RESUMO
BACKGROUND: Previously, a novel multiplex system of 64 loci was constructed based on capillary electrophoresis platform, including 59 autosomal insertion/deletions (A-InDels), two Y-chromosome InDels, two mini short tandem repeats (miniSTRs), and an Amelogenin gene. The aim of this study is to evaluate the efficiencies of this multiplex system for individual identification, paternity testing and biogeographic ancestry inference in Chinese Hezhou Han (CHH) and Hubei Tujia (CTH) groups, providing valuable insights for forensic anthropology and population genetics research. RESULTS: The cumulative values of power of discrimination (CDP) and probability of exclusion (CPE) for the 59 A-InDels and two miniSTRs were 0.99999999999999999999999999754, 0.99999905; and 0.99999999999999999999999999998, 0.99999898 in CTH and CHH groups, respectively. When the likelihood ratio thresholds were set to 1 or 10, more than 95% of the full sibling pairs could be identified from unrelated individual pairs, and the false positive rates were less than 1.2% in both CTH and CHH groups. Biogeographic ancestry inference models based on 35 populations were constructed with three algorithms: random forest, adaptive boosting and extreme gradient boosting, and then 10-fold cross-validation analyses were applied to test these three models with the average accuracies of 86.59%, 84.22% and 87.80%, respectively. In addition, we also investigated the genetic relationships between the two studied groups with 33 reference populations using population statistical methods of FST, DA, phylogenetic tree, PCA, STRUCTURE and TreeMix analyses. The present results showed that compared to other continental populations, the CTH and CHH groups had closer genetic affinities to East Asian populations. CONCLUSIONS: This novel multiplex system has high CDP and CPE in CTH and CHH groups, which can be used as a powerful tool for individual identification and paternity testing. According to various genetic analysis methods, the genetic structures of CTH and CHH groups are relatively similar to the reference East Asian populations.
Assuntos
Genética Populacional , Irmãos , Humanos , Filogenia , China , Mutação INDEL , Repetições de Microssatélites , Genética Forense/métodos , Frequência do GeneRESUMO
AIMS: Nuclear protein 1 (Nupr1) is a multifunctional stress-induced protein involved in the regulation of tumorigenesis, apoptosis, and autophagy. However, its role in pulmonary hypertension (PH) after METH exposure remains unexplored. In this study, we aimed to investigate whether METH can induce PH and describe the role and mechanism of Nupr1 in the development of PH. METHODS AND RESULTS: Mice were made to induce pulmonary hypertension (PH) upon chronic intermittent treatment with METH. Their right ventricular systolic pressure (RVSP) was measured to assess pulmonary artery pressure. Pulmonary artery morphometry was determined by H&E staining and Masson staining. Nupr1 expression and function were detected in human lungs, mice lungs exposed to METH, and cultured pulmonary arterial smooth muscle cells (PASMCs) with METH treatment. Our results showed that chronic intermittent METH treatment successfully induced PH in mice. Nupr1 expression was increased in the cultured PASMCs, pulmonary arterial media from METH-exposed mice, and METH-ingested human specimens compared with control. Elevated Nupr1 expression promoted PASMC phenotype change from contractile to synthetic, which triggered pulmonary artery remodeling and resulted in PH formation. Mechanistically, Nupr1 mediated the opening of store-operated calcium entry (SOCE) by activating the expression of STIM1, thereby promoting Ca2+ influx and inducing phenotypic conversion of PASMCs. CONCLUSIONS: Nupr1 activation could promote Ca2+ influx through STIM1-mediated SOCE opening, which promoted METH-induced pulmonary artery remodeling and led to PH formation. These results suggested that Nupr1 played an important role in METH-induced PH and might be a potential target for METH-related PH therapy.
Assuntos
Hipertensão Pulmonar , Metanfetamina , Camundongos , Humanos , Animais , Hipertensão Pulmonar/induzido quimicamente , Hipertensão Pulmonar/genética , Hipertensão Pulmonar/metabolismo , Metanfetamina/metabolismo , Músculo Liso Vascular/metabolismo , Proteínas Nucleares/metabolismo , Células Cultivadas , Artéria Pulmonar/metabolismo , Miócitos de Músculo Liso/metabolismo , Proliferação de CélulasRESUMO
Deletion/insertion polymorphism (DIP) is one of the more promising genetic markers in the field of forensic genetics for personal identification and biogeographic ancestry inference. In this research, we used an in-house developed ancestry-informative marker-DIP system, including 56 autosomal diallelic DIPs, three Y-chromosomal DIPs, and an Amelogenin gene, to analyze the genetic polymorphism and ancestral composition of the Chinese Korean group, as well as to explore its genetic relationships with the 26 reference populations. The results showed that this novel panel exhibited high genetic polymorphism in the studied Korean group and could be effectively applied for forensic individual identification in the Korean group. In addition, the results of multiple population genetic analyses indicated that the ancestral component of the Korean group was dominated by northern East Asia. Moreover, the Korean group was more closely related to the East Asian populations, especially to the Japanese population in Tokyo. This study enriched the genetic data of the Korean ethnic group in China and provided information on the ancestry of the Korean group from the perspective of population genetics.
