Hemochromatosis: Evaluation of the dietary iron model and regulation of hepcidin

Our knowledge of iron homeostasis has increased steadily over the last two decades; much of this has been made possible through the study of animal models of iron-related disease. Analysis of transgenic mice with deletions or perturbations in genes known to be involved in systemic or local regulation of iron metabolism has been particularly informative. The effect of these genes on iron accumulation and hepcidin regulation is traditionally compared with wildtype mice fed a high iron diet, most often a 2% carbonyl iron diet. Recent studies have indicated that a very high iron diet could be detrimental to the health of the mice and could potentially affect homeostasis of other metals, for example zinc and copper. We analyzed mice fed a diet containing either 0.25%, 0.5%, 1% or 2% carbonyl iron for two weeks and compared them with mice on a control diet. Our results indicate that a 0.25% carbonyl iron diet is sufficient to induce maximal hepatic hepcidin response. Importantly these results also demonstrate that in a chronic setting of iron administration, the amount of excess hepatic iron may not further influence hepcidin regulation and that expression of hepcidin plateaus at lower hepatic iron levels. These studies provide further insights into the regulation of this important hormone.     

Endoplasmic reticulum stress response is involved in Mycobacterium tuberculosis protein ESAT-6-mediated apoptosis

Mycobacterium tuberculosis (Mtb) infection leads to the induction of the apoptotic response, which is associated with bacilli killing. The early secreted mycobacterial antigen ESAT-6 of Mtb has been shown to induce apoptosis in human macrophages and epithelial cells. In the present study, we demonstrate that the stimulation of human epithelial A549 cells by ESAT-6 induces the endoplasmic reticulum (ER) stress response. We observed that ESAT-6 treatment increases intracellular Ca²⁺ concentration, which results in ROS accumulation, and therefore induces the onset of ER stress-induced apoptosis. Our results uncover a novel apoptotic mechanism of ESAT-6 through ER stress responses in pathologic conditions such as tuberculosis.     

Protective effect of catechin in type I Gaucher disease cells by reducing endoplasmic reticulum stress

Gaucher disease (GD) is the most common lysosomal storage disorder (LSD) and is divided into three phenotypes, I, II, and III. Type I is the most prevalent form and has its onset in adulthood. The degree of endoplasmic reticulum (ER) stress is one of the factors that determine GD severity. It has recently been reported that antioxidants reduce ER stress and apoptosis by scavenging the oxidants that cause oxidative stress. For this report, we investigated the possibility that catechin can act on type I GD patient cells to alleviate the pathogenic conditions of GD. We treated GD cells with catechin and examined the expression level of GRP78/BiP (an ER stress marker) by western blots and fluorescence microscopy, the proliferation rate of GD cells, and scratch-induced wound healing activity. Our results show that catechin reduces the expression level of GRP78/BiP, leads to cell proliferation rates of GD cells similar levels to normal cells, and improves wound healing activity. We conclude that catechin protects against ER stress in GD cells and catechin-mediated reductions in ER stress may be associated with enhanced cell survival.     

Apoptosis induced by endoplasmic reticulum stress involved in diabetic kidney disease

Endoplasmic reticulum stress has been suggested to play a crucial role in the pathogenesis of diabetic complications. However, whether it is involved in the renal injury of diabetic nephropathy is still not known. We investigated the involvement of ER-associated apoptosis in kidney disease of streptozocin (STZ)-induced diabetic rats. We used albuminuria examination, hematoxylin & eosin (H&E) staining and TUNEL analysis to identify the existence of diabetic nephropathy and enhanced apoptosis. We performed immunohistochemistry, Western blot, and real-time PCR to analyze indicators of ER molecule chaperone and ER-associated apoptosis. GRP78, the ER chaperone, was up-regulated significantly in diabetic kidney compared to control. Furthermore, three hallmarks of ER-associated apoptosis, C/EBP homologous protein (CHOP), c-JUN NH2-terminal kinase (JNK) and caspase-12, were found to have activated in the diabetic kidney. Taken together, those results suggested that apoptosis induced by ER stress occurred in diabetic kidney, which may contribute to the development of diabetic nephropathy.     

