Cell Biology International ISSN 1065-6995 doi: 10.1002/cbin.10272
SHORT COMMUNICATION
Nuclear factor-kB as a link between endoplasmic reticulum stress and inflammation during cardiomyocyte hypoxia/reoxygenation Qin Wu1, Qiqi Wang2*, Zhidong Guo3, Yunpeng Shang2, Lei Zhang2 and Shikun Gong4 1 2 3 4
Department Department Department Department
of Ophthalmology, The First Affiliated Hospital, College of Medicine, Zhejiang University, Hangzhou 310003, China of Cardiology, The First Affiliated Hospital, College of Medicine, Zhejiang University, 79 Qingchun Road, Hangzhou 310003, China of Emergency, First People’s Hospital of Hangzhou, Hangzhou 310006, China of Cardiology, NingBo BeiLun Hospital of TCM, Ningbo 315806, China
Abstract Endoplasmic reticulum stress (ERS) can initiate inflammation, and the coupling of these responses is thought to be fundamental to the pathogenesis of cardiovascular disease. However, the mechanism linking ERS and inflammation in myocardial ischemia/reperfusion needs further investigation. Cultured cardiomyocytes were pretreated with SP600125 or salubrinal, followed by tunicamycin to clarify the involvement of the IRE1a and PERK pathways in ERS inflammation. The cardiomyocytes were given hypoxia/reoxygenation (H/R), and the effects of the NF-kB inhibitor, SN50, were followed. GRP78 protein levels were similar in the tunicamycin (Tm), salubrinal, and SP600125 groups, but were lower in cells treated with SN50. SN50 might effectively block the H/R-induced link between ERS and inflammation in cardiomyocytes by decreasing GRP78. This knowledge will aid in the development of therapies for myocardial ischemia/reperfusion injury. Keywords: endoplasmic reticulum stress; inflammation; hypoxia/reoxygenation
Introduction Endoplasmic reticulum stress (ERS) mediates the unfoldedprotein response (UPR) to the accumulation of unfolded or misfolded proteins. UPR can initiate inflammation, and the coupling of these responses may be fundamental to the pathogenesis of inflammatory diseases (Zhang and Kaufman, 2008). Prolonged or chronic inflammation is detrimental and affects the development of diseases, such as Alzheimer’s disease, type 1 and type 2 diabetes, and cardiovascular disease (Charo and Ransohoff, 2006; Hansson and Libby, 2006; Hotamisligil, 2006). The molecular link between ERS responses and inflammatory responses might be mediated by the inositol-requiring transmembrane kinase and endonuclease 1a (IRE1a) and the ER-localized protein kinase PERK pathways. IRE1a from the ER might be involved in integrating ERS signaling with inflammatoryresponse signaling. Autophosphorylation of IRE1a in
response to ERS induces a conformational change in its cytosolic domain, which can then bind to the adaptor protein, tumor-necrosis factor-a (TNF-a)-receptor-associated factor 2 (TRAF2) (Urano et al., 2000; Hu et al., 2006), with production of the inflammatory cytokine TNF-a. The formation of the IRE1a–TRAF2 complex seems to be crucial for activating both JUN N-terminal kinase (JNK) and nuclear factor (NF)-kB in response to ERS. JNK and NF-kB might interact and synergize to regulate inflammation. UPR can directly promote NF-kB activation in response to ERS through PERK–eIF2a-mediated attenuation of translation. NF-kB (Kaufman, 2002; Yamamuro et al., 2006). Activation of the transcription factor NF-kB and the mitogen-activated protein kinase, JNK, appear to be crucial factors in ERS inflammation, and signaling pathways in the UPR and inflammation are interconnected through several mechanisms. Some progress has been made in understanding the signaling pathways that integrate ERS and inflammation, and the physiological significance of this connection in
Corresponding author: e-mail: [email protected] Abbreviations: ER, endoplasmic reticulum; UPR, unfolded-protein response; IRE1a, inositol-requiring transmembrane kinase and endonuclease 1a; TNF-a, tumor-necrosis factor-a; JNK, JUN N-terminal kinase; NF, nuclear factor; Tm, tunicamycin; MCs, myocardial cells; FCS, fetal calf serum; DMEM, Dulbecco’s modified Eagle medium; H/R, hypoxia/reoxygenation; CCK-8, cell counting kit-8; CBA, cytometric bead array; IkB, inhibitors of NF-kB
Cell Biol Int 38 (2014) 881–887 © 2014 International Federation for Cell Biology
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cardiovascular diseases. ERS and inflammation underlie the development of atherosclerotic lesions (Li et al., 2005). ERS in myocardial ischemia/reperfusion has been researched, but few studies have attempted to clarify the mechanism of ERS inflammation during this process. We therefore investigated the way in which the ERS-induced UPR triggered inflammation in cultured cardiomyocytes after hypoxia/ reoxygenation (H/R), and also examined the potential role of NF-kB in connecting the ERS and inflammation during cardiomyocyte H/R. The knowledge gained will aid in the development of therapies for myocardial ischemia/reperfusion injury. Materials and methods The protocol was approved by the Animal Care and Use Committee of Zhejiang University (Hangzhou, China). All procedures were carried out in strict accordance with the guidelines set by the National Institutes of Health (Bethesda, MD, USA).
the tubes containing the supernatant were transferred to a Sorvall TJ-25 table-top centrifuge (Sorvall, Newton, CT, USA) set at 4 C and were centrifuged at 1,000 rpm for 10 min at 4 C. Cells were pre-plated for 1 h in Dulbecco’s modified Eagle medium (DMEM, Jinuo Biotechnology, Hangzhou, China) with 10% FCS, and attached cells were discarded. Supernatants were aspirated, and this MC-containing medium was diluted to 200 mL. Ten microliters of MC-containing medium was placed in a hemocytometer, and the number of cells in a 5 5 square in the hemocytometer was counted twice under a light microscope. The concentration of MCs was adjusted to 1 106/mL. MCs were plated onto gelatincoated culture dishes and cultured with DMEM with 10% FCS and 100 mM 5-bromo-2-deoxyuridine (BrdU; Sigma– Aldrich Co., St. Louis, MO, USA). At 36 h after plating, the medium was changed to DMEM without BrdU. Cells were incubated at 37 C in an incubator containing air þ 5% CO2. DMEM was changed every 3 days. Cells were collected on day 5 and their morphology and phenotypic markers were determined (Wang et al., 2013).
