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GRP78 and induces unfolded protein response in HepG2 cells
Chemico-Biological Interactions 176 (2008) 196–203
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La3+ binds to BiP/GRP78 and induces unfolded protein response in HepG2 cells Chenxi Shen a , Zaiquan Li b,∗ , Xiaoda Yang a,∗∗ , Kui Wang a a
State Key Laboratories for Natural and Biomimetic Drugs and Department of Chemical Biology, School of Pharmaceutical Science, Peking University, Beijing 100191, PR China b Department of Biochemistry and Molecular Biology, School of Basic Medical Sciences, Peking University, Beijing 100191, PR China
a r t i c l e
i n f o
Article history: Received 29 April 2008 Received in revised form 10 July 2008 Accepted 29 July 2008 Available online 19 August 2008 Keywords: Lanthanum Endoplasmic reticulum Unfolded protein response BiP/GRP78
a b s t r a c t The effects of La3+ on the unfolded protein response signaling pathways were investigated in human hepatoblastoma HepG2 cells. Our data showed that La3+ could induce unfolded protein response in HepG2 cells, including a significant increase of BiP/GRP78 level, which is an important ER residential chaperone and an ER stress hallmark, in a concentration and time-dependent manner, UPR transducer IRE1 phosphorylation and splicing activation IRE1 downstream substrate XBP1 mRNA. By using La3+ -affinity chromatography, the possible cellular target of La3+ leading to UPR events was shown to be the ER residential chaperone BiP/GRP78. BiP/GRP78 was shown to be a La3+ binding protein and the interaction of La3+ with BiP/GRP78 resulted in dissociation of BiP–IRE1 complexes. La3+ induced dissociation of the BiP/GRP78-IRE1 complex was in a time and concentration manner. The apparent dissociation constant was estimated to be 4 nM. In addition, La3+ was observed to slightly stimulate the production of cellular ROS and cause alteration of intracellular Ca2+ , indicating the possible involvement of ROS and Ca2+ alteration in La3+ induced UPR. The present work provides a new perspective for understanding the biological and toxicological effects of La3+ . © 2008 Published by Elsevier Ireland Ltd.
1. Introduction Abbreviations: ER, endoplasmic reticulum; BiP/GRP78, immunoglobulin heavy-chain binding protein/78 kDa glucose-regulated protein; IRE1, inositol-requiring kinase 1; XBP1, X-box binding protein 1; PERK, PKR (protein kinase regulated by RNA)-like ER-associated kinase; ATF6, activating transcription factor 6; NLD, N-terminal luminal domain; UPR, unfolded protein response; eIF2␣, eukaryotic translation initiation factor 2 subunit ␣; ERK, extracellular signal-regulated kinase; Ln, lanthanides; La, lanthanum; Tg, thapsigargin; EDTA, ethylenediamineN,N,N ,N -tetraacetic acid; PMSF, phenylmethysulfonyl fluoride; DCFH-DA, 2 7 -dichlorofluorescein; ROS, reactive oxygen species; Fluo 3-AM, 1-[2-Amino-5-(2,7-dichloro-6-hydroxy-3-oxo-9-xanthenyl)phenoxy]2-(2-amino-5-methylphenoxy)ethane-N,N,N’,N’-tetraacetic acid, pentaacetoxymethyl ester; FBS, fetal bovine serum; ICP-MS, inductively coupled plasma-mass spectrometry. ∗ Corresponding author. Tel.: +86 10 8280 1631. ∗∗ Corresponding author. Tel.: +86 10 8280 1539; fax: +86 10 6601 5584. E-mail addresses: [email protected] (Z. Li), [email protected] (X. Yang). 0009-2797/$ – see front matter © 2008 Published by Elsevier Ireland Ltd. doi:10.1016/j.cbi.2008.07.014
The endoplasmic reticulum (ER) is an organelle responsible for the synthesis, initial post-translational modification, folding, export, and secretion of membrane proteins [1]. It also functions as a Ca2+ storage organelle and resource of calcium signals [2,3]. ER is rich in Ca2+ dependent molecular chaperones, such as GRP78, GRP94, and calreticulin, which assist properly folding of proteins [4]. However, those ER chaperone function are easily affected by Ca2+ fluctuation in the ER. Disruption of Ca2+ homeostasis, is one of the important factors, that can result in ER stress. Of the Ca2+ -dependent chaperones, the glucoseregulated protein 78 (GRP78, also known as immunoglobin-binding protein, BiP) is a major functional molecule regulated by the mammalian ER stress response, and its
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up-regulation has been shown to be important in protecting cells from cytotoxic agents. Also, GRP78 participates in the storage of Ca2+ within the ER lumen and that the fluctuation of the protein can induce adjustments of the cellular Ca2+ homeostasis [5]. GRP78 can bind one molecule of Ca2+ , ATP, or ADP for each in a cooperative manner [6]. The Ca2+ -binding property of GRP78 may be part of a signal transduction pathway that modulates complex interactions between GRP78, ATP/ADP, secretary proteins, and caspases, and thus ultimately has important consequences for cell viability and apoptosis [7,8]. Lanthanide (Ln) is a group of metal ions with similar properties, which have been known for their diversity in biological effects and of great potential in medical applications [9]. In Chinese agricultural practices, Ln compounds have been used to increase the production of crops and to promote the growth of livestocks for many years [10], thus their profound effects on cell proliferation and apoptosis are of particular interest [9,11]. Among Ln members, La3+ , has been used extensively as a calcium channel blocker or antagonist in cellular studies and other biological systems [12]. It has been of considerable interest after lanthanum carbonate was approved by FDA as a new drug for the treatment of hyperphosphatemia of chronic renal failure (CRF) [13,14]. The effect of La3+ on cell proliferation and apoptosis has been extensively studied [11,15–17]. La3+ (and other lanthanide ions) were shown to enter the cells via multiple pathways [18–20] and even accumulated in some types of cells [21,22]. In NIH3T3 cells, La3+ can induce ERK signaling via a metal-sensing mechanism linking proliferation and apoptosis [15,16]. In other cell types, La3+ -induced cell apoptosis was regarded to be via mitochondrial pathway and highly related to reactive oxygen species (ROS) induction [17]. However, La3+ was also observed to inhibit ROS formation in some in vitro studies [23]. Studies of new La3+ targets are appropriate for elucidating the mechanisms of the biological effects of La3+ (and other lanthanide ions). As described above, a growing body of evidence suggests an active role for the ER in regulating cell viability and apoptosis. In addition, recent studies have indicated cross-talk between ER stress and oxidative stress [24]. As a Ca2+ antagonist, La3+ was reported to inhibit sarcoplasmic/endoplasmic reticulum ATPase and cause the depletion of calcium [25,26]. Since ER is abundant of Ca2+ -dependent chaperones, e.g. GRP78, it is quite possible that ER may be a novel direct intracellular target for La3+ . In the present work, we investigated the effects of La3+ on HepG2 cell lines focusing on its interaction with GRP78 protein and related ER stress signaling. We found that La3+ could induce unfolded protein response involving dissociation of BiP from BiP–IRE1 complex by La3+ . 2. Materials and methods 2.1. Materials Modified Eagle’s Medium (MEM) was obtained GibcoBRL; fetal bovine serum (FBS) was purchased Hyclone; trypsin, thapsigargin (Tg) were purchased Sigma. DCFH-DA and Fluo 3-AM were purchased
