2 Department of Pharmacology, Univerity of Texas Southwestern Medical Center, 5323 Harry Hines Blvd., Dallas, TX 75390-9041, USA; 3 Department of Biochemistry II, Heinrich-Duker-Weg 12, Gottingen D-37073, Germany; and 4 Glycobiology and Carbohydrate Chemistry Program, Burnham Institute, 10901 N. Torrey Pines Rd., La Jolla, CA 92037, USA
Received on November 6, 2001; revised on January 29, 2002; accepted on February 4, 2002.
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Abstract |
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Type I congenital disorders of glycosylation (CDGs) are characterized by diminished LLO synthesis and aberrant N-glycosylation. Such defects would be predicted to cause chronic ER stress with continuous UPR activation. We employed a quantitative pharmacological approach with dermal fibroblasts to show that (1) compared with three other well-known UPR aspects (transcriptional activation, inhibition of translation, and cell death), LLO extension was the most sensitive to ER stress; and (2) Type I CDG cells had a mild form of chronic ER stress in which LLO extension was continuously stress-activated, but other aspects of the UPR were unchanged. To our knowledge, Type I CDGs are the only human diseases shown to have chronic ER stress resulting from genetic defects in the ER quality control system.
In conclusion, LLO extension has a high priority in the UPR of dermal fibroblasts. This suggests that cells stimulate N-glycosylation as part of a first line of defense against ER dysfunction. The broader implications of these results for the biological significance of the UPR are discussed.
Key words: congenital disorder of glycosylation/endoplasmic reticulum stress/glycosylation/lipid-linked oligosaccharide/unfolded protein response
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Introduction |
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Considerable progress has been made in the identification of UPR signaling components in both the yeast Saccharomyces cerevisiae and mammalian cells, but a number of important questions remain unanswered. One unresolved issue arises from the fact that in S. cerevisiae the UPR is a single response that controls transcription of stress-responsive genes, whereas in mammalian cells the UPR is actually a group of complex effects. These are referred to here as UPR aspects (Table I). As with S. cerevisiae, one aspect of the UPR in mammals is the stimulation of transcription of genes whose translation products are able to counteract ER stress, such as ER chaperones (for example, GRP78/BiP) and folding enzymes. The ER membrane proteins Ire1p and ATF6 have pivotal roles in this regulation. However, in mammals another UPR aspect is activation of the PKR-like ER kinase (PERK), causing phosphorylation of eukaryotic inhibition factor 2 (eIF2
) and inhibition of protein synthesis (Harding et al., 1999
). Paradoxically, this inhibits translation of most mRNAs, including those encoded by UPR-responsive genes. (In a few cases, though, abundant mRNA increases may partially compensate for loss of translational efficiency, and in the case of the transcription factor ATF4 translational efficiency is enhanced; Harding et al., 2000a
). Nonetheless, both transcriptional activation and translation inhibition help cells survive ER stress (Harding et al., 2000b
). It is therefore puzzling that the UPR in mammals is also reported to initiate apoptotic cell death (Zinszner et al., 1998
; Nakagawa et al., 2000
). Thus, it appears that these well-known aspects of the UPR are potentially counteractive. It has remained unclear what strategies, if any, are used by mammalian cells to solve this problem.
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In this study we used a dual strategy to assess the biological significance of activation of LLO extension by the UPR. First, we employed a quantitative pharmacological approach to determine whether LLO extension in normal dermal fibroblasts has priority over the other well-known aspects of the UPR, i.e., transcriptional activation, inhibition of translation, and cell death. Surprisingly, LLO extension had the highest priority in the UPR and could be preferentially activated. Second, we examined the UPR in fibroblasts from patients with Types Ia, Ib, and Ic congenital disorders of glycosylation (CDGs), a family of genetic diseases characterized by multisystem defects due to deficient synthesis of Glc3Man9GlcNAc2-P-P-dolichol (Marquardt and Freeze, 2001), to test the prediction that the resulting hypoglycosylation would activate the UPR. We found that Type I CDG cells had a mild form of chronic ER stress in which LLO extension was continuously stress-activated, but there was no evidence that the other UPR aspects were activated. Together, these results support the role of LLO extension as part of a first line of defense against ER stress and indicate that this level of response is sufficient to handle the ER stress resulting from Type I CDG mutations.