Protocols Step 1: Primary culture of neonatal rat myocardial cells (MCs). Step 2: ERS positive control cultures were established using tunicamycin (Tm, 5 mg/mL). Cells were cultured with SP600125 (JNK inhibitor, 10 mmol/L for 1 h) or salubrinal (eIF2a phosphatase inhibitor, 10 mmol/L for 1 h), then cultured with Tm. We followed the effects of these inhibitors on ERS-inflammation injury induced by Tm. Step 3: Cells treated with H/R, Tm, H/R þ SN50 (NF-kB inhibitor of nuclear factor-kB, 50 mg/mL), and Tm þ SN50 were compared to determine the importance of NF-kB in ERS inflammation. The ERS protein GRP78, inflammation (TNF-a), myocardial injury, and cell viability were assessed.
Primary culture of neonatal rat MCs One day-old Sprague–Dawley rats were provided by the Shanghai Laboratory Animal Center (Chinese Academy of Sciences, Shanghai, China). The chest wall was cut and the heart was isolated and washed three times in ice-cold D-Hanks solution. Six hearts were dissected, being careful not to press down on the hearts while cutting, and excising them as quickly as possible. The heart tissue was minced and placed into sterilized flasks. Ten microliters of digestion buffer was added, and the mixture was incubated at 37 C for 10 min, using a magnetic stirrer at 80 rpm. After stirring, the supernatant was removed, a further 10 mL of digestion buffer was added, and the supernatant was transferred to 50-mL tubes containing 5 mL of 10% fetal calf serum (FCS; Gibco, Billings, MT, USA). This process was repeated four times to release single cells. Once digestion was complete, 882
Hypoxia/reoxygenation (H/R) of MCs Oxygen was completely replaced using a sustained inflow of hypoxic gas into the sealed chamber for 45 min. Fifth-day cultured myocytes were put into hypoxic solution, put into the chamber, and incubated at 37 C in a thermostatically controlled water bath, with an inflow of hypoxic gas for 12 h. The hypoxia was stopped and the DMEM with 10% FCS introduced. The myocytes were placed in a air plus CO2 incubator for a further 12 h. Cells were divided into the following groups: (1) control group; (2) Tm group, 1 mg/L; (3) H/R group, hypoxia 12 h, reoxygenation 12 h; (4) Tm þ SN50 group, Tm 1 mg/L þ SN50 50 mg/mL; (5) H/R þ SN50 group, SN50 50 mg/mL.
Assessment of MC viability Heart rates were counted at the end of experiment. Cell viability was assessed using a Cell Counting Kit-8 (CCK-8 Tongren, Japan; Ishiyama et al., 1997). The CCK-8 test is a dehydrogenase-dependent colorimetric assay that indirectly measures cellular metabolic activity.
Western blot analysis Proteins were separated by sodium dodecyl sulfate– polyacrylamide gel electrophoresis, transferred to a nitrocellulose membrane, and incubated with monoclonal antibodies to GRP78 (diluted 1:500, sc-1050, Santa Cruz Biotechnology, Santa Cruz, CA, USA) at 4 C overnight. Cell Biol Int 38 (2014) 881–887 © 2014 International Federation for Cell Biology
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Membranes were incubated with secondary antibody (diluted 1:5,000, Lianke Biosciences, China) for 2 h and developed with BeyoECL reagent (Biyuntian Biosciences, China). The blots were exposed for development (Chomczynski and Sacchin, 1987).
RNA extraction and reverse transcription-polymerase chain reaction (RT-PCR) analysis Chemicals and reagents were purchased from TaKaRa Biotechnology (Shiga, Japan), unless otherwise specified.
RNA isolation and cDNA synthesis Total RNA was extracted from the cultured MCs using RNAiso Plus (TaKaRa Biotechnology) in accordance with manufacturer’s instructions. RNA concentrations were determined by absorbance readings at 260 nm using a SmartSpec Plus Spectrophotometer (Bio-Rad, Tokyo, Japan). Reverse transcription of RNA was performed using PrimeScript1 Reverse Transcriptase (RNase H) (TaKaRa Biotechnology) as outlined in the instruction manual. cDNA served as a template for PCR (Wang et al., 2013).
Quantitative PCR The primer sequences were: 50 -CTCCACGGCTTCCGATAATCA-30 and 50 -TCCAGTCAGATCAAATGTACCCAGA-30 for GRP-78; and 50 -AAATGGTGAAGGTCGGTGTGAAC-30 and 50 -CAACAATCTCCACTTTGCCACTG-30 for glyceraldehyde 3-phosphate dehydrogenase (GAPDH). Real-time PCR was carried out using a Reverse Transcriptase (RNase H) kit (TaKaRa Biotechnology) in an ABI 7500 Fast System (Applied Biosystems, Foster City, CA, USA) with the following universal cycling conditions: denaturation for 30 s at 95 C, amplification for 40 cycles, with denaturation for 5 s at 95 C, annealing for 60 s at 60 C and extension for 34 s at 72 C. Samples were tested in triplicate and mean values were used for quantification. mRNA expression levels were normalized to GAPDH. Relative mRNA levels are shown as unit values of 2DDCt, where Ct is the threshold cycle value (defined as the fractional cycle number at which the target fluorescent signal passes a fixed threshold above the baseline).
