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Endocrinology Vol. 143, No. 6 2169-2177
Copyright © 2002 by The Endocrine Society

INTRACELLULAR SIGNAL SYSTEMS

Endoplasmic Reticulum Stress Causes Thyroglobulin Retention in this Organelle and Triggers Activation of Nuclear Factor-{kappa}B Via Tumor Necrosis Factor Receptor-Associated Factor 2

Antonio Leonardi, Pasquale Vito, Claudio Mauro, Francesco Pacifico, Luca Ulianich, Eduardo Consiglio, Silvestro Formisano and Bruno Di Jeso

Dipartimento di Biologia e Patologia Cellulare e Molecolare (A.L., C.M., L.U., S.F.), BioGem Consortium (P.V.), Centro di Endocrinologia e Oncologia Sperimentale (F.P., E.C.), Federico II, University of Naples, 80100 Naples, Italy; and Laboratorio di Patologia Generale, Dipartimento di Scienze e Tecnologie Biologiche ed Ambientali, University of Lecce (B.D.J.), 73100 Lecce, Italy

Address all correspondence and requests for reprints to: Dr. Bruno Di Jeso, Laboratorio di Patologia Generale, Dipartimento di Scienze e Tecnologie Biologiche ed Ambientali, Facoltà di Scienze MFN, Strada Lecce-Monteroni, Università degli Studi di Lecce, 73100 Lecce, Italy. E-mail: . bdijeso{at}ilenic.unile.it


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Perturbing the endoplasmic reticulum homeostasis of thyroid cell lines with thapsigargin, a specific inhibitor of the sarco-endoplasmic reticulum Ca2+ adenosine triphosphatases, and tunicamycin, an inhibitor of the N-linked glycosylation, blocked Tg in the endoplasmic reticulum. This event was signaled outside the endoplasmic reticulum and resulted in activation of the c-Jun N-terminal kinase (JNK)/stress-activated protein kinase and nuclear factor-{kappa}B (NF-{kappa}B) stress response pathways. Activation of the JNK/stress-activated protein kinase signaling pathway was assessed by measuring the amount of phospho-JNK and the activity of JNK by kinase assays. Activation of the NF-{kappa}B signaling pathway was assessed by measuring the level of inhibitory subunit I{kappa}B{alpha}, DNA binding, and transcriptional activity of NF-{kappa}B. Cycloheximide treatment, at a dose able to profoundly inhibit protein synthesis in FRTL-5 cells, obliterated the decrease in the level of the inhibitory subunit I{kappa}B{alpha} produced by thapsigargin and tunicamycin. Therefore, protein synthesis was required to generate a signal from stressed endoplasmic reticulum. This substantiates the hypothesis that endoplasmic reticulum retention of newly synthesized Tg and other cargo (secretory and membrane) proteins functions upstream of signal activation. Dominant negative TNF receptor-associated factor 2 (TRAF2) inhibited activation of NF-{kappa}B, which was also inhibited in embryonic fibroblasts derived from TRAF2-/- mice, respect to their normal counterpart. These data extend the recent demonstration that TRAF2 mediated JNK activation in response to endoplasmic reticulum stress and strongly strengthened the idea that endogenous stress signals initiated in the endoplasmic reticulum proceed by a pathway similar to that initiated by plasma membrane receptors in response to extracellular signals.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
MEMBRANE AND SECRETORY proteins are cotranslationally translocated in the lumen of the endoplasmic reticulum where addition of N-linked oligosaccharide chains, folding, and assembly occur. Protein folding and processing within the lumen of the endoplasmic reticulum are dependent upon the maintenance of a oxidizing environment, a high Ca2+ concentration, and a battery of endoplasmic reticulum-resident enzymes and chaperones. Alterations in the endoplasmic reticulum redox potential, glycosylation machinery, or Ca2+ levels of the endoplasmic reticulum secondary to various pathophysiological conditions or pharmacological manipulations, as well as many diseases, such as cystic fibrosis, Alzheimer’s disease, and primary congenital hypothyroidism, cause misfolded proteins to accumulate in the endoplasmic reticulum lumen. These retained proteins, which are eventually degraded, trigger a complex chains of events termed the unfolded protein response (UPR) (1). Activation of mammalian UPR is characterized in part by increased transcription of several genes encoding endoplasmic reticulum molecular chaperones, as well as induction of CHOP/GADD153, a transcription factor that regulates growth arrest and apoptosis (2). These changes occur concomitantly with a marked decrease in the rate of protein synthesis (3). Stress of the endoplasmic reticulum induced by agents that cause accumulation of misfolded proteins in that compartment also activates c-Jun N-terminal kinases (JNKs)/stress-activated protein kinases (SAPKs) (4). In mammals it has been shown that an overload of the endoplasmic reticulum by protein overexpression (major histocompatibility complex class I, adenovirus E3/19K) activates nuclear factor-{kappa}B (NF-{kappa}B), and this phenomenon has been termed the endoplasmic reticulum overload response (EOR) (5). However, many stimuli, such as tunicamycin, thapsigargin, and brefeldin A, trigger both the UPR and the EOR (6).