from from from from
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Molecular Probes. Antibodies against IRE1 were purchased form Abcam. Antibodies against GRP78 and the corresponding secondary antibodies were obtained from Santa Cruz. Other reagents were of analytical grade. Lanthanum chloride solution was prepared by dissolving La2 O3 (99.9% purity) in hydrochloric acid. The solution was evaporated to remove the excess HCl, redissolved in 1 mM HCl and the LaCl3 solution was diluted to 100 mM. 2.2. Cell culture HepG2, human hepatoblastoma cell line, was purchased from ATCC (Cat. HB-8065). The cells were cultured in Modified Eagle’s Medium supplemented with 10% FBS (GibcoBRL) and 100 units penicillin-100 g streptomycin/mL at 37 ◦ C in a humidified 5% CO2 atmosphere. All experiments were performed using cells within 30 passages. 2.3. Immunoprecipitation Immunoprecipitation assays were conducted using a modified method of Klenova et al. [27]. Briefly, the cells were resuspended in 20 volumes (packed cell) of NP-40 lysis buffer (20 mM Tris–HCl, pH 7.4, 150 mM NaCl, 2 mM EDTA, 1%NP-40, 1 mM NaF, 1 mM Na2 VO3 , 1 mM PMSF, 5 g/mL aprotinin, 5 g/mL leupeptin [28]). The lysate was incubated on ice for 15–20 min with occasional, gentle mixing and then centrifuged at 10,000 × g for 15 min at 4 ◦ C. The supernatant was collected for subsequent assays. The protein A-Sepharose (Amersham, Cat. 17-0974-01) was added to the supernatant at the amount of 10 L per 500 g total protein to pre-clean the sample. After incubation at room temperature for 30 min with gentle mixing, the samples were centrifuged at 12,000 × g for 10 min at 4 ◦ C and the supernatant was recovered. A pilot experiment was conducted to determine the amount of antibody required. Then needed antibody was added along with 20 L of protein A-Sepharose per mL of the pre-cleaned lysate. The mixture was incubated at 4 ◦ C overnight and then centrifuged at 12,000 × g to recover the immune complex associated with the beads. The beads were gently washed three times with lysis buffer followed by centrifugation at 12,000 × g. 2.4. RT-PCR analysis Activation of IRE1 was determined by quantitatively measuring the splicing of its substrate, the mRNA encoding the XBP1 transcription factor using a modified method of Shang [29]. RNA was harvested immediately after cells were exposed to different concentrations of La3+ for desired period of time. First-strand cDNA synthesis was performed with the Superscript First-Strand Synthesis System for RT-PCR (Promega). To amplify XBP1 (NM 005080) mRNA, PCR was conducted for 30 cycles (94 ◦ C for 30 s; 58 ◦ C for 30 s; and 72 ◦ C for 1 min or 10 min incubation in final cycle) using a primer pair 5 -CTGGAAAGCAAGTGGTAGA-3 and 5 -CTGGGTCCTTCTGGGTAGAC-3 with Taq DNA Polymerase (Dingguo Biotechnologies Inc., Beijing, China). The spliced (XBP1s, 398 bp) and unspliced (XBP1u, 424 bp) XBP1 were analyzed with 2.5% agarose gels and the band
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density were read on a United-Bio GK-330C Plus densitometer after staining with ethidium bromide. Partly, XBP1 fragments were further confirmed with a second primer pair 5 -CCTTGTAGTTGAGAACCAGG-3 and 5 -GGGGCTTGGTATATATGTGG-3 , which yielded a 442 bp fragment for unspliced form of XBP1. 2.5. Western blot analysis HepG2 cell lysates were prepared with NP-40 lysis buffer [28]. Proteins were separated on an 8% acryamide SDS–PAGE and electroblotted to PVDF immunoblotting membrane. Then the membrane was incubated overnight at 4 ◦ C with a polyclonal rabbit antibodies against IRE1 (1:1000, Abcam, USA) for analysis of IRE1, or a rabbit polyclonal anti-GRP78 antibody GRP78 (H129) (1:500, Santa Cruz Biotechnology, Santa Cruz, CA) for detection of GRP78 [30], followed by the incubation for 1 h at room temperature with goat anti-rabbit IgG (1:10,000) that was conjugated to horseradish peroxidase (Santa Cruz, USA). The membrane was washed three times with TBS-T buffer between each step. The bands were visualized using an enhanced chemiluminescent detection kit (Applygen Technologies Inc., Beijing, China). 2.6. Protein isolation with La3+ -affinity column Proteins in the cytosol were obtained from the homogenates of La3+ -treated HepG2 cells by centrifugation at 16,000 × g for 15 min. The supernatant was dialyzed against 10 volumes of 0.05 M phosphate buffer (pH 7.4) using a dialysis tube with 12–14 kDa retention size. The dialyzed proteins were applied to a La-affinity column equilibrated with 0.05 M phosphate buffer (pH 7.4) and 0.5 M NaCl. Proteins weakly adhering to the affinity column were eluted with phosphate-buffered saline (PBS) (0.05 M K2 HPO4 –Na2 HPO4 and 0.5 M NaCl, pH 7.4), followed by a imidazole gradient by linear mixture of buffer 1 (0.05 M K2 HPO4 –Na2 HPO4 , 0.5 M NaCl, pH 7.4) and buffer 2(0.05 M K2 HPO4 –Na2 HPO4 , 0.5 M NaCl and 0.01 M imidazole, pH 7.4). Finally, proteins were completely washed off using 0.05 M Na2 -EDTA, pH 8.0. The La-affinity column was prepared by removing Ni2+ from a commercial Ni-affinity column (Wsac Technologies Inc., Beijing, China) with 0.05 M EDTA (twice, three volumes each) and then saturating the naked column with 0.2 M LaCl3 . The column was then washed with three volumes of double-distilled water to remove unbound La3+ ions and equilibrated with PBS. An EDTA-treated Ni column was used as a control. No proteins were eluted with 0.05 M EDTA from the control column.
2.8. Detection of intracellular Ca2+ ([Ca2+ ]i ) HepG2 cells (1 × 105 ) were seeded in a Ø35 mm Petri dish and cultured at 37 ◦ C for 24 h. After exposure to different concentrations of La3+ for desired period of time, the cells was incubated with 5 mol/L Fluo 3-AM at 37 ◦ C for 30 min in dark. After three times of wash with Ca2+ free Hank’s buffer solution, the cells were observed under a Laser scanning confocal microscope (TCSNT LEICA, Germany) with the wavelength of excitation and emission light at 488 and 526 nm, respectively. The fluorescence intensity indicating the concentration of Ca2+ was analyzed with the Leica TCSNT software. 2.9. Determination of La3+ in HepG2 cells HepG2 cells in 24 well plates were incubated with MEM media containing different concentrations of La3+ for 3 h. Then the cells were washed three times with ice-cold 50 mM of EDTA/PBS (pH 7.2) solution to remove residual medium and surface-bound metal ions. Then the cells were collected and completely digested with ultrapure nitric acid. The amounts of lanthanide in the collected samples were determined with ICP-MS (Leeman Labs Inc., USA). 3. Results 3.1. UPR events in HepG2 cells upon incubation with La3+ A series of UPR events were observed in La3+ -treated HepG2 cell. As in Fig. 1, incubation of HepG2 cells with LaCl3 resulted in significant increase of BiP/GRP78 level, which is an important ER residential chaperone and an ER stress hallmark, in both a concentration (Fig. 1A) and time (Fig. 1B) -dependent manner. Phosphorylation of IRE1 was
2.7. Assays of cellular ROS Levels of ROS inside the cells were monitored by DCFHDA. The cells were planted in a six-well plate and treated with LaCl3 as described above. DCFH-DA (2.5 g/ml) was added into the wells and the staining was carried out at 37 ◦ C for 20 min. The cells were then washed with PBS and harvested for fluorospectrophotometer (Schimadzu RF-5310 PC) analysis.
