• Users Online: 641
  • Home
  • Print this page
  • Email this page


 
 Table of Contents  
RESEARCH ARTICLE
Year : 2016  |  Volume : 11  |  Issue : 7  |  Page : 1134-1140

Heat shock protein 70 protects PC12 cells against ischemia-hypoxia/reoxygenation by maintaining intracellular Ca2+ homeostasis


1 Department of Intensive Care Unit, Affiliated Qingdao Municipal Hospital of Qingdao University, Qingdao, Shandong Province, China
2 Department of Intensive Care Unit, Affiliated Hospital of Jining Medical University, Jining, Shandong Province, China

Date of Acceptance29-Apr-2016
Date of Web Publication16-Aug-2016

Correspondence Address:
Yan Qu
Department of Intensive Care Unit, Affiliated Qingdao Municipal Hospital of Qingdao University, Qingdao, Shandong Province
China
Login to access the Email id

Source of Support: None, Conflict of Interest: None


DOI: 10.4103/1673-5374.187051

Rights and Permissions
  Abstract 

Heat shock protein 70 (HSP70) maintains Ca2+ homeostasis in PC12 cells, which may protect against apoptosis; however, the mechanisms of neuroprotection are unclear. Therefore, in this study, we examined Ca2+ levels in PC12 cells transfected with an exogenous lentiviral HSP70 gene expression construct, and we subsequently subjected the cells to ischemia-hypoxia/reoxygenation injury. HSP70 overexpression increased neuronal viability and ATPase activity, and it decreased cellular reactive oxygen species levels and intracellular Ca2+ concentration after hypoxia/reoxygenation. HSP70 overexpression enhanced the protein and mRNA expression levels of sarcoplasmic/endoplasmic reticulum Ca2+-ATPase (SERCA), but it decreased the protein and mRNA levels of inositol 1,4,5-trisphosphate receptor (IP3R), thereby leading to decreased intracellular Ca2+ concentration after ischemia-hypoxia/reoxygenation. These results suggest that exogenous HSP70 protects against ischemia-hypoxia/reoxygenation injury, at least in part, by maintaining cellular Ca2+ homeostasis, by upregulating SERCA expression and by downregulating IP3R expression.

Keywords: nerve regeneration; exogenous heat shock protein 70; lentivirus transfection; ischemia-hypoxia/reoxygenation; PC12 cells; Ca2+; endoplasmic reticulum; inositol 1,4,5-trisphosphate receptor; sarcoplasmic/endoplasmic reticulum Ca2+-ATPase; neural regeneration


How to cite this article:
Liu Y, Wang Xc, Hu D, Huang Sr, Li Qs, Li Z, Qu Y. Heat shock protein 70 protects PC12 cells against ischemia-hypoxia/reoxygenation by maintaining intracellular Ca2+ homeostasis. Neural Regen Res 2016;11:1134-40

How to cite this URL:
Liu Y, Wang Xc, Hu D, Huang Sr, Li Qs, Li Z, Qu Y. Heat shock protein 70 protects PC12 cells against ischemia-hypoxia/reoxygenation by maintaining intracellular Ca2+ homeostasis. Neural Regen Res [serial online] 2016 [cited 2018 Oct 23];11:1134-40. Available from: http://www.nrronline.org/text.asp?2016/11/7/1134/187051


  Introduction Top


Cerebrovascular ischemia is a condition in which there is insufficient blood flow to the brain to meet metabolic demand. This results in cerebral hypoxia/reoxygenation and neuronal cell death. Heat shock proteins (HSPs) are a group of conserved stress proteins found in eukaryotic and prokaryotic cells. These proteins are encoded by heat shock genes, and their expression is induced by heat stress or other adverse conditions (Pignataro et al., 2007; Fei et al., 2008). HSP70 is a 70-kDa stress protein of the inducible form, the most abundant and conserved member of the HSPs. A previous study found that HSP70 can be used as a sensitive marker of cerebral hypoxia/reoxygenation at the early stage (Riezzo et al., 2010). Hypoxia/reoxygenation induces the expression of HSP70 in many organs, such as the liver, heart and intestine (Ramaglia and Buck, 2004; Liu et al., 2007; Sazontova et al., 2007; Orsenigo et al., 2012). Recent studies have shown that HSP70 protects the kidney, the mucosa of stress-induced gastric ulcers, and intestinal epithelial cells during hypoxia/reoxygenation (Bedirli et al., 2004; Oyake et al., 2006; Yuan et al., 2008). HSP70 mediates neuroprotection induced by ischemic preconditioning (Liu et al., 2004). Increased expression of HSP70 in the human brain has been suggested to prevent cell death in pathophysiological conditions (Radons and Multhoff, 2005). HSP70 tightly regulates Ca2+ homeostasis in PC12 cells and appears to have a strong anti-apoptotic function (Hu et al., 2015). However, the effects of HSP70 in the human brain against hypoxic damage and Ca2+ overload are unclear.