The signal transmission pathways that mediate the different responses in the UPR and EOR have been extensively studied over the past several years. Mammalian cells contain at least three endoplasmic reticulum signaling proteins. Inositol requiring (IRE) 1{alpha} and IRE1ß, encoded by different genes, are transmembrane proteins that act as folding sensors with their luminal domain and initiate the signal transmission with their cytosolic domain containing both an essential serine/threonine kinase and a ribonuclease module (7, 8). The induction of both endoplasmic reticulum chaperone genes and CHOP/GADD153 involves IRE1 activation (7, 8). The third identified endoplasmic reticulum signaling protein is PERK (PKR-ER-related kinase), which shows a luminal domain and a cytosolic domain homologous to the cytosolic RNA-dependent protein kinase (9). PERK mediates the repression of protein synthesis through phosphorylation of eIF-2a (9). Finally, the pathway that leads to NF-{kappa}B involves, in sequence, Ca2+ loss from the endoplasmic reticulum lumen, increased production of reactive oxygen intermediates, and activation of NF-{kappa}B (10). Very recently, the pathway that leads to JNK activation has been elucidated. After an endoplasmic reticulum stress, IRE1 becomes oligomerized and activated, and this causes the clustering of TRAF2 to the cytoplasmic portion of IRE1 and JNK activation (11). It appears, therefore, that the activation of JNK by endoplasmic reticulum stress proceeds by a pathway similar to that used by cells to respond to extracellular signals such as TNF{alpha}. In fact, TNF{alpha}-induced receptor trimerization results in recruitment of adapter proteins to their cytosolic side, among which are the TRAF proteins, in particular TRAF2 (12, 13). The TRAFs activate proximal kinases to initiate a kinase cascade, causing JNK activation (14, 15). Notably, TRAF recruitment to TNF receptors activates not only JNK, but also NF-{kappa}B (12, 16). Therefore, we reasoned that if TRAF2 recruitment by TNF receptors causes both JNK and NF-{kappa}B activation, perhaps TRAF2 recruitment by IRE1 after an endoplasmic reticulum stress activates not only JNK, but also NF-{kappa}B.

We have been studying a cellular system of fully differentiated thyroid cells in continuous culture, the FRTL-5 and PC-Cl3 cell lines. These cell lines synthesize and secrete very large amounts of Tg, a high molecular weight glycoprotein that constitutes the molecular site of synthesis and storage of thyroid hormones. We have shown that treatment of FRTL-5 cells with thapsigargin in a low Ca2+ medium (0.1 mM) dramatically inhibits the secretion of Tg that is trapped in the endoplasmic reticulum and alters its folding and oligomerization (17). Moreover, we directly tested the action of thapsigargin on the endoplasmic reticulum Ca2+ stores of FRTL-5 cells (17). Therefore, we decided to use this cellular system to test the hypothesis that an endoplasmic reticulum stress, chiefly a Ca2+ loss from the endoplasmic reticulum lumen caused by thapsigargin, activates through TRAF2 not only JNK, but also NF-{kappa}B.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Cell culture and biological reagents
FRTL-5 cells (CRL 8305, American Type Culture Collection, Manassas, VA) are a continuous cloned line of thyroid differentiated cells (18). These cells were maintained as previously described (19). WT and TRAF2-/- mouse embryonic fibroblast (MEF) were provided by Drs. T. W. Mak and W. C. Yeh (20). These cells were cultured in MEM-Glutamax (Life Technologies, Inc., Gaithersburg, MD) supplemented with 10% FCS and 0.1 mM sodium pyruvate. Polyclonal anti-Tg rabbit antibodies were raised against rat Tg as previously described (21). All other antibodies were purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). Thapsigargin was obtained from Calbiochem (La Jolla, CA); tunicamycin was purchased from Roche (Indianapolis, IN). The dominant negative form of TRAF6 {Delta}N275 was generated by PCR. The dominant negative form of TRAF2 {Delta}N105 was previously described (22).

Metabolic labeling and immunoprecipitation
FRTL-5 cells (2 x 106) were labeled for 5 min at 37 C in medium without cysteine and methionine containing [35S]cysteine and [35S]methionine (50 µCi L-35S in vitro cell labeling mix/ml; Amersham Pharmacia Biotech, Piscataway, NJ). The labeled cells were washed with cold PBS and lysed in 1 ml 1% Triton X-100 lysis buffer [25 mM HEPES (pH 7.4), 150 mM NaCl, 10% glycerol, 5 mM EDTA, 2 mM dithiothreitol, and Complete protease inhibitor mixture (Roche)]. Nuclear and cellular debris were removed by centrifugation at 10,000 x g for 10 min at 4 C. The lysate was incubated for 2 h at 4 C with 2 µl anti-Tg polyclonal antibodies and was collected with 20 µl protein G-Sepharose beads (Amersham Pharmacia Biotech). Beads were washed extensively with lysis buffer and boiled in SDS sample buffer, and the supernatant was subjected to 5% SDS-PAGE and autoradiography.