Fig. 1. La3+ increased BiP/GRP78 level and resulted in phosphorylation of IRE1. (A) GRP78 level in HepG2 cells treated with different concentrations of LaCl3 for 3 h (lanes 1–5: 0, 1.6, 8, 40 or 200 M of LaCl3 ). (B) GRP78 induction upon incubation with 8 M of LaCl3 for indicated period of time. (C) Phosphorylation of IRE1 upon incubation with 8 M of LaCl3 for 3 h (lane 1: control; lane 2: 8 M LaCl3 ).
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Fig. 2. RT-PCR analysis of XBP1 mRNA splicing in HepG2 cell. (A) Agarose gel electrophoresis of XBP1 mRNA from HepG2 cells treated with different stimulus for 3 h. Lane 1: control; lane 2: cells treated with 200 M of LaCl3 ; lane 3: cell treated with DMSO solvent; lane 4: cells treated with 800 pM of thapsigargin (Tg) (dissolved in DMSO). (B) Agarose gel electrophoresis of XBP1 mRNA from HepG2 cells treated with different concentrations of LaCl3 (lanes 1–4: control, 1.6, 8, and 40 M of LaCl3 ). (C) Illustration of XBP1 mRNA splicing activation.
also observed (Fig. 1C). Activation of IRE1 signaling pathway would result in splicing of XBP1 mRNA. As shown in Fig. 2, La3+ resulted in formation of spliced XBP1 mRNA (Fig. 2A), however, weaker than the strong ER stress inducer thapsigargin. 3.2. La3+ resulted in dissociation of BiP–IRE1 complexes As shown in Fig. 3A, upon incubation of HepG2 cells with La3+ , BiP/GRP78 disappeared from BiP–IRE1 complexes in a time-dependent manner, indicating that La3+ resulted in dissociation of BiP–IRE1 complexes. To investigate whether binding of La3+ to BiP/GRP78 was involved in the dissociation of BiP–IRE1 complexes, the direct action of La3+ to the cell lysate was studied. Results in Fig. 3B showed that BiP/GRP78 band totally disappeared from the complexes after incubation for 15 and 30 min. In contrast, by removing of La3+ with EDTA, the binding of BiP/GRP78 to BiP–IRE1 complexes recovered. In addition, La3+ resulted in dissociation of BiP–IRE1 complexes in a concentration dependent manner (Fig. 3C). 3.3. BiP/GRP78 is a La3+ binding protein To investigate if BiP/GRP78 was a cellular target for La3+ , a La3+ -affinity column was prepared as described and the results are shown in Fig. 4. There was primarily one band at about 75 kDa observed upon elution of La3+ -binding pro-
Fig. 3. Western blot analysis of BiP–IRE1 complex using antibodies against Ire1 (upper panel) and BiP (lower panel). (A) BiP–IRE1 complexes were precipitated from cell lysate upon incubation of HepG2 cells with 8 M of La3+ for the indicated time periods using antibodies against the IRE1 as described in Section 2. An anti-actin IgG was used as a negative control. (B) To HepG2 cell lysate, La3+ was added to a final concentration of 40 M and incubated for 0, 15, and 30 min. Then BiP–IRE1 complexes were isolated as described above. To on sample after 30 min, an aliquot of EDTA was added to a final concentration of 20 mM and incubated for additional 10 min. (C) Free La3+ concentration dependency of BiP–IRE1 complex dissociation induced by La3+ . Various concentrations of LaCl3 were added into the BiP–IRE1 complex in the presence of 5 mM malic acid, and then the amount of BiP in the complex was detected as described in Section 2. The free La3+ concentrations were calculated according to the binding constants of La3+ to malic acid. The blots were quantified and plotted against the free La3+ concentration, and then the data were fitted into a Hill model with a dissociation constant (Kd ) of 3.78 ± 0.7 nmol/L and a Hill coefficient (n) of 0.77 ± 0.12.
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Fig. 5. Detection of cellular ROS level. ROS level in HepG2 cells treated with different concentrations of LaCl3 for 3 h (lanes 1–6: 0, 0.32, 1.6, 8, 40 or 200 M of LaCl3 ). *Compared with the control group, P < 0.05; **compared with the control group, P < 0.01.
increasing extracellular La3+ concentrations and reached a platform by 40 M. 4. Discussion
Fig. 4. Identification of BiP/GRP78 as a La3+ binding protein. (A) Electrophoresis of fraction eluted from La3+ -affinity column. Lane 1: cell lysate; lane 2: fraction eluted with sample buffer (0.02 M phosphate, 0.5 M NaCl, pH 7.4); lanes 3–6: fractions eluted with 0.02 M phosphate buffer pH 7.4 containing 0.5 M NaCl and 50, 100, 200, or 400 mM imidazole, respectively; lanes 7 and 8: fraction eluted with 50 mM or 20 mM of EDTA, pH 8.0; lane 9: molecular weight marker. (B) Western blot analysis of EDTA elution fractions using an antibody against BiP/GRP78.