Detection of plasma cytokine with cytometric bead array (CBA) Analysis of TNF-a used a rat inflammation CBA kit (Bender Med Systems Products, Vienna, Austria) and a FACS Calibur flow cytometer (Becton Dickinson, Bedford, MA). Standard curve for each cytokine from each kit was generated by using the reference cytokine concentrations with a range of 27–20,000 pg/mL. The minimum detection Cell Biol Int 38 (2014) 881–887 © 2014 International Federation for Cell Biology
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of CBA is 4.3 pg/mL (for TNF-a). CBA raw data exported from the FACS Calibur flow cytometer was analyzed by Flow Cytomix Pro2.1 software (Bender MedSystems GmbH).
Pathology of cultured MCs Cultured MCs were stained with hematoxylin and eosin and examined by an observer blinded to the protocol. The following morphological criteria (Zingarelli et al., 1998; Wang et al., 2013) were used to determine histopathological changes: (A) Normal: abundant contoured cardiomyocytes with several antennae; nuclei of identical size; cytoplasm enriched and well-stained. (B) Injured: few cardiomyocytes; contoured with few antennae; nuclei small; cytoplasm not enriched; acidophilic staining. Factors associated with group A had a score of 1, and those associated with group B had a score of zero. The score (per high-magnification view) was used as the basis for assessing lesion severity on this semiquantitative scoring scale.
Statistical analysis Results are given as mean SD. Five parallels were used to calculate SD. Data were analyzed using analysis of variance followed by the Scheffe’s test for multiple comparisons. P < 0.05 was considered significant. Results
Effects of IRE1a and PERK pathway inhibition on GRP78 concentration determined by western blotting We established positive ERS control MC cultures using Tm, and compared GRP78 concentrations with cells treated with IRE1a and PERK inhibitors, SP60012 and salubrinal. The concentrations of GRP78 were higher in the positive control, SP60012 and salubrinal groups compared with the untreated control cells (P < 0.001). This suggests that single inhibition of the IRE1a or PERK pathway had no obvious influence on ERS inflammation, and implies that other important factors may control this mechanism (Figure 1).
Heart rate and CCK-8 test Heart rate decreased in MCs subjected to H/R, while heart rate increased in cells treated with the NF-kB inhibitor SN50. By the CCK-8 test, cells in H/R had lower viability, which recovered after culture with SN50 (Figure 2).
Effect of SN50 on GRP78 protein expression GRP78 expression increased in MCs subjected to H/R, but decreased in cells cultured with SN50. This suggests that H/R 883
From endoplasmic reticulum stress to inflammation
Figure 1 Effects of IRE1a and PERK pathway on GRP78 determined by Western blotting. (A) GRP78 expression by Western blotting (left to right: control; Tm 5 mg/mL for 6 h; Tm 5 mg/mL for 6 h þ 10 mmol Salubrinal for 1 h; Tm 5 mg/mL for 6 h þ SP600125 10 mmol/L for 1 h). (B) GAPDH expression by Western blotting. With SP60012 and Salubrinal culture, the concentration of GRP78 was similar with the Tm only group. *Compared to control group, the concentration of GRP78 was higher in the other three groups (P < 0.001).
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Figure 2 Heart rate and CCK-8 test at different groups. (A) CCK-8 test: *P < 0.01 compared with Tm, H/R, Tm þ SN50, H/R þ SN50 groups. **P < 0.05 compared with Tm group. #P < 0.05 compared with H/R group. (B) Heart rate: *P < 0.01 compared with Tm, H/R, Tm þ SN50, H/R þ SN50 groups. **P < 0.05 compared with Tm group. #P < 0.05 compared with H/R group.
might be associated with ERS, and that NF-kB might be an important marker of the ERS-inflammation mechanism (Figure 3).
Effect of SN50 on GRP78 gene expression GRP78 mRNA levels increased in MCs subjected to H/R, but decreased in cells cultured with SN50. As for GRP78 protein levels, the idea that H/R might be associated with ERS by NF-kB is supported (Figure 4).
TNF-a levels measured by CBA Compared with the H/R group, TNF-a concentrations decreased (8.10 1.12 pg/mL vs. 11.70 1.31 pg/mL) by treatment with SN50. This suggests that H/R might be associated with ERS inflammation, and that SN50 could effectively inhibit important markers in the ERSinflammation pathway (Figure 5).
Effect of SN50 on morphological injury Cardiomyocytes in H/R were severely damaged, which was alleviated by SN50. The morphological score showed that 884
Figure 3 Effect of SN50 on GRP78 expression at different groups by Western blot. (A) GRP78 expression by western blotting (left to right: Control, Tm, H/R, Tm þ SN50, H/R þ SN50). (B) GAPDH expression by Western blotting. (C) Relative expression of GRP78 at different groups. *P < 0.001 compared to control group, the relative expression of GRP78 was higher in all the other groups. **P < 0.05 compared with Tm group. # P < 05 compared with H/R group.
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Figure 4 Effect of SN50 on GRP78 expression at different groups by RT-PCR. (A) Image of the mRNA product. (B) Relative level of GRP78 at different groups. *P < 0.001 compared to control group, the relative expression of GRP78 was higher in all the other groups. **P < 0.01 compared with Tm group. #P < 0.001 compared with H/R group.