Kinase assay
Anti-JNK immunoprecipitates were used for the immune complex kinase assay that was performed at 30 C for 10 min with 2 µg substrate, 10 µCi [{gamma}-32P]ATP, and 50 µM ATP in a total of 20 µl kinase buffer [20 mM HEPES (pH 7.4), 10 mM MgCl2, 25 mM ß-glycerophosphate, 50 µM Na3VO4, and 50 µM dithiothreitol]. The substrate was GST-c-Jun (amino acids 1–79). The reactions were terminated by boiling in SDS sample buffer, and the products were resolved by 12% SDS-PAGE. Phosphorylated proteins were detected by autoradiography.

Reporter assay
FRTL-5 cells (4 x 105 cells/well) were seeded in six-well (35-mm) plates. After 12 h cells were transfected with 0.5 µg Ig-{kappa}B-luciferase reporter gene plasmid and various amount of each expression plasmid. Lipofectamine-mediated transfections were performed according to the manufacturer’s instructions (Life Technologies, Inc.). Total amounts of transfected DNA were kept constant by supplementing empty expression vector plasmids as needed. Twenty-four hours after transfection cells were stimulated with thapsigargin or tunicamycin for 8 h, cell extracts were prepared, and reporter gene activity was determined via the luciferase assay system (Promega Corp.). Expression of the Rous sarcoma virus promoter-ß-galactosidase vector (0.2 µg) was used to normalize transfection efficiencies.

EMSA
Total cell extracts were prepared using a detergent lysis buffer [50 mM Tris (pH 7.4), 250 mM NaCl, 50 mM NaF, 1 mM Na3VO4, 0.5% Nonidet P-40, 0.5 mM dithiotheritol, and Complete protease inhibitor mixture (Roche)]. Cells were harvested by centrifugation, washed once in cold PBS, and resuspended in detergent lysis buffer (30 µl/5 x 106 cells). The cell lysate was incubated on ice for 30 min, then centrifuged for 5 min at 10,000 x g at 4 C. The protein content of the supernatant was determined, and equal amounts of protein (10 µg) were added to a reaction mixture containing 20 µg BSA, 2 µg poly(dI-dC), 10 µl binding buffer [20 mM HEPES (pH 7.9), 10 mM MgCl2, 20% glycerol, 100 mM KCl, 0.2 mM EDTA, 0.5 mM dithiothreitol, and 0.5 mM phenylmethylsulfonylfluoride] and 100,000 cpm of a 32P-labeled oligonucleotide in a final volume of 20 µl. Samples were incubated at room temperature for 30 min and run on a 4% acrylamide gel.

Other procedures
Endo-ß-N-acetylglucosaminidase H (EndoH) and peptide N-glycohydrolase F (PNGase F) digestions were performed as previously reported (19).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Thapsigargin and tunicamycin caused retention of Tg in the endoplasmic reticulum
The ability of TRAF2 to transduce signals from both the plasma membrane and the endoplasmic reticulum prompted us to investigate its role in mediating NF-{kappa}B activation after an endoplasmic reticulum stress. To this end we used a thyroid cell line system in which an endoplasmic reticulum stress was evaluated by monitoring the intracellular fate of newly synthesized Tg. Treatment of FRTL-5 cells with thapsigargin, a specific inhibitor of the sarco-endoplasmic reticulum Ca2+-adenosine triphosphatases, in medium containing 0.1 mM Ca2+ led to depletion of Ca2+ from endoplasmic reticulum stores and dramatically inhibited the secretion of Tg. Tg was trapped in the endoplasmic reticulum and showed alteration in its folding and oligomerization (17). To improve the endoplasmic reticulum-stressing effect of thapsigargin, we decided to perform the thapsigargin treatments in a nominally Ca2+-free medium instead of a 0.1 mM Ca2+ medium. FRTL-5 cells were pulse-labeled with [35S]methionine and chased in a nominally Ca2+-free medium in the absence or presence of thapsigargin. At the indicated time of chase, the amounts of secreted and intracellular Tg were evaluated by immunoprecipitation and SDS-PAGE. As shown in Fig. 1AGo, treatment with thapsigargin dramatically inhibited Tg secretion in the culture medium. This suggested that depletion of Ca2+ from the endoplasmic reticulum caused intracellular retention of Tg. As there were no differences between the total Tg (secreted plus retained) immunoprecipitated from cells incubated with or without thapsigargin in either media (not shown), the differences observed were due to inhibition of secretion and not to intracellular degradation. This effect was not specific for thapsigargin, as a 2-h treatment of cells with 10 µg/ml tunicamycin, an inhibitor of protein glycosylation, caused Tg to be retained in FRTL-5 cells, as well (data not shown).



