Basal autophagy maintains pancreatic acinar cell homeostasis and protein synthesis and prevents ER stress

Pancreatic acinar cells possess very high protein synthetic rates as they need to produce and secrete large amounts of digestive enzymes. Acinar cell damage and dysfunction cause malnutrition and pancreatitis, and inflammation of the exocrine pancreas that promotes development of pancreatic ductal adenocarcinoma (PDAC), a deadly pancreatic neoplasm. The cellular and molecular mechanisms that maintain acinar cell function and whose dysregulation can lead to tissue damage and chronic pancreatitis are poorly understood. It was suggested that autophagy, the principal cellular degradative pathway, is impaired in pancreatitis, but it is unknown whether impaired autophagy is a cause or a consequence of pancreatitis. To address this question, we generated Atg7^Δpan mice that lack the essential autophagy-related protein 7 (ATG7) in pancreatic epithelial cells. Atg7^Δpan mice exhibit severe acinar cell degeneration, leading to pancreatic inflammation and extensive fibrosis. Whereas ATG7 loss leads to the expected decrease in autophagic flux, it also results in endoplasmic reticulum (ER) stress, accumulation of dysfunctional mitochondria, oxidative stress, activation of AMPK, and a marked decrease in protein synthetic capacity that is accompanied by loss of rough ER. Atg7^Δpan mice also exhibit spontaneous activation of regenerative mechanisms that initiate acinar-to-ductal metaplasia (ADM), a process that replaces damaged acinar cells with duct-like structures.The pancreatic acinar cell is responsible for production and secretion of numerous digestive enzymes, including amylase, lipase, and various proteases. To cope with the high daily demand for these enzymes, the acinar cell possesses one of the highest protein biosynthetic rates of all cells, together with an extensive rough endoplasmic reticulum (RER) network (1). Due to its high protein synthetic rates, the acinar cell is prone to the accumulation of misfolded proteins and subsequent induction of ER stress (2, 3). ER stress was suggested to be involved in the pathogenesis of pancreatitis, a potentially fatal inflammatory disease of the exocrine pancreas (2, 4). By progressing from acute (sudden onset; duration <6 mo), to recurrent acute (>1 episode of acute pancreatitis), and chronic (duration >6 mo) disease (5), pancreatitis increases the risk of pancreatic ductal adenocarcinoma (PDAC), the fourth deadliest cancer worldwide, with a median survival of 6 mo (6). The molecular mechanisms mediating the progression of pancreatitis from acinar cell damage and inflammation to formation of pancreatic intraepithelial neoplasia (PanIN) and PDAC are not fully understood. Recent studies suggest that in addition to ER stress, insufficient autophagy also contributes to development of pancreatitis (7).Autophagy is an evolutionarily conserved, catabolic quality control process that maintains cellular homeostasis by degrading damaged organelles, misfolded protein aggregates, and foreign organisms (8). Autophagy is also important for generation of amino acids and other building blocks during starvation (9). There are three classes of autophagy: macroautophagy, microautophagy, and chaperone-mediated autophagy (9). Macroautophagy, the major type of autophagy (hereafter referred to as autophagy), entails formation of double-membrane vesicles (autophagosomes) that sequester damaged organelles and biomolecules and recycle them after transport into lysosomes, where they are degraded. The rate of autophagy is increased in response to diverse stress conditions, including nutrient deprivation, viral infection, and genotoxic stress. In this way, autophagy controls the cross-talk between the intracellular demand for energy, building blocks, and external stimuli (9). Autophagy is critically involved in mammalian development, cell survival, and longevity (10), and its impairment correlates with many pathological conditions (11), including pancreatitis (7). Notably, the mammalian exocrine pancreas exhibits a higher autophagy rate (or autophagic flux) than the liver, kidney, heart, or endocrine pancreas (12), underscoring the likely importance of autophagy in maintaining acinar cell homeostasis and function. Until recently, however, the role of autophagy in pancreatitis has been controversial. On one hand, the genetic inhibition of autophagy [Autophagy Related 5 (Atg5) gene ablation] was found to reduce trypsinogen activation and attenuate pancreatic damage in mice challenged with the pancreatic enzyme secretagogue cerulein (13); but selective and protective autophagy can sequester and degrade potentially deleterious activated zymogens during early pancreatitis (14). More recent findings from experimental models (cerulein-induced pancreatitis), and genetically altered mice (Pdx-Cre; Ikkα^F/F, also known as Ikkα^Δpan, and Ptf1a-Cre; Atg5^F/F) have demonstrated that insufficient autophagy, at least in mice, can lead to the onset of pancreatitis (15–17). The mechanisms by which disruption of autophagy triggers pancreatitis are poorly understood.Degradation of long-lived proteins, a major function of autophagy, is impaired during experimental pancreatitis, especially after administration of cerulein, which causes acinar cell vacuolization and excessive trypsinogen activation (18). Moreover, autophagic flux is reduced during pancreatitis due to defective cathepsin-mediated processing of lysosomal proteases (18). Similarly, pancreas-specific ablation of inhibitor of IκB kinase (IKK)α results in acinar damage ranging from vacuole accumulation to chronic pancreatitis (15). IKKα deficiency impairs the completion of autophagy in acinar cells, with accumulation of the chaperon and autophagy substrate ubiquitin-binding protein p62/SQSTM1 as the key pathogenic mechanism (15). However, ablation of ATG5 was reported to either inhibit (13) or promote (16) pancreatitis. In addition, inhibition of autophagy can either accelerate the development of early malignant lesions in mice lacking the transformation-related protein 53 (p53) (19) or cause the death of established pancreatic cancer (20). The latter results gave rise to several ongoing clinical trials (clinicaltrials.gov numbers {"type":"clinical-trial","attrs":{"text":"NCT01494155","term_id":"NCT01494155"}}NCT01494155, {"type":"clinical-trial","attrs":{"text":"NCT01978184","term_id":"NCT01978184"}}NCT01978184, {"type":"clinical-trial","attrs":{"text":"NCT01506973","term_id":"NCT01506973"}}NCT01506973, {"type":"clinical-trial","attrs":{"text":"NCT01128296","term_id":"NCT01128296"}}NCT01128296, and {"type":"clinical-trial","attrs":{"text":"NCT01273805","term_id":"NCT01273805"}}NCT01273805) that intend to evaluate the impact of autophagy inhibitors on human pancreatic cancer. However, a recent commentary has raised concerns about the safety of this therapeutic approach (21). Given all of these questions, we decided to take a closer look at the impact of autophagy inhibition on pancreatic health and function by generating mice that lack autophagy-related protein 7 (ATG7) in pancreatic epithelial cells. These mice, termed Atg7^Δpan, exhibit striking acinar cell degeneration, which is followed by pronounced pancreatic inflammation and fibrosis. Whereas loss of ATG7 leads to the expected decrease in autophagic flux, it also results in ER stress, accumulation of dysfunctional mitochondria, oxidative stress, and a marked reduction in protein synthetic ability.     