Assuntos
Etnicidade , Polimorfismo Genético , Humanos , Etnicidade/genética , Genética Populacional , China , República da Coreia , Frequência do Gene , Polimorfismo de Nucleotídeo ÚnicoRESUMO
Renal interstitial fibrosis (RIF) is a pathological process that fibrotic components are excessively deposited in the renal interstitial space due to kidney injury, resulting in impaired renal function and chronic kidney disease. The molecular mechanisms controlling renal fibrosis are not fully understood. In this present study, we identified Nuclear protein 1 (Nupr1), a transcription factor also called p8, as a novel regulator promoting renal fibrosis. Unilateral ureteral obstruction (UUO) time-dependently induced Nupr1 mRNA and protein expression in mouse kidneys while causing renal damage and fibrosis. Nupr1 deficiency (Nupr1-/- ) attenuated the renal tubule dilatation, tubular epithelial cell atrophy, and interstitial collagen accumulation caused by UUO. Consistently, Nupr1-/- significantly decreased the expression of type I collagen, myofibroblast markers smooth muscle α-actin (α-SMA), fibroblast-specific protein 1 (FSP-1), and vimentin in mouse kidney that were upregulated by UUO. These results suggest that Nupr1 protein was essential for fibroblast activation and/or epithelial-mesenchymal transition (EMT) during renal fibrogenesis. Indeed, Nupr1 was indispensable for TGF-ß-induced myofibroblast activation of kidney interstitial NRK-49F fibroblasts, multipotent mesenchymal C3H10T1/2 cells, and the EMT of kidney epithelial NRK-52E cells. It appears that Nupr1 mediated TGF-ß-induced α-SMA expression and collagen synthesis by initiating Smad3 signaling pathway. Importantly, trifluoperazine (TFP), a Nupr1 inhibitor, alleviated UUO-induced renal fibrosis. Taken together, our results demonstrate that Nupr1 promotes renal fibrosis by activating myofibroblast transformation from both fibroblasts and tubular epithelial cells.
Assuntos
Proteínas de Ligação a DNA/fisiologia , Transição Epitelial-Mesenquimal , Fibroblastos/fisiologia , Rim/patologia , Proteínas de Neoplasias/fisiologia , Animais , Células Cultivadas , Colágeno/metabolismo , Matriz Extracelular/metabolismo , Fibrose , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Miofibroblastos/fisiologia , Ratos , Transdução de Sinais/fisiologia , Proteína Smad3/fisiologia , Fatores de Transcrição da Família Snail/fisiologia , Trifluoperazina/farmacologiaRESUMO
The biological role of vacuolar protein sorting 33B (VPS33B) has not been examined in colorectal cancer (CRC). We report that VPS33B was downregulated in dextran sulfate sodium/azoxymethane (DSS/AOM) -induced CRC mice models and nicotine-treated CRC cells via the PI3K/AKT/c-Jun pathway. Reduced VPS33B is an unfavorable factor promoting poor prognosis in human CRC patients. VPS33B overexpression suppressed CRC proliferation, intrahepatic metastasis and chemoresistance of cisplatin (DDP) in vivo and in vitro through modulating the epidermal growth factor receptor (EGFR)/RAS/ERK/c-Myc/p53/miR-133a-3p feedback loop and the downstream cell cycle or EMT-related factors. Furthermore, NESG1 as a newly identified tumor suppressor interacted with VPS33B via colocalization in the cytoplasm, and it was stimulated by VPS33B through the downregulation of RAS/ERK/c-Jun-mediated transcription. NESG1 also activated VPS33B expression via the RAS/ERK/c-Jun pathway. Suppression of NESG1 increased cell growth, migration and invasion via the reversion of the VPS33B-modulating signal in VPS33B-overexpressed cells. Taken together, VPS33B as a tumor suppressor is easily dysregulated by chemical carcinogens and it interacts with NESG1 to modulate the EGFR/RAS/ERK/c-Myc/p53/miR-133a-3p feedback loop and thus suppress the malignant phenotype of CRC.
Assuntos
Neoplasias Colorretais/tratamento farmacológico , Neoplasias Colorretais/genética , Genes Supressores de Tumor/efeitos dos fármacos , Nicotina/farmacologia , Transdução de Sinais/efeitos dos fármacos , Proteínas de Transporte Vesicular/genética , Animais , Ciclo Celular/efeitos dos fármacos , Ciclo Celular/genética , Linhagem Celular Tumoral , Movimento Celular/efeitos dos fármacos , Movimento Celular/genética , Proliferação de Células/efeitos dos fármacos , Proliferação de Células/genética , Proteínas do Citoesqueleto/genética , Regulação para Baixo/efeitos dos fármacos , Regulação para Baixo/genética , Receptores ErbB/genética , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Regulação Neoplásica da Expressão Gênica/genética , Células HT29 , Humanos , Camundongos , Transdução de Sinais/genética , Transcrição Gênica/efeitos dos fármacos , Transcrição Gênica/genéticaRESUMO
Methamphetamine (METH) is an amphetamine-type drug that is highly addictive and widely abused. Many studies have shown that METH exposure causes severe damage not only to the nervous system but also to the cardiovascular system. Melusin protein is a mechanotransducer that plays an important role in maintaining normal heart function. However, the role of melusin in METH-induced cardiotoxicity has not yet been reported. We hypothesized that methamphetamine can produce cardiac damage and apoptosis by decreasing the quantity of melusin. To test this hypothesis, we determined the protein expression of melusin and apoptosis markers in METH-treated rats and primary rat cardiomyocytes. We also established a melusin-overexpressing cell model to assess the importance of melusin in maintaining antiapoptotic pathways. To confirm our findings from the in vitro and animal models, we also evaluated the apoptotic index of cardiomyocytes and the protein expression of apoptotic markers in postmortem heart tissues from deceased METH abusers and age-matched control subjects. The results showed that the apoptosis of cardiomyocytes was increased significantly and that the protein expression of melusin was decreased after exposure to METH in primary rat cardiomyocytes, in rats and in humans. METH treatment also decreased the expression of the downstream proteins FAK, IQGAP1, p-AKT, p-GSK3ß, and p-ERK in primary rat cardiomyocytes and in vivo. After overexpression of melusin, the above effects were partially reversed in primary rat cardiomyocytes. We conclude that METH can produce cardiac damage and apoptosis by decreasing melusin, while melusin-activated signaling by phosphorylated AKT, phosphorylated GSK3ß, and ERK may be resistant to methamphetamine-induced myocardial apoptosis.
