Reduced endoplasmic reticulum luminal calcium links saturated fatty acid-mediated endoplasmic reticulum stress and cell death in liver cells

Chronic exposure to elevated free fatty acids, in particular long chain saturated fatty acids, provokes endoplasmic reticulum (ER) stress and cell death in a number of cell types. The perturbations to the ER that instigate ER stress and activation of the unfolded protein in response to fatty acids in hepatocytes have not been identified. The present study employed H4IIE liver cells and primary rat hepatocytes to examine the hypothesis that saturated fatty acids induce ER stress via effects on ER luminal calcium stores. Exposure of H4IIE liver cells and primary hepatocytes to palmitate and stearate reduced thapsigargin-sensitive calcium stores and increased biochemical markers of ER stress over similar time courses (6 h). These changes preceded cell death, which was only observed at later time points (16 h). Co-incubation with oleate prevented the reduction in calcium stores, induction of ER stress markers and cell death observed in response to palmitate. Inclusion of calcium chelators, BAPTA-AM or EGTA, reduced palmitate- and stearate-mediated enrichment of cytochrome c in post-mitochondrial supernatant fractions and cell death. These data suggest that redistribution of ER luminal calcium contributes to long chain saturated fatty acid-mediated ER stress and cell death.     

疱疹病毒与内质网应激

内质网是分泌型蛋白和膜蛋白折叠及翻译后修饰的主要场所.病毒感染所引起的宿主细胞内环境的改变可使细胞或病毒的未折叠和/或错误折叠蛋白在内质网中大量聚集,使内质网处于生理功能紊乱的应激状态.为了缓解这种应激压力,细胞会启动未折叠蛋白反应(UPR),并通过一系列分子的信号转导维持内质网稳态;同时病毒也会通过对UPR的精密调控...     

Mitochondria and endoplasmic reticulum: mitochondria-endoplasmic reticulum interplay in type 2 diabetes pathophysiology

Mitochondria and endoplasmic reticulum (ER) are two important metabolic organelles for the maintenance of cellular homeostasis and their functional defects are suspected to participate to the aetiology of type 2 diabetes (T2D). Particularly, excessive lipid intake and/or ectopic lipid accumulation in tissues (referred as lipotoxicity) are involved in alterations of both organelles and are closely linked to peripheral insulin resistance and defective insulin secretion. Since, mitochondria and ER are physically and functionally interconnected, their respective alterations during T2D could be interrelated. However, the mechanisms that coordinate the interplay between mitochondrial dysfunction and ER stress, and its relevance in the control of glucose homeostasis are unknown. Among these mechanisms, we will discuss on the potential role of altered mitochondria/ER crosstalk in organelle dysfunctions and in T2D pathophysiology.     

The endoplasmic reticulum and calcium storage

Calcium storage is one of the functions commonly attributed to the endoplasmic reticulum (ER) in nonmuscle cells. Several recent studies have added support to this concept. Analysis of reticuloplasm, the luminal ER content, has shown that it contains several proteins (reticuloplasmins) which are prospective calcium storage proteins. One of these, calreticulin, is also present in the sarcoplasmic reticulum (SR). In sea urchin eggs, a calsequestrin-like protein has been clearly localised to the ER. The recent demonstration that the IP3 receptor, which has similarities with the calcium release channel in the SR is also localised in the ER membrane suggests that calcium stored in the ER is important for intracellular signalling. The alternative view, that the physiologically important calcium store is a specialised organelle, the calciosome, is not supported by these observations. Recent evidence also suggests that ER calcium might be important in ER structure and in the retention of the luminal ER proteins.     

Depletion of oxidative and endoplasmic reticulum stress regulators in Pick disease

Both oxidative and endoplasmic reticulum (ER) stress is associated with multiple neurodegenerative, age-related diseases. The rare disorder Pick disease (PiD) shares some pathological hallmarks of other neurodegenerative diseases that may be related to oxidative stress. Importantly, activation of an ER stress response, which is also involved in aging, has not yet been investigated in PiD. In this study, we assessed the implication of ER stress associated with oxidative stress in PiD as a potential mechanism involved in its pathogenesis. Samples from morphologically affected frontal cortex and apparently pathologically preserved occipital cortex showed region-dependent increases in different protein oxidative damage pathways. The oxidative modifications targeted antioxidant enzymes, proteases, heat shock proteins, and synaptic proteins. These effects were associated with compromised proteasomal function and ER stress in frontal cortex samples. In addition, we observed a depletion in ER chaperones (glucose-regulated proteins Grp78/BiP and glucose-regulated protein 94) and differences in tissue content and distribution of nuclear factor-erythroid 2 p45-related respiratory 2, required for cell survival during the unfolded protein response. These results demonstrate increased region-specific protein oxidative damage in PiD, with proteasomal alteration and dysfunctional ER stress response. We suggest this was caused by complete and specific depletion of Grp78/BiP, contributing to the pathophysiology of this neurodegenerative disease.     