神经变性构象病及其分子基础

构象病的概念被广泛用于命名与蛋白质的构象异常相关的疾病。随着生命科学的进步,人们对神经变性疾病发病的分子机制有了较好的认识,发现几乎所有的此类疾病,诸如阿尔采末病（AD）、帕金森病（PD）、亨廷顿病（HD）以及朊蛋白病（PrD）等都具有一个共同的特征,即病变细胞中蓄积有大量错误折叠并易于聚合的蛋白质,这符合构象病的特点,所以又派生了神经变性构象病的新概念。近年来,人们在神经变性构象病的蛋白质错误折叠和聚合以及其细胞毒性方面的认识越来越走向深入,这将对寻找有效的治疗方法起到极大的推动作用。     

Functional polymorphisms of HSPA5: possible association with bipolar disorder

Altered endoplasmic reticulum stress (ER) response signaling is suggested in bipolar disorder. Previously, we preliminarily reported the genetic association of HSPA5 (GRP78/BiP) with bipolar disorder. Here, we extended our analysis by increasing the number of Japanese case-control samples and NIMH Genetics Initiative bipolar trio samples (NIMH trios), and also analyzed schizophrenia samples. In Japanese, nominally significant association of one haplotype was observed in extended samples of bipolar disorder but not in schizophrenia. In NIMH trios, no association was found in total samples. However, an exploratory analysis suggested that the other haplotype was significantly over-transmitted to probands only from the paternal side. The associated haplotype in Japanese or NIMH pedigrees shared three common polymorphisms in the promotor, which was found to alter promotor activity. These findings suggested promotor polymorphisms of HSPA5 may affect the interindividual variability of ER stress response and may confer a genetic risk factor for bipolar disorder.     

Autophagy modulates endoplasmic reticulum stress-induced cell death in podocytes: A protective role

Endoplasmic reticulum stress occurs in a variety of patho-physiological mechanisms and there has been great interest in managing this pathway for the treatment of clinical diseases. Autophagy is closely interconnected with endoplasmic reticulum stress to counteract the possible injurious effects related with the impairment of protein folding. Studies have shown that glomerular podocytes exhibit high rate of autophagy to maintain as terminally differentiated cells. In this study, podocytes were exposed to tunicamycin and thapsigargin to induce endoplasmic reticulum stress. Thapsigargin/tunicamycin treatment induced a significant increase in endoplasmic reticulum stress and of cell death, represented by higher GADD153 and GRP78 expression and propidium iodide flow cytometry, respectively. However, thapsigargin/tunicamycin stimulation also enhanced autophagy development, demonstrated by monodansylcadaverine assay and LC3 conversion. To evaluate the regulatory effects of autophagy on endoplasmic reticulum stress-induced cell death, rapamycin (Rap) or 3-methyladenine (3-MA) was added to enhance or inhibit autophagosome formation. Endoplasmic reticulum stress-induced cell death was decreased at 6 h, but was not reduced at 24 h after Rap+TG or Rap+TM treatment. In contrast, endoplasmic reticulum stress-induced cell death increased at 6 and 24 h after 3-MA+TG or 3-MA+TM treatment. Our study demonstrated that thapsigargin/tunicamycin treatment induced endoplasmic reticulum stress which resulted in podocytes death. Autophagy, which counteracted the induced endoplasmic reticulum stress, was simultaneously enhanced. The salvational role of autophagy was supported by adding Rap/3-MA to mechanistically regulate the expression of autophagy and autophagosome formation. In summary, autophagy helps the podocytes from cell death and may contribute to sustain the longevity as a highly differentiated cell lineage.     

