Heat shock induces expression of OSTC/DC2, a novel subunit of oligosaccharyltransferase, in vitro and in vivo

Abstract

Mammalian oligosaccharyltransferase complex subunit OSTC/DC2 protein has recently been shown to be a new subunit of the oligosaccharyltransferase; however, its physiological role is still unclear. Here, we report the expression pattern of OSTC/DC2 protein in the context of heat shock stress. Its upregulation was detected both in cells treated with heat shock in vitro and in an animal model of heat shock in vivo. Northern blot analysis indicated that OSTC/DC2 mRNA is ubiquitously expressed in various human tissues, with abundant expression in the placenta and liver. The temporal changes of OSTC/DC2 protein expression following acute heat shock in human malignant glioblastoma cell line U87MG and mice were analyzed by Western blot assay. In general, expression of OSTC/DC2 protein was elevated after heat shock; however, the time courses of the change of OSTC/DC2 protein expression varied in different tissues. In the cerebellum, heat shock induction of OSTC/DC2 protein and activation of AKT, a key regulator of stress response, followed a similar time course. These results suggest that the upregulation of OSTC/DC2, a novel component of the oligosaccharyltransferase complex, is part of the mammalian heat shock response.

Introduction

Heat stress is any combination of environmental parameters producing temperature conditions that are higher than the upper range of an animal's thermal neutral zone [[1], [2]]. When cells are treated with stress, such as heat shock, genes related to growth are downregulated and expression of proteins believed to increase cellular survival are selectively upregulated [[3], [4]]. In addition, accumulation of misfolded protein due to heat shock requires enhanced unfolding protein response to alleviate endoplasmic reticulum (ER) stress [[5], [6]].

The oligosaccharyltransferase of yeast (Saccharomyces cerevisiae) is a multiunit, eight‐protein transmembrane complex in the ER [7]. This complex associates with the rough ER and other translational machinery, and selectively catalyzes post‐translational N‐linked glycosylation of nascent proteins [[8], [9]]. These complexes are highly conserved from yeast to human reflecting their essential roles [[8], [10]], such as inhibition of apoptosis in yeast [11]. Using blue native polyacrylamide gel electrophoresis and mass spectrometry, Shibatani et al. [12] found a novel subunit, OSTC/DC2, copurified with the mammalian oligosaccharyltransferase complex. Sequence alignment reveals that OSTC/DC2 is weakly homologous to the C‐terminal halves of the yeast oligosaccharyltransferase components Ost3p and Ost6p, which suggests that OSTC/DC2 is a mammalian counterpart of the oligosaccharyltransferase complex subunit involved with glycosylation during protein synthesis [12].

Recently, OSTC/DC2 has been reported to enhance the processing of amyloid beta (Aβ) peptide from the amyloid precursor protein [13]; however, its physiological role is still not clear. In a suppression subtractive hybridization screening, we identified OSTC/DC2 as a differentially regulated gene during lipopolysaccharide‐induced brainstem death of rat (see Fig. S1 and Table S1 in the supplementary material online). Here, we show that OSTC/DC2 protein is upregulated by heat shock both in vitro and in vivo, suggesting that OSTC/DC2 is part of a defense mechanism against stress.

Materials and methods

Animals and cell cultures

Male New Zealand rabbits and BALB/c mice were kept in a conventional animal facility with 12 hour light/dark cycles and controlled temperature at 25°C. Experimental protocol was approved by the Animal Care and Use Committee of the National Sun Yat‐sen University, Kaohsiung, Taiwan. J5, a human hepatoma cell line, and U87MG, a human glioblastoma cell line, were cultured in Dulbecco's modified Eagle's medium supplemented with 10% calf serum (Hyclone, Logan, UT, USA), 1% penicillin/streptomycin at 37°C in humidified air with 5% CO₂.

Northern blot analysis

The coding sequence of OSTC/DC2 mRNA was PCR‐amplified, gel‐purified, and used as the probe. The sequence was confirmed by sequencing and was in agreement with that obtained from GenBank. The probe was labeled with [α‐³²P] dCTP by a Rediprime II random primer labeling system (Amersham Pharmacia, Buckinghamshire, UK). A Multiple Tissue Northern Blot (BD Biosciences, San Jose, CA, USA) was incubated with the ³²P‐dCTP labeled probe at 68°C. Radioactivity was analyzed using phosphoimaging instrumentation (Fujifilm FLA‐3000, Tokyo, Japan).

Western blot analysis

In brief, samples containing 50 μg of protein were resolved on 12% sodium dodecyl sulfate‐polyacrylamide gel electrophoresis and electroblotted onto a nitrocellulose membrane. The membranes were probed with an in‐house generated anti‐DC2 (see Fig. S1 in the supplementary material online), anti‐actin, anti‐AKT, or anti‐p‐AKT (S473) antibodies (all from R&D Systems, Minneapolis, MN, USA). The blots were developed with the Enhanced Chemiluminescence Detection System (Amersham Pharmacia). Chemiluminescent signals were captured using the Fujifilm LAS‐3000 system (Fujifilm). Band intensities were quantitated by ImageJ (http://rsbweb.nih.gov/ij/) and normalized to that of β‐actin to obtain relative expression levels.