PC12 cells are a clonal cell line derived from rat adrenal medulla pheochromocytoma. PC12 cells have similar characteristics to neurons in vitro, and consequently, they have been widely used to study neuronal biology and pharmacology (Dijkmans et al., 2008). In the present study, we used PC12 cells to study neuronal Ca2+ homeostasis, as in a previous study (Smaili et al., 2001).

Ca2+ overload is involved in the pathology of cerebral hypoxia/reoxygenation. In the brain, ryanodine receptor (RyR) and inositol 1, 4, 5-trisphosphate receptor (IP3R) are Ca2+ release channels located on the endoplasmic and/or sarcoplasmic reticulum. Sarcoplasmic/endoplasmic reticulum Ca2+-ATPase (SERCA) resides in the sarcoplasmic reticulum and transfers Ca2+ from the cytosol to the lumen of the sarcoplasmic reticulum at the expense of ATP hydrolysis. Na+/Ca2+ exchanger (NCX) is a critically important membrane antiporter that removes Ca2+ from cells. NCX removes a single Ca2+ ion in exchange for three Na+ ions. NCX is present in many different cell types and species (Brustovetsky et al., 2010).

Ca2+ accumulation in ischemia-hypoxia/reoxygenation is either abolished or significantly attenuated by overexpression of HSP70 (Hu et al., 2015). However, the mechanisms by which HSP70 maintains Ca2+ homeostasis have not been elucidated. Therefore, in the present study, we investigated the changes in Ca2+ levels in PC12 cells transfected with an exogenous lentiviral HSP70 gene expression construct and subjected to hypoxia/reoxygenation injury.


  Materials and Methods Top


Cell culture

Differentiated PC12 cells, provided by the Department of Physiology, Qingdao University, Qingdao, Shandong Province, China, were cultured in Dulbecco's modified Eagle's medium supplemented with 5% horse serum, 10% fetal bovine serum, penicillin (100 U/mL) and streptomycin (100 μg/mL), under 5% CO2 and 95% air (20% O2) at 37°C (Yuan et al., 2005). An optical microscope (Olympus BX51, Tokyo, Japan) was used to observe cellular morphology. The final concentration of cells in each group was 5 × 105 cells/mL. The cells were divided into three groups: lentiviral infection group (lentiviral HSP70 gene delivery vector; Shanghai R&S Biotechnology Co., Ltd., Shanghai, China), lentivirus control group (empty vector, only containing lentivirus without the HSP70 gene; Shanghai R&S Biotechnology Co., Ltd.) and non-infection group. Technology for lentiviral infection was provided by Gene Chemical Company, Shanghai, China. Virus was screened with puromycin.

Cell culture model of ischemia-hypoxia/reoxygenation

The three different groups of cells were incubated with serum-free medium under sterile conditions. The cells were covered with culture solution and placed in a sealed container under 95% N2 and 5% CO2 at a flow rate of 10 L/min for 4–5 minutes. Samples were incubated in a hypoxic environment (approximately 1% O2) in a 37°C incubator for 8 hours. Afterwards, normal medium was added, and the cells were cultured for 24 hours to simulate reoxygenation (Galán-Cobo et al., 2013).

In vitro cell viability assay

Neuronal cell viability was measured using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. PC12 cells were seeded in 96-well plates at 5 × 103 cells/well for 24 hours, and incubated with serum-free medium under sterile conditions for 8 hours. At the end of the incubation period, 20 μL of MTT solution, 5 mg/mL, was added to each well and incubated for 4 hours. After removal of the medium, 150 μL dimethyl sulfoxide was added to each well to dissolve the formazan crystals. The optical density of each well was measured at 490 nm with a microplate reader (Biotek Synergy H1, Winooski, VT, USA).

