Alveolar epithelial cells undergo epithelial-to-mesenchymal transition in response to endoplasmic reticulum stress

Expression of mutant surfactant protein C (SFTPC) results in endoplasmic reticulum (ER) stress in type II alveolar epithelial cells (AECs). AECs have been implicated as a source of lung fibroblasts via epithelial-to-mesenchymal transition (EMT); therefore, we investigated whether ER stress contributes to EMT as a possible mechanism for fibrotic remodeling. ER stress was induced by tunicamyin administration or stable expression of mutant (L188Q) SFTPC in type II AEC lines. Both tunicamycin treatment and mutant SFTPC expression induced ER stress and the unfolded protein response. With tunicamycin or mutant SFTPC expression, phase contrast imaging revealed a change to a fibroblast-like appearance. During ER stress, expression of epithelial markers E-cadherin and Zonula occludens-1 decreased while expression of mesenchymal markers S100A4 and α-smooth muscle actin increased. Following induction of ER stress, we found activation of a number of pathways, including MAPK, Smad, β-catenin, and Src kinase. Using specific inhibitors, the combination of a Smad2/3 inhibitor (SB431542) and a Src kinase inhibitor (PP2) blocked EMT with maintenance of epithelial appearance and epithelial marker expression. Similar results were noted with siRNA targeting Smad2 and Src kinase. Together, these studies reveal that induction of ER stress leads to EMT in lung epithelial cells, suggesting possible cross-talk between Smad and Src kinase pathways. Dissecting pathways involved in ER stress-induced EMT may lead to new treatment strategies to limit fibrosis.     

Sulfatase modifying factor 1 trafficking through the cells: from endoplasmic reticulum to the endoplasmic reticulum

Sulfatase modifying factor 1 (SUMF1) is the gene mutated in multiple sulfatase deficiency (MSD) that encodes the formylglycine-generating enzyme, an essential activator of all the sulfatases. SUMF1 is a glycosylated enzyme that is resident in the endoplasmic reticulum (ER), although it is also secreted. Here, we demonstrate that upon secretion, SUMF1 can be taken up from the medium by several cell lines. Furthermore, the in vivo engineering of mice liver to produce SUMF1 shows its secretion into the blood serum and its uptake into different tissues. Additionally, we show that non-glycosylated forms of SUMF1 can still be secreted, while only the glycosylated SUMF1 enters cells, via a receptor-mediated mechanism. Surprisingly, following its uptake, SUMF1 shuttles from the plasma membrane to the ER, a route that has to date only been well characterized for some of the toxins. Remarkably, once taken up and relocalized into the ER, SUMF1 is still active, enhancing the sulfatase activities in both cultured cells and mice tissues.     

Reactive oxygen species-mediated endoplasmic reticulum stress contributes to aldosterone-induced apoptosis in tubular epithelial cells

Apoptosis contributes to tubular epithelial cell death and atrophy in aldosterone (Aldo)-induced renal injury. This study aimed to determine mechanisms underlying Aldo-induced reactive oxygen species (ROS) and endoplasmic reticulum (ER) stress in tubular epithelial cells. Intracellular ROS generation was evaluated by 2',7'-dichlorofluorescin diacetate fluorescence. Apoptosis was detected by annexin V/propidium iodide staining and flow cytometry. ER stress induced protein and mRNA were evaluated by Western blot and real-time PCR, respectively. Aldo promoted tubular epithelial cell apoptosis, increased intracellular ROS production and induced ER stress, as evidenced by increased expression of glucose-regulated protein 78 (GRP78) and CAAT/enhancer-binding protein homologous protein (CHOP) in a dose- and time-dependent manner. Additionally, siRNA knockdown of CHOP and antioxidant N-acetyl-l-cysteine (NAC) attenuated ER stress-mediated apoptosis. NAC also could inhibit Aldo-induced expression of GRP78 and CHOP. Altogether, these observations suggest that Aldo induces apoptosis via ROS-mediated, CHOP-dependent activation in renal tubular epithelial cells.     

The ultrastructural differentiation of the endoplasmic reticulum in choroidal epithelial cells of the chick embryo

During the development of the choroidal epithelium in the chick embryo, a substantial concentration of granular endoplasmic reticulum differentiates in the subnuclear cytoplasm of the epithelial cells. The formation of the membranous components of this organelle is preceded by the appearance of a dense, localized population of small, free polyribosomes. Subsequently, numerous membrane-bound vesicles appear in the perinuclear cytoplasm. These primordial ER vesicles measure from 0.1 μ to 0.5 μ or more and they originate from evaginations of the outer nuclear membrane. These vesicles commonly occur in successive rows situated around the margin of the nucleus, and they expand and/or interconnect to form incipient ER tubules. Most vesicles and early tubules are smooth to nearly smooth in appearance. With continued development nuclear evaginations cease, and ER tubules expand in Situ to form an elaborate, laminated system of 7–12 ‘bag-like’ cisternae. Throughout this period of expansive growth, small polyribosomes attach to the developing ER cisternae. As the ER cisternae progressively attain their granular appearance, the number of small, free polyribosomes diminishes. During later stages of development larger polyribosomes appear in association with the subnuclear concentration of ER, and the first accumulations of electron-dense material develop within cisternal spaces.     