Assuntos
Apoptose/efeitos dos fármacos , Proteínas do Citoesqueleto/metabolismo , Coração/efeitos dos fármacos , Metanfetamina/efeitos adversos , Proteínas Musculares/metabolismo , Miócitos Cardíacos/efeitos dos fármacos , Animais , Células Cultivadas , Masculino , Miocárdio/metabolismo , Miócitos Cardíacos/metabolismo , Ratos , Ratos Sprague-Dawley , Transdução de Sinais/efeitos dos fármacosRESUMO
Methamphetamine (Meth) is a widely abused psychoactive drug that primarily damages the nervous system, notably causing dopaminergic neuronal apoptosis. CCAAT-enhancer binding protein (C/EBPß) is a transcription factor and an important regulator of cell apoptosis and autophagy. Insulin-like growth factor binding protein (IGFBP5) is a proapoptotic factor that mediates Meth-induced neuronal apoptosis, and Trib3 (tribbles pseudokinase 3) is an endoplasmic reticulum (ER) stress-inducible gene involved in autophagic cell death through the mammalian target of rapamycin (mTOR) signaling pathway. To test the hypothesis that C/EBPß is involved in Meth-induced IGFBP5-mediated neuronal apoptosis and Trib3-mediated neuronal autophagy, we measured the protein expression of C/EBPß after Meth exposure and evaluated the effects of silencing C/EBPß, IGFBP5, or Trib3 on Meth-induced apoptosis and autophagy in neuronal cells and in the rat striatum after intrastriatal Meth injection. We found that, at relatively high doses, Meth exposure increased C/EBPß protein expression, which was accompanied by increased neuronal apoptosis and autophagy; triggered the IGFBP5-mediated, p53-up-regulated modulator of apoptosis (PUMA)-related mitochondrial apoptotic signaling pathway; and stimulated the Trib3-mediated ER stress signaling pathway through the Akt-mTOR signaling axis. We also found that autophagy is an early response to Meth-induced stress upstream of apoptosis and plays a detrimental role in Meth-induced neuronal cell death. These results suggest that Meth exposure induces C/EBPß expression, which plays an essential role in the neuronal apoptosis and autophagy induced by relatively high doses of Meth; however, relatively low concentrations of Meth did not change the expression of C/EBPß in vitro. Further studies are needed to elucidate the role of C/EBPß in low-dose Meth-induced neurotoxicity.-Xu, X., Huang, E., Luo, B., Cai, D., Zhao, X., Luo, Q., Jin, Y., Chen, L., Wang, Q., Liu, C., Lin, Z., Xie, W.-B., Wang, H. Methamphetamine exposure triggers apoptosis and autophagy in neuronal cells by activating the C/EBPß-related signaling pathway.
RESUMO
N-myc downstream-regulated gene 1 (NDRG1) has been implicated in tumorigenesis and metastasis in different cancers. However, its role in nasopharyngeal carcinoma remains unknown. We found that NDRG1 expression level was high in nasopharyngeal cancer 5-8F cells but low in 5-8F-LN cells with lymphatic metastasis potential. Knockdown of NDRG1 by shRNA promoted 5-8F cell proliferation, migration, and invasion in vitro and its tumorigenesis in vivo. Moreover, NDRG1 deficiency induced an epithelial-mesenchymal transition (EMT) of 5-8F cells as shown by an attenuation of E-cadherin and an induction of N-cadherin and vimentin expression. NDRG1 knockdown also enhanced Smad2 expression and phosphorylation. Smad2 signaling was attenuated in 5-8F cells but was significantly activated in 5-8F-LN cells. Knockdown of Smad2 restored E-cadherin but attenuated N-cadherin expression in NDRG1-deficient 5-8F cells, suggesting a reduction of EMT. Consistently, blockade of Smad2 in 5-8F-LN cells increased E-cadherin while diminishing N-cadherin and vimentin expression. These data indicate that Smad2 mediates the NDRG1 deficiency-induced EMT of 5-8F cells. In tumors derived from NDRG1-deficient 5-8F cells, E-cadherin expression was inhibited while vimentin and Smad2 were increased in a large number of cancer cells. Most importantly, NDRG1 expression was attenuated in human nasopharyngeal carcinoma tissues, resulted in a lower survival rate in patients. The NDRG1 was further decreased in the detached nasopharyngeal cancer cells, which was associated with a further reduced survival rate in patients with lymphatic metastasis. Taken together, these results demonstrated that NDRG1 prevents nasopharyngeal tumorigenesis and metastasis via inhibiting Smad2-mediated EMT of nasopharyngeal cells.