Association analysis of single nucleotide polymorphisms in autophagy related 7 (ATG7) gene in patients with coronary artery disease

Recent experimental studies sparked the involvement of autophagy-related 7 (ATG7) in the development of atherosclerosis. However, the genetic variants and their association with coronary artery disease (CAD) are still to be unveiled. Therefore, we aimed to design a retrospective case-control study for the analysis of ATG7 gene polymorphisms and their association with CAD among the subjects originating from Pakistan.The ATG7 noncoding polymorphisms (rs1375206; Chr3:11297643 C/G and rs550744886; Chr3:11272004 C/G) were examined in 600 subjects, including 300 individuals diagnosed with CAD. Arginase-1 (ARG1) and nitric oxide metabolites were measured by the colorimetric enzymatic assay. Genotyping of noncoding ATG7 polymorphisms was accomplished by the polymerase chain reaction–restriction fragment length polymorphism method.A significant association of ATG7 (rs1375206 and rs550744886) was observed in individuals exhibiting CAD (P < .0001, for each single-nucleotide polymorphism). Moreover, variant allele G at both loci showed high occurrence and significant association with the disease phenotype as compared to the wild-type allele (odds ratio [OR] = 2.03, P < .0001 and OR = 2.08, P < .001, respectively). Variant genotypes at ATG7 rs1375206 and rs550744886 showed significant association with high concentrations of ARG1 and low nitric oxide metabolites among the patients (P < .0001 for each). A significant difference was noted in the distribution of the haplotype G-G, mapped at Chr3:11297643-11272004 between cases and controls (P < .0001).The study concludes that ATG7 polymorphisms are among the risk factors for CAD in the subjects from Pakistan. The study thus highlights the novel risk factors for high incidents of the disease and reported for the first time to the best of our knowledge.     