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  		Figure 1. A, Inhibition of Tg secretion by thapsigargin in FRTL-5 cells cultured in a nominally Ca2+-free medium. B, Thapsigargin blocks the processing of Tg high mannose oligosaccharides into complex carbohydrates. C, Tunicamycin profoundly inhibits the N-linked glycosylation of Tg. A, FRTL-5 cells were labeled with [35S]cysteine and [35S]methionine as outlined in Materials and Methods and chased in a nominally Ca2+-free medium in the absence (Co) or presence (Thaps) of 5 µM thapsigargin for the indicated times. Secreted and intracellular Tg were immunoprecipitated, resolved by SDS-PAGE, and quantified by scanning densitometry. The mean labeled secreted Tg in three independent experiments (±SD) is plotted against the time of chase. B, FRTL-5 cells were labeled with [35S]cysteine and [35S]methionine as outlined in Materials and Methods and chased in a nominally Ca2+-free medium in the absence (Co) or presence (Thaps) of 5 µM thapsigargin for the indicated times. Cells were lysed, and intracellular Tg was immunoprecipitated, divided in two aliquots, digested with or without EndoH, and resolved by SDS-PAGE. C, FRTL-5 cells were mock-treated or treated with 10 µg/ml tunicamycin for 60 or 120 min in a nominally Ca2+-free medium and labeled with [35S]cysteine and [35S]methionine as outlined in Materials and Methods. Intracellular Tg was immunoprecipitated, digested or mock-digested with PNGase F as outlined in Materials and Methods, and resolved by SDS-PAGE.

 
To confirm that thapsigargin also blocked Tg in the endoplasmic reticulum in a nominally Ca2+-free medium, we decided to repeat in this condition the experiment exploring the EndoH sensitivity of the retained Tg. Newly synthesized Tg is N-linked glycosylated in the endoplasmic reticulum, where high mannose chains are attached to the protein. The carbohydrate chains are then processed, and they become EndoH resistant upon transport of the protein to the medial Golgi (23). FRTL-5 cells were pulse-labeled with [35S]methionine and chased for different times in a nominally Ca2+-free medium in the absence or presence of thapsigargin. Cells were lysed, and intracellular Tg was immunoprecipitated and digested with EndoH. The appearance of a slowly migrating, EndoH-resistant Tg species in untreated cells indicated that Tg moved from the endoplasmic reticulum to the medial Golgi (Fig. 1BGo). Tg immunoprecipitated from FRTL-5 cells treated with thapsigargin did not show any slowly migrating, EndoH-resistant form, thus demonstrating that a loss of Ca2+ from the endoplasmic reticulum caused Tg to be retained at a point before the medial Golgi (Fig. 1BGo). To show that 10 µg/ml tunicamycin inhibited N-linked glycosylation of Tg we explored the sensitivity of Tg, after tunicamycin treatments, to PNGase F, which cleaves all N-linked chains. As shown in Fig. 1CGo, treatment of FRTL-5 cells with 10 µg/ml tunicamycin for 1 h inhibited the N-linked glycosylation of the newly synthesized Tg by 50%, and treatment for 2 h almost completely inhibited the N-linked glycosylation of newly synthesized Tg. These data demonstrated that thapsigargin had a similar, perhaps more pronounced, effect on FRTL-5 cells in a nominally Ca2+-free medium compared with a 0.1 mM Ca2+ medium and that tunicamycin, in our experimental conditions, strongly inhibited the N-linked glycosylation of newly synthesized Tg. We conclude, therefore, that both agents caused a block of Tg in the endoplasmic reticulum very likely because of a folding defect (17), probably causing an endoplasmic reticulum stress.