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Methamphetamine (METH) is an amphetamine-like psychostimulant that is commonly abused. Previous studies have shown that METH can induce damages to the nervous system and recent studies suggest that METH can also cause adverse and potentially lethal effects on the cardiovascular system. Recently, we demonstrated that DNA damage-inducible transcript 4 (DDIT4) regulates METH-induced neurotoxicity. However, the role of DDIT4 in METH-induced cardiotoxicity remains unknown. We hypothesized that DDIT4 may mediate METH-induced autophagy and apoptosis in cardiomyocytes. To test the hypothesis, we examined DDIT4 protein expression in cardiomyocytes and in heart tissues of rats exposed to METH with Western blotting. We also determined the effects on METH-induced autophagy and apoptosis after silencing DDIT4 expression with synthetic siRNA with or without pretreatment of a mTOR inhibitor rapamycin in cardiomyocytes using Western blot analysis, fluorescence microscopy and TUNEL staining. Our results showed that METH exposure increased DDIT4 expression and decreased phosphorylation of mTOR that was accompanied with increased autophagy and apoptosis both in vitro and in vivo. These effects were normalized after silencing DDIT4. On the other hand, rapamycin promoted METH-induced autophagy and apoptosis in DDIT4 knockdown cardiomyocytes. These results suggest that DDIT4 mediates METH-induced autophagy and apoptosis through mTOR signaling pathway in cardiomyocytes.
Assuntos
Estimulantes do Sistema Nervoso Central/farmacologia , Metanfetamina/farmacologia , Miocárdio/metabolismo , Miócitos Cardíacos/metabolismo , Fatores de Transcrição/biossíntese , Animais , Apoptose/efeitos dos fármacos , Autofagia/efeitos dos fármacos , Técnicas de Cultura de Células , Expressão Gênica , Masculino , RNA Interferente Pequeno , Ratos , Ratos Sprague-Dawley , Transdução de Sinais , Serina-Treonina Quinases TORRESUMO
RATIONALE: Vascular smooth muscle cell (VSMC) differentiation from neural crest cells (NCCs) is critical for cardiovascular development, but the mechanisms remain largely unknown. OBJECTIVE: Transforming growth factor-ß (TGF-ß) function in VSMC differentiation from NCCs is controversial. Therefore, we determined the role and mechanism of a TGF-ß downstream signaling intermediate Smad2 in NCC differentiation to VSMCs. METHODS AND RESULTS: By using Cre/loxP system, we generated a NCC tissue-specific Smad2 knockout mouse model and found that Smad2 deletion resulted in defective NCC differentiation to VSMCs in aortic arch arteries during embryonic development and caused vessel wall abnormality in adult carotid arteries where the VSMCs are derived from NCCs. The abnormalities included 1 layer of VSMCs missing in the media of the arteries with distorted and thinner elastic lamina, leading to a thinner vessel wall compared with wild-type vessel. Mechanistically, Smad2 interacted with myocardin-related transcription factor B (MRTFB) to regulate VSMC marker gene expression. Smad2 was required for TGF-ß-induced MRTFB nuclear translocation, whereas MRTFB enhanced Smad2 binding to VSMC marker promoter. Furthermore, we found that Smad2, but not Smad3, was a progenitor-specific transcription factor mediating TGF-ß-induced VSMC differentiation from NCCs. Smad2 also seemed to be involved in determining the physiological differences between NCC-derived and mesoderm-derived VSMCs. CONCLUSIONS: Smad2 is an important factor in regulating progenitor-specific VSMC development and physiological differences between NCC-derived and mesoderm-derived VSMCs.
Assuntos
Diferenciação Celular , Desenvolvimento Muscular , Músculo Liso Vascular/metabolismo , Miócitos de Músculo Liso/metabolismo , Crista Neural/metabolismo , Proteína Smad2/metabolismo , Células-Tronco/metabolismo , Fatores de Transcrição/metabolismo , Transporte Ativo do Núcleo Celular , Animais , Aorta Torácica/anormalidades , Aorta Torácica/metabolismo , Sítios de Ligação , Artérias Carótidas/anormalidades , Artérias Carótidas/metabolismo , Linhagem Celular , Regulação da Expressão Gênica no Desenvolvimento , Masculino , Camundongos , Camundongos Endogâmicos C3H , Camundongos Knockout , Músculo Liso Vascular/anormalidades , Miócitos de Músculo Liso/patologia , Regiões Promotoras Genéticas , Interferência de RNA , Transdução de Sinais , Proteína Smad2/deficiência , Proteína Smad2/genética , Fatores de Transcrição/genética , TransfecçãoRESUMO
AIM: Methamphetamine (METH) chronic exposure is an important risk factor for hypertension development. However, the mechanisms behind METH-induced hypertension remain unclear. Therefore, we aimed to reveal the potential mechanisms underlying METH-induced hypertension. METHODS AND RESULTS: We structured the mouse hypertension model by METH, and observed that METH-treated mice have presented vascular remodeling (large-and small-size arteries) with collagen deposit around the vessel and increasing blood pressure (BP) and Sigma1 receptor (Sigmar1) in vascular tissue. We hypothesized that Sigmar1 is crucial in METH-induced hypertension and vascular remodeling. Sigmar1 knockout (KO) mice and antagonist (BD1047) pretreated mice exposed to METH for six-week showed higher BP and more collagen deposited around vessels than wild-type (WT) mice exposed to METH for six-week, in contrast, mice pretreated with Sigmar1 agonist (PRE-084) had unchanged BP and perivascular collagen despite the six-week METH exposure. Furthermore, we found that METH exposure induced vascular smooth muscle cells (VSMCs) and mesenchymal stem cells to differentiate into the myofibroblast-like cell and secrete collagen into surrounding vessels. Mechanically, Sigmar1 can suppress the COL1A1 expression by blocking the classical fibrotic TGF-ß/Smad2/3 signaling pathway in METH-exposed VSMCs and mesenchymal stem cells. CONCLUSION: Our results suggest that Sigmar1 is involved in METH-induced hypertension and vascular fibrosis by blocking the activation of the TGF-ß/Smad2/3 signaling pathway. Accordingly, Sigmar1 may be a novel therapeutic target for METH-induced hypertension and vascular fibrosis.