ATG7 is a haploinsufficient repressor of tumor progression and promoter of metastasis

The role of autophagy in cancer is complex. Both tumor-promoting and tumor-suppressive effects are reported, with tumor type, stage and specific genetic lesions dictating the role. This calls for analysis in models that best recapitulate each tumor type, from initiation to metastatic disease, to specifically understand the contribution of autophagy in each context. Here, we report the effects of deleting the essential autophagy gene Atg7 in a model of pancreatic ductal adenocarcinoma (PDAC), in which mutant Kras^G12D and mutant Trp53^172H are induced in adult tissue leading to metastatic PDAC. This revealed that Atg7 loss in the presence of Kras^G12D/+ and Trp53^172H/+ was tumor promoting, similar to previous observations in tumors driven by embryonic Kras^G12D/+ and deletion of Trp53. However, Atg7 hemizygosity also enhanced tumor initiation and progression, even though this did not ablate autophagy. Moreover, despite this enhanced progression, fewer Atg7 hemizygous mice had metastases compared with animals wild type for this allele, indicating that ATG7 is a promoter of metastasis. We show, in addition, that Atg7^+/− tumors have comparatively lower levels of succinate, and that cells derived from Atg7^+/− tumors are also less invasive than those from Atg7^+/+ tumors. This effect on invasion can be rescued by ectopic expression of Atg7 in Atg7^+/− cells, without affecting the autophagic capacity of the cells, or by treatment with a cell-permeable analog of succinate. These findings therefore show that ATG7 has roles in invasion and metastasis that are not related to the role of the protein in the regulation of autophagy.

The preservation of cellular integrity and the ability to adapt to different forms of cellular stress are key in the prevention of various forms of disease. Autophagy constitutes a group of processes that facilitates these goals by delivering cellular constituents to lysosomes for degradation and recycling (1). The cargoes destined for degradation can either be targeted specifically to promote cellular health and integrity, or simply digested nonselectively in response to cellular cues such as nutrient deprivation or tissue remodeling (1).The best characterized form of autophagy is macroautophagy, which is often and hereafter referred to more simply as autophagy. Autophagy involves a number of evolutionarily conserved ATG proteins that orchestrate the formation of a double-membraned structure called an autophagosome which sequesters cellular cargo (2, 3). Autophagosomes then traffic to and fuse with lysosomes to form another structure termed the autolysosome, within which degradation occurs (1). The ability to understand the function of autophagy has been facilitated by the identification that a number of ATG proteins, including ATG7 and ATG5, are essential for autophagosome formation, and mice containing floxed alleles for the genes encoding these proteins have been fundamental in understanding the role of autophagy in various forms of mammalian physiology and disease (2–5).It is now clear that autophagy has a major role in both preventing and sustaining cancer (6). Several autophagy genes have been shown to be mutated in major forms of cancer, and mice lacking autophagy genes have been shown to be tumor prone (7–9). Studies have indicated, however, that the role of autophagy in cancer can depend on tumor type, the stage of tumor development, and the specific genetic mutations associated with disease progression (10–13). The current consensus is that autophagy has a tumor suppressive role before or in the early stages of tumor development when protecting cellular fidelity is fundamental in preventing cancer initiation and early progression (6, 14). In established tumors, however, autophagy generally appears to have a tumor supportive role (6, 14). Within the highly stressed environment of an established tumor, it is considered that autophagy mitigates this stress to permit tumor cell survival under these conditions.A tumor type in which autophagy has been studied extensively is pancreatic ductal adenocarcinoma (PDAC). Using a genetically engineered mouse model (GEMM) of PDAC, it was reported that PDAC development driven by mutant KRAS was dependent on autophagy (10, 15, 16). This was, however, found to be different in PDAC driven by KRAS and the absence of p53 in which the loss of autophagy was tumor-promoting (10). In contrast to these findings, studies of patient-derived xenografts (PDX) formed in immuno-compromised mice indicated that inhibition of autophagy impaired tumor formation irrespective of p53 status (16). In human PDAC when p53 is perturbed, the TP53 gene is usually mutated and retained rather than deleted/silenced, and the genetic lesions associated with the disease occur at focal sites within the adult pancreas rather than being expressed from embryonic development as is the case with most GEMM models (17). We therefore decided to analyze autophagy—by conditional deletion of the essential autophagy gene Atg7—in a mouse model of PDAC involving alleles for mutant Kras^G12D and mutant Trp53^172H, which are recombined in the adult using an inducible Cre recombinase driven by the pancreas selective promoter Pdx1. These mice undergo the full spectrum of tumor development from initiation to metastatic disease and our studies using these animals provide further insights into the relationship between Trp53 and loss of Atg7 and also highlight disparate roles for ATG7 and autophagy during tumor development.     