teins from the affinity column with 20 mM EDTA; while more bands were observed when elution with 50 mM EDTA (Fig. 4A). The 75 kDa band was identified to be BiP/GRP78 by Western blot assay (Fig. 4B). 3.4. La3+ slightly elevated intracellular levels of reactive oxygen species (ROS) As shown in Fig. 5, the intracellular ROS levels increased up to 1–1.5-fold of normal upon incubation of HepG2 with La3+ for 3 h. The concentration dependency for ROS induction was not clearly observed. 3.5. Effect of La3+ on [Ca2+ ]i in HepG2 cells As shown in Fig. 6, 0.32 M La3+ just slightly reduced intracellular Ca2+ , however, La3+ , by 8 M and over, elevated [Ca2+ ]i significantly. For contrast, Tg (0.8 M), a strong ER inducer, resulted in a slightly higher level of Ca2+ , which is consistent with previous observation [31]. 3.6. La3+ content in HepG2 cells The result shown in Fig. 7 revealed that upon incubation for 3 h, the La3+ content in HepG2 cells increased with
Previously, we found that Ln3+ can result in cell apoptosis associated with mitochondrial alterations (i.e. MT swelling, decrease of m and release of Cyt-c) and elevated ROS levels in isolated mouse hepatocytes and several other cell lines [17]. Both primary cells and tumor cells exhibited similar response pattern to Ln3+ incubation, however, the primary cells need relatively higher concentrations of Ln3+ possibly due to higher metabolic capacities. Since tumor cell lines could be better for clearly observing the intrinsic toxicity/effects [32] and additionally in order to avoid possible problem of stability for high concentration of La3+ in culture media, we investigated effects of La3+ on HepG2 cell lines focusing on its interaction with GRP78 protein and related ER stress signaling. The experimental results clearly revealed that treatment with La3+ resulted in UPR which is evidenced by a series of UPR events, including: (i) time and concentrationdependent expression induction of BiP/GRP78 (Fig. 1A and B), which is widely used as an indicator for ER stress and the onset of the UPR; (ii) XBP1 mRNA splicing (Fig. 2A and B); (iii) phosphorylation of IRE1 (Fig. 1C); and dissociation of BiP–IRE1 complexes (Fig. 3). It was shown that the ER transducer IRE1, when dissociated from the BiP–IRE1 complex, would be activated by autophosphorylation. Activated IRE1 would splice its downstream substrate XBP1 mRNA and thus produced the transcription factor XBP1 (Fig. 2A–C) [33,34]. The observation of above result indicated that La3+ could activate the IRE1/XBP1 signaling transduction, resulting in expression of ER chaperones, e.g. BiP/GRP78. The possible cellular targets of La3+ were investigated using a La3+ -affinity column (Fig. 4A). Among the bands of the potential La3+ binding proteins, a ∼75 kDa major protein was identified. By immunoassay, this protein is confirmed to be BiP/GRP78 (Fig. 4B), suggesting that BiP/GRP78, as a
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Fig. 6. The intracellular Ca2+ levels ([Ca2+ ]i ) in HepG2 cells in HepG2 cells upon treatment with La3+ . (A) Fluorescent image of HepG2 cells under LCM. (B) Quantization of Ca2+ level indicated by Fluo 3-AM. HepG2 cells were incubated with different concentrations of LaCl3 (a–f: 0, 0.32, 1.6, 8, 40 or 200 M of LaCl3 ; g: 0.8 M Tg for contrast) for 3 h, then [Ca2+ ]i was stained with Ca2+ probe, Fluo 3-AM as described in Section 2. *Compared with the control group, P < 0.05, **compared with the control group, P < 0.01.
Ca2+ -binding protein, could be a major direct target of La3+ inside cells. To test whether the dissociation of BiP–IRE1 complexes was caused by La3+ binding, The direct effect of La3+ was investigated by the direct binding to the cell lysate. The association state of BiP–IRE1 complex was observed. As shown in Fig. 3, BiP/GRP78 was observed releasing from BiP–IRE1 and this dissociation could be reversed upon the addition of La3+ strong chelator EDTA. The apparent binding constant was estimated to be 4 nM (Fig. 3D). Shown in Fig. 7, La3+ was observed to enter HepG2 cells upon
incubation with La3+ -containing media. Assuming cells are small balls with ∼10 m diameter, data in Fig. 7 suggested a range of M to mM of total La3+ concentrations inside cells. Considering La3+ would bind to a variety of proteins, the present result suggested the potential of nM to M range of intracellular free La3+ concentrations; this is in consistence with previous report [20] that the intracellular concentration of free lanthanum ion ([La3+ ]i ) could be over 80 nM upon incubation of keratinocytes with 300 M of La3+ . The results above clearly indicated that binding of La3+ to BiP/GRP78 could cause release of BiP/GRP78
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Fig. 7. The cellular content of La3+ upon incubation of HepG2 cells with La3+ . HepG2 cells in 24 well plates were incubated with La3+ for 3 h. Then the cells were collected after wash with PBS solution containing 50 mM EDTA to remove surface-bound metal ions. Then the cells were digested and the amounts of La3+ were determined with ICP-MS. Data are average of triplicate measurements.
from the protein complexes, accounting primarily for the downstream signal transduction. Previously, accumulation of ROS and subsequent oxidative stress [35] were proposed to be important result for ER stress. Since La3+ was observed to cause slightly elevated level of cellular ROS (Fig. 5), therefore, it cannot be ruled out that ROS may also be involved in La3+ -induced UPR. However, La3+ -induced UPR may explain at least part of the reason for the relatively low levels (1–1.5-fold of the control) of ROS induction for La3+ as observed previously [17] and in the present work. Since slight elevation of ROS levels was observed upon incubation of La3+ (<1 M) with isolated mitochondria (data not published), possibly mitochondria could be a cellular source of ROS; nevertheless, other source of ROS (e.g. lysosomes) cannot be excluded so far. Although sustained elevation of [Ca2+ ]i was previously proposed not involved in ER stress induction [36], we observed elevation of [Ca2+ ]i upon incubation of HepG2 cells with La3+ over 8 M (Fig. 6). When taking a look at ROS and [Ca2+ ]i together (Fig. 8), it was observed that induction of ROS was prior to [Ca2+ ]i elevation, but in parallel with the elevation of [Ca2+ ]i . Recently, mitochondrial ROS was proposed to modulate bidirectionally Ca2+ spark activity; in the physiological regime, ROS promote the Ca2+ spark [37]. Therefore, variation of intracellular Ca2+ concentration in HepG2 cells would be closely associated with La3+ -induced ROS and might be involved in the subsequent events. Overall, the general pathway for La3+ to induce UPR could be illustrated in Scheme 1. The present results suggest further studies on the effects of La3+ on PERK and ATF6 as described above would be appropriate. Three transmembrane proteins, known for the transduction of UPR signals, IRE1, PERK and ATF6, are regulated in similar manner by binding with BiP/GRP78 [38–44]. The prevailing model is that the chaperone BiP/GRP78, as a negative regulator, binds to the NLD of IRE1, thus preventing IRE1 activation [40]. Upon ER stress, BiP/GRP78 binds
Fig. 8. Plot of [Ca2+ ]i versus ROS level in HepG2 cells upon incubation with La3+ . Data are from Figs. 5 and 6.
Scheme 1.
to unfolded proteins, permitting the release of NLD and its dimerization. This leads to the activation of the protein kinase and RNase activities in the cytosolic domain of IRE1 [45,46]. Since similar regulation mechanism was expected for activation of PERK and ATF6 signaling [40], it is possible that La3+ may induce the corresponding downstream signal transduction. In summary, the effect of La3+ on UPR in HepG2 cells was investigated. The results showed that BiP/GRP78 could be a major direct target of La3+ inside cells. Binding of La3+ to BiP/GRP78 (possibly in combination with the action of ROS and Ca2+ induced by La3+ ) resulted in dissociation of BiP/GRP78 from BiP–IRE1 complexes, thus activating the IRE1/XBP1 signal pathway. This result suggested that BiP/GRP78 as well as a variety of ER-related cell responses should be involved in the action of La3+ , which provides a new insight for understanding the biological and toxicological effects of La3+ . Acknowledgements This work was supported by NSFC (#20671008 and #20637010). We thank Prof. John J. Hefferren for editing the manuscript. References [1] K. Zhang, R.J. Kaufman, Protein folding in the endoplasmic reticulum and the unfolded protein response, Handb. Exp. Pharmacol. (2006) 69–91. [2] M.C. Ashby, A.V. Tepikin, ER calcium and the functions of intracellular organelles, Semin. Cell Dev. Biol. 12 (2001) 11–17.