Assuntos
Hipertensão , Metanfetamina , Músculo Liso Vascular , Receptores sigma , Receptor Sigma-1 , Animais , Masculino , Camundongos , Pressão Sanguínea/efeitos dos fármacos , Colágeno/metabolismo , Modelos Animais de Doenças , Hipertensão/induzido quimicamente , Hipertensão/metabolismo , Hipertensão/patologia , Hipertensão/genética , Células-Tronco Mesenquimais/metabolismo , Metanfetamina/efeitos adversos , Metanfetamina/toxicidade , Camundongos Endogâmicos C57BL , Camundongos Knockout , Músculo Liso Vascular/metabolismo , Músculo Liso Vascular/patologia , Músculo Liso Vascular/efeitos dos fármacos , Miócitos de Músculo Liso/metabolismo , Miócitos de Músculo Liso/patologia , Miócitos de Músculo Liso/efeitos dos fármacos , Receptores sigma/metabolismo , Receptores sigma/genética , Transdução de Sinais/efeitos dos fármacos , Remodelação Vascular/efeitos dos fármacosRESUMO
Methamphetamine (METH) is a widely abused amphetamine-type psychoactive drug that causes serious health problems. Previous studies have demonstrated that METH can induce neuron autophagy and apoptosis in vivo and in vitro. However, the molecular mechanisms underlying METH-induced neuron autophagy and apoptosis remain poorly understood. Stromal interacting molecule 1 (STIM1) was hypothesized to be involved in METH-induced neuron autophagy and apoptosis. Therefore, the expression of STIM1 protein was measured and the effect of blocking STIM1 expression with siRNA was investigated in cultured neuronal cells, and the hippocampus and striatum of mice exposed to METH. Furthermore, intracellular calcium concentration and endoplasmic reticulum (ER) stress-related proteins were determined in vitro and in vivo in cells treated with METH. The results suggested that STIM1 mediates METH-induced neuron autophagy by activating the p-Akt/p-mTOR pathway. METH exposure also resulted in increased expression of Orai1, which was reversed after STIM1 silencing. Moreover, the disruption of intracellular calcium homeostasis induced ER stress and up-regulated the expression of pro-apoptotic protein CCAAT/enhancer-binding protein homologous protein (CHOP), resulting in classic mitochondria apoptosis. METH exposure can cause neuronal autophagy and apoptosis by increasing the expression of STIM1 protein; thus, STIM1 may be a potential gene target for therapeutics in METH-caused neurotoxicity.
Assuntos
Apoptose , Autofagia , Estresse do Retículo Endoplasmático , Metanfetamina , Neurônios , Molécula 1 de Interação Estromal , Metanfetamina/toxicidade , Animais , Molécula 1 de Interação Estromal/metabolismo , Molécula 1 de Interação Estromal/genética , Autofagia/efeitos dos fármacos , Apoptose/efeitos dos fármacos , Neurônios/efeitos dos fármacos , Neurônios/patologia , Neurônios/metabolismo , Estresse do Retículo Endoplasmático/efeitos dos fármacos , Estresse do Retículo Endoplasmático/fisiologia , Camundongos , Camundongos Endogâmicos C57BL , Estimulantes do Sistema Nervoso Central/toxicidade , Cálcio/metabolismo , Células Cultivadas , Masculino , Hipocampo/efeitos dos fármacos , Hipocampo/metabolismo , Hipocampo/patologia , Proteína ORAI1/metabolismo , Proteína ORAI1/genética , Serina-Treonina Quinases TOR/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Fator de Transcrição CHOP/metabolismo , Fator de Transcrição CHOP/genética , Corpo Estriado/efeitos dos fármacos , Corpo Estriado/metabolismo , Corpo Estriado/patologiaRESUMO
Smooth muscle cell (SMC) differentiation and proliferation occur simultaneously during embryonic development. The underlying mechanisms especially common factors regulating both processes, however, remain largely unknown. The present study has identified cell division cycle 7 (Cdc7) as one of the factors mediating both the proliferation and SMC differentiation. TGF-ß induces Cdc7 expression and phosphorylation in the initial phase of SMC differentiation of pluripotent mesenchymal C3H10T1/2 cells. Cdc7 specific inhibitor or shRNA knockdown suppresses TGF-ß-induced expression of SMC early markers including α-SMA, SM22α, and calponin. Cdc7 overexpression, on the other hand, enhances SMC marker expression. Cdc7 function in inducing SMC differentiation is independent of Dumbbell former 4 or Dbf4, the catalytic subunit of Cdc7 critical for cell proliferation, suggesting that Cdc7 mediates SMC differentiation through a mechanism distinct from cell proliferation. Cdc7 regulates SMC differentiation via activating SMC marker gene transcription. Knockdown of Cdc7 by shRNA inhibits SMC marker gene promoter activities. Mechanistically, Cdc7 interacts with Smad3 to induce SMC differentiation. Smad3 is required for Cdc7 function in inducing SMC promoter activities and marker gene expression. Likewise, Cdc7 enhances Smad3 binding to SMC marker promoter via supporting Smad3 nuclear retention and physically interacting with Smad3. Taken together, our studies have demonstrated a novel role of Cdc7 in SMC differentiation.