内质网应激与衰老相关性房颤的研究进展

心房颤动（房颤）是临床上最常见的心律失常之一,表现为心房重构和收缩功能障碍,且其发病率随年龄增长而增加。近年来的研究显示,内质网应激在房颤的发生发展、病理生理过程中扮演着重要角色,内质网应激是指蛋白质分泌增加或内质网蛋白折叠中断可导致内质网腔内未折叠或错误折叠的蛋白质积累,有效的内质网应激可以延缓心肌细胞的衰老,阻止房颤的发生发展。     

The Emerging Roles of Viroporins in ER Stress Response and Autophagy Induction during Virus Infection

Viroporins are small hydrophobic viral proteins that oligomerize to form aqueous pores on cellular membranes. Studies in recent years have demonstrated that viroporins serve important functions during virus replication and contribute to viral pathogenicity. A number of viroporins have also been shown to localize to the endoplasmic reticulum (ER) and/or its associated membranous organelles. In fact, replication of most RNA viruses is closely linked to the ER, and has been found to cause ER stress in the infected cells. On the other hand, autophagy is an evolutionarily conserved “self-eating” mechanism that is also observed in cells infected with RNA viruses. Both ER stress and autophagy are also known to modulate a wide variety of signaling pathways including pro-inflammatory and innate immune response, thereby constituting a major aspect of host-virus interactions. In this review, the potential involvement of viroporins in virus-induced ER stress and autophagy will be discussed.     

The role of autophagy in anticancer therapy: promises and uncertainties

Autophagy (literally self-eating) is a catabolic mechanism involved in the recycling and turnover of cytoplasmic constituents. Although often referred to as type II programmed cell death, autophagy is primarily a survival rather than a cell death mechanism in response to different stress stimuli. Autophagy is a process in which part of the cytoplasm or entire organelles are sequestered into double-membrane vesicles, called autophagosomes, which ultimately fuse with lysosomes to degrade their contents. Studies show that autophagy is associated with a number of pathological conditions, including cancer, infectious diseases, myopathies and neurodegenerative disorders. With respect to cancer, it has been suggested that the early stages of tumourigenesis are associated with downregulation of autophagy-related (ATG) genes. Indeed, several ATG genes display tumour suppressor function, including Beclin1, which is frequently hemizygously deleted in breast cancer cells. Conversely, in advanced stages of tumourigenesis or during anticancer therapy, autophagy may promote survival of tumour cells in adverse environmental conditions. Therefore, a thorough understanding of autophagy in different cancer types and stages is a prerequisite to determine an autophagy-activating or autophagy-inhibiting treatment strategy.     