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La3+ binds to BiP/GRP78 and induces unfolded protein response in HepG2 cells Chenxi Shen a , Zaiquan Li b,∗ , Xiaoda Yang a,∗∗ , Kui Wang a a
State Key Laboratories for Natural and Biomimetic Drugs and Department of Chemical Biology, School of Pharmaceutical Science, Peking University, Beijing 100191, PR China b Department of Biochemistry and Molecular Biology, School of Basic Medical Sciences, Peking University, Beijing 100191, PR China
a r t i c l e
i n f o
Article history: Received 29 April 2008 Received in revised form 10 July 2008 Accepted 29 July 2008 Available online 19 August 2008 Keywords: Lanthanum Endoplasmic reticulum Unfolded protein response BiP/GRP78
a b s t r a c t The effects of La3+ on the unfolded protein response signaling pathways were investigated in human hepatoblastoma HepG2 cells. Our data showed that La3+ could induce unfolded protein response in HepG2 cells, including a significant increase of BiP/GRP78 level, which is an important ER residential chaperone and an ER stress hallmark, in a concentration and time-dependent manner, UPR transducer IRE1 phosphorylation and splicing activation IRE1 downstream substrate XBP1 mRNA. By using La3+ -affinity chromatography, the possible cellular target of La3+ leading to UPR events was shown to be the ER residential chaperone BiP/GRP78. BiP/GRP78 was shown to be a La3+ binding protein and the interaction of La3+ with BiP/GRP78 resulted in dissociation of BiP–IRE1 complexes. La3+ induced dissociation of the BiP/GRP78-IRE1 complex was in a time and concentration manner. The apparent dissociation constant was estimated to be 4 nM. In addition, La3+ was observed to slightly stimulate the production of cellular ROS and cause alteration of intracellular Ca2+ , indicating the possible involvement of ROS and Ca2+ alteration in La3+ induced UPR. The present work provides a new perspective for understanding the biological and toxicological effects of La3+ . © 2008 Published by Elsevier Ireland Ltd.
1. Introduction Abbreviations: ER, endoplasmic reticulum; BiP/GRP78, immunoglobulin heavy-chain binding protein/78 kDa glucose-regulated protein; IRE1, inositol-requiring kinase 1; XBP1, X-box binding protein 1; PERK, PKR (protein kinase regulated by RNA)-like ER-associated kinase; ATF6, activating transcription factor 6; NLD, N-terminal luminal domain; UPR, unfolded protein response; eIF2␣, eukaryotic translation initiation factor 2 subunit ␣; ERK, extracellular signal-regulated kinase; Ln, lanthanides; La, lanthanum; Tg, thapsigargin; EDTA, ethylenediamineN,N,N ,N -tetraacetic acid; PMSF, phenylmethysulfonyl fluoride; DCFH-DA, 2 7 -dichlorofluorescein; ROS, reactive oxygen species; Fluo 3-AM, 1-[2-Amino-5-(2,7-dichloro-6-hydroxy-3-oxo-9-xanthenyl)phenoxy]2-(2-amino-5-methylphenoxy)ethane-N,N,N’,N’-tetraacetic acid, pentaacetoxymethyl ester; FBS, fetal bovine serum; ICP-MS, inductively coupled plasma-mass spectrometry. ∗ Corresponding author. Tel.: +86 10 8280 1631. ∗∗ Corresponding author. Tel.: +86 10 8280 1539; fax: +86 10 6601 5584. E-mail addresses: [email protected] (Z. Li), [email protected] (X. Yang). 0009-2797/$ – see front matter © 2008 Published by Elsevier Ireland Ltd. doi:10.1016/j.cbi.2008.07.014
The endoplasmic reticulum (ER) is an organelle responsible for the synthesis, initial post-translational modification, folding, export, and secretion of membrane proteins [1]. It also functions as a Ca2+ storage organelle and resource of calcium signals [2,3]. ER is rich in Ca2+ dependent molecular chaperones, such as GRP78, GRP94, and calreticulin, which assist properly folding of proteins [4]. However, those ER chaperone function are easily affected by Ca2+ fluctuation in the ER. Disruption of Ca2+ homeostasis, is one of the important factors, that can result in ER stress. Of the Ca2+ -dependent chaperones, the glucoseregulated protein 78 (GRP78, also known as immunoglobin-binding protein, BiP) is a major functional molecule regulated by the mammalian ER stress response, and its
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up-regulation has been shown to be important in protecting cells from cytotoxic agents. Also, GRP78 participates in the storage of Ca2+ within the ER lumen and that the fluctuation of the protein can induce adjustments of the cellular Ca2+ homeostasis [5]. GRP78 can bind one molecule of Ca2+ , ATP, or ADP for each in a cooperative manner [6]. The Ca2+ -binding property of GRP78 may be part of a signal transduction pathway that modulates complex interactions between GRP78, ATP/ADP, secretary proteins, and caspases, and thus ultimately has important consequences for cell viability and apoptosis [7,8]. Lanthanide (Ln) is a group of metal ions with similar properties, which have been known for their diversity in biological effects and of great potential in medical applications [9]. In Chinese agricultural practices, Ln compounds have been used to increase the production of crops and to promote the growth of livestocks for many years [10], thus their profound effects on cell proliferation and apoptosis are of particular interest [9,11]. Among Ln members, La3+ , has been used extensively as a calcium channel blocker or antagonist in cellular studies and other biological systems [12]. It has been of considerable interest after lanthanum carbonate was approved by FDA as a new drug for the treatment of hyperphosphatemia of chronic renal failure (CRF) [13,14]. The effect of La3+ on cell proliferation and apoptosis has been extensively studied [11,15–17]. La3+ (and other lanthanide ions) were shown to enter the cells via multiple pathways [18–20] and even accumulated in some types of cells [21,22]. In NIH3T3 cells, La3+ can induce ERK signaling via a metal-sensing mechanism linking proliferation and apoptosis [15,16]. In other cell types, La3+ -induced cell apoptosis was regarded to be via mitochondrial pathway and highly related to reactive oxygen species (ROS) induction [17]. However, La3+ was also observed to inhibit ROS formation in some in vitro studies [23]. Studies of new La3+ targets are appropriate for elucidating the mechanisms of the biological effects of La3+ (and other lanthanide ions). As described above, a growing body of evidence suggests an active role for the ER in regulating cell viability and apoptosis. In addition, recent studies have indicated cross-talk between ER stress and oxidative stress [24]. As a Ca2+ antagonist, La3+ was reported to inhibit sarcoplasmic/endoplasmic reticulum ATPase and cause the depletion of calcium [25,26]. Since ER is abundant of Ca2+ -dependent chaperones, e.g. GRP78, it is quite possible that ER may be a novel direct intracellular target for La3+ . In the present work, we investigated the effects of La3+ on HepG2 cell lines focusing on its interaction with GRP78 protein and related ER stress signaling. We found that La3+ could induce unfolded protein response involving dissociation of BiP from BiP–IRE1 complex by La3+ . 2. Materials and methods 2.1. Materials Modified Eagle’s Medium (MEM) was obtained GibcoBRL; fetal bovine serum (FBS) was purchased Hyclone; trypsin, thapsigargin (Tg) were purchased Sigma. DCFH-DA and Fluo 3-AM were purchased
from from from from