Endoplasmic reticulum stress causes activation of the JNK signaling pathway
It is well known that accumulation of proteins in the lumen of the endoplasmic reticulum initiates a stress response known as UPR/EOR. One of the pathways activated after endoplasmic reticulum stress is the SAPK/JNK pathway (11). To investigate whether the endoplasmic reticulum stress triggered by thapsigargin and tunicamycin was able to activate the SAPK/JNK pathway in FRTL-5 cells, we measured the levels of phospho-JNK, which is the active form of JNK, in cells treated with tunicamycin or thapsigargin. FRTL-5 cells were treated with tunicamycin for 60 and 120 min or with thapsigargin for 30 and 60 min. Cells were lysed, and the amount of phospho-JNK was measured by Western blot using phospho-specific anti-JNK antibodies. Both tunicamycin and thapsigargin increased the amount of phosphorylated JNK (Fig. 2AGo). Treatment of FRTL-5 cells with thapsigargin consistently caused activation of JNK within 60 min, whereas the effect of tunicamycin was clearly visible only after 120 min of treatment. Although it is possible that a higher dose of tunicamycin would shorten the signaling time, it is important to note that the time required for tunicamycin to activate JNK correlated well with the time required for tunicamycin to inhibit the N-linked glycosylation of newly synthesized Tg, which is complete after 120 min (Fig. 1CGo). To further demonstrate that the activity of JNK was increased after induction of endoplasmic reticulum stress, we also measured the relative levels of JNK kinase activity in cells treated with thapsigargin or tunicamycin by using an immune complex kinase assay. FRTL-5 cells were treated with tunicamycin for 120 min or with thapsigargin for 60 min. The cell lysates were immunoprecipitated with anti-JNK antibodies, and the activity of endogenous JNK was measured in an in vitro kinase assay using GST-c-Jun as substrate. Lysate from endoplasmic reticulum-stressed FRTL-5 cells all exhibited increased JNK kinase activity (Fig. 2BGo).



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  		Figure 2. Thapsigargin and tunicamycin activate the JNK signaling pathway. A, FRTL-5 cells were treated with thapsigargin (Thaps; 5 µM) for 30 and 60 min and with tunicamycin (Tun; 10 µg/ml) for 60 and 120 min in a nominally Ca2+-free medium. Cells were lysed, and the amount of phospho-JNK was measured by Western blot using phospho-specific anti-JNK antibodies. The amounts of cell lysate loaded were normalized using polyclonal antitubulin antibodies. B, FRTL-5 cells were treated with tunicamycin (10 µg/ml) for 120 min or thapsigargin (5 µM) for 60 min in a nominally Ca2+-free medium. Cell lysates were immunoprecipitated with anti-JNK antibodies, and the activity of endogenous JNK was measured by using GST-c-Jun (amino acids 1–79). To normalize the amounts of cell lysate used in the kinase assay, one tenth of each cell lysate was separately analyzed for tubulin content by Western blot.

 
Endoplasmic reticulum stress causes activation of NF-{kappa}B
NF-{kappa}B is a ubiquitously expressed family of transcription factors controlling the expression of numerous genes involved in inflammation, immune responses, and protection from apoptosis (24, 25). In most cell types NF-{kappa}B is present in an inactive form bound to its inhibitory subunit, I{kappa}B. Upon stimulation of cells with a variety of agents, such as inflammatory cytokines, UV irradiation, as well as bacterial and viral infection, I{kappa}B is phosphorylated on specific serine residues and degraded through a proteosome-dependent pathway (26). The released NF-{kappa}B dimer rapidly translocates to the nucleus, where it activates transcription of target genes.

To investigate whether the endoplasmic reticulum stress triggered by thapsigargin and tunicamycin was able to activate NF-{kappa}B in FRTL-5 cells, we treated cells with thapsigargin and tunicamycin and monitored the level of the inhibitory subunit I{kappa}B{alpha} by Western blot. As shown in Fig. 3AGo, the level of I{kappa}B{alpha} in the whole cell lysate was decreased after treatment with thapsigargin and tunicamycin. As for JNK activation, in the NF-{kappa}B activation there was a striking correlation between the time required to inhibit glycosylation of Tg and the time required to signal outside the endoplasmic reticulum. To further demonstrate the activation of NF-{kappa}B after endoplasmic reticulum stress, we performed an EMSA on nuclear extract from FRTL-5 cells treated in the same way. Both drugs caused the nuclear translocation of NF-{kappa}B and induced its DNA-binding activity (Fig. 3BGo). The specificity of the protein-DNA complex was confirmed by a competition assay. Binding was competed by the addition of nonradioactive {kappa}B oligonucleotide (Fig. 3BGo).



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  		Figure 3. Thapsigargin and tunicamycin activate NF-{kappa}B. A, FRTL-5 cells were treated with thapsigargin (Thaps; 5 µM) and tunicamycin (Tun; 10 µg/ml) for the indicated period of time. Cell extracts were Western blotted with anti-I{kappa}B{alpha} antibodies. The lower panel shows a Western blot antitubulin as control for protein loading. B, FRTL-5 cells were treated with thapsigargin (Thaps; 5 µM) and tunicamycin (Tun; 10 µg/ml) for the indicated period of time. Total cell extracts were prepared and analyzed by EMSA using a 32P-labeled oligonucleotide probe containing an NF-{kappa}B-binding site. The right part of the autoradiograph shows the same cell extracts incubated with a 50-fold molar excess of unlabeled NF-{kappa}B oligonucleotide. C, Relative luciferase activity observed in FRTL-5 cells transfected in triplicate with 0.5 µg Ig-{kappa}B luciferase reporter plasmid. Twenty-four hours after transfection cells were stimulated with thapsigargin (Thaps; 5 µM) or tunicamycin (Tun; 10 µg/ml) for 12 h or were left untreated (Co) and then harvested. Measurements were normalized for ß-galactosidase of a cotransfected Rous sarcoma virus-ß-galactosidase plasmid. Values shown (in arbitrary units) represent the mean (±SD) of at least three independent experiments.