Melatonin enhances the occurrence of autophagy induced by oxidative stress in Arabidopsis seedlings

The beneficial effect that melatonin has against mitochondrial dysfunctioning seems to be linked to mitophagy. Roles for melatonin have been demonstrated in promoting health and preventing disease, as well as activating the process of autophagy in general. However, no reports have been made about how the application of melatonin regulates that process when plants are exposed to oxidative stress. We investigated the influence of different concentrations of melatonin (0.0, 0.5, 5.0, 10.0, or 50.0 μm ) on autophagy under methyl viologen (MV)‐induced oxidative stress. Arabidopsis seedlings that were pretreated with 5 or 10 μm melatonin underwent relatively strong induction of autophagy, as evidenced by the number of monodansylcadaverine (MDC)‐stained autophagosomes in root samples. Pretreatment with 10 μm melatonin also alleviated MV‐induced photo‐oxidation damage and significantly reduced the accumulation of oxidized proteins. Those responses might have been due to the strong upregulation of genes that involved in AtATG8‐PE conjugation pathway, which enhanced the capacity for autophagy. Histochemical staining revealed that both and H₂O₂ were highly accumulated upon MV exposure, although the response did not differ significantly between control and melatonin‐pretreated seedlings. By contrast, exogenous melatonin upregulated the expression of two genes for H₂O₂‐scavenging enzymes, that is, AtAPX1 and AtCATs. The activation of autophagy by melatonin without an alteration in ROS production may be part of a survival mechanism that is enhanced by melatonin after cellular damage. Therefore, it represents a second level of defense to remove damaged proteins when antioxidant activities are compromised.     

氧化应激与自噬在肝细胞癌发生发展中的作用

肝细胞癌(HCC)是肝脏的原发恶性肿瘤,其发生过程十分复杂,氧化应激学说是其发生发展机制研究的众多重要学说之一。自噬是细胞清除胞内错误折叠的蛋白质或受损细胞器,维持内环境稳态的重要方式。越来越多的证据显示自噬在肝纤维化和HCC中发挥重要作用,且与氧化应激有密切关系。结合当前国内外最新研究成果,分别从自噬与HCC的关系、氧化应激与HCC的关系来分析二者在HCC发病机制中的相互作用。指出自噬在HCC发病进程中调控氧化应激的分子机制可能成为今后的研究热点,若能够激活或阻断自噬调控氧化应激的某个关键通路,或可为HCC的早期诊断及治疗提供新的手段。     

Differential activation of myocardial ER stress response: A possible role in hypoxic tolerance

Background

Low oxygen availability in the high altitude milieu causes adverse physiological and pathological consequences to the cardiopulmonary system. A key role is played by proteins in maintaining optimal cardiac function under stress. Differential response to hypoxia may be linked to the susceptibility of proteins to free radical induced modifications. The present study was designed to understand the significance of protein oxidation and ER stress in the myocardial response to hostile environments.

Methods

Sprague–Dawley rats were exposed to simulated hypoxia equivalent to 223 mm Hg pressure, screened on the basis of time taken for onset of a characteristic hyperventilatory response and categorized as susceptible (< 10 min), normal (10–25 min) or tolerant (> 25 min). Protein modifications and activity of cellular proteolytic enzymes were assayed in myocardial tissue extracts to identify alterations in protein homeostasis. To evaluate the ER stress response, expression of various ER marker chaperones was investigated.

Results

Susceptible animals displayed a distinct increase in protein oxidation and intracellular thiol content. They showed higher expression of ER stress hallmarks, GRP78, PDI and ERO1α, and exhibited a greater activation of the proteasome and calpain proteolytic systems, associated with elevated oxidized proteins. While a marked upregulation in the prosurvival signaling cascade PI3K/Akt/mTOR was observed in tolerant animals, the expression of pro-apoptotic caspase-3 and CHOP remained unaltered.

Conclusion

Thus, higher susceptibility to hypoxia is linked to a disruption in the proteostasis and activation of the ER stress response. Enhanced tolerance to hostile environments may be contributed by better maintenance of protein folding homeostasis.     