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Molecular Probes. Antibodies against IRE1 were purchased form Abcam. Antibodies against GRP78 and the corresponding secondary antibodies were obtained from Santa Cruz. Other reagents were of analytical grade. Lanthanum chloride solution was prepared by dissolving La2 O3 (99.9% purity) in hydrochloric acid. The solution was evaporated to remove the excess HCl, redissolved in 1 mM HCl and the LaCl3 solution was diluted to 100 mM. 2.2. Cell culture HepG2, human hepatoblastoma cell line, was purchased from ATCC (Cat. HB-8065). The cells were cultured in Modified Eagle’s Medium supplemented with 10% FBS (GibcoBRL) and 100 units penicillin-100 g streptomycin/mL at 37 ◦ C in a humidified 5% CO2 atmosphere. All experiments were performed using cells within 30 passages. 2.3. Immunoprecipitation Immunoprecipitation assays were conducted using a modified method of Klenova et al. [27]. Briefly, the cells were resuspended in 20 volumes (packed cell) of NP-40 lysis buffer (20 mM Tris–HCl, pH 7.4, 150 mM NaCl, 2 mM EDTA, 1%NP-40, 1 mM NaF, 1 mM Na2 VO3 , 1 mM PMSF, 5 g/mL aprotinin, 5 g/mL leupeptin [28]). The lysate was incubated on ice for 15–20 min with occasional, gentle mixing and then centrifuged at 10,000 × g for 15 min at 4 ◦ C. The supernatant was collected for subsequent assays. The protein A-Sepharose (Amersham, Cat. 17-0974-01) was added to the supernatant at the amount of 10 L per 500 g total protein to pre-clean the sample. After incubation at room temperature for 30 min with gentle mixing, the samples were centrifuged at 12,000 × g for 10 min at 4 ◦ C and the supernatant was recovered. A pilot experiment was conducted to determine the amount of antibody required. Then needed antibody was added along with 20 L of protein A-Sepharose per mL of the pre-cleaned lysate. The mixture was incubated at 4 ◦ C overnight and then centrifuged at 12,000 × g to recover the immune complex associated with the beads. The beads were gently washed three times with lysis buffer followed by centrifugation at 12,000 × g. 2.4. RT-PCR analysis Activation of IRE1 was determined by quantitatively measuring the splicing of its substrate, the mRNA encoding the XBP1 transcription factor using a modified method of Shang [29]. RNA was harvested immediately after cells were exposed to different concentrations of La3+ for desired period of time. First-strand cDNA synthesis was performed with the Superscript First-Strand Synthesis System for RT-PCR (Promega). To amplify XBP1 (NM 005080) mRNA, PCR was conducted for 30 cycles (94 ◦ C for 30 s; 58 ◦ C for 30 s; and 72 ◦ C for 1 min or 10 min incubation in final cycle) using a primer pair 5 -CTGGAAAGCAAGTGGTAGA-3 and 5 -CTGGGTCCTTCTGGGTAGAC-3 with Taq DNA Polymerase (Dingguo Biotechnologies Inc., Beijing, China). The spliced (XBP1s, 398 bp) and unspliced (XBP1u, 424 bp) XBP1 were analyzed with 2.5% agarose gels and the band
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density were read on a United-Bio GK-330C Plus densitometer after staining with ethidium bromide. Partly, XBP1 fragments were further confirmed with a second primer pair 5 -CCTTGTAGTTGAGAACCAGG-3 and 5 -GGGGCTTGGTATATATGTGG-3 , which yielded a 442 bp fragment for unspliced form of XBP1. 2.5. Western blot analysis HepG2 cell lysates were prepared with NP-40 lysis buffer [28]. Proteins were separated on an 8% acryamide SDS–PAGE and electroblotted to PVDF immunoblotting membrane. Then the membrane was incubated overnight at 4 ◦ C with a polyclonal rabbit antibodies against IRE1 (1:1000, Abcam, USA) for analysis of IRE1, or a rabbit polyclonal anti-GRP78 antibody GRP78 (H129) (1:500, Santa Cruz Biotechnology, Santa Cruz, CA) for detection of GRP78 [30], followed by the incubation for 1 h at room temperature with goat anti-rabbit IgG (1:10,000) that was conjugated to horseradish peroxidase (Santa Cruz, USA). The membrane was washed three times with TBS-T buffer between each step. The bands were visualized using an enhanced chemiluminescent detection kit (Applygen Technologies Inc., Beijing, China). 2.6. Protein isolation with La3+ -affinity column Proteins in the cytosol were obtained from the homogenates of La3+ -treated HepG2 cells by centrifugation at 16,000 × g for 15 min. The supernatant was dialyzed against 10 volumes of 0.05 M phosphate buffer (pH 7.4) using a dialysis tube with 12–14 kDa retention size. The dialyzed proteins were applied to a La-affinity column equilibrated with 0.05 M phosphate buffer (pH 7.4) and 0.5 M NaCl. Proteins weakly adhering to the affinity column were eluted with phosphate-buffered saline (PBS) (0.05 M K2 HPO4 –Na2 HPO4 and 0.5 M NaCl, pH 7.4), followed by a imidazole gradient by linear mixture of buffer 1 (0.05 M K2 HPO4 –Na2 HPO4 , 0.5 M NaCl, pH 7.4) and buffer 2(0.05 M K2 HPO4 –Na2 HPO4 , 0.5 M NaCl and 0.01 M imidazole, pH 7.4). Finally, proteins were completely washed off using 0.05 M Na2 -EDTA, pH 8.0. The La-affinity column was prepared by removing Ni2+ from a commercial Ni-affinity column (Wsac Technologies Inc., Beijing, China) with 0.05 M EDTA (twice, three volumes each) and then saturating the naked column with 0.2 M LaCl3 . The column was then washed with three volumes of double-distilled water to remove unbound La3+ ions and equilibrated with PBS. An EDTA-treated Ni column was used as a control. No proteins were eluted with 0.05 M EDTA from the control column.
2.8. Detection of intracellular Ca2+ ([Ca2+ ]i ) HepG2 cells (1 × 105 ) were seeded in a Ø35 mm Petri dish and cultured at 37 ◦ C for 24 h. After exposure to different concentrations of La3+ for desired period of time, the cells was incubated with 5 mol/L Fluo 3-AM at 37 ◦ C for 30 min in dark. After three times of wash with Ca2+ free Hank’s buffer solution, the cells were observed under a Laser scanning confocal microscope (TCSNT LEICA, Germany) with the wavelength of excitation and emission light at 488 and 526 nm, respectively. The fluorescence intensity indicating the concentration of Ca2+ was analyzed with the Leica TCSNT software. 2.9. Determination of La3+ in HepG2 cells HepG2 cells in 24 well plates were incubated with MEM media containing different concentrations of La3+ for 3 h. Then the cells were washed three times with ice-cold 50 mM of EDTA/PBS (pH 7.2) solution to remove residual medium and surface-bound metal ions. Then the cells were collected and completely digested with ultrapure nitric acid. The amounts of lanthanide in the collected samples were determined with ICP-MS (Leeman Labs Inc., USA). 3. Results 3.1. UPR events in HepG2 cells upon incubation with La3+ A series of UPR events were observed in La3+ -treated HepG2 cell. As in Fig. 1, incubation of HepG2 cells with LaCl3 resulted in significant increase of BiP/GRP78 level, which is an important ER residential chaperone and an ER stress hallmark, in both a concentration (Fig. 1A) and time (Fig. 1B) -dependent manner. Phosphorylation of IRE1 was
2.7. Assays of cellular ROS Levels of ROS inside the cells were monitored by DCFHDA. The cells were planted in a six-well plate and treated with LaCl3 as described above. DCFH-DA (2.5 g/ml) was added into the wells and the staining was carried out at 37 ◦ C for 20 min. The cells were then washed with PBS and harvested for fluorospectrophotometer (Schimadzu RF-5310 PC) analysis.