Trafficking of the NMDAR2B receptor subunit distal cytoplasmic tail from endoplasmic reticulum to the synapse

NMDA receptor NR2A/B subunits have PDZ-binding domains on their extreme C-termini that are known to interact with the PSD-95 family and other PDZ proteins. We explore the interactions between PSD-95 family proteins and the NR2A/B cytoplasmic tails, and the consequences of these interactions, from the endoplasmic reticulum (ER) through delivery to the synapse in primary rat hippocampal and cortical cultured neurons. We find that the NR2A/B cytoplasmic tails cluster very early in the secretory pathway and interact serially with SAP102 beginning at the intermediate compartment, and then PSD-95. We further establish that colocalization of the distal C-terminus of NR2B and PSD-95 begins at the trans-Golgi Network (TGN). Formation of NR2B/PSD-95/SAP102 complexes is dependent on the PDZ binding domain of NR2B subunits, but association with SAP102 and PSD-95 plays no distinguishable role in cluster pre-formation or initial targeting to the vicinity of the synapse. Instead the PDZ binding domain plays a role in restricting cell-surface clusters to postsynaptic targets.     

Targeting aequorin to the endoplasmic reticulum of living cells.

The photoprotein aequorin has been engineered with an ER targeting sequence at the N-terminus, with and without KDEL at the C-terminus, so that it locates in the ER-secretory pathway. For the first time the free Ca2+ has been quantified inside the ER and shown to be 5-20 times that in the cytosol. In COS cells free Ca2+ in the ER ranged from 1-5mM at 37 degrees C, decreasing 2-5-fold within 1 min of exposure to the Ca2+ ionophore ionomycin in the absence of external Ca2+.     

Xanthurenic acid provokes formation of unfolded proteins in endoplasmic reticulum of the lens epithelial cells

The role of xanthurenic acid in a cell is unknown, but it is suspected to provoke several diseases. This study shows that accumulation of xanthurenic acid in the lens epithelial cells leads to an overexpression of endoplasmic reticulum (ER) resident stress chaperones proteins, glucose-regulated protein (Grp94), and calreticulin. Both chaperones proteins are overexpressed in the presence of unfolded proteins. A formation of the unfolded protein in the presence of xanthurenic acid may take place due to covalent binding of xanthurenic acid to protein. Grp94 is responsible for scavenging of the unfolded proteins. The results suggest that Grp94 scavenged xanthurenic acid-modified proteins, and for this reason become preferentially yellow-stained in the presence of yellow xanthurenic acid. Such a modified Grp94 is weakly recognized by anti-Grp94 antibody. An end point of the xanthurenic acid accumulation in the cell is the cell death. In conclusion xanthurenic acid can lead to cell pathology.     

Differentiated murine airway epithelial cells synthesize a leukocyte-adhesive hyaluronan matrix in response to endoplasmic reticulum stress

In this report, we describe a novel method for culturing murine trachea epithelial cells on a native basement membrane at an air-liquid interface to produce a pseudostratified, differentiated airway epithelium composed of ciliated and nonciliated cells. This model was used to examine hyaluronan synthesis by the airway epithelial cells (AECs) in response to poly(I,C) and tunicamycin. The former induces a response similar to viral infection, and the latter is a bacterial toxin known to induce endoplasmic reticulum (ER) stress. We found significant accumulation of hyaluronan on the apical surface of the AECs in response to ER stress, but, unlike previously reported results with smooth muscle cells, no increase in hyaluronan was observed in response to poly(I,C). Monocytic U937 cells adhered at 4 degrees C to the apical surface of the AECs subjected to ER stress by a mechanism almost entirely mediated by hyaluronan. The U937 cells spontaneously released themselves from the abnormal hyaluronan matrix when their metabolism was restored by shifting the temperature from 4 to 37 degrees C in a custom-made flow chamber. Time lapse confocal microscopy permitted live imaging of this interaction between the U937 cells and the hyaluronan matrix and their subsequent response at 37 degrees C. Within 45 min, we observed dynamic protrusions of the U937 cell plasma membrane into nearby hyaluronan matrix, resulting in the degradation of this matrix. Simultaneously, we observed some reorganization of the hyaluronan matrix, from a generalized, apical distribution to localized regions around the AEC tight junctions. We discuss the implications these results might have for the airway epithelium and its relation to airway inflammation and hyperresponsiveness associated with asthma and other airway diseases.     