Measurements of Na+/K+-ATPase, Ca2+/Mg2+-ATPase and total-ATPase activities

Cells were seeded in 24-well plates after hypoxia/reoxygenation treatment for 8 hours. A 3-mL aliquot of 1 × 106 cells/mL cell suspension was disrupted with an ultrasonic disrupter (Solarbio, Shanghai, China) (parameter settings: 160 Hz; pulse duration, 6 seconds; interval, 10 seconds; total of 20 pulses). Cells were centrifuged at 45 × g and 4°C for 10 minutes. The supernatant was collected for protein quantification. Na+/K+-ATPase, Ca2+/Mg2+-ATPase and total-ATPase activities were measured in accordance with the instructions in the assay kit (Beyotime, Haimen, Jiangsu Province, China). Optical density was measured at 636 nm in a spectrophotometer (NanoDrop 2000, Thermo Scientific, Waltham, MA, USA).

Quantitative real time-polymerase chain reaction (qRT-PCR)

Total cellular RNA was extracted using the TRIzol one-step method after hypoxia/reoxygenation treatment for 8 hours. A 2-μg sample of RNA was reverse-transcribed using oligo(dT) primers and the Roche Reverse Transcriptase Kit (Roche, Basel, Switzerland) according to the manufacturer's protocol. The mRNA expression levels of the various genes were normalized to the expression level of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) in the same cDNA sample. Quantitative real-time PCR was performed with a LightCycler96 instrument (Roche) (Yan et al., 2014). The PCR reaction contained 2× FastStart Essential DNA Green Master Mix (10 μL), upstream and downstream primers (0.4 μM each), cDNA template (2 μL; ≤ 0.1 μg), and RNase-free water to a final volume of 20 μL. The reaction conditions were as follows: pre-denaturation at 95°C for 5 minutes; 35 cycles of 94°C for 15 seconds, annealing at 60°C for 30 seconds, extension at 70°C for 30 seconds. Specificity of the PCR products was verified by melting curve analysis. Differential expression of mRNA was calculated using the 2–ΔΔCt method. The primers are listed in [Table 1].
Table 1: Primers for quantitative real time-polymerase chain reaction

Click here to view


Western blot assay

HSP70, IP3R and SERCA protein levels were determined using a previously reported method (Honisch et al., 2015). Briefly, cells were washed three times with ice-cold phosphate buffered saline and suspended in 400 μL ice-cold radioimmune precipitation assay lysis buffer and 4 μL ice-cold phenylmethyl sulfonylfluoride lysis buffer (Thermo Fisher). Protein concentration was determined using the Bradford assay (BioRad, München, Germany). Samples containing 50 μg of total protein were solubilized in sample buffer at 100 °C for 5 minutes. The samples were subjected to 10% sodium dodecyl sulfate polyacrylamide gel electropheresis analysis and then electro-transferred onto polyvinylidene fluoride membranes and blocked with 5% non-fat milk in Tris-buffered saline/0.10% Tween 20 at room temperature for 2 hours. The membranes were incubated with primary antibodies; rabbit anti-rat HSP70 (1:1,000; Abcam, Cambridge, UK), rabbit anti-rat SERCA (1:1,000; Cell Signaling Technology, Danvers, MA, USA), rabbit anti-rat IP3R (1:1,000; Cell Signaling Technology) or rabbit anti-rat β-actin (1:2,000; Cell Signaling Technology) at 4°C overnight. After washing with Tris-buffered saline/0.10% Tween 20, the blots were incubated with secondary goat anti-rabbit and anti-mouse antibodies (1:4,000; Boster, Wuhan, Hubei Province, China) for 2 hours at room temperature. Signals were visualized with enhanced chemiluminescence (Beyotime).

Measurements of reactive oxygen species (ROS)

To analyze the kinetics of ROS generation (Xu et al., 2009), PC12 cells were exposed to ischemia-hypoxia/reoxygenation for 8 hours, and then incubated in normal medium at 37°C for 24 hours. ROS were detected using the fluorescent probe dihydroethidium (Beyotime), dihydrorhodamine 123 (Molecular Probes) and 3-amino-4-aminomethyl-29,79-difluorescein diacetate (Molecular Probes). Cells were incubated with 2 mM dihydroethidium, 5 mM dihydrorhodamine 123 and 5 mM 3-amino-4-aminomethyl-29,79-difluorescein diacetate for 30 minutes at 37°C in the dark. The fluorescence intensity of ROS probes was analyzed by flow cytometric analysis (CyFlow® Counter, PARTEC, Munster, Germany).