H/R might be associated with cell damage, and that SN50 could protect against H/R injury in myocytes. These results show that NF-kB as an important marker of the ERSinflammation mechanism (Figure 6). Discussion H/R-induced ERS inflammation in MCs. Salubrinal selectively inhibits the dephosphorylation of eIF2a to reduce the phosphorylation of PERK and IRE1a, while SP600125 inhibits JNK pathways. However, neither of these inhibitors prevented ERS inflammation induced by Tm, represented by
From endoplasmic reticulum stress to inflammation
GRP78 levels. The NF-kB inhibitor SN50effectively reduced cardiomyocyte ERS inflammation after H/R. SN50 can protect cells against ERS-induced inflammation by selectively inhibiting the translocation of NF-kB to the nucleus, suggesting that NF-kB acts as a link between the ERS and inflammation during cardiomyocyte H/R. Salubrinal protected cells against ERS-induced apoptosis by selectively inhibiting the dephosphorylation of eIF2a, thereby inhibiting protein synthesis and accumulation in the ER (Boyce et al., 2005). Although the PERK pathway might be crucial to apoptosis, it is not the only link between ERS and inflammation; activated JNK induces the expression of inflammatory genes by phosphorylating transcription factor activator protein 1 (Davis, 2000). We found ERS activation was not inhibited by the JNK inhibitor, SP600125, indicating that JNK does not provide a link between ERS and inflammation. Overall, the results suggest that ERS-induced inflammation after H/R in cardiomyocytes is related to NF-kB, which regulates inflammation and cell survival. NF-kB is a key transcriptional regulator in the onset of inflammation (Rius et al., 2008). In the absence of inflammatory stimuli, NF-kB remains in an inactive state through binding to a member of the constitutively-expressed family of inhibitors of NF-kB (IkB). Activation of NF-kB is initiated by signal-induced phosphorylation of IkB, which is subsequently degraded. Degradation of IkB exposes a nuclear-localization signal in NF-kB, allowing it to translocate to the nucleus, where it induces the transcription of numerous inflammatory genes (Rius et al., 2008). However, the details of the mechanism by which NF-kB is activated by H/R in MCs are poorly understood. An increase in the ER protein-folding load results in the activation of NF-kB (Meyer et al., 1992; Pahl and Baeuerle, 1995). Calcium chelators and antioxidants contribute to the activation of NF-kB in response to ERS (Pahl and Baeuerle, 1996). ERassociated NF-kB activation might thus result from the oxidative stress of excessive protein folding, and/or from ERS-mediated leakage of calcium into the cytosol (Deniaud et al., 2008). UPR also directly promotes NF-kB activation in response to ERS through PERK-eIF2a-mediated attenuation of translation (Urano et al., 2000; Hu et al., 2006). Activation of NF-kB after ERS can thus occur via several mechanisms, implying that NF-kB might represent a crucial link between ERS and inflammation. In conclusion, our findings confirmed the occurrence of ERS-inflammation injury in cardiomyocytes during H/R and established a role for NF-kB as a crucial link between ERS and inflammation. Acknowledgment and funding
Figure 5 TNF-a levels measured by CBA. *P < 0.001 compared to control group, the relative expression of GRP78 was higher in all the other groups. #P < 0.001 compared with H/R group.
Cell Biol Int 38 (2014) 881–887 © 2014 International Federation for Cell Biology
This paper was supported by the National Science Foundation for Young Scholars of China (grant no. 30900612), the Grant of Medical Science Research Foundation of 885
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Figure 6 Effect of SN50 on morphological injury. (A–E) Tm, Tm þ SN50, H/R, H/R þ SN50, control group, respectively (400 magnification). (F) Morphological score. *P < 0.001 compared to control group, the score was higher in all the other groups. **P < 0.001 compared with H/R group. # P < 0.01 compared with Tm group.
Zhejiang Province (grant no. 2009B127, 2009A072), and funded by the Science and Technology Department of Zhejiang Province (grant no. 2010C33036, 2012C37104), People’s Republic of China. Conflicts of interest The authors report no conflict of interest. 886
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Chomczynski P, Sacchin N (1987) Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction. Ann Biochem 162: 156–9. Davis RJ (2000) Signal transduction by the JNK group of MAP kinases. Cell 103: 239–52. Deniaud A, Sharaf el dein O, Maillier E, Poncet D, Kroemer G, Lemaire C, Brenner C (2008) Endoplasmic reticulum stress induces calcium-dependent permeability transition, mitochondrial outer membrane permeabilization and apoptosis. Oncogene 27: 285–99. Hansson GK, Libby P (2006) The immune response in atherosclerosis: a double-edged sword. Nature Rev Immunol 6: 508–19. Hotamisligil GS (2006) Inflammation and metabolic disorders. Nature 444: 860–7. Hu P, Han Z, Couvillon AD, Kaufman RJ, Exton JH (2006) Autocrine tumor necrosis factor a links endoplasmic reticulum stress to the membrane death receptor pathway through IRE1a-mediated NF-kB activation and down-regulation of TRAF2 expression. Mol Cell Biol 26: 3071–84. Ishiyama M, Miyazono Y, Sasamoto K, Ohkura Y, Ueno K (1997) A highly water-soluble disulfonated tetrazolium salt as a chromogenic indicator for ADH as well as cell viability. Talanta 44: 1299–305. Kaufman RJ (2002) Orchestrating the unfolded protein response in health and disease. J Clin Invest 110: 1389–98. Li Y, Schwabe RF, DeVries-Seimon T, Yao PM, GerbodGiannone MC, Tall AR, Davis RJ, Flavell R, Brenner DA, Tabas I (2005) Free cholesterol-loaded macrophages are an abundant source of tumor necrosis factor-a and interleukin-6: model of NF-kB- and MAP kinase-dependent inflammation in advanced atherosclerosis. J Biol Chem 280: 21763–72. Meyer M, Caselmann WH, Schlüter V, Schreck R, Hofschneider PH, Baeuerle PA (1992) Hepatitis B virus transactivator MHBst: activation of NF-kB, selective inhibition by