Trafficking of lipids from the endoplasmic reticulum to the Golgi apparatus in a cell-free system from rat liver

Trafficking and sorting of lipids during transport from the endoplasmic reticulum to the Golgi apparatus was studied using a cell-free system from rat liver. Transitional elements of the endoplasmic reticulum were prepared from liver slices prelabeled with [14C]- or [3H]acetate as the donor fraction. Non-radioactive Golgi apparatus were immobilized on nitrocellulose as the acceptor. When reconstituted, the radiolabeled donor retained a capacity to transfer labeled lipids to the non-radioactive Golgi apparatus acceptor. Transfer exhibited two kinetically different components. One was stimulated by ATP, facilitated by cytosol and inhibited by guanosine 5'-O-(thiotriphosphate) and N-ethylmaleimide. In parallel with protein transport, the ATP-dependent lipid transfer occurred with a temperature transition at about 20 degrees C. The other was not stimulated by ATP, did not require cytosol, was acceptor unspecific, was unaffected by inhibitors and, while temperature dependent, did not exhibit a sharp temperature transition. The ATP-independent transfer was non-vesicular. In contrast, the ATP-dependent transfer was vesicular. Transition vesicles isolated by preparative free-flow electrophoresis, when used as the donor fraction, transferred lipids to Golgi apparatus acceptor with a 5-6-fold greater efficiency than that exhibited by the unfractionated transitional endoplasmic reticulum. Formation of transition vesicles was ATP-dependent. Transferred lipids were chiefly phosphatidylcholine and cholesterol. Membrane triglycerides, major constituents of the transitional endoplasmic reticulum membranes, were both depleted in the transition vesicle-enriched fractions and not transferred to Golgi apparatus suggestive of lipid sorting prior to or during transition vesicle formation. The characteristics of the ATP plus cytosol-dependent transfer were similar to those for protein transfer mediated by transition vesicles. Thus, the 50-70-nm vesicles derived from transitional endoplasmic reticulum appear to function in the trafficking of both newly synthesized proteins and lipids from the endoplasmic reticulum to the Golgi apparatus.     

Lipolysis response to endoplasmic reticulum stress in adipose cells

In obesity and diabetes, adipocytes show significant endoplasmic reticulum (ER) stress, which triggers a series of responses. This study aimed to investigate the lipolysis response to ER stress in rat adipocytes. Thapsigargin, tunicamycin, and brefeldin A, which induce ER stress through different pathways, efficiently activated a time-dependent lipolytic reaction. The lipolytic effect of ER stress occurred with elevated cAMP production and protein kinase A (PKA) activity. Inhibition of PKA reduced PKA phosphosubstrates and attenuated the lipolysis. Although both ERK1/2 and JNK are activated during ER stress, lipolysis is partially suppressed by inhibiting ERK1/2 but not JNK and p38 MAPK and PKC. Thus, ER stress induces lipolysis by activating cAMP/PKA and ERK1/2. In the downstream lipolytic cascade, phosphorylation of lipid droplet-associated protein perilipin was significantly promoted during ER stress but attenuated on PKA inhibition. Furthermore, ER stress stimuli did not alter the levels of hormone-sensitive lipase and adipose triglyceride lipase but caused Ser-563 and Ser-660 phosphorylation of hormone-sensitive lipase and moderately elevated its translocation from the cytosol to lipid droplets. Accompanying these changes, total activity of cellular lipases was promoted to confer the lipolysis. These findings suggest a novel pathway of the lipolysis response to ER stress in adipocytes. This lipolytic activation may be an adaptive response that regulates energy homeostasis but with sustained ER stress challenge could contribute to lipotoxicity, dyslipidemia, and insulin resistance because of persistently accelerated free fatty acid efflux from adipocytes to the bloodstream and other tissues.     

Trafficking of oligomannosides released during N-glycosylation: a clearing mechanism of the rough endoplasmic reticulum

The main reaction of N-glycosylation of proteins is the transfer 'en bloc' of the oligosaccharide moieties of lipid intermediates to an asparagine residue of the nascent protein. For the past 15 years, a few laboratories including ours have shown that the process was accompanied by the release of oligosaccharide-phosphates and of neutral oligosaccharides possessing one GlcNAc (OS-Gn(1)) or two GlcNAc (OS-Gn(2)) at the reducing end. The aim of this review is to gather the evidence for the different origins of these soluble oligomannosides, to examine their subcellular location and intracellular trafficking. Furthermore, using Brefeldin A we demonstrated that this released oligomannoside material could be the substrate for the Golgi glycosidases and glycosyltransferases. Indeed, released oligomannoside never reach the Golgi vesicles either because they are directly produced in the cytosol as has been demonstrated for oligosaccharide-phosphates and for neutral oligosaccharides possessing one GlcNAc at the reducing end or because they are actively transported out of the rough endoplasmic reticulum to the cytosol. One of the functions of oligomannoside trafficking between rough endoplasmic reticulum, cytosol and lysosomes could be to prevent these oligosaccharides for competing with glycosylation in the Golgi.     