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Results |
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As shown in Figure 1, panel A, extension of LLO intermediates was the UPR aspect most sensitive to DTT. With 0.4 mM DTT there was full stimulation (approximately 30-fold), with essentially no activation of the remaining UPR aspects. Treatments with 210 mM DTT were needed to strongly activate glucose regulated protein (GRP) 78 transcription (20-fold) and cell death (35%). DTT treatment of 2 mM also strongly decreased incorporation of [35S]methionine into total cellular protein (98% inhibition) and increased eIF2 phosphorylation (Table II), but as demonstrated the effect of DTT on translation was apparently independent of the UPR. Similar results were obtained with three different fibroblast sources from the American Type Culture Collection (ATCC). In the case of LLO extension, marked stimulations were repeatedly noted with small increases of the DTT concentration in the 0.4 mM range. This suggests that small changes in DTT concentration can result in large increases in protein misfolding, that there is a threshold for activation, or that activation of UPR signals is cooperative.
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A summary of these experiments is presented in Figure 1, panel E. The UPR aspects that were appreciably activated by each form of ER stress are indicated, and it can be seen that DTT, AZC, CSN, and TN each gave different results. TG gave a hybrid result because the AZC and TN responses were similar to those obtained by treatments with low and high concentrations of TG, respectively. Two important conclusions come from these data. First, depending on the exact type of ER stress, vastly different UPRs can be triggered. In other words, ER stresses are not necessarily interchangeable (see Discussion). Second, the UPRs are organized according to a hierarchy: LLO extension had the highest priority, being activated by all ER stresses, followed by transcriptional activation, translational inhibition, and cell death.
The effects of DTT on LLO extension and BiP transcription, but not its effect on translation, are due to the UPR
DTT (2 mM) activated GRP78 transcription 510-fold, inhibited translation almost completely (Figure 1), and enhanced phosphorylation of eIF2 2.5-fold. However, concentrations of AZC, CSN, TN, and TG that caused similar increases of GRP78 transcription had much milder effects on translation (Figure 1) and phosphorylation of eIF2
(data not shown). Although the effect of DTT on translation in embryonic fibroblasts is clearly due to PERK activation by the UPR (Harding et al., 2000b
), it appeared that the effect of DTT on translation in dermal fibroblasts was greater than could be explained by UPR activation.
We reasoned that UPR-specific effects should be dampened in cells allowed to adapt to ER stress by application of a prestress. Presumably, this dampening is due to accumulation of protective factors. In contrast, UPR-independent effects should not be affected by prestress. As shown in Table II, a prestress in which dermal fibroblasts were subjected to a 40-min treatment with 2 mM DTT 16 h prior to analysis mitigated the ability of a subsequent DTT stress to stimulate both LLO extension and GRP78 transcription. Thus, both of these aspects were UPR-dependent, as anticipated. This also shows that these UPR aspects were not stimulated as a direct result of the chemical activity of DTT, such as by modification of a critical cysteine residue in a regulatory enzyme. However, the prestress treatment had almost no effect on incorporation of [35S]-methionine into protein or phosphorylation of eIF2. Thus, in dermal fibroblasts, DTT stimulates phosphorylation of eIF2
and inhibits translation predominantly by a mechanism(s) that does not appear to require the UPR, implicating a direct chemical effect of DTT.
UPR activation of GRP78 transcription is mitigated in CDG Type I dermal fibroblasts
The results of the preceding sections gave insights into the dermal fibroblast UPR that were essential for assessment of Type I CDG mutations. Since the nature of the UPR depended upon the type of ER stress, not every known UPR aspect would necessarily be a valid indicator. In addition, the results predicted that any UPR activation would include stimulation of LLO extension. For these studies, dermal fibroblasts from patients with three sub-classes of Type I CDG were used: Type Ia (phosphomannomutase deficient), Type Ib (phosphomannose isomerase deficient), or Type Ic (glucose-P-dolichol:Man9GlcNAc2-P-P-dolichol glucosyltransferase I deficient). In all cases, transfer of Glc3Man9GlcNAc2 necessary to form glycoproteins is reduced (Table IV). This is due to limited synthesis of Glc3Man9GlcNAc2-P-P-dolichol as well as accumulation of dolichol-P-P linked oligosaccharide intermediates that are poor donor substrates for oligosaccharyltransferase. Although some protein glycosylation can occur with such intermediates, they lack the critical sugar residues needed for efficient ER quality control (Lehrman, 2001). Because CDG cells are viable and have normal protein synthesis (Körner et al., 1998b
), chronic inhibition of translation and initiation of apoptosis was unlikely. Therefore, LLO extension and GRP78 transcription were examined; the latter is generally considered to be a standard marker of ER stress.