Cardiac overexpression of metallothionein rescues cardiac contractile dysfunction and endoplasmic reticulum stress but not autophagy in sepsis

Sepsis is characterized by systematic inflammation where oxidative damage plays a key role in organ failure. This study was designed to examine the impact of the antioxidant metallothionein (MT) on lipopolysaccharide (LPS)-induced cardiac contractile and intracellular Ca²⁺ dysfunction, oxidative stress, endoplasmic reticulum (ER) stress and autophagy. Mechanical and intracellular Ca²⁺ properties were examined in hearts from FVB and cardiac-specific MT overexpression mice treated with LPS. Oxidative stress, activation of mitogen-activated protein kinase pathways (ERK, JNK and p38), ER stress, autophagy and inflammatory markers iNOS and TNFα were evaluated. Our data revealed enlarged end systolic diameter, decreased fractional shortening, myocyte peak shortening and maximal velocity of shortening/relengthening as well as prolonged duration of relengthening in LPS-treated FVB mice associated with reduced intracellular Ca²⁺ release and decay. LPS treatment promoted oxidative stress (reduced glutathione/glutathione disulfide ratio and ROS generation). Western blot analysis revealed greater iNOS and TNFα, activation of ERK, JNK and p38, upregulation of ER stress markers GRP78, Gadd153, PERK and IRE1α, as well as the autophagy markers Beclin-1, LCB3 and Atg7 in LPS-treated mouse hearts without any change in total ERK, JNK and p38. Interestingly, these LPS-induced changes in echocardiographic, cardiomyocyte mechanical and intracellular Ca²⁺ properties, ROS, stress signaling and ER stress (but not autophagy, iNOS and TNFα) were ablated by MT. Antioxidant N-acetylcysteine and the ER stress inhibitor tauroursodeoxycholic acid reversed LPS-elicited depression in cardiomyocyte contractile function. LPS activated AMPK and its downstream signaling ACC in conjunction with an elevated AMP/ATP ratio, which was unaffected by MT. Taken together, our data favor a beneficial effect of MT in the management of cardiac dysfunction in sepsis.     

Melatonin reduces endoplasmic reticulum stress and autophagy in liver of leptin‐deficient mice

The sedentary lifestyle of modern society along with the high intake of energetic food has made obesity a current worldwide health problem. Despite great efforts to study the obesity and its related diseases, the mechanisms underlying the development of these diseases are not well understood. Therefore, identifying novel strategies to slow the progression of these diseases is urgently needed. Experimental observations indicate that melatonin has an important role in energy metabolism and cell signalling; thus, the use of this molecule may counteract the pathologies of obesity. In this study, wild‐type and obese (ob/ob) mice received daily intraperitoneal injections of melatonin at a dose of 500 μg/kg body weight for 4 weeks, and the livers of these mice were used to evaluate the oxidative stress status, proteolytic (autophagy and proteasome) activity, unfolded protein response, inflammation and insulin signalling. Our results show, for the first time, that melatonin could significantly reduce endoplasmic reticulum stress in leptin‐deficient obese animals and ameliorate several symptoms that characterize this disease. Our study supports the potential of melatonin as a therapeutic treatment for the most common type of obesity and its liver‐associated disorders.     

AMPK involvement in endoplasmic reticulum stress and autophagy modulation after fatty liver graft preservation: a role for melatonin and trimetazidine cocktail

Ischemia/reperfusion injury (IRI) associated with liver transplantation plays an important role in the induction of graft injury. Prolonged cold storage remains a risk factor for liver graft outcome, especially when steatosis is present. Steatotic livers exhibit exacerbated endoplasmic reticulum (ER) stress that occurs in response to cold IRI. In addition, a defective liver autophagy correlates well with liver damage. Here, we evaluated the combined effect of melatonin and trimetazidine as additives to IGL‐1 solution in the modulation of ER stress and autophagy in steatotic liver grafts through activation of AMPK. Steatotic livers were preserved for 24 hr (4°C) in UW or IGL‐1 solutions with or without MEL + TMZ and subjected to 2‐hr reperfusion (37°C). We assessed hepatic injury (ALT and AST) and function (bile production). We evaluated ER stress (GRP78, PERK, and CHOP) and autophagy (beclin‐1, ATG7, LC3B, and P62). Steatotic livers preserved in IGL‐1 + MEL + TMZ showed lower injury and better function as compared to those preserved in IGL‐1 alone. IGL‐1 + MEL + TMZ induced a significant decrease in GRP78, pPERK, and CHOP activation after reperfusion. This was consistent with a major activation of autophagic parameters (beclin‐1, ATG7, and LC3B) and AMPK phosphorylation. The inhibition of AMPK induced an increase in ER stress and a significant reduction in autophagy. These data confirm the close relationship between AMPK activation and ER stress and autophagy after cold IRI. The addition of melatonin and TMZ to IGL‐1 solution improved steatotic liver graft preservation through AMPK activation, which reduces ER stress and increases autophagy.     