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antioxidants and integral membrane localization. EMBO J 11: 2991–3001. Pahl HL, Baeuerle PA (1995) Expression of influenza virus hemagglutinin activates transcription factor NF-kB. J Virol 69: 1480–4. Pahl HL, Baeuerle PA (1996) Activation of NF-kB by ER stress requires both Ca2þ and reactive oxygen intermediates as messengers. FEBS Lett 392: 129–36. Rius J, Guma M, Schachtrup C, Akassoglou K, Zinkernagel AS, Nizet V, Johnson RS, Haddad GG, Karin M (2008) NFkappaB links innate immunity to the hypoxic response through transcriptional regulation of HIF-1alpha. Nature 453: 807–11. Urano F, Wang X, Bertolotti A, Zhang Y, Chung P, Harding HP, Ron D (2000) Coupling of stress in the ER to activation of JNK protein kinases by transmembrane protein kinase IRE1. Science 287: 664–6. Wang QQ, Jin JM, Guo ZD, Chen FX, Qiu YG, Zhu JH, Shang YP (2013) Improvements in the primary culture of neonate rat myocardial cells by study of the mechanism of endoplasmic reticulum stress. Cell Stress Chaperones 18: 367–75. Yamamuro A, Yoshioka Y, Ogita K, Maeda S (2006) Involvement of endoplasmic reticulum stress on the cell death induced by 6-hydroxydopamine in human neuroblastoma SH-SY5Y cells. Neurochem Res 31: 657–64. Zhang KZ, Kaufman RJ (2008) From endoplasmic-reticulum stress to the inflammatory response. Nature 454: 455–62. Zingarelli B, Salzman AL, Szabo C (1998) Genetic disruption of poly (ADP-Ribose) synthetase inhibits the expression of P-selectin and intercellular adhesion molecule-1 in myocardial ischemia/reperfusion injury. Circ Res 83: 85–94. Received 13 December 2013; accepted 7 February 2014. Final version published online 17 March 2014.
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SHORT COMMUNICATION
Nuclear factor-kB as a link between endoplasmic reticulum stress and inflammation during cardiomyocyte hypoxia/reoxygenation Qin Wu1, Qiqi Wang2*, Zhidong Guo3, Yunpeng Shang2, Lei Zhang2 and Shikun Gong4 1 2 3 4
Department Department Department Department
of Ophthalmology, The First Affiliated Hospital, College of Medicine, Zhejiang University, Hangzhou 310003, China of Cardiology, The First Affiliated Hospital, College of Medicine, Zhejiang University, 79 Qingchun Road, Hangzhou 310003, China of Emergency, First People’s Hospital of Hangzhou, Hangzhou 310006, China of Cardiology, NingBo BeiLun Hospital of TCM, Ningbo 315806, China
Abstract Endoplasmic reticulum stress (ERS) can initiate inflammation, and the coupling of these responses is thought to be fundamental to the pathogenesis of cardiovascular disease. However, the mechanism linking ERS and inflammation in myocardial ischemia/reperfusion needs further investigation. Cultured cardiomyocytes were pretreated with SP600125 or salubrinal, followed by tunicamycin to clarify the involvement of the IRE1a and PERK pathways in ERS inflammation. The cardiomyocytes were given hypoxia/reoxygenation (H/R), and the effects of the NF-kB inhibitor, SN50, were followed. GRP78 protein levels were similar in the tunicamycin (Tm), salubrinal, and SP600125 groups, but were lower in cells treated with SN50. SN50 might effectively block the H/R-induced link between ERS and inflammation in cardiomyocytes by decreasing GRP78. This knowledge will aid in the development of therapies for myocardial ischemia/reperfusion injury. Keywords: endoplasmic reticulum stress; inflammation; hypoxia/reoxygenation
Introduction Endoplasmic reticulum stress (ERS) mediates the unfoldedprotein response (UPR) to the accumulation of unfolded or misfolded proteins. UPR can initiate inflammation, and the coupling of these responses may be fundamental to the pathogenesis of inflammatory diseases (Zhang and Kaufman, 2008). Prolonged or chronic inflammation is detrimental and affects the development of diseases, such as Alzheimer’s disease, type 1 and type 2 diabetes, and cardiovascular disease (Charo and Ransohoff, 2006; Hansson and Libby, 2006; Hotamisligil, 2006). The molecular link between ERS responses and inflammatory responses might be mediated by the inositol-requiring transmembrane kinase and endonuclease 1a (IRE1a) and the ER-localized protein kinase PERK pathways. IRE1a from the ER might be involved in integrating ERS signaling with inflammatoryresponse signaling. Autophosphorylation of IRE1a in
response to ERS induces a conformational change in its cytosolic domain, which can then bind to the adaptor protein, tumor-necrosis factor-a (TNF-a)-receptor-associated factor 2 (TRAF2) (Urano et al., 2000; Hu et al., 2006), with production of the inflammatory cytokine TNF-a. The formation of the IRE1a–TRAF2 complex seems to be crucial for activating both JUN N-terminal kinase (JNK) and nuclear factor (NF)-kB in response to ERS. JNK and NF-kB might interact and synergize to regulate inflammation. UPR can directly promote NF-kB activation in response to ERS through PERK–eIF2a-mediated attenuation of translation. NF-kB (Kaufman, 2002; Yamamuro et al., 2006). Activation of the transcription factor NF-kB and the mitogen-activated protein kinase, JNK, appear to be crucial factors in ERS inflammation, and signaling pathways in the UPR and inflammation are interconnected through several mechanisms. Some progress has been made in understanding the signaling pathways that integrate ERS and inflammation, and the physiological significance of this connection in
Corresponding author: e-mail: [email protected] Abbreviations: ER, endoplasmic reticulum; UPR, unfolded-protein response; IRE1a, inositol-requiring transmembrane kinase and endonuclease 1a; TNF-a, tumor-necrosis factor-a; JNK, JUN N-terminal kinase; NF, nuclear factor; Tm, tunicamycin; MCs, myocardial cells; FCS, fetal calf serum; DMEM, Dulbecco’s modified Eagle medium; H/R, hypoxia/reoxygenation; CCK-8, cell counting kit-8; CBA, cytometric bead array; IkB, inhibitors of NF-kB
Cell Biol Int 38 (2014) 881–887 © 2014 International Federation for Cell Biology
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From endoplasmic reticulum stress to inflammation
cardiovascular diseases. ERS and inflammation underlie the development of atherosclerotic lesions (Li et al., 2005). ERS in myocardial ischemia/reperfusion has been researched, but few studies have attempted to clarify the mechanism of ERS inflammation during this process. We therefore investigated the way in which the ERS-induced UPR triggered inflammation in cultured cardiomyocytes after hypoxia/ reoxygenation (H/R), and also examined the potential role of NF-kB in connecting the ERS and inflammation during cardiomyocyte H/R. The knowledge gained will aid in the development of therapies for myocardial ischemia/reperfusion injury. Materials and methods The protocol was approved by the Animal Care and Use Committee of Zhejiang University (Hangzhou, China). All procedures were carried out in strict accordance with the guidelines set by the National Institutes of Health (Bethesda, MD, USA).