Heat shock treatment of cell lines and animals

U87MG cells were cultured in dishes and placed in a water bath set at 42°C for 1 hour. Then, the cells were returned to the incubator for recovery of the indicated periods of time. Mice were anesthetized by intraperitoneal injection of 25% urethane (30 μL/g). An electronic thermometer was inserted approximately 1‐cm depth into the anus after the mouse was fully anesthetized. The mouse was placed in a floating plate made of Styrofoam with the head and upper limbs fixed by Scotch tape and then half‐submerged in a 40°C water bath such that only its head was above the water surface. After the anal temperature had reached 40°C, a count for 5 minutes was started. During the heat shock treatment, the mouse was intraperitoneally injected with 0.85% saline (20 μL/g) to prevent dehydration. Mice organs were collected at time points of 0 minutes, 5 minutes, 30 minutes, 1 hour, 3 hours, 12 hours, and 24 hours after heat shock treatment.

Results

Expression of OSTC/DC2 mRNA in normal human tissues

To examine the expression profile of OSTC/DC2 mRNA, expression levels in normal human tissues were analyzed with Northern blotting. OSTC/DC2 mRNA expression was relatively lower in the brain, colon, and thymus; slightly higher in the spleen, leukocytes, small intestine, lung, and kidney; and highly expressed in the placenta and liver (Fig. 1). The differential expression pattern of OSTC/DC2 mRNA suggests that, as a subunit of a basic protein enzyme complex, the function of OSTC/DC2 may be regulatory and organ‐specific.

Figure 1.

Expression pattern of OSTC/DC2 mRNA in normal human tissues. Northern blot analysis of Multiple Tissue Northern Blot showing tissue‐specific expression of OSTC/DC2 mRNA in human tissues. Probing for β‐actin mRNA provided an internal control.

Change in OSTC/DC2 protein expression in heat shocked U87MG cells and mice

To examine the protein expression pattern of OSTC/DC2 in heat shock response, U87MG cells were subjected to heat shock in a water bath, and protein level changes in OSTC/DC2 and stress‐related proteins such as HSP90, HSP70, and the activation of AKT at various recovery time points were measured. After heat shock at 42°C for 1 hour, expression of OSTC/DC2, HSP90, HSP70, and AKT phosphorylation in U87MG cells increased after 1–12 hours of recovery (Fig. 2A).

Figure 2.

Induction of OSTC/DC2 protein expression by heat shock. (A) Changes in OSTC/DC2 protein expression following heat shock of human glioblastoma cells. U87MG cells were heat shocked in a water bath at 42°C for 1 hour and the expression of OSTC/DC2, HSP90, HSP70, and the activation of AKT at indicated recovery time points were detected by Western blotting. (B) Alterations of OSTC/DC2 protein expression in the cerebral cortex, cerebral medulla, brain stem and lung of mice following heat shock. BALB/c mice in the control group were compared with mice that were subjected to hyperthermia for 5 minutes at 40°C, and organs were harvested at the indicated time periods. Representative results from three experiments are shown.

After ascertaining that OSTC/DC2 was upregulated in heat shocked cells, we then asked whether OSTC/DC2 is also upregulated in tissues or organs of systemically heat shocked mice. The mice were subjected to 40°C heat shock for 5 minutes and the expression of OSTC/DC2 protein in the cortex, cerebral medulla, brain stem, and lung was examined by Western blotting. Consistent with the OSTC/DC2 mRNA expression pattern in human tissues (Fig. 1), OSTC/DC2 protein expression was relatively higher in the lung and lower in nervous tissue of control mice (Fig. 2B). Intriguingly, OSTC/DC2 protein expression was also differentially regulated by heat shock in the lung and nervous tissue. After heat shock, OSTC/DC2 was first increased and then gradually decreased to a normal level in the cortex, cerebral medulla, and brain stem, whereas in the lung OSTC/DC2 expression decreased below a normal level at 30 minutes–12 hours after heat shock (Fig. 2B; see Fig. S2A in the supplementary material online). The most drastic change of OSTC/DC2 expression was seen in the cerebral cortex, where OSTC/DC2 expression was strongly upregulated immediately after heat shock and then quickly returned to normal level (Fig. 2B, the cerebral cortex; see Fig. S2A in the supplementary material online). This result further supports the heat shock‐inducible property of OSTC/DC2, and it also suggests that cells respond to heat shock differently in different tissues.

Parallel expression of OSTC/DC2 and phosphorylated AKT in the cerebellum after heat shock

Activation of AKT by stress has previously been reported to increase neuronal survival by inhibiting cellular apoptosis inducers [[14], [15]]. Maroni et al. [16] reported that the phosphorylation of AKT significantly increased after heat shock in the cerebellum of rats. To test if OSTC/DC2 expression correlates with AKT activation in vivo, the expression of OSTC/DC2 and p‐AKT in mice cerebellum after heat shock was examined by Western blotting. Both OSTC/DC2 and p‐AKT expression were upregulated 5 minutes after heat shock treatment and gradually reduced over time (Fig. 3; see Fig. S2B in the supplementary material online). This result suggests that OSTC/DC2 is regulated in concert with the stress response pathway that mediates AKT activation.