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Discussion |
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The CDG-Ic cells had a somewhat mild degree of chronic ER stress (Figure 2), and as summarized in Table IV, CDG-Ic N-linked glycans would be expected to be partially effective for ER quality control because they are fully mannosylated. In addition to facilitating mannosylation of Man25GlcNAc2-P-P-dolichol intermediates, UPR stress also unexpectedly enhanced glucosylation of Man9GlcNAc2-P-P-dolichol in CDG-Ic cells. Though the cellular concentrations of UDP-glucose and glucose-P-dolichol are normally enough for efficient LLO glucosylation, these amounts are not sufficient to overcome the reduced activity of the leaky glucosyltransferase in CDG-Ic (Körner et al., 1998a). Thus one effect of ER stress might be increased production of glucose-P-dolichol.
Other explanations for the CDG results reported here were considered. CDG-Ia, Ib, and Ic affect genes encoding three distinct enzymes (Table IV), so the minimal stress effects on CDG LLOs cannot be explained by a direct genetic block at the metabolic step (which remains to be identified but appears likely to involve hexose metabolism; Doerrler and Lehrman, 1999) responsible for stimulating LLO extension. CDG is a pediatric disease, and it was possible that the extension of LLOs in CDG cells failed to respond to ER stress because such regulation might occur only in adult cells. Therefore, LLOs in dermal fibroblasts from four non-CDG pediatric donors, age 2 through 7 years, were examined in the absence or presence of ER stress (0.5 mM DTT). In all cases the basal and UPR-stimulated oligosaccharide profiles were indistinguishable from those of normal adult fibroblasts (data not shown).
The characteristics of CDG Type I reported here can be contrasted with other human diseases in which defects in the folding of specific membrane or secretory proteins would be expected to exhibit chronic activation of the UPR. A number of such diseases are known, but stable elevation of GRP78/BiP has been documented in very few instances (for example, Medeiros-Neto et al., 1996; Kim and Arvan, 1998
). It would be interesting to determine whether the ER stresses in these other diseases are sufficient to stimulate LLO extension. As a result of this study, the Type I CDGs are the only human genetic diseases reported to have chronic ER stress due to defects in the ER quality control machinery.
Relevant properties of dermal fibroblast cells
It remains to be determined how the information reported here for dermal fibroblasts applies to the UPRs in other differentiated cells. However, as pointed out in the Introduction, the dermal fibroblasts are highly sensitive to stress, and the results of any future studies should be interpreted with caution. For example, in the course of the current studies we noted that several freshly thawed aliquots of dermal fibroblasts exhibited enhanced LLO extension under control conditions for up to 2 weeks of culture. No dependence on the actual passage number was detected. Furthermore, two independent dermal fibroblast cultures expressing the catalytic subunit of telomerase (not shown) were examined in an attempt to identify cultures that retained UPR responsiveness of LLO extension, yet were capable of immortal growth suitable for genetic studies. Early passages of such cultures exhibited the desired regulation of LLO extension. However, continued passage for 23 months resulted in partial activation of LLO extension under basal conditions (data not shown). Perhaps extended periods in culture favor more proliferative cells with greater needs for ER lipid and protein synthesis and consequently greater ER stress.
LLO extension in permanent cell lines was unresponsive to ER stress (Doerrler and Lehrman, 1999). Thus, compared with dermal fibroblasts, permanent cell lines may have a weak, chronic form of ER stress. Such an idea is easily reconciled with the observation that transcription of UPR-sensitive genes in cell lines under basal conditions is growth-factor dependent and not due to conventional UPR signaling (Brewer et al., 1997
). Based on results with 0.4 mM DTT-treated dermal fibroblasts and untreated CDG cells, weak ER stress can stimulate oligosaccharide synthesis while having little effect on GRP78 transcription (Figure 1).
The high sensitivity of LLO extension to ER stress may also explain the differences observed among our three laboratories regarding the LLO compositions in normal dermal fibroblasts in the absence of deliberate ER stress. Each laboratory grows the cells in complete glucose medium (510 mM glucose) and examines the LLO after labeling cells for 2030 min with [2-3H]-mannose in the presence of 0.5 mM glucose. In the M.L. laboratory, where all of the experiments in the current study were performed, cells under these conditions show an abundance of LLO intermediates with three to five mannose residues. Mild ER stress changes this pattern to the one consistently observed under control conditions in the H.F. and C. K. laboratories, with an abundance of Glc3Man9GlcNAc2-P-P-dolichol (Doerrler and Lehrman, 1999; Figure 2). An exchange of cells (normal dermal fibroblast culture 12F), medium, and serum between the M.L. and H.F. labs showed that the discrepancy is due to a subtle difference in laboratory technique or environment, not reagents or cell samples (data not shown).