内质网应激与自噬的交互作用对血管钙化的影响

目的证实内质网应激(ERS)和自噬的交互作用对血管钙化(VC)的影响。方法维生素D3肌注和尼古丁灌胃制备大鼠在体血管钙化模型,取主动脉行茜素红染色和钙含量检测,Western blot检测相关蛋白的表达水平。结果与对照组相比,钙化组大鼠主动脉管壁钙沉积显著增加,血管平滑肌细胞(VSMC)收缩表型标志蛋白SM-22α和Calponin表达显著降低,而成骨细胞样表型标志蛋白骨形态发生蛋白2(BMP-2)和Runt相关转录因子2(RUNX2)表达显著升高,ERS标志蛋白葡萄糖调节蛋白(GRP78)和C/EBP同源蛋白(CHOP)以及自噬标志蛋白轻链3(LC3Ⅱ)和Beclin-1表达显著升高。钙化大鼠应用ERS激动剂衣霉素[10μg/(kg·d)]可进一步增加血管壁钙沉积及BMP-2和RUNX2表达水平,而SM-22α和Calponin表达进一步减少,GRP78和CHOP以及LC3Ⅱ和Beclin-1表达水平进一步增加。钙化大鼠应用自噬抑制剂3-甲基腺嘌呤[10 mg/(kg·d)]可降低LC3Ⅱ和Beclin-1水平,同时GRP78和CHOP表达升高,增加血管壁钙沉积及BMP-2和RUNX2表达水平,降低SM-22α和Calponin表达。结论内质网应激与自噬的交互作用影响血管钙化的发展。     

硫化氢对大鼠肝星状细胞增殖及Ca2+浓度的影响

目的 探讨硫化氢(H2S)对大鼠原代肝星状细胞(HsC)增殖及Ca2+浓度的影响及其作用机制.方法 大鼠肝星状原代细胞作为研究对象,将过氧化氢(H2O2)作用于大鼠原代HSC制造肝纤维化的氧化应激模型,用钙离子荧光探针Fluo-3/AM负载细胞,并在此基础上应用不同剂量的NaSH(H2S供体)和KATP通道抑制剂(格列本脲)对各组细胞进行干预,用激光扫描共聚焦显微镜(LSCM)和CCK-8的方法分别检测不同刺激条件对细胞内Ca2+浓度改变及细胞增殖情况的影响.结果 低浓度H2S(100μmo/L NaSH)明显降低HSC细胞内Ca2+浓度(P＜0.05),抑制细胞增殖;K离子通道阻断剂——格列本脲可阻断H2S的作用.高浓度H2S(1mmol/L NaSH)使HSC细胞内Ca2+浓度增加,促进细胞增殖.结论 低浓度H2S通过激活HSC细胞KATP通道,降低细胞内Ca2+浓度,从而抑制细胞增殖;高浓度H2S使HSC细胞内Ca2+浓度增加,促进细胞增殖.     

线粒体相关内质网膜:心血管疾病治疗的新靶点

线粒体相关内质网膜是指内质网和线粒体之间高度动态的紧密连接部分,参与维持内质网和线粒体的正常功能,与细胞脂质代谢、钙稳态、线粒体动力学、自噬和凋亡、内质网应激和炎症等密切相关。研究显示线粒体相关内质网膜功能异常或者数量和结构改变参与心血管疾病的发生发展。本文总结了线粒体相关内质网膜的功能,阐述了其在心血管疾病中的作用及可能机制,为线粒体相关内质网膜成为心血管疾病治疗的新靶点提供理论参考。     