the tubes containing the supernatant were transferred to a Sorvall TJ-25 table-top centrifuge (Sorvall, Newton, CT, USA) set at 4 C and were centrifuged at 1,000 rpm for 10 min at 4 C. Cells were pre-plated for 1 h in Dulbecco’s modified Eagle medium (DMEM, Jinuo Biotechnology, Hangzhou, China) with 10% FCS, and attached cells were discarded. Supernatants were aspirated, and this MC-containing medium was diluted to 200 mL. Ten microliters of MC-containing medium was placed in a hemocytometer, and the number of cells in a 5 5 square in the hemocytometer was counted twice under a light microscope. The concentration of MCs was adjusted to 1 106/mL. MCs were plated onto gelatincoated culture dishes and cultured with DMEM with 10% FCS and 100 mM 5-bromo-2-deoxyuridine (BrdU; Sigma– Aldrich Co., St. Louis, MO, USA). At 36 h after plating, the medium was changed to DMEM without BrdU. Cells were incubated at 37 C in an incubator containing air þ 5% CO2. DMEM was changed every 3 days. Cells were collected on day 5 and their morphology and phenotypic markers were determined (Wang et al., 2013).
Protocols Step 1: Primary culture of neonatal rat myocardial cells (MCs). Step 2: ERS positive control cultures were established using tunicamycin (Tm, 5 mg/mL). Cells were cultured with SP600125 (JNK inhibitor, 10 mmol/L for 1 h) or salubrinal (eIF2a phosphatase inhibitor, 10 mmol/L for 1 h), then cultured with Tm. We followed the effects of these inhibitors on ERS-inflammation injury induced by Tm. Step 3: Cells treated with H/R, Tm, H/R þ SN50 (NF-kB inhibitor of nuclear factor-kB, 50 mg/mL), and Tm þ SN50 were compared to determine the importance of NF-kB in ERS inflammation. The ERS protein GRP78, inflammation (TNF-a), myocardial injury, and cell viability were assessed.
Primary culture of neonatal rat MCs One day-old Sprague–Dawley rats were provided by the Shanghai Laboratory Animal Center (Chinese Academy of Sciences, Shanghai, China). The chest wall was cut and the heart was isolated and washed three times in ice-cold D-Hanks solution. Six hearts were dissected, being careful not to press down on the hearts while cutting, and excising them as quickly as possible. The heart tissue was minced and placed into sterilized flasks. Ten microliters of digestion buffer was added, and the mixture was incubated at 37 C for 10 min, using a magnetic stirrer at 80 rpm. After stirring, the supernatant was removed, a further 10 mL of digestion buffer was added, and the supernatant was transferred to 50-mL tubes containing 5 mL of 10% fetal calf serum (FCS; Gibco, Billings, MT, USA). This process was repeated four times to release single cells. Once digestion was complete, 882
Hypoxia/reoxygenation (H/R) of MCs Oxygen was completely replaced using a sustained inflow of hypoxic gas into the sealed chamber for 45 min. Fifth-day cultured myocytes were put into hypoxic solution, put into the chamber, and incubated at 37 C in a thermostatically controlled water bath, with an inflow of hypoxic gas for 12 h. The hypoxia was stopped and the DMEM with 10% FCS introduced. The myocytes were placed in a air plus CO2 incubator for a further 12 h. Cells were divided into the following groups: (1) control group; (2) Tm group, 1 mg/L; (3) H/R group, hypoxia 12 h, reoxygenation 12 h; (4) Tm þ SN50 group, Tm 1 mg/L þ SN50 50 mg/mL; (5) H/R þ SN50 group, SN50 50 mg/mL.
Assessment of MC viability Heart rates were counted at the end of experiment. Cell viability was assessed using a Cell Counting Kit-8 (CCK-8 Tongren, Japan; Ishiyama et al., 1997). The CCK-8 test is a dehydrogenase-dependent colorimetric assay that indirectly measures cellular metabolic activity.