Misfolded proteins, endoplasmic reticulum stress and neurodegeneration

The accumulation of misfolded proteins (e.g. mutant or damaged proteins) triggers cellular stress responses that protect cells against the toxic buildup of such proteins. However, prolonged stress due to the buildup of these toxic proteins induces specific death pathways. Dissecting these pathways should be valuable in understanding the pathogenesis of, and ultimately in designing therapy for, neurodegenerative diseases that feature misfolded proteins.     

Connecting endoplasmic reticulum stress to autophagy by unfolded protein response and calcium

Eukaryotic cells respond to the accumulation of unfolded proteins in the endoplasmic reticulum (ER) either by unfolded protein response that leads to an increase in the capacity of the ER to fold its client proteins or by apoptosis when the function of ER cannot be restored. Emerging data now indicate that ER stress is also a potent inducer of macroautophagy, a process whereby eukaryotic cells recycle their macromolecules and organelles. Depending on the context, autophagy counterbalances ER stress-induced ER expansion, enhances cell survival or commits the cell to non-apoptotic death. Here, we discuss the signaling pathways linking ER stress to autophagy and possibilities for their clinical exploitation.     

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.     

The endoplasmic reticulum and neuronal calcium signalling

The endoplasmic reticulum (ER) is a multifunctional signalling organelle regulating a wide range of neuronal functional responses. The ER is intimately involved in intracellular Ca(2+) signalling, producing local or global cytosolic calcium fluctuations via Ca(2+)-induced Ca(2+) release (CICR) or inositol-1,4,5-trisphosphate-induced Ca(2+) release (IICR). The CICR and IICR are controlled by two subsets of Ca(2+) release channels residing in the ER membrane, the Ca(2+)-gated Ca(2+) release channels, generally known as ryanodine receptors (RyRs) and InsP(3)-gated Ca(2+) release channels, referred to as InsP(3)-receptors (InsP(3)Rs). Both types of Ca(2+) release channels are expressed abundantly in nerve cells and their activation triggers cytoplasmic Ca(2+) signals important for synaptic transmission and plasticity. The RyRs and InsP(3)Rs show heterogeneous localisation in distinct cellular sub-compartments, conferring thus specificity in local Ca(2+) signals. At the same time, the ER Ca(2+) store emerges as a single interconnected pool fenced by the endomembrane. The continuity of the ER Ca(2+) store could play an important role in various aspects of neuronal signalling. For example, Ca(2+) ions may diffuse within the ER lumen with comparative ease, endowing this organelle with the capacity for "Ca(2+) tunnelling". Thus, continuous intra-ER Ca(2+) highways may be very important for the rapid replenishment of parts of the pool subjected to excessive stimulation (e.g. in small compartments within dendritic spines), the facilitated removal of localised Ca(2+) loads, and finally in conveying Ca(2+) signals from the site of entry towards the cell interior and nucleus.     

Prion protein misfolding affects calcium homeostasis and sensitizes cells to endoplasmic reticulum stress

Prion-related disorders (PrDs) are fatal neurodegenerative disorders characterized by progressive neuronal impairment as well as the accumulation of an abnormally folded and protease resistant form of the cellular prion protein, termed PrP(RES). Altered endoplasmic reticulum (ER) homeostasis is associated with the occurrence of neurodegeneration in sporadic, infectious and familial forms of PrDs. The ER operates as a major intracellular calcium store, playing a crucial role in pathological events related to neuronal dysfunction and death. Here we investigated the possible impact of PrP misfolding on ER calcium homeostasis in infectious and familial models of PrDs. Neuro2A cells chronically infected with scrapie prions showed decreased ER-calcium content that correlated with a stronger upregulation of UPR-inducible chaperones, and a higher sensitivity to ER stress-induced cell death. Overexpression of the calcium pump SERCA stimulated calcium release and increased the neurotoxicity observed after exposure of cells to brain-derived infectious PrP(RES). Furthermore, expression of PrP mutants that cause hereditary Creutzfeldt-Jakob disease or fatal familial insomnia led to accumulation of PrP(RES) and their partial retention at the ER, associated with a drastic decrease of ER calcium content and higher susceptibility to ER stress. Finally, similar results were observed when a transmembrane form of PrP was expressed, which is proposed as a neurotoxic intermediate. Our results suggest that alterations in calcium homeostasis and increased susceptibility to ER stress are common pathological features of both infectious and familial PrD models.     