内质网应激／未折叠蛋白反应与非酒精性脂肪性肝病

近年来,由于内质网应激／未折叠蛋白反应可影响物质代谢途径中的许多环节,故在非酒精性脂肪性肝病中起的作用越来越受到重视。现就内质网应激／未折叠蛋白反应在非酒精性脂肪性肝病中的作用和影响作一综述。     

Intracellular calcium dysregulation in autism spectrum disorder: An analysis of converging organelle signaling pathways

Autism spectrum disorder (ASD) is a group of complex, neurological disorders that affect early cognitive, social, and verbal development. Our understanding of ASD has vastly improved with advances in genomic sequencing technology and genetic models that have identified >800 loci with variants that increase susceptibility to ASD. Although these findings have confirmed its high heritability, the underlying mechanisms by which these genes produce the ASD phenotypes have not been defined. Current efforts have begun to “functionalize” many of these variants and envisage how these susceptibility factors converge at key biochemical and biophysical pathways. In this review, we discuss recent work on intracellular calcium signaling in ASD, including our own work, which begins to suggest it as a compelling candidate mechanism in the pathophysiology of autism and a potential therapeutic target. We consider how known variants in the calcium signaling genomic architecture of ASD may exert their deleterious effects along pathways particularly involving organelle dysfunction including the endoplasmic reticulum (ER), a major calcium store, and the mitochondria, a major calcium ion buffer, and theorize how many of these pathways intersect.     

Hereditary hemochromatosis: An update vision of the laboratory diagnosis

Haemochromatosis (HC) is an inherited disorder of iron metabolism. The 85–90% of Hereditary hemochromatosis cases are caused by mutations in HFE gene (HC type 1). The remaining 10–15% of HC cases are caused by mutations in other non-HFE genes (HJV, HAMP, TRF2, SLC40A1, BMP6). The study of patients for the diagnosis of HC has an important laboratory approached: analysis of biochemical parameters and genetic studies. To confirm a case, it is necessary to carry out a genetic study of the C282Y and H63D mutations. The presence of C282Y mutation in homozygosis is compatible with the diagnosis of HC type 1. Due to the incomplete penetrance of this mutation and the variable phenotypic expression, the severe forms of the disease are relatively rare. The study of variants in non-HFE genes allows more detailed study of both non-classic HC cases and those with more severe clinical expression. The genotype characterization of a patient not always justified the phenotype expression of the symptoms in this disease. All laboratory clinicians must consider recommendation provide by the experts in the Materia.     

SelK is a novel ER stress-regulated protein and protects HepG2 cells from ER stress agent-induced apoptosis

Selenoprotein K (SelK), an endoplasmic reticulum (ER) resident protein, its biological function has been less-well studied. To investigate the role of SelK in the ER stress response, effects of SelK gene silence and ER stress agents on expression of SelK and cell apoptosis in HepG2 cells were studied. The results showed that SelK was regulated by ER stress agents, Tunicamycin (Tm) and β-Mercaptoethanol (β-ME), in HepG2 cells. Moreover, the SelK gene silence by RNA interference could significantly aggravate HepG2 cell death and apoptosis induced by the ER stress agents. These results suggest that SelK is an ER stress-regulated protein and plays an important role in protecting HepG2 cells from ER stress agent-induced apoptosis.     

Overexpression of calreticulin sensitizes SERCA2a to oxidative stress

Calreticulin (CRT), a Ca(2+)-binding molecular chaperone in the endoplasmic reticulum, plays a vital role in cardiac physiology and pathology. Oxidative stress is a main cause of myocardiac disorder in the ischemic heart, but the function of CRT under oxidative stress is not fully understood. In this study, the effect of overexpression of CRT on sarcoplasmic/endoplasmic reticulum Ca(2+)-ATPase (SERCA) 2a under oxidative stress was examined using myocardiac H9c2 cells transfected with the CRT gene. The in vitro activity of SERCA2a and uptake of (45)Ca(2+) into isolated microsomes were suppressed by H(2)O(2) in CRT-overexpressing cells compared with controls. Moreover, SERCA2a protein was degraded via a proteasome-dependent pathway following the formation of a complex with CRT under the stress with H(2)O(2). Thus, we conclude that overexpression of CRT enhances the inactivation and degradation of SERCA2a in the cells under oxidative stress, suggesting some pathophysiological functions of CRT in Ca(2+) homeostasis of myocardiac disease.     