Western blot analysis Proteins were separated by sodium dodecyl sulfate– polyacrylamide gel electrophoresis, transferred to a nitrocellulose membrane, and incubated with monoclonal antibodies to GRP78 (diluted 1:500, sc-1050, Santa Cruz Biotechnology, Santa Cruz, CA, USA) at 4 C overnight. Cell Biol Int 38 (2014) 881–887 © 2014 International Federation for Cell Biology
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Membranes were incubated with secondary antibody (diluted 1:5,000, Lianke Biosciences, China) for 2 h and developed with BeyoECL reagent (Biyuntian Biosciences, China). The blots were exposed for development (Chomczynski and Sacchin, 1987).
RNA extraction and reverse transcription-polymerase chain reaction (RT-PCR) analysis Chemicals and reagents were purchased from TaKaRa Biotechnology (Shiga, Japan), unless otherwise specified.
RNA isolation and cDNA synthesis Total RNA was extracted from the cultured MCs using RNAiso Plus (TaKaRa Biotechnology) in accordance with manufacturer’s instructions. RNA concentrations were determined by absorbance readings at 260 nm using a SmartSpec Plus Spectrophotometer (Bio-Rad, Tokyo, Japan). Reverse transcription of RNA was performed using PrimeScript1 Reverse Transcriptase (RNase H) (TaKaRa Biotechnology) as outlined in the instruction manual. cDNA served as a template for PCR (Wang et al., 2013).
Quantitative PCR The primer sequences were: 50 -CTCCACGGCTTCCGATAATCA-30 and 50 -TCCAGTCAGATCAAATGTACCCAGA-30 for GRP-78; and 50 -AAATGGTGAAGGTCGGTGTGAAC-30 and 50 -CAACAATCTCCACTTTGCCACTG-30 for glyceraldehyde 3-phosphate dehydrogenase (GAPDH). Real-time PCR was carried out using a Reverse Transcriptase (RNase H) kit (TaKaRa Biotechnology) in an ABI 7500 Fast System (Applied Biosystems, Foster City, CA, USA) with the following universal cycling conditions: denaturation for 30 s at 95 C, amplification for 40 cycles, with denaturation for 5 s at 95 C, annealing for 60 s at 60 C and extension for 34 s at 72 C. Samples were tested in triplicate and mean values were used for quantification. mRNA expression levels were normalized to GAPDH. Relative mRNA levels are shown as unit values of 2DDCt, where Ct is the threshold cycle value (defined as the fractional cycle number at which the target fluorescent signal passes a fixed threshold above the baseline).
Detection of plasma cytokine with cytometric bead array (CBA) Analysis of TNF-a used a rat inflammation CBA kit (Bender Med Systems Products, Vienna, Austria) and a FACS Calibur flow cytometer (Becton Dickinson, Bedford, MA). Standard curve for each cytokine from each kit was generated by using the reference cytokine concentrations with a range of 27–20,000 pg/mL. The minimum detection Cell Biol Int 38 (2014) 881–887 © 2014 International Federation for Cell Biology
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of CBA is 4.3 pg/mL (for TNF-a). CBA raw data exported from the FACS Calibur flow cytometer was analyzed by Flow Cytomix Pro2.1 software (Bender MedSystems GmbH).
Pathology of cultured MCs Cultured MCs were stained with hematoxylin and eosin and examined by an observer blinded to the protocol. The following morphological criteria (Zingarelli et al., 1998; Wang et al., 2013) were used to determine histopathological changes: (A) Normal: abundant contoured cardiomyocytes with several antennae; nuclei of identical size; cytoplasm enriched and well-stained. (B) Injured: few cardiomyocytes; contoured with few antennae; nuclei small; cytoplasm not enriched; acidophilic staining. Factors associated with group A had a score of 1, and those associated with group B had a score of zero. The score (per high-magnification view) was used as the basis for assessing lesion severity on this semiquantitative scoring scale.
Statistical analysis Results are given as mean SD. Five parallels were used to calculate SD. Data were analyzed using analysis of variance followed by the Scheffe’s test for multiple comparisons. P < 0.05 was considered significant. Results
Effects of IRE1a and PERK pathway inhibition on GRP78 concentration determined by western blotting We established positive ERS control MC cultures using Tm, and compared GRP78 concentrations with cells treated with IRE1a and PERK inhibitors, SP60012 and salubrinal. The concentrations of GRP78 were higher in the positive control, SP60012 and salubrinal groups compared with the untreated control cells (P < 0.001). This suggests that single inhibition of the IRE1a or PERK pathway had no obvious influence on ERS inflammation, and implies that other important factors may control this mechanism (Figure 1).
Heart rate and CCK-8 test Heart rate decreased in MCs subjected to H/R, while heart rate increased in cells treated with the NF-kB inhibitor SN50. By the CCK-8 test, cells in H/R had lower viability, which recovered after culture with SN50 (Figure 2).
Effect of SN50 on GRP78 protein expression GRP78 expression increased in MCs subjected to H/R, but decreased in cells cultured with SN50. This suggests that H/R 883
From endoplasmic reticulum stress to inflammation
Figure 1 Effects of IRE1a and PERK pathway on GRP78 determined by Western blotting. (A) GRP78 expression by Western blotting (left to right: control; Tm 5 mg/mL for 6 h; Tm 5 mg/mL for 6 h þ 10 mmol Salubrinal for 1 h; Tm 5 mg/mL for 6 h þ SP600125 10 mmol/L for 1 h). (B) GAPDH expression by Western blotting. With SP60012 and Salubrinal culture, the concentration of GRP78 was similar with the Tm only group. *Compared to control group, the concentration of GRP78 was higher in the other three groups (P < 0.001).