ER stresses are not interchangeable
An interesting outcome of this study is that various types of ER stress are not generally interchangeable as UPR inducers in mammalian cells. This is probably due to the abilities of different types of misfolded proteins to interact with the various UPR signaling systems that take part in each of the UPR aspects. Some indications of this had been reported earlier: DTT and TG differed in their abilities to activate signaling mediated by the lumenal domains of Ire1p and PERK (Bertolotti et al., 2000), and CSN had no detrimental effects on translation (Prostko et al., 1993
) or cell viability (Lehrman and Zeng, 1989
). However, in the majority of studies on UPR signaling, little consideration is typically given to the possibility that differences in the type and concentration of UPR inducer might result in differential signaling. At least in dermal fibroblasts, this is clearly the case. Another example of the need for such caution pertains to conflicting studies regarding the potential role of presenilin-1 in UPR signaling. It was recently noted (Imaizumi et al., 2001
) that these conflicts may be explained by variations in the time (15 min vs. 60 min) and concentration of TG (1 µM vs. 5 µM) used to activate the UPR. These investigators also reported UPR variability with cell type and the quality of the medium, as we found earlier (Doerrler and Lehrman, 1999
).
Versatility of the UPR
The idea that UPR aspects are organized in a hierarchy suggests a solution to the problem described in the Introductionthat the UPR is composed of potentially counterproductive aspects. LLO extension, and perhaps other high-priority aspects that remain to be identifiedseems to be an economical first step toward reducing ER stress. Previous work indicated that activation is probably due to modulation of sugar metabolism (Doerrler and Lehrman, 1999). Although the exact control point remains to be determined, it is likely controlled by a rapid, posttranslational regulatory step because full activation occurs within only 20 min of DTT treatment. If activation involves a simple reversible posttranslational modification, such as phosphorylation, rapid deactivation and return to the basal state would be possible. Activation of chaperone synthesis, on the other hand, requires a higher degree of ER stress and the production of additional mRNA and protein molecules. Such effects would be more difficult to reverse.
Simultaneous activation of LLO extension and new chaperone synthesis should be synergistic because both promote the folding of nascent ER proteins, and this can occur without inhibition of translation or promotion of cell death. However, some ER stresses also cause inhibition of protein synthesis. This inhibition is transient, with maximal effects lasting approximately 2 h in dermal fibroblast cells (data not shown) but not completely resolved by 16 h (Table II). Thus cells may use this strategy to temporarily halt production of folding substrates. Such a drastic measure may come at a price, however. The benefits of increased production of LLOs (which modify nascent proteins) and chaperones (requiring translation) cannot be realized during periods of translational inhibition, and other cellular processes requiring new protein synthesis would be hindered.
The UPR aspects involving oligosaccharide synthesis, transcriptional control, and translational inhibition have protective value, have no apparent long-term toxic effects, and are most likely involved in normal physiology. For example, the UPR has been implicated in metabolic control, i.e., translational regulation of glucose metabolism (Harding et al., 2001; Scheuner et al., 2001
) and transcriptional regulation of lipid metabolism (Werstuck et al., 2001
). However, some ER stresses can also promote cell death. In such cases stress may be due to failure of the protective UPR aspects to restore normal physiology. Extensive ER stress would be anticipated under severe pathological conditions, and in these cases death of the cells might help preserve the organism.
During review of this manuscript, the crucial role of regulated splicing of the mRNA encoding the transcription factor XBP-1 in the mammalian UPR was reported (Yoshida et al., 2001; Shen et al., 2001
; Calfon et al., 2002
). This discovery links the functions of the ER stress sensors ATF6 and Ire1p (reviewed by Ma and Hendershot, 2001
). It will therefore be an important goal of future research to determine to what degree each of these molecules detects the various ER stress conditions examined here.
Conclusion
The UPR in primary cultures of human dermal fibroblasts is versatile and differs depending on the specific type of ER stress. UPR aspects are organized in a hierarchy in which potentially counterproductive interactions are minimized. An understanding of this hierarchy was essential for identifying ER stress in Type I CDG. Among the four UPR aspects examined, extension of LLOs has the highest priority, consistent with a role in a first line of defense against ER dysfunction.