Fig. 1. La3+ increased BiP/GRP78 level and resulted in phosphorylation of IRE1. (A) GRP78 level in HepG2 cells treated with different concentrations of LaCl3 for 3 h (lanes 1–5: 0, 1.6, 8, 40 or 200 M of LaCl3 ). (B) GRP78 induction upon incubation with 8 M of LaCl3 for indicated period of time. (C) Phosphorylation of IRE1 upon incubation with 8 M of LaCl3 for 3 h (lane 1: control; lane 2: 8 M LaCl3 ).
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Fig. 2. RT-PCR analysis of XBP1 mRNA splicing in HepG2 cell. (A) Agarose gel electrophoresis of XBP1 mRNA from HepG2 cells treated with different stimulus for 3 h. Lane 1: control; lane 2: cells treated with 200 M of LaCl3 ; lane 3: cell treated with DMSO solvent; lane 4: cells treated with 800 pM of thapsigargin (Tg) (dissolved in DMSO). (B) Agarose gel electrophoresis of XBP1 mRNA from HepG2 cells treated with different concentrations of LaCl3 (lanes 1–4: control, 1.6, 8, and 40 M of LaCl3 ). (C) Illustration of XBP1 mRNA splicing activation.
also observed (Fig. 1C). Activation of IRE1 signaling pathway would result in splicing of XBP1 mRNA. As shown in Fig. 2, La3+ resulted in formation of spliced XBP1 mRNA (Fig. 2A), however, weaker than the strong ER stress inducer thapsigargin. 3.2. La3+ resulted in dissociation of BiP–IRE1 complexes As shown in Fig. 3A, upon incubation of HepG2 cells with La3+ , BiP/GRP78 disappeared from BiP–IRE1 complexes in a time-dependent manner, indicating that La3+ resulted in dissociation of BiP–IRE1 complexes. To investigate whether binding of La3+ to BiP/GRP78 was involved in the dissociation of BiP–IRE1 complexes, the direct action of La3+ to the cell lysate was studied. Results in Fig. 3B showed that BiP/GRP78 band totally disappeared from the complexes after incubation for 15 and 30 min. In contrast, by removing of La3+ with EDTA, the binding of BiP/GRP78 to BiP–IRE1 complexes recovered. In addition, La3+ resulted in dissociation of BiP–IRE1 complexes in a concentration dependent manner (Fig. 3C). 3.3. BiP/GRP78 is a La3+ binding protein To investigate if BiP/GRP78 was a cellular target for La3+ , a La3+ -affinity column was prepared as described and the results are shown in Fig. 4. There was primarily one band at about 75 kDa observed upon elution of La3+ -binding pro-
Fig. 3. Western blot analysis of BiP–IRE1 complex using antibodies against Ire1 (upper panel) and BiP (lower panel). (A) BiP–IRE1 complexes were precipitated from cell lysate upon incubation of HepG2 cells with 8 M of La3+ for the indicated time periods using antibodies against the IRE1 as described in Section 2. An anti-actin IgG was used as a negative control. (B) To HepG2 cell lysate, La3+ was added to a final concentration of 40 M and incubated for 0, 15, and 30 min. Then BiP–IRE1 complexes were isolated as described above. To on sample after 30 min, an aliquot of EDTA was added to a final concentration of 20 mM and incubated for additional 10 min. (C) Free La3+ concentration dependency of BiP–IRE1 complex dissociation induced by La3+ . Various concentrations of LaCl3 were added into the BiP–IRE1 complex in the presence of 5 mM malic acid, and then the amount of BiP in the complex was detected as described in Section 2. The free La3+ concentrations were calculated according to the binding constants of La3+ to malic acid. The blots were quantified and plotted against the free La3+ concentration, and then the data were fitted into a Hill model with a dissociation constant (Kd ) of 3.78 ± 0.7 nmol/L and a Hill coefficient (n) of 0.77 ± 0.12.
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Fig. 5. Detection of cellular ROS level. ROS level in HepG2 cells treated with different concentrations of LaCl3 for 3 h (lanes 1–6: 0, 0.32, 1.6, 8, 40 or 200 M of LaCl3 ). *Compared with the control group, P < 0.05; **compared with the control group, P < 0.01.
increasing extracellular La3+ concentrations and reached a platform by 40 M. 4. Discussion
Fig. 4. Identification of BiP/GRP78 as a La3+ binding protein. (A) Electrophoresis of fraction eluted from La3+ -affinity column. Lane 1: cell lysate; lane 2: fraction eluted with sample buffer (0.02 M phosphate, 0.5 M NaCl, pH 7.4); lanes 3–6: fractions eluted with 0.02 M phosphate buffer pH 7.4 containing 0.5 M NaCl and 50, 100, 200, or 400 mM imidazole, respectively; lanes 7 and 8: fraction eluted with 50 mM or 20 mM of EDTA, pH 8.0; lane 9: molecular weight marker. (B) Western blot analysis of EDTA elution fractions using an antibody against BiP/GRP78.