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Figure 2 Heart rate and CCK-8 test at different groups. (A) CCK-8 test: *P < 0.01 compared with Tm, H/R, Tm þ SN50, H/R þ SN50 groups. **P < 0.05 compared with Tm group. #P < 0.05 compared with H/R group. (B) Heart rate: *P < 0.01 compared with Tm, H/R, Tm þ SN50, H/R þ SN50 groups. **P < 0.05 compared with Tm group. #P < 0.05 compared with H/R group.
might be associated with ERS, and that NF-kB might be an important marker of the ERS-inflammation mechanism (Figure 3).
Effect of SN50 on GRP78 gene expression GRP78 mRNA levels increased in MCs subjected to H/R, but decreased in cells cultured with SN50. As for GRP78 protein levels, the idea that H/R might be associated with ERS by NF-kB is supported (Figure 4).
TNF-a levels measured by CBA Compared with the H/R group, TNF-a concentrations decreased (8.10 1.12 pg/mL vs. 11.70 1.31 pg/mL) by treatment with SN50. This suggests that H/R might be associated with ERS inflammation, and that SN50 could effectively inhibit important markers in the ERSinflammation pathway (Figure 5).
Effect of SN50 on morphological injury Cardiomyocytes in H/R were severely damaged, which was alleviated by SN50. The morphological score showed that 884
Figure 3 Effect of SN50 on GRP78 expression at different groups by Western blot. (A) GRP78 expression by western blotting (left to right: Control, Tm, H/R, Tm þ SN50, H/R þ SN50). (B) GAPDH expression by Western blotting. (C) Relative expression of GRP78 at different groups. *P < 0.001 compared to control group, the relative expression of GRP78 was higher in all the other groups. **P < 0.05 compared with Tm group. # P < 05 compared with H/R group.
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Figure 4 Effect of SN50 on GRP78 expression at different groups by RT-PCR. (A) Image of the mRNA product. (B) Relative level of GRP78 at different groups. *P < 0.001 compared to control group, the relative expression of GRP78 was higher in all the other groups. **P < 0.01 compared with Tm group. #P < 0.001 compared with H/R group.
H/R might be associated with cell damage, and that SN50 could protect against H/R injury in myocytes. These results show that NF-kB as an important marker of the ERSinflammation mechanism (Figure 6). Discussion H/R-induced ERS inflammation in MCs. Salubrinal selectively inhibits the dephosphorylation of eIF2a to reduce the phosphorylation of PERK and IRE1a, while SP600125 inhibits JNK pathways. However, neither of these inhibitors prevented ERS inflammation induced by Tm, represented by
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GRP78 levels. The NF-kB inhibitor SN50effectively reduced cardiomyocyte ERS inflammation after H/R. SN50 can protect cells against ERS-induced inflammation by selectively inhibiting the translocation of NF-kB to the nucleus, suggesting that NF-kB acts as a link between the ERS and inflammation during cardiomyocyte H/R. Salubrinal protected cells against ERS-induced apoptosis by selectively inhibiting the dephosphorylation of eIF2a, thereby inhibiting protein synthesis and accumulation in the ER (Boyce et al., 2005). Although the PERK pathway might be crucial to apoptosis, it is not the only link between ERS and inflammation; activated JNK induces the expression of inflammatory genes by phosphorylating transcription factor activator protein 1 (Davis, 2000). We found ERS activation was not inhibited by the JNK inhibitor, SP600125, indicating that JNK does not provide a link between ERS and inflammation. Overall, the results suggest that ERS-induced inflammation after H/R in cardiomyocytes is related to NF-kB, which regulates inflammation and cell survival. NF-kB is a key transcriptional regulator in the onset of inflammation (Rius et al., 2008). In the absence of inflammatory stimuli, NF-kB remains in an inactive state through binding to a member of the constitutively-expressed family of inhibitors of NF-kB (IkB). Activation of NF-kB is initiated by signal-induced phosphorylation of IkB, which is subsequently degraded. Degradation of IkB exposes a nuclear-localization signal in NF-kB, allowing it to translocate to the nucleus, where it induces the transcription of numerous inflammatory genes (Rius et al., 2008). However, the details of the mechanism by which NF-kB is activated by H/R in MCs are poorly understood. An increase in the ER protein-folding load results in the activation of NF-kB (Meyer et al., 1992; Pahl and Baeuerle, 1995). Calcium chelators and antioxidants contribute to the activation of NF-kB in response to ERS (Pahl and Baeuerle, 1996). ERassociated NF-kB activation might thus result from the oxidative stress of excessive protein folding, and/or from ERS-mediated leakage of calcium into the cytosol (Deniaud et al., 2008). UPR also directly promotes NF-kB activation in response to ERS through PERK-eIF2a-mediated attenuation of translation (Urano et al., 2000; Hu et al., 2006). Activation of NF-kB after ERS can thus occur via several mechanisms, implying that NF-kB might represent a crucial link between ERS and inflammation. In conclusion, our findings confirmed the occurrence of ERS-inflammation injury in cardiomyocytes during H/R and established a role for NF-kB as a crucial link between ERS and inflammation. Acknowledgment and funding
Figure 5 TNF-a levels measured by CBA. *P < 0.001 compared to control group, the relative expression of GRP78 was higher in all the other groups. #P < 0.001 compared with H/R group.
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This paper was supported by the National Science Foundation for Young Scholars of China (grant no. 30900612), the Grant of Medical Science Research Foundation of 885
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Figure 6 Effect of SN50 on morphological injury. (A–E) Tm, Tm þ SN50, H/R, H/R þ SN50, control group, respectively (400 magnification). (F) Morphological score. *P < 0.001 compared to control group, the score was higher in all the other groups. **P < 0.001 compared with H/R group. # P < 0.01 compared with Tm group.
Zhejiang Province (grant no. 2009B127, 2009A072), and funded by the Science and Technology Department of Zhejiang Province (grant no. 2010C33036, 2012C37104), People’s Republic of China. Conflicts of interest The authors report no conflict of interest. 886
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