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Materials and methods |
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Activation of the UPR
The following UPR inducers (Doerrler and Lehrman, 1999; Lehrman, 2001
) were used as described in the figure legends and Table I: dithiothreitol (DTT; Sigma, St. Louis, MO), castanospermine (CSN; from Matreya, Pleasant Gap, PA, or Dr. Alan Elbein, University of Arkansas for Medical Science, Little Rock, AR), L-azetidine-2-carboxylic acid (AZC; Sigma), thapsigargin (TG; Sigma), and tunicamycin (TN; Sigma). None caused appreciable cytoplasmic stress in dermal fibroblasts as judged by HSP70 gene transcription, while two cytoplasmic stress inducers, arsenite (Harding et al., 2000b
) and diamide (Kosower et al., 1969
), activated HSP70 transcription without effect on GRP78 transcription (data not shown).
Measurement of stimulation of LLO extension
Either during (DTT) or immediately after (AZC, TG, CSN) treatment with UPR inducers, fibroblasts were metabolically labeled with D-[2-3H] mannose (Amersham Pharmacia Biotech, NJ) for 20 min in medium with 0.5 mM D-glucose. Isolation of total LLOs, release of [3H]-oligosaccharides from the dolichol-P-P carrier with mild acid, and high-pressure liquid chromatography (HPLC) analyses were carried out as described (Doerrler and Lehrman, 1999). Peak heights for Man35GlcNAc2 and Glc3Man9GlcNAc2 were measured and then normalized to mannose content to reflect molar quantities of each oligosaccharide. The molar percentage of Glc3Man9GlcNAc2 in each sample was then calculated. The percentage for stressed cells (maximally 5070%) was divided by that for unstressed cells (typically 13%) to determine the fold change.
Measurement of stimulation of GRP78/BiP transcription
Total RNA was isolated from cells 5 h after treatment with UPR inducers or from untreated controls and analyzed with northern blots with randomly primed [32P]probes (Amersham Pharmacia Biotech) corresponding to GRP78/BiP and actin (Doerrler and Lehrman, 1999). GRP78 RNA signals were measured with a Fuji (CT) phosphorimager and normalized to actin signals. The fold enhancements of normalized GRP78 signals in treated cells compared to those in untreated controls were determined.
Measurement of cell death
After treatment with UPR-inducing agents, dishes were rinsed twice in sterile phosphate buffered saline (PBS) and cells were allowed to grow in complete RPMI 1640 medium with 10% fetal bovine serum for 7 days. After removal of medium and rinsing with PBS, cells were stained with Gram Crystal Violet (Difco Laboratories, MI) for 30 min, washed thoroughly with water, and air-dried. After visual assessment of results, dishes were treated with 0.2% NP40 for 30 min with gentle shaking to elute-bound dye (Lehrman and Zeng, 1989). The quantity of eluted dye was estimated by the absorbance at 590 nm and used as a measure of cell mass. The cell death in treated cells was expressed as percentage loss of crystal violet staining compared to untreated controls.
Measurement of inhibition of protein synthesis
Immediately after treatment with UPR inducers, metabolic labeling of fibroblasts with [35S]-L-methionine (Amersham Pharmacia Biotech) in complete RPMI 1640 medium was carried out for 20 min as described (Harding et al., 2000b). However, to avoid potential stress due to nutrient deprivation, no preincubation period in methionine-free medium was used. Whole cell lysates were prepared in RIPA buffer (150 mM NaCl, 1.0% [w/v] Nonidet P-40, 0.5% deoxycholate, 0.1% sodium dodecyl sulfate [SDS], 50 mM TrisCl [pH 8.0], 1 mM phenylmethylsulfonylfluoride, 10 µg/ml aprotinin, 10 µg/ml leupeptin, all from Sigma) and resolved by SDSpolyacrylamide gel electrophoresis (PAGE). The total protein radioactivity in each lane was measured with a phosphorimager. The translation inhibition in treated cells was expressed as the percentage decrease of incorporation of [35S]-methionine into protein compared with untreated controls.
Measurement of phosphorylation of eIF2
Cell lysates in RIPA buffer were resolved by SDSPAGE, transferred to nitrocellulose membranes (Schleicher & Schuell), and subjected to immunoblot analysis by with a rabbit polyclonal antibody specific for the phosphorylated form of eIF2 (Research Genetics, AL). Autoradiographs were scanned with a BioRad Fluor-S MultiImager to determine signal intensities.
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Acknowledgments |
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Abbreviations |
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Footnotes |
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References |
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