphism of FTO gene is associated with PCOS risk, and that A allele is a risk factor for PCOS susceptibility simultaneously.