ER stress stimulates production of the key antimicrobial peptide,cathelicidin, by forming a previously unidentified intracellular S1P signaling complex

We recently identified a previously unidentified sphingosine-1-phosphate (S1P) signaling mechanism that stimulates production of a key innate immune element, cathelicidin antimicrobial peptide (CAMP), in mammalian cells exposed to external perturbations, such as UVB irradiation and other oxidative stressors that provoke subapoptotic levels of endoplasmic reticulum (ER) stress, independent of the well-known vitamin D receptor-dependent mechanism. ER stress increases cellular ceramide and one of its distal metabolites, S1P, which activates NF-κB followed by C/EBPα activation, leading to CAMP production, but in a S1P receptor-independent fashion. We now show that S1P activates NF-κB through formation of a previously unidentified signaling complex, consisting of S1P, TRAF2, and RIP1 that further associates with three stress-responsive proteins; i.e., heat shock proteins (GRP94 and HSP90α) and IRE1α. S1P specifically interacts with the N-terminal domain of heat shock proteins. Because this ER stress-initiated mechanism is operative in both epithelial cells and macrophages, it appears to be a universal, highly conserved response, broadly protective against diverse external perturbations that lead to increased ER stress. Finally, these studies further illuminate how ER stress and S1P orchestrate critical stress-specific signals that regulate production of one protective response by stimulating production of the key innate immune element, CAMP.Mammalian epithelial tissues face hostile external environments, where they are repeatedly bombarded by external perturbants, such as UV irradiation, oxidative stress and microbial pathogens that potentially threaten the integrity of epithelial and nonepithelial tissues/cells. Antimicrobial peptides (AMPs) represent highly conserved, innate immune elements that protect the host from microbial pathogens, while also signaling a variety of downstream responses that further fortify innate and adaptive immunity (1, 2). We have shown that AMP production increases not only in response to microbial challenges, but also in response to a wide variety of other external perturbations that converge on the endoplasmic reticulum (ER), where AMP orchestrate a host of ER-initiated stress responses (3). Although high doses of external perturbants can cause excessive ER stress, leading to increased production of the proapoptotic lipid ceramide, threatening cells with apoptosis (4, 5), subtoxic levels of the same perturbants instead provoke lower levels of ER stress, with incrementally reduced ceramide production that rescues cells from apoptosis in part through metabolic conversion of ceramide to sphingosine-1-phosphate (S1P) (6). S1P in turn stimulates production of the key AMP, cathelicidin AMP (CAMP), and its downstream proteolytic product, LL-37, through transactivation of NF-κB–C/EBPα (7). This mechanism operates independently of the well-known vitamin D receptor-dependent mechanism, which instead regulates CAMP synthesis under basal conditions (3, 7). However, CAMP production is not only always beneficial: Although transient increases defend against microbial pathogens, sustained production of this AMP can stimulate downstream inflammatory responses, and even tumorigenesis (1).S1P modulates a variety of cellular functions (e.g., cell proliferation, differentiation and motility) through well-known G protein coupled, S1P receptor-dependent mechanisms (8). However, we showed recently that ER stress-stimulated CAMP production is likely receptor-independent (7). Although prior studies showed that ER stress activates NF-κB via plasma membrane-localized S1P1, S1P2, and S1P3 receptor activation (9, 10), using specific activators/agonists and inhibitors/antagonists of all five identified S1P receptors (S1P1–S1P5), we showed that these pharmacological interventions did not modify the ER stress-induced increase in CAMP expression (7). Although prior study shows that TNFα receptor activation can initiate S1P binding to TRAF2 on plasma membrane, forming a signaling complex (S1P–TRAF2–TRADD–RIP1) that activates NF-κB (11), this mechanism does not appear to regulate CAMP synthesis. Instead, we identify and delineate a previously unidentified TNFα receptor- and a S1P receptor-independent mechanism that regulate CAMP production through intracellular assembly of a S1P–TRAF2-stress-responsive protein signaling complex that forms in response to ER stress. Elucidation of this previously unidentified regulatory mechanism could point to potentially previously undeveloped therapeutic approaches to either enhance innate immunity or to suppress excessive CAMP production causing inflammation and tumorigenesis.