Assuntos
Proteínas de Ciclo Celular/metabolismo , Fibroblastos/citologia , Miócitos de Músculo Liso/citologia , Proteínas Serina-Treonina Quinases/metabolismo , Transdução de Sinais/fisiologia , Fator de Crescimento Transformador beta1/metabolismo , Animais , Biomarcadores/metabolismo , Proteínas de Ciclo Celular/genética , Diferenciação Celular/efeitos dos fármacos , Diferenciação Celular/fisiologia , Divisão Celular/efeitos dos fármacos , Divisão Celular/fisiologia , Linhagem Celular , Fibroblastos/fisiologia , Regulação da Expressão Gênica no Desenvolvimento/fisiologia , Camundongos , Miócitos de Músculo Liso/fisiologia , Células-Tronco Pluripotentes/citologia , Células-Tronco Pluripotentes/fisiologia , Regiões Promotoras Genéticas/fisiologia , Proteínas Serina-Treonina Quinases/genética , RNA Interferente Pequeno/genética , Transdução de Sinais/efeitos dos fármacos , Proteína Smad3/metabolismo , Fator de Crescimento Transformador beta1/farmacologiaRESUMO
The neuroinflammatory hypothesis of Alzheimer's disease (AD) posits that amyloid-beta (Aß) phagocytosis along with subsequent lysosomal damage and NLRP3 inflammasome activation plays important roles in Aß-induced microglia activation and microglia-induced neurotoxicity. Sulforaphane (SFN) has neuroprotective effects for AD. However, whether SFN can inhibit its cytotoxic autophagy and NLRP3 inflammasome activation in microglia remain unknown. In this study, results showed SFN played an indirect, protective role on neurons via a series of impacts on Aß-activated microglia, including inhibition of autophagy initiation as well as autophagic lysosomal membrane permeability and subsequent NLRP3/caspase-1 inflammasomes activation. M1 phenotype polarization was also inhibited. Our results demonstrated that SFN could inhibit the cytostatic autophagy-induced NLRP3 signaling pathway in Aß-activated microglia by decreasing reactive oxygen species (ROS) production. These results provide novel insight into the potential role of SFN in AD therapy.
Assuntos
Doença de Alzheimer , Microglia , Humanos , Microglia/metabolismo , Proteína 3 que Contém Domínio de Pirina da Família NLR/metabolismo , Inflamassomos/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Peptídeos beta-Amiloides/metabolismo , Autofagia , Doença de Alzheimer/metabolismo , Neurônios/metabolismoRESUMO
Both TGF-ß and myocardin (MYOCD) are important for smooth muscle cell (SMC) differentiation, but their precise role in regulating the initiation of SMC development is less clear. In TGF-ß-induced SMC differentiation of pluripotent C3H10T1/2 progenitors, we found that TGF-ß did not significantly induce Myocd mRNA expression until 18 h of stimulation. On the other hand, early SMC markers such as SM α-actin, SM22α, and SM calponin were detectable beginning 2 or 4 h after TGF-ß treatment. These results suggest that Myocd expression is blocked during the initiation of TGF-ß-induced SMC differentiation. Consistent with its endogenous expression, Myocd promoter activity was not elevated until 18 h following TGF-ß stimulation. Surprisingly, Smad signaling was inhibitory to Myocd expression because blockade of Smad signaling enhanced Myocd promoter activity. Overexpression of Smad3, but not Smad2, inhibited Myocd promoter activity. Conversely, shRNA knockdown of Smad3 allowed TGF-ß to activate the Myocd promoter in the initial phase of induction. Myocd was activated by PI3 kinase signaling and its downstream target Nkx2.5. Interestingly, Smad3 did not affect PI3 kinase activity. However, Smad3 physically interacted with Nkx2.5. This interaction blocked Nkx2.5 binding to the Myocd promoter in the early stage of TGF-ß induction, leading to inhibition of Myocd mRNA expression. Moreover, Smad3 inhibited Nkx2.5-activated Myocd promoter activity in a dose-dependent manner. Taken together, our results reveal a novel mechanism for Smad3-mediated inhibition of Myocd in the initiation phase of SMC differentiation.