teins from the affinity column with 20 mM EDTA; while more bands were observed when elution with 50 mM EDTA (Fig. 4A). The 75 kDa band was identified to be BiP/GRP78 by Western blot assay (Fig. 4B). 3.4. La3+ slightly elevated intracellular levels of reactive oxygen species (ROS) As shown in Fig. 5, the intracellular ROS levels increased up to 1–1.5-fold of normal upon incubation of HepG2 with La3+ for 3 h. The concentration dependency for ROS induction was not clearly observed. 3.5. Effect of La3+ on [Ca2+ ]i in HepG2 cells As shown in Fig. 6, 0.32 M La3+ just slightly reduced intracellular Ca2+ , however, La3+ , by 8 M and over, elevated [Ca2+ ]i significantly. For contrast, Tg (0.8 M), a strong ER inducer, resulted in a slightly higher level of Ca2+ , which is consistent with previous observation [31]. 3.6. La3+ content in HepG2 cells The result shown in Fig. 7 revealed that upon incubation for 3 h, the La3+ content in HepG2 cells increased with
Previously, we found that Ln3+ can result in cell apoptosis associated with mitochondrial alterations (i.e. MT swelling, decrease of m and release of Cyt-c) and elevated ROS levels in isolated mouse hepatocytes and several other cell lines [17]. Both primary cells and tumor cells exhibited similar response pattern to Ln3+ incubation, however, the primary cells need relatively higher concentrations of Ln3+ possibly due to higher metabolic capacities. Since tumor cell lines could be better for clearly observing the intrinsic toxicity/effects [32] and additionally in order to avoid possible problem of stability for high concentration of La3+ in culture media, we investigated effects of La3+ on HepG2 cell lines focusing on its interaction with GRP78 protein and related ER stress signaling. The experimental results clearly revealed that treatment with La3+ resulted in UPR which is evidenced by a series of UPR events, including: (i) time and concentrationdependent expression induction of BiP/GRP78 (Fig. 1A and B), which is widely used as an indicator for ER stress and the onset of the UPR; (ii) XBP1 mRNA splicing (Fig. 2A and B); (iii) phosphorylation of IRE1 (Fig. 1C); and dissociation of BiP–IRE1 complexes (Fig. 3). It was shown that the ER transducer IRE1, when dissociated from the BiP–IRE1 complex, would be activated by autophosphorylation. Activated IRE1 would splice its downstream substrate XBP1 mRNA and thus produced the transcription factor XBP1 (Fig. 2A–C) [33,34]. The observation of above result indicated that La3+ could activate the IRE1/XBP1 signaling transduction, resulting in expression of ER chaperones, e.g. BiP/GRP78. The possible cellular targets of La3+ were investigated using a La3+ -affinity column (Fig. 4A). Among the bands of the potential La3+ binding proteins, a ∼75 kDa major protein was identified. By immunoassay, this protein is confirmed to be BiP/GRP78 (Fig. 4B), suggesting that BiP/GRP78, as a
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Fig. 6. The intracellular Ca2+ levels ([Ca2+ ]i ) in HepG2 cells in HepG2 cells upon treatment with La3+ . (A) Fluorescent image of HepG2 cells under LCM. (B) Quantization of Ca2+ level indicated by Fluo 3-AM. HepG2 cells were incubated with different concentrations of LaCl3 (a–f: 0, 0.32, 1.6, 8, 40 or 200 M of LaCl3 ; g: 0.8 M Tg for contrast) for 3 h, then [Ca2+ ]i was stained with Ca2+ probe, Fluo 3-AM as described in Section 2. *Compared with the control group, P < 0.05, **compared with the control group, P < 0.01.
Ca2+ -binding protein, could be a major direct target of La3+ inside cells. To test whether the dissociation of BiP–IRE1 complexes was caused by La3+ binding, The direct effect of La3+ was investigated by the direct binding to the cell lysate. The association state of BiP–IRE1 complex was observed. As shown in Fig. 3, BiP/GRP78 was observed releasing from BiP–IRE1 and this dissociation could be reversed upon the addition of La3+ strong chelator EDTA. The apparent binding constant was estimated to be 4 nM (Fig. 3D). Shown in Fig. 7, La3+ was observed to enter HepG2 cells upon
incubation with La3+ -containing media. Assuming cells are small balls with ∼10 m diameter, data in Fig. 7 suggested a range of M to mM of total La3+ concentrations inside cells. Considering La3+ would bind to a variety of proteins, the present result suggested the potential of nM to M range of intracellular free La3+ concentrations; this is in consistence with previous report [20] that the intracellular concentration of free lanthanum ion ([La3+ ]i ) could be over 80 nM upon incubation of keratinocytes with 300 M of La3+ . The results above clearly indicated that binding of La3+ to BiP/GRP78 could cause release of BiP/GRP78
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Fig. 7. The cellular content of La3+ upon incubation of HepG2 cells with La3+ . HepG2 cells in 24 well plates were incubated with La3+ for 3 h. Then the cells were collected after wash with PBS solution containing 50 mM EDTA to remove surface-bound metal ions. Then the cells were digested and the amounts of La3+ were determined with ICP-MS. Data are average of triplicate measurements.
from the protein complexes, accounting primarily for the downstream signal transduction. Previously, accumulation of ROS and subsequent oxidative stress [35] were proposed to be important result for ER stress. Since La3+ was observed to cause slightly elevated level of cellular ROS (Fig. 5), therefore, it cannot be ruled out that ROS may also be involved in La3+ -induced UPR. However, La3+ -induced UPR may explain at least part of the reason for the relatively low levels (1–1.5-fold of the control) of ROS induction for La3+ as observed previously [17] and in the present work. Since slight elevation of ROS levels was observed upon incubation of La3+ (<1 M) with isolated mitochondria (data not published), possibly mitochondria could be a cellular source of ROS; nevertheless, other source of ROS (e.g. lysosomes) cannot be excluded so far. Although sustained elevation of [Ca2+ ]i was previously proposed not involved in ER stress induction [36], we observed elevation of [Ca2+ ]i upon incubation of HepG2 cells with La3+ over 8 M (Fig. 6). When taking a look at ROS and [Ca2+ ]i together (Fig. 8), it was observed that induction of ROS was prior to [Ca2+ ]i elevation, but in parallel with the elevation of [Ca2+ ]i . Recently, mitochondrial ROS was proposed to modulate bidirectionally Ca2+ spark activity; in the physiological regime, ROS promote the Ca2+ spark [37]. Therefore, variation of intracellular Ca2+ concentration in HepG2 cells would be closely associated with La3+ -induced ROS and might be involved in the subsequent events. Overall, the general pathway for La3+ to induce UPR could be illustrated in Scheme 1. The present results suggest further studies on the effects of La3+ on PERK and ATF6 as described above would be appropriate. Three transmembrane proteins, known for the transduction of UPR signals, IRE1, PERK and ATF6, are regulated in similar manner by binding with BiP/GRP78 [38–44]. The prevailing model is that the chaperone BiP/GRP78, as a negative regulator, binds to the NLD of IRE1, thus preventing IRE1 activation [40]. Upon ER stress, BiP/GRP78 binds
Fig. 8. Plot of [Ca2+ ]i versus ROS level in HepG2 cells upon incubation with La3+ . Data are from Figs. 5 and 6.
Scheme 1.
to unfolded proteins, permitting the release of NLD and its dimerization. This leads to the activation of the protein kinase and RNase activities in the cytosolic domain of IRE1 [45,46]. Since similar regulation mechanism was expected for activation of PERK and ATF6 signaling [40], it is possible that La3+ may induce the corresponding downstream signal transduction. In summary, the effect of La3+ on UPR in HepG2 cells was investigated. The results showed that BiP/GRP78 could be a major direct target of La3+ inside cells. Binding of La3+ to BiP/GRP78 (possibly in combination with the action of ROS and Ca2+ induced by La3+ ) resulted in dissociation of BiP/GRP78 from BiP–IRE1 complexes, thus activating the IRE1/XBP1 signal pathway. This result suggested that BiP/GRP78 as well as a variety of ER-related cell responses should be involved in the action of La3+ , which provides a new insight for understanding the biological and toxicological effects of La3+ . Acknowledgements This work was supported by NSFC (#20671008 and #20637010). We thank Prof. John J. Hefferren for editing the manuscript. References [1] K. Zhang, R.J. Kaufman, Protein folding in the endoplasmic reticulum and the unfolded protein response, Handb. Exp. Pharmacol. (2006) 69–91. [2] M.C. Ashby, A.V. Tepikin, ER calcium and the functions of intracellular organelles, Semin. Cell Dev. Biol. 12 (2001) 11–17.
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