Figure 3.

Effect of in vivo heat shock on protein expression of OSTC/DC2 and phosphorylation of AKT in mouse cerebellum. Mice were mildly heat shocked at 40°C for 5 minutes and then allowed to recover for the indicated time. Cerebellum tissue from control (sham‐treated) mice and mice recovered at indicated times after heat shock were analyzed for the expression patterns of OSTC/DC2, phosphorylated AKT, total AKT, and β‐actin. Representative immunoblots from three experiments are shown.

Discussion

Research on stress and hyperthermia has derived a general principle that physiological stress situations often lead to prolonged changes in gene activities and physiology for days or weeks after treatment of the initial acute stress [[17], [18], [19]]. Stress may upregulate protective proteins in ischemic stress of animal models, and these proteins could modulate protective or pro‐life responses to further stress [[20], [21], [22], [23]]. At the whole animal level, these delayed responses also tend to change the body's physiological responses to stress situations [[19], [21], [24]].

Since the identification of OSTC/DC2 as a novel subunit of mammalian oligosaccharyltransferase by Shibatani et al. [12], little has been determined about the physiological function of OSTC/DC2. Our study identifies a potential role of OSTC/DC2: a regulator/mediator of mammalian heat shock response. In agreement with our finding, other components of the oligosaccharyltransferase complex have been reported to be associated with a variety of stress responses. The OST3/6 subunit is required for Arabidopsis to tolerate abiotic stress and the protein underglycosylation due to OST3/6 deficiency results in the onset of ER stress [25]. A mammalian component of the oligosaccharyltransferase complex, DAD1, is intimately involved in regulating apoptosis [26]. Abnormal N‐glycosylation and apoptotic cell death are increased in DAD1 knockout mice [[27], [28]]. A DAD1 homolog in the spider Araneus ventricosus has also been observed to be upregulated when the spider is hypothermically or hyperthermically stressed [29]. These findings indicate that changes in gene expression of subunits of the oligosaccharyltransferase complex and N‐glycosylation activity are critical in the response of an organism to a stressful environment. In addition, the strikingly immediate upregulation of OSTC/DC2 in response to heat shock is only observed in the cortex (Fig. 2B), suggesting that OSTC/DC2 may have unique functions in neurons, which is consistent with a recent report that OSTC/DC2 is involved in the processing of amyloid precursor protein [13]. We believe that OSTC/DC2 may play more than one role. One of the major roles is likely to assist the expression of glycosylated proteins. The abundant expression of OSTC/DC2 mRNA in the liver and placenta (Fig. 1), organs that rely heavily on exocytosis and membrane transport to perform their physiological functions, may reflect the role of OSTC/DC2 to assist the maturation of secretory glycoproteins or glycosylated membrane proteins.

OSTC/DC2 induction is synchronous to increased expression of HSP70 and activation of AKT after heat shock (2, 3), supporting the notion that OSTC/DC2 may play a role in heat shock response. However, induction of OSTC/DC2 expression is independent of AKT activation (see Fig. S3 in the supplementary material online). The signaling pathway that mediates heat shock induction of OSTC/DC2 remains to be determined. Furthermore, although OSTC/DC2 was upregulated by heat shock both in vivo and in vitro, OSTC/DC2 expression was induced at different phases of the heat shock recovery course. OSTC/DC2 expression was probably already increased during heat shock in various tissues of the heat shocked animal (Figs. 2B and 3), whereas its expression in heat shocked U87MG cells was elevated at 1 hour into the recovery phase (Fig. 2A). This may suggest that OSTC/DC2 expression in an in vivo heat shock response is subject to central homeostatic control.

In conclusion, we observed increased OSTC/DC2 protein expression in mice subjected to systemic heat shock. The results fit into a picture of physiological response to stress that involves changes in molecular physiology over hours, days, and weeks following the initial stress. At the molecular and cellular levels, this includes upregulation of members of the oligosaccharyltransferase complex and stress signaling pathway. Future research may focus on the effects of OSTC/DC2 downregulation on either N‐glycosylation or apoptosis‐related signaling pathways after heat shock to shock stress response.

Acknowledgments

The authors thank Dr Chen‐Fu Saw, Department of Biological Sciences, National Sun Yat‐Sen University, for his technical consultation on mice brain work. This work was supported by grants NSC 95‐2311‐B‐110‐005 (to C.‐L.C.) and NSC 98‐2321‐B‐037‐061 (to Y.‐R.H.) from the National Science Council, Taiwan; by grants DOH102‐TD‐C‐111‐002 from the Department of Health, Executive Yuan, (Taiwan, R.O.C.) and KMUCOP‐102‐05 from Kaohsiung Medical University Research Foundation (to C.‐N.T.); and NSYSU‐KMU Joint Research Project, NSYSU‐KMU 2013‐P019 (to C.‐L.C. and C.‐N.T.) Taiwan.

Supporting information

Supplementary data

Supplementary data

Supplementary data related to this article can be found at https://doi.org/10.1016/j.kjms.2014.01.003.