 
We subsequently tested whether the NF-{kappa}B activated by treatment with thapsigargin and tunicamycin was transcriptionally active. FRTL-5 cells were transfected with an Ig-{kappa}B luciferase reporter plasmid and 24 h after transfection were treated with thapsigargin and tunicamycin for 8 h. Both thapsigargin and tunicamycin activated the NF-{kappa}B-driven luciferase activity (Fig. 3CGo).

Thus, endoplasmic reticulum stress after a luminal Ca2+ loss or a block of glycosylation activated a functional NF-{kappa}B complex.

Protein synthesis is required to activate NF-{kappa}B from the endoplasmic reticulum
Our data suggest that the stressing effect of thapsigargin and tunicamycin is linked to the retention in the endoplasmic reticulum of Tg and other cargo proteins. To further support this contention we tested whether a block of protein synthesis by decreasing the level of newly synthesized Tg and other cargo (membrane and secretory) proteins was able to inhibit generation of the signal from the endoplasmic reticulum. We monitored the activation of NF-{kappa}B by assaying the level of I{kappa}B{alpha} by Western blot after cycloheximide treatment. Preliminary experiments showed that in FRTL-5 cells, cycloheximide (0.5 µg/ml for 1 h) was the optimal treatment, because in these conditions protein synthesis was inhibited by 70–80%, and a higher concentration did not give any significant additional inhibition (not shown). Cells were treated with cycloheximide (0.5 µg/ml for 1 h), then with thapsigargin for 60 and 90 min and with tunicamycin for 60 and 120 min, in the presence of cycloheximide, and the levels of I{kappa}B{alpha} were assayed by Western blot. As shown in Fig. 4Go, A and B, under control conditions thapsigargin and tunicamycin decreased I{kappa}B{alpha} by about 50%; in presence of cycloheximide there was no reduction in I{kappa}B{alpha}. In addition, the TNF-induced inhibition of I{kappa}B{alpha} was cycloheximide insensitive. In these experiments we observed a reduction in I{kappa}B{alpha} in the controls in the presence of cycloheximide compared with the control level in the absence of cycloheximide. This was probably caused by the action of cycloheximide to decrease the steady state levels of I{kappa}B{alpha}.



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  		Figure 4. Protein synthesis is required to activate NF-{kappa}B from the endoplasmic reticulum. A, FRTL-5 cells were treated with or without 0.5 µg/ml cycloheximide for 1 h then with thapsigargin (Thaps; 5 µM) and tunicamycin (Tun; 10 µg/ml) for the indicated period of time in the presence or absence of 0.5 µg/ml cycloheximide. Cell extracts were Western blotted with anti-I{kappa}B{alpha} antibodies. The lower panel shows a Western blot antitubulin as a control for protein loading. B, I{kappa}B{alpha}/tubulin ratios from two different experiments were plotted as a percentage of the control in the absence ({square}) and in the presence ({blacksquare}) of cycloheximide.

 
We concluded, therefore, that protein synthesis is specifically required to generate a signal from the endoplasmic reticulum, thus suggesting that signaling is a downstream event of endoplasmic reticulum retention of newly synthesized Tg and other cargo proteins.

TRAF2 mediates the endoplasmic reticulum stress-induced activation of NF-{kappa}B
Recently, it has been demonstrated that endoplasmic reticulum stress activates JNK via the IRE1/TRAF2 complex (11). Given the central role of TRAF2 in mediating endoplasmic reticulum responses, we investigated whether TRAF2 was able to also mediate the endoplasmic reticulum stress-induced NF-{kappa}B activation. We tested the effect of an N-terminal deletion mutant of TRAF2, which acts as a dominant negative form of TRAF2 (22), on thapsigargin-induced {kappa}B reporter expression using a transient transfection assay in FRTL5 cells. FRTL5 cells were transiently transfected with a NF-{kappa}B-driven luciferase reporter plasmid in the presence of increasing amount of TRAF2 {Delta}N105. This dominant negative form of TRAF2 was able to block the activation of NF-{kappa}B after thapsigargin treatment in a dose-dependent manner (Fig. 5Go). Overexpression of a dominant negative form of another member of the TRAF family, TRAF6 {Delta}N275, did not affect the NF-{kappa}B activation after thapsigargin treatment (Fig. 5Go). The dominant negative effect of TRAF2 {Delta}N105 on TRAF2-mediated signal transmission and that of TRAF6 {Delta}N275 on TRAF6-mediated signal transmission were demonstrated by the ability of TRAF2 {Delta}N105 and TRAF6 {Delta}N275 to block TNF- and IL-1-induced NF-{kappa}B activation, respectively (Fig. 5Go).