抑制或激活泛素-蛋白酶体途径对大鼠肺泡巨噬细胞内质网应激的影响

目的:研究肺泡巨噬细胞(NR8383)不同蛋白酶体激活程度对内质网应激的影响。方法:构建UbG76V-GFP融合蛋白,将含有UbG76V-GFP的质粒导入NR8383细胞,筛选出可稳定表达UbG76V-GFP的细胞系,通过蛋白酶体抑制剂(MG132)、蛋白酶体激活剂(阿霉素)干预蛋白酶体活性。荧光显微镜观察不同蛋白酶体活性下大鼠肺泡巨噬细胞在缺氧复氧2 h、4 h、6 h时蛋白酶体活性,Western blot及PCR技术检测不同蛋白酶体活性下大鼠肺泡巨噬细胞在缺氧复氧2 h、4 h、6 h时泛素化蛋白及内质网应激相关基因的表达。结果:在缺氧复氧2 h、4 h、6 h这3个时间点,加入MG132组大鼠肺泡巨噬细胞绿色荧光及泛素化蛋白(Ubiquitin)表达明显降低(P0.05),而PCR及Western blot示内质网应激基因BIP(免疫球蛋白结合蛋白)、XBP-1(X-盒结合蛋白)和CHOP(C/EBP同源蛋白)平均扩增量及蛋白表达量明显增加(P0.05);加入阿霉素组大鼠肺泡巨噬细胞在缺氧复氧2 h、4h、6 h表现出相反的实验结果,绿色荧光及Ubiquitin蛋白相对表达均明显增加(P0.05),而PCR及Western blot示内质网应激基因BIP、XBP-1和CHOP平均扩增量及蛋白表达量明显增加(P0.05)。结论:本实验结果表明活细胞泛素-蛋白酶体活性程度与内质网应激存在紧密联系,外源性增强泛素蛋白酶体活性会抑制内质网应激,外源性减弱泛素蛋白酶体活性会增强内质网应激。     

内质网应激与肝细胞脂类代谢

肝细胞担负大量的代谢功能,包括脂肪酸的合成与类固醇的代谢。内质网应激反应（ERstressresponse）作为内质网中特殊的机制用以保证内质网内部的稳态和功能正常。有研究指出内质网应激诱导的信号通路及其通路上的关键蛋白参与肝细胞的脂类代谢过程。本文主要讨论内质网应激反应影响肝细胞脂类代谢的机制,以及内质网应激与脂类代谢紊乱疾病的相关性。     

Endoplasmic reticulum stress-mediated apoptosis is activated in vascular calcification

Apoptosis of vascular smooth muscle cells plays an important role in vascular calcification (VC). However, the potential mechanism remains poorly understood. Previous studies showed that apoptosis mediated by endoplasmic reticulum stress (ERS) participates in several diseases with VC. We prepared two rat models of calcification, vitamin D₃ plus nicotine (VDN) and rapid calcification (RC), to investigate whether ERS-mediated apoptosis is activated in VC. TUNEL staining and cleaved caspase 3 protein levels illustrated enhanced apoptosis in calcification groups. Western blot analysis revealed the ERS hallmarks GRP78 and GRP94 increased by 43.9% and 91.7%, respectively, in the VDN group and GRP78 elevated by 84.0% in the RC group (all P < 0.05) as compared with controls. Moreover, two molecules of ERS-induced apoptosis, caspase 12 and C/EBP homologous protein, were up-regulated nearly 3-fold (P < 0.05) in the VDN group and 10-fold (P < 0.01) in the RC group. Our results indicated that ERS-induced apoptosis may be involved in VC, and amelioration of ERS could be a novel strategy to prevent and treat the related diseases.     