Assuntos
Diferenciação Celular , Inativação Gênica , Miócitos de Músculo Liso/citologia , Proteínas Nucleares/genética , Células-Tronco Pluripotentes/citologia , Proteína Smad3/fisiologia , Transativadores/genética , Animais , Linhagem Celular , Proteína Homeobox Nkx-2.5 , Proteínas de Homeodomínio/fisiologia , Camundongos , Regiões Promotoras Genéticas , Proteína Smad3/genética , Fatores de Tempo , Fatores de Transcrição/fisiologia , Transcrição Gênica , Fator de Crescimento Transformador beta/farmacologiaRESUMO
Response gene to complement 32 (RGC-32) is a downstream target of transforming growth factor-ß (TGF-ß). TGF-ß is known to play a pathogenic role in renal fibrosis. In this study, we investigated RGC-32 function in renal fibrosis following unilateral ureteral obstruction (UUO) in mice, a model of progressive tubulointerstitial fibrosis. RGC-32 is normally expressed only in blood vessels of mouse kidney. However, UUO induces RGC-32 expression in renal interstitial cells at the early stage of kidney injury, suggesting that RGC-32 is involved in interstitial fibroblast activation. Indeed, expression of smooth muscle α-actin (α-SMA), an indicator of fibroblast activation, is limited to the interstitial cells at the early stage, and became apparent later in both interstitial and tubular cells. RGC-32 knockdown by shRNA significantly inhibits UUO-induced renal structural damage, α-SMA expression and collagen deposition, suggesting that RGC-32 is essential for the onset of renal interstitial fibrosis. In vitro studies indicate that RGC-32 mediates TGF-ß-induced fibroblast activation. Mechanistically, RGC-32 interacts with Smad3 and enhances Smad3 binding to the Smad binding element in α-SMA promoter as demonstrated by DNA affinity assay. In the chromatin setting, Smad3, but not Smad2, binds to α-SMA promoter in fibroblasts. RGC-32 appears to be essential for Smad3 interaction with the promoters of fibroblast activation-related genes in vivo. Functionally, RGC-32 is crucial for Smad3-mediated α-SMA promoter activity. Taken together, we identify RGC-32 as a novel fibrogenic factor contributing to the pathogenesis of renal fibrosis through fibroblast activation.
Assuntos
Fibroblastos/metabolismo , Regulação da Expressão Gênica , Nefropatias/metabolismo , Rim/metabolismo , Proteínas Nucleares/biossíntese , Actinas/biossíntese , Actinas/genética , Animais , Linhagem Celular , Colágeno/biossíntese , Colágeno/genética , Modelos Animais de Doenças , Fibroblastos/patologia , Fibrose , Humanos , Rim/patologia , Nefropatias/genética , Nefropatias/patologia , Masculino , Camundongos , Proteínas Nucleares/genética , Elementos de Resposta/genética , Proteína Smad2/genética , Proteína Smad2/metabolismo , Proteína Smad3/genética , Proteína Smad3/metabolismoRESUMO
OBJECTIVE: The objectives of this study were to determine the role of response gene to complement 32 (RGC-32) in vascular lesion formation after experimental angioplasty and to explore the underlying mechanisms. METHODS AND RESULTS: Using a rat carotid artery balloon-injury model, we documented for the first time that neointima formation was closely associated with a significantly increased expression of RGC-32 protein. Short hairpin RNA knockdown of RGC-32 via adenovirus-mediated gene delivery dramatically inhibited the lesion formation by 62% as compared with control groups 14 days after injury. Conversely, RGC-32 overexpression significantly promoted the neointima formation by 33%. Gain- and loss-of-function studies in primary culture of rat aortic smooth muscle cells (RASMCs) indicated that RGC-32 is essential for both the proliferation and migration of RASMCs. RGC-32 induced RASMC proliferation by enhancing p34(CDC2) activity. RGC-32 stimulated the migration of RASMC by inducing focal adhesion contact and stress fiber formation. These effects were caused by the enhanced rho kinase II-α activity due to RGC-32-induced downregulation of Rad GTPase. CONCLUSIONS: RGC-32 plays an important role in vascular lesion formation following vascular injury. Increased RGC-32 expression in vascular injury appears to be a novel mechanism underlying the migration and proliferation of vascular smooth muscle cells. Therefore, targeting RGC-32 is a potential therapeutic strategy for the prevention of vascular remodeling in proliferative vascular diseases.
Assuntos
Lesões das Artérias Carótidas/patologia , Lesões das Artérias Carótidas/fisiopatologia , Proteínas de Ciclo Celular/fisiologia , Proteínas Musculares/fisiologia , Miócitos de Músculo Liso/patologia , Miócitos de Músculo Liso/fisiologia , Proteínas do Tecido Nervoso/fisiologia , Animais , Sequência de Bases , Lesões das Artérias Carótidas/etiologia , Lesões das Artérias Carótidas/genética , Proteínas de Ciclo Celular/antagonistas & inibidores , Proteínas de Ciclo Celular/genética , Movimento Celular/genética , Movimento Celular/fisiologia , Proliferação de Células , Células Cultivadas , Expressão Gênica , Técnicas de Silenciamento de Genes , Masculino , Proteínas Musculares/antagonistas & inibidores , Proteínas Musculares/genética , Neointima/patologia , Neointima/fisiopatologia , Proteínas do Tecido Nervoso/antagonistas & inibidores , Proteínas do Tecido Nervoso/genética , RNA Interferente Pequeno/genética , Ratos , Ratos Sprague-Dawley , Proteínas ras/genética , Proteínas ras/fisiologiaRESUMO
AIMS: Thoracic aortic aneurysm/dissection (TAAD) is a life-threatening disease with diverse clinical manifestations. Although the association between methamphetamine (METH) and TAAD is frequently observed, the causal relationship between METH abuse and aortic aneurysm/dissection has not been established. This study was designed to determine if METH causes aortic aneurysm/dissection and delineate the underlying mechanism. METHODS AND RESULTS: A new TAAD model was developed by exposing METH to SD rats pre-treated with lysyl oxidase inhibitor ß-aminopropionitrile (BAPN). Combination of METH and BAPN caused thoracic aortic aneurysm/dissection in 60% of rats. BAPN+METH significantly increased the expression and activities of both matrix metalloproteinase MMP2 and MMP9, consistent with the severe elastin breakage and dissection. Mechanistically, METH increased CCAAT-enhancer binding protein ß (C/EBPß) expression by enhancing mothers against decapentaplegic homolog 3 (Smad3) and extracellular regulated protein kinase (ERK1/2) signaling. METH also promoted C/EBPß binding to MMP2 and MMP9 promoters. Blocking C/EBPß significantly attenuated METH+BAPN-induced TAAD and MMP2/MMP9 expression. Moreover, BAPN+METH promoted aortic medial smooth muscle cell (SMC) apoptosis through C/EBPß-mediated IGFBP5/p53/PUMA signaling pathways. More importantly, the expression of C/EBPß, MMP2/MMP9, and apoptosis-promoting proteins was increased in the aorta of human patients with thoracic aortic dissection, suggesting that the mechanisms identified in animal study could be relevant to human disease. CONCLUSIONS: Our study demonstrated that METH exposure has a casual effect on TAAD. C/EBPß mediates METH-introduced TAAD formation by causing elastin breakage, medial cell loss and degeneration. Therefore, C/EBPß may be a potential factor for TAAD clinical diagnosis or treatment.