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  		Figure 5. Dominant negative TRAF2 blocks thapsigargin-dependent NF-{kappa}B activation. FRTL-5 cells (4 x 105) were transfected in triplicate with 0.5 mg Ig-{kappa}B luciferase reporter plasmid together with increasing amounts of dominant negative forms of TRAF2 (T2{Delta}N) or TRAF6 (T6{Delta}N). Twenty-four hours after transfection cells were either stimulated with thapsigargin (THAPS; 5 µM) for 8 h or were left untreated (Co) and then harvested. Measurement were normalized for ß-galactosidase activity, and data (±SD) are shown as arbitrary units.

 
To further demonstrate that TRAF2 is involved in signaling from endoplasmic reticulum, we used TRAF2-/- MEF. The TRAF2-/- phenotype is characterized by sensitivity to TNF-induced cell death, almost complete reduction of JNK activation, and delayed kinetics of activation of NF-{kappa}B upon TNF treatment (20, 27). Treatment of TRAF2-/- MEF with tunicamycin and thapsigargin did not caused a decrease in I{kappa}B{alpha} (Fig. 6Go). In contrast, treatment of control MEF with the same drugs resulted in strong decrease in I{kappa}B{alpha} (Fig. 6Go). These results indicate that NF-{kappa}B is activated in endoplasmic reticulum-initiated signal transduction and suggest that TRAF2 is involved in this pathway.



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  		Figure 6. In mouse embryonic fibroblasts TRAF2-/- thapsigargin and tunicamycin were unable to activate NF-{kappa}B. MEF and MEF TRAF2-/- cells were treated with thapsigargin (Thaps; 5 µM) and tunicamycin (Tun; 10 µg/ml) for the indicated periods of time. Cell extracts were Western blotted with anti-I{kappa}B{alpha} antibodies. The lower panel shows a Western blot antitubulin as a control for protein loading.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
In this paper we show that an alteration in endoplasmic reticulum homeostasis causes retention of Tg in this organelle and activation of SAPKs and NF-{kappa}B signal transmission pathways. Most importantly, we show that NF-{kappa}B activation involves, at least partially, the adaptor protein TRAF2.

Alterations in the endoplasmic reticulum homeostasis cause misfolded proteins to accumulate in the endoplasmic reticulum lumen. These retained proteins, which are eventually degraded, trigger a complex chains of events termed the UPR (1). Although the final effects of the UPR are well known, the involved signal transmission pathways are less understood and actively studied over the past several years. It has been recently shown in pancreatic acinar epithelial AR42J cells and in 293T cells that an endoplasmic reticulum stress causes activation of the SAPK pathway and that this involves TRAF2 (11). TRAF2 is a member of the TRAF family of adapter molecules that link members of the TNF receptor superfamily, IL-1 receptors, and Toll receptors to NF-{kappa}B and SAPK/JNK (16). In particular, TRAF2 appears to be central to signaling via TNF receptors I and II. On the other hand, the pathway originating from the endoplasmic reticulum that leads to NF-{kappa}B involves, in sequence, a Ca2+ loss from the endoplasmic reticulum lumen, an increased production of reactive oxygen intermediates, and activation of NF-{kappa}B (10). Our results suggest the existence of a common mechanism coupling endoplasmic reticulum stress to SAPKs and NF-{kappa}B activation, in that both activation pathways use TRAF2. Notably, the time required to activate NF-{kappa}B and SAPK was essentially the same, 1–2 h, which is considerably shorter than the time required to up-regulate immunoglobulin heavy chain binding protein mRNA in FRTL-5 cells after treatments with thapsigargin and tunicamycin (6 h; data not shown). Moreover, our data strengthened the idea that endogenous stress signals initiated in the endoplasmic reticulum proceed by a pathway similar to that initiated by plasma membrane receptors in response to extracellular signals. In fact, it is well know that the signal transmission pathway originating from TNF receptors I and II activates both SAPKs and NF-{kappa}B via TRAF2. However, our results are compatible with the fact that in response to an endoplasmic reticulum stress, NF-{kappa}B could also be activated by other signal transmission mechanisms, for instance that described by Pahl and Bauerle (10). If this is indeed the case, the similarity of the pathways originating from TNF receptors and IRE1 is further corroborated. In fact, TRAF2 is critically important in TNF-initiated activation of SAPK/JNK. This has been confirmed by the failure of TNF to activate this signaling path in cells of mice lacking TRAF2 or expressing a dominant negative form of TRAF2 (20, 27) However, NF-{kappa}B activation is only partially affected in the same cells. It was suggested that other TRAF proteins, such as TRAF5, might have compensated for the lack of TRAF2 (20). The latter hypothesis is supported by the lack of NF-{kappa}B activation after TNF stimulation in MEF derived from mice lacking both TRAF2 and TRAF5 (28). It also possible, however, that an independent mechanism exists by which the TNF receptor may activate NF-{kappa}B.