Three-dimensional organization of a transcellular tubulocisternal endoplasmic reticulum in epithelial cells of Reissner's membrane in the guinea-pig

Summary The ultrastructure of the epithelial cells of Reissner's membrane (membrana vestibularis) in the guinea-pig is described following vascular perfusion with glutaraldehyde of live, anaesthetised and artificially respirated animals. Postfixation in a solution containing O_sO₄ and potassium ferricyanide revealed a well-developed tubulocisternal endoplasmic reticulum, not previously described, the continuity of which has been mapped by serial sectioning and reconstruction. Large disc-shaped subsurface cisternae lining the cell membrane, but separated from it by a space approximately 10 nm wide, are in continuity with the smooth endoplasmic reticulum, forming an elaborated transcellular canalicular pathway. This structure is compared to that found in solute-transporting epithelia, e.g., renal proximal tubule, gall bladder, small intestine and choroid plexus. The fixation method used in the present study is compared to other techniques used for preservation of Reissner's membrane. Each epithelial cell of Reissner's membrane is endowed with one kinocilium, one to four multivesicular bodies, and a number of intercalated bodies. The functional significance of the canalicular pathway is discussed.     

The endoplasmic reticulum in regenerating liver cells

Summary Light-microscopic analysis of mouse liver homogenates six days after partial hepatectomy, showed a higher percentage of nuclei with adherent cytoplasm than homogenates from normal liver. This observation was true for animals with either a slow or rapid recovery of body weight after the operation. The phenomenon was not a function of the changes in the proportions of parenchymal and non-parenchymal tissue in the regenerating liver.Electron-microscopic analysis of random samples from normal and regenerating livers indicated an increase in the perinuclear rough endoplasmic reticulum, and a displacefment of the glycogen depots within the regenerating cells six days after partial hepatectomy.The marked resistance towards homogenization, shown by the cytoplasm of the regenerating cells, may have been due to the observed increase of perinuclear membranes. However, qualitative changes of the cell membranes and a general decrease of proteolytic activity connected with liver regeneration may also have contributed.     

Specializations of endoplasmic reticulum in bovine placental cells

Summary The endoplasmic reticulum of mononuclear placental cells from 10 cows in different stages of pregnancy has been studied with the electron microscope. Basicaly the cryptal cells are provided with rough surfaced endoplasmic reticulum. In addition, whorls of rough or smooth cytomembranes encircle lipid droplets or plain cytoplasmic matrix. The trophoblastic cells also contain rough surfaced endoplasmic reticulum. Furthermore, skein-like conglomerations of smooth tubules are encountered in some cells. The significance of the membranous structures is discussed.This work was supported by the Swedish Medical Research Council (Project no K 68-12x-2494-01) and NIH General Research Support Grant FR05462.     

Dynamic behavior of endoplasmic reticulum in living cells

Endoplasmic reticulum (ER) was studied by fluorescence microscopy of living CV-1 cells treated with the fluorescent carbocyanine dye DiOC6(3). Using video recording and image processing techniques, several distinct forms of highly localized movements of ER were documented, categorized, and analyzed in terms of mechanism and structural implications. These include tubule branching, ring closure, and sliding. These localized movements have been observed to generate the basic elements of ER: linear tubules, polygonal reticulum, and triple junctions. We propose that as such they act as the mechanism for constructing the polygonal lattice of interconnected membrane tubules that constitutes ER. The nature of these movements suggests possible involvement of the cytoskeleton, and, in view of the close correlations in the distributions of ER and microtubules, and the accompanying paper (Dabora and Sheetz), it is possible that microtubules may play a role in generating ER motility and in constructing and maintaining the ER network in living cells.     

Retrieval of transmembrane proteins to the endoplasmic reticulum

A COOH-terminal double lysine motif maintains type I transmembrane proteins in the ER. Proteins tagged with this motif, eg., CD8/E19 and CD4/E19, rapidly receive post-translational modifications characteristic of the intermediate compartment and partially colocalized to this organelle. These proteins also received modifications characteristic of the Golgi but much more slowly. Lectin staining localized these Golgi modified proteins to ER indicating that this motif is a retrieval signal. Differences in the subcellular distribution and rate of post-translational modification of CD8 maintained in the ER by sequences derived from a variety of ER resident proteins suggested that the efficiency of retrieval was dependent on the sequence context of the double lysine motif and that retrieval may be initiated from multiple positions along the exocytotic pathway.