Darier's disease: from dyskeratosis to endoplasmic reticulum calcium ATPase deficiency

The skin is the body's largest organ and has an essential barrier protective function against physical, chemical, and pathogen aggressions and prevents fluid loss. The outer layer of the skin, known as the epidermis, plays a key role in this protection, through a tightly regulated differentiation programme from basal keratinocytes to the stratum corneum at the skin surface. During this process, keratinocytes from the base of the epidermis undergo major morphological and functional changes during their migration through the spinous and granular layers, to become terminally differentiated corneocytes which will be shed from the skin's surface. The role of extracellular Ca2+ in cell-to-cell adhesion and in epidermal differentiation was known to be important, but the identification of the sarco/endoplasmic reticulum Ca2+ transport ATPase (ATP2A2) as the defective gene in a rare genetic skin disease known as Darier's disease, came as a surprise and shed light on the key role of Ca2+ signaling in the homeostasis of the epidermis.     

Mitochondria maintain maturation and secretion of lipoprotein lipase in the endoplasmic reticulum

Considering the physiological Ca2+ dynamics within the ER (endoplasmic reticulum), it remains unclear how efficient protein folding is maintained in living cells. Thus, utilizing the strictly folding-dependent activity and secretion of LPL (lipoprotein lipase), we evaluated the impact of ER Ca2+ content and mitochondrial contribution to Ca2+-dependent protein folding. Exhaustive ER Ca2+ depletion by inhibition of sarcoplasmic/endoplasmic reticulum Ca2+-ATPases caused strong, but reversible, reduction of cell-associated and released activity of constitutive and adenovirus-encoded human LPL in CHO-K1 (Chinese-hamster ovary K1) and endothelial cells respectively, which was not due to decline of mRNA or intracellular protein levels. In contrast, stimulation with the IP3 (inositol 1,4,5-trisphosphate)-generating agonist histamine only moderately and transiently affected LPL maturation in endothelial cells that paralleled a basically preserved ER Ca2+ content. However, in the absence of extracellular Ca2+ or upon prevention of transmitochondrial Ca2+ flux, LPL maturation discontinued upon histamine stimulation. Collectively, these data indicate that Ca2+-dependent protein folding in the ER is predominantly controlled by intraluminal Ca2+ and is largely maintained during physiological cell stimulation owing to efficient ER Ca2+ refilling. Since Ca2+ entry and mitochondrial Ca2+ homoeostasis are crucial for continuous Ca2+-dependent protein maturation in the ER, their pathological alterations may result in dysfunctional protein folding.     

Mitochondria recycle Ca(2+) to the endoplasmic reticulum and prevent the depletion of neighboring endoplasmic reticulum regions

To study Ca(2+) fluxes between mitochondria and the endoplasmic reticulum (ER), we used "cameleon" indicators targeted to the cytosol, the ER lumen, and the mitochondrial matrix. High affinity mitochondrial probes saturated in approximately 20% of mitochondria during histamine stimulation of HeLa cells, whereas a low affinity probe reported averaged peak values of 106 +/- 5 microm, indicating that Ca(2+) transients reach high levels in a fraction of mitochondria. In concurrent ER measurements, [Ca(2+)](ER) averaged 371 +/- 21 microm at rest and decreased to 133 +/- 14 microm and 59 +/- 5 microm upon stimulation with histamine and thapsigargin, respectively, indicating that substantial ER refilling occur during agonist stimulation. A larger ER depletion was observed when mitochondrial Ca(2+) uptake was prevented by oligomycin and rotenone or when Ca(2+) efflux from mitochondria was blocked by CGP 37157, indicating that some of the Ca(2+) taken up by mitochondria is re-used for ER refilling. Accordingly, ER regions close to mitochondria released less Ca(2+) than ER regions lacking mitochondria. The ER heterogeneity was abolished by thapsigargin, oligomycin/rotenone, or CGP 37157, indicating that mitochondrial Ca(2+) uptake locally modulate ER refilling. These observations indicate that some mitochondria are very close to the sites of Ca(2+) release and recycle a substantial portion of the captured Ca(2+) back to vicinal ER domains. The distance between the two organelles thus determines both the amplitude of mitochondrial Ca(2+) signals and the filling state of neighboring ER regions.     

内质网应激在肾脏疾病进展中的作用

内质网应激是机体对有害刺激的一种自身应答机制,细胞是存活还是死亡取决于刺激信号的强弱,适宜的内质网应激可保护细胞免受各种刺激的损害作用,而过强或过长时间的内质网应激使保护机制不能与损伤抗衡则扰乱内质网稳态,诱导细胞凋亡发生。内质网应激作为多种应激过程的共同通路,与多种肾脏疾病的进展密切相关,例如:肾小球疾病、肾小管间质损伤、肾缺血再灌注损伤、糖尿病肾病等。本文就内质网应激在肾脏疾病进展中作用的研究进展作一综述。