Accumulation of cargo proteins in the endoplasmic reticulum occurs not only in various pharmacological manipulations, but also in a variety of diseases, such as viral infections, primary congenital hypothyroidism (29), cystic fibrosis (30), juvenile pulmonary emphysema (31), osteogenesis imperfecta (32), autosomal dominant neurohypophyseal diabetes insipidus (33), and familial hypercholesterolemia (34). In about 20% of primary congenital hypothyroidism a mutation is present in one of the genes encoding proteins essential in the biosynthesis of thyroid hormones (29). One of these proteins is Tg. Mutations of the Tg gene have been found in both animals (35, 36, 37, 38) and humans (39, 40, 41, 42, 43). Among these mutations, missense mutations of the mouse gene (36), the rat gene (38), and the human gene (42, 43) cause impaired intracellular transport of Tg. In these cases there are greatly enlarged thyroids, with the exception of the rdw/rdw rat (38), and enlarged thyreocyte endoplasmic reticulum membranes. The enlargement of the endoplasmic reticulum is due to the expression of compensatory levels of endoplasmic reticulum chaperones (triggered by accumulation of Tg), whereas the increase in thyroid volume is caused by increased levels of TSH (42). Using this feedback mechanism, cells maintain a chaperone reserve to further binding to unfolded/misfolded proteins that enter the endoplasmic reticulum. However, less clear is the physiological role of NF-{kappa}B in the UPR. NF-{kappa}B could trigger chaperone and/or endoplasmic reticulum membrane formation to relieve the congestion of the endoplasmic reticulum. These hypotheses remain to be investigated. On the other hand, it is well known that NF-{kappa}B triggers an inflammatory response. This induction could play a role during viral infections where large quantities of viral proteins are produced, causing an endoplasmic reticulum overload. In fact, interferons and cytokines, induced by NF-{kappa}B, can act as antiviral agents. Moreover, inflammation is well documented in a subset of cystic fibrosis patients with the PiZ variant of the {alpha}1-antitrypsin that have extensive hepatic damage and early cirrhosis of the liver (44). An analogous case could be represented by the rdw/rdw rat, which dramatically contrasts with most human patients and animal models of congenital hypothyroid goiter. In this case a Tg mutation causing impaired intracellular transport does not exist in goiter, but, rather, in a hypoplastic thyroid gland (38). In these cases NF-{kappa}B could play a role in controlling endoplasmic reticulum stress-induced apoptosis in a way analogous to TNF receptor-triggered apoptosis. Our results showing that NF-{kappa}B activation from the endoplasmic reticulum involves the adaptor protein TRAF2, as NF-{kappa}B activation from TNF receptors does, further suggest this hypothesis. This possibility is currently under investigation. Indeed, the recent finding that caspase-12, an endoplasmic reticulum-resident caspase, physically and functionally interacts with TRAF2 (45) also substantiates the central role of TRAF2 as a key switch of the endoplasmic reticulum controlling the alternative adaptation/apoptosis.

In conclusion, in this study we demonstrate that an endoplasmic reticulum stress caused by thapsigargin and tunicamycin triggers activation of SAPK/JNK and NF-{kappa}B stress response pathways, very likely acting through a retention of Tg and other cargo proteins in the endoplasmic reticulum. Moreover, we provide further evidence for the central role that the adapter molecule TRAF2 plays in the transduction of signals from the endoplasmic reticulum to the cytosol during endoplasmic reticulum stress.


   Acknowledgments
 


   Footnotes
 
This work was supported in part by a grant from Associazione Italiana Ricerca sul Cancro (to A.L.) and Ministero della Universita’e Ricerca Scientifica Grant 2001065217 (to S.F.).

Abbreviations: EndoH, Endo-ß-N-acetylglucosaminidase H; EOR, endoplasmic reticulum overload response; IRE, inositol requiring; JNK, c-Jun N-terminal kinase; MEF, mouse embryonic fibroblast; NF-{kappa}B, nuclear factor-{kappa}B; PERK, PKR-ER-related kinase; PNGase F, Peptide N- glycohydrolase F; SAPK, stress-activated protein kinase; THAPS, thapsigargin; TRAF, TNF receptor-associated factor; Tun, tunicamycin; UPR, unfolded protein response.

Received October 29, 2001.

Accepted for publication February 11, 2002.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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