Assuntos
Aneurisma da Aorta Torácica , Dissecção Aórtica , Metanfetamina , Aminopropionitrilo , Dissecção Aórtica/induzido quimicamente , Dissecção Aórtica/metabolismo , Animais , Aneurisma da Aorta Torácica/induzido quimicamente , Aneurisma da Aorta Torácica/metabolismo , Proteína beta Intensificadora de Ligação a CCAAT/metabolismo , Elastina , Humanos , Metaloproteinase 2 da Matriz/genética , Metaloproteinase 9 da Matriz/metabolismo , Ratos , Ratos Sprague-DawleyRESUMO
Response gene to complement 32 (RGC-32) is activated by transforming growth factor- ß (TGF-ß) and plays an important role in smooth muscle cell (SMC) differentiation from neural crest Monc-1 cells. The molecular mechanism governing TGF-ß activation of RGC-32, however, remains to be determined. The present studies indicate that TGF-ß regulates RGC-32 gene transcription. Sequence analysis revealed a Smad binding element (SBE) located in the region from -1344 to -1337 bp upstream of the transcription start site of RGC-32 gene. A polyomavirus enhancer activator (PEA3) binding site is adjacent to the SBE. Mutation at either SBE or PEA3 site significantly inhibited RGC-32 promoter activity. Mutations at both sites completely abolished TGF-ß-induced promoter activity. Biochemically, TGF-ß stimulated recruitment of Smad2, Smad4, and PEA3 to the RGC-32 promoter, as revealed by gel shift and chromatin immunoprecipitation analyses. Functionally, Smad2, but not Smad3, activated RGC-32 promoter. PEA3 appeared to enhance Smad2 activity. In agreement with their function, Smad2, but not Smad3, physically interacted with PEA3. In TGF-ß-induced SMC differentiation of Monc-1 cells, knockdown of Smad2 by short hairpin RNA resulted in downregulation of RGC-32 and SMC marker genes. The downregulation of SMC markers, however, was rescued by exogenously introduced RGC-32. These results demonstrate that Smad2 regulation of RGC-32 transcription is essential for SMC differentiation from neural crest cells.
Assuntos
Diferenciação Celular , Células-Tronco Embrionárias/metabolismo , Miócitos de Músculo Liso/metabolismo , Crista Neural/metabolismo , Proteínas Nucleares/metabolismo , Proteína Smad2/metabolismo , Fatores de Transcrição/metabolismo , Fator de Crescimento Transformador beta1/metabolismo , Animais , Sítios de Ligação , Biomarcadores/metabolismo , Linhagem Celular , Imunoprecipitação da Cromatina , Ensaio de Desvio de Mobilidade Eletroforética , Regulação da Expressão Gênica no Desenvolvimento , Camundongos , Mutação , Crista Neural/citologia , Proteínas Nucleares/genética , Regiões Promotoras Genéticas , Interferência de RNA , Transdução de Sinais , Proteína Smad2/genética , Proteína Smad3/metabolismo , Fatores de Transcrição/genética , Ativação Transcricional , TransfecçãoRESUMO
Methamphetamine (METH) is a highly addictive amphetamine-type drug that has caused persistent harm to society and human health in recent years. Most studies have shown that METH severely damages the central nervous system, and this drug has been found to be toxic to the cardiovascular system in recent years. Therefore, we hypothesized that METH may also damage vascular smooth muscle. We examined the expression of the apoptosis-related proteins Caspase 3 and PARP after METH treatment in vivo and in vitro and detected the expression of endoplasmic reticulum stress-related proteins. After treatment with the endoplasmic reticulum stress inhibitor 4-PBA, changes in the above indicators were examined. C/EBP homologous protein (Chop) expression was also detected, and the relationship between endoplasmic reticulum stress and apoptosis was further determined by siRNA silencing of Chop. The results indicated that METH can induce apoptosis of vascular smooth muscle cells (VSMCs) and upregulate the expression of Chop and endoplasmic reticulum stress-related proteins. Chop inhibits protein kinase B phosphorylation and further inhibits forkhead box class O3a (Foxo3a) dephosphorylation, resulting in increased p53 upregulated molecular of apoptosis (PUMA) transcription. Increased PUMA induces apoptosis through the mitochondrial pathway. These results indicate that Chop is involved in the METH-induced endoplasmic reticulum stress and apoptosis in VSMCs and may be a potential therapeutic target for METH-induced VSMC injury.