The unfolded protein response in human corneal endothelial cells following hypothermic storage: implications of a novel stress pathway

Human corneal endothelial cells (HCEC) have become increasingly important for a range of eye disease treatment therapies. Accordingly, a more detailed understanding of the processing and preservation associated stresses experienced by corneal cells might contribute to improved therapeutic outcomes. To this end, the unfolded protein response (UPR) pathway was investigated as a potential mediator of corneal cell death in response to hypothermic storage. Once preservation-induced failure had begun in HCECs stored at 4 °C, it was noted that necrosis accounted for the majority of cell death but with significant apoptotic involvement, peaking at several hours post-storage (4–8 h). Western blot analysis demonstrated changes associated with apoptotic activation (caspase 9, caspase 3, and PARP cleavage). Further, the activation of the UPR pathway was observed through increased and sustained levels of ER folding and chaperone proteins (Bip, PDI, and ERO1-Lα) in samples experiencing significant cell death. Modulation of the UPR pathway using the specific inhibitor, salubrinal, resulted in a 2-fold increase in cell survival in samples experiencing profound cold-induced failure. Furthermore, this increased cell survival was associated with increased membrane integrity, cell attachment, and decreased necrotic cell death populations. Conversely, addition of the UPR inducer, tunicamycin, during cold exposure resulted in a significant decrease in HCEC survival during the recovery period. These data implicate for the first time that this novel cell stress pathway may be activated in HCEC as a result of the complex stresses associated with hypothermic exposure. The data suggest that the targeted control of the UPR pathway during both processing and preservation protocols may improve cell survival and function of HCEC thus improving the clinical utility of these cells as well as whole human corneas.     

SRO9基因参与调控酿酒酵母内质网应激反应

【目的】研究酵母SRO9基因在内质网应激(Endoplasmic reticulum stress,ERS)中的作用。【方法】利用PCR介导的同源重组方法构建SRO9基因缺失菌株,检测其在内质网应激诱导剂衣霉素处理条件下的克隆形成能力;通过比色法检测细胞内的H2O2含量,超氧化物歧化酶SOD活性和细胞增殖能力;通过实时荧光定量PCR检测内质网应激靶基因和超氧化物歧化酶编码基因SOD1及SOD2的转录水平。【结果】相对于野生型酵母菌株,SRO9基因缺失酵母菌株对内质网应激诱导剂衣霉素的抗性增强,参与内质网应激反应的靶基因转录上调;细胞内H2O2含量下降,SOD1、SOD2转录水平降低,总SOD活性降低;对氧化剂CHP和VK3的抵抗性减弱,复制寿命明显缩短。【结论】SRO9基因缺失酵母细胞对内质网应激诱导剂衣霉素的抗性增强,原因可能是由于SRO9基因缺失激活了细胞的内质网应激反应。     

2-脱氧葡萄糖诱导大鼠内质网应激预处理模型的最佳剂量

目的探讨2-脱氧葡萄糖(2-deoxy-glucose,2-DG)诱导大鼠内质网应激模型的最佳剂量。方法选择Wistar雄性大鼠108只,体质量240～260 g,采用不同剂量2-DG建立大鼠内质网应激的模型,随机分为6组:2-DG 50、100、150、200 mg/kg组,假手术组,缺血再灌注组。2-DG组按工作浓度为50 mg/mL溶于双蒸水腹腔注射7d,假手术组和缺血再灌注组腹腔注射双蒸水7 d,于脑缺血再灌注后12 h处死,对各组大鼠进行神经行为学评分,采用HE染色观察脑组织的病理形态,免疫组化法和Westernblot法测定GRP78蛋白的表达,用PCR法检测GRP78mRNA的表达。结果与脑缺血再灌注组相比,2-DG各剂量组大鼠的神经行为学评分明显减低(P<0.01),而以100 mg/kg组评分减低最为明显(P<0.05);2-DG各剂量组能不同程度的改善大鼠脑海马CA1区神经细胞的核深染、核固缩程度,减少细胞及间质的水肿,使胞膜趋于清楚、形态接近正常、核仁清晰可见的神经细胞数目增多,而以2-DG100 mg/kg组的效果最为明显。2-DG各剂量组GRP78蛋白表达明显增加,与脑缺血再灌注组比较,差异有显著性(P<0.01),而以100 mg/kg剂量组的GRP78蛋白表达最高,与其他2-DG剂量组比较差异有显著性(P<0.05)。结论 2-DG对脑缺血再灌注所致的神经细胞损伤具有保护作用,其最佳剂量为100 mg/kg。2-DG具有诱导大鼠内质网应激的作用,其最佳剂量是100 mg/kg。