Ca2+ assay

The levels of free cytosolic Ca2+ were measured using the cell-permeable Ca2+-sensitive fluorescent dye Fluo-3/AM. PC12 cells were exposed to hypoxia/reoxygenation for 8 hours, and then incubated in normal meduim at 37°C for 24 hours. PC12 cells were incubated with 5 mM Fluo-3/AM (Beyotime) for 30 minutes at 37°C. The fluorescence intensity of Fluo-3/AM probes was analyzed by flow cytometric analysis (CyFlow® Counter, PARTEC).

Statistical analysis

Data were analyzed with SPSS 17.0 software (SPSS, Chicago, IL, USA) and were expressed as the mean ± SEM. One-way analysis of variance followed by the least significant difference test was used to compare differences between groups. Intergroup differences in cell viability measurements were compared using two-way analysis of variance followed by the Student-Newman-Keuls test. P < 0.05 was considered statistically significant.


  Results Top


Effects of ischemia-hypoxia/reoxygenation on PC12 cell morphology

Under an optical microscope, PC12 cells were small and translucent immediately after passage in suspension. At 24 hours, most of the adherent cells showed the emergence of processes. At 48 hours, the cells were plump, and formed a network ([Figure 1]A). After an 8-hour period of ischemia-hypoxia/reoxygenation, PC12 cells exhibited no obvious morphological changes ([Figure 1]B).
Figure 1: Morphology of differentiated PC12 cells after ischemia- hypoxia/reoxygenation for 8 hours (× 200).
(A) After plating for 48 hours, normal PC12 cells were plump and interconnected, forming a network. (B) After ischemia-hypoxia/reoxygenation for 8 hours, PC12 cells displayed no obvious morphological changes.


Click here to view


HSP70 overexpression increased neuronal viability after ischemia-hypoxia/reoxygenation

MTT assay showed that PC12 cell viability was significantly higher in the lentiviral infection group than in the lentivirus control and non-infection groups after ischemia-hypoxia/reoxygenation for 8 hours (P < 0.05). There was no difference in cell viability between the lentivirus control group and non-infection group (P > 0.05; [Figure 2]A). After ischemia-hypoxia/reoxygenation for 8 hours, the three groups underwent reoxygenation for 7 days. Cell viability was significantly higher in the lentiviral infection group than in the lentivirus control group or the non-infection group (P < 0.05; [Figure 2]B).
Figure 2: Effect of HSP70 overexpression on neuronal viability after ischemia-hypoxia/reoxygenation.
(A) Effects of exogenous HSP70 on cell viability of PC12 cells after 8 hours of ischemia-hypoxia/reoxygenation. (B) PC12 cells were then incubated for 1–7 days to simulate reoxygenation. Percent cell viability was assessed by MTT assay. Data are expressed as the mean ± SEM. *P < 0.05, vs. lentivirus control group; #P < 0.05, vs. non-infection group (two-way analysis of variance followed by Student-Newman-Keuls test). The experiment was performed in triplicate. Lentiviral infection group: Lentivirus-mediated HSP70 gene transfected PC12 cells; lentivirus control group: empty vector transfected PC12 cells; non-infection group: untransfected PC12 cells. Cells were exposed to ischemia-hypoxia/reoxygenation for 8 hours. HSP70: Heat shock protein 70; MTT: 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide.


Click here to view


HSP70 overexpression increased ATPase activities in PC12 cells after ischemia-hypoxia/reoxygenation

The activities of Na+/K+-ATPase, Ca2+/Mg2+-ATPase and total-ATPase were significantly higher in the lentiviral infection group than in the lentivirus control group or the non-infection group after ischemia-hypoxia/reoxygenation for 8 hours in PC12 cells (P < 0.01). There was no difference between the lentivirus control group and the non-infection group (P > 0.05; [Figure 3]).
Figure 3: Effects of exogenous HSP70 on Na+/K+-ATPase, Ca2+/Mg2+- ATPase and total-ATPase activities in PC12 cells after ischemia-hypoxia/reoxygenation for 8 hours.
Data are presented as the mean ± SEM and were analyzed by analysis of variance followed by the least significant difference test. **P < 0.01, vs. lentivirus control group, ##P < 0.01, vs. non-infection group. The experiment was performed in triplicate. Lentiviral infection group: Lentivirus-mediated HSP70 gene transfected PC12 cells; lentivirus control group: empty vector transfected PC12 cells; non-infection group: untransfected PC12 cells. Cells were exposed to ischemia/hypoxia/reoxygenation for 8 hours. HSP70: Heat shock protein 70.


Click here to view


HSP70 overexpression upregulated SERCA2a and SERCA2b mRNA expression and downregulated IP3R mRNA expression in PC12 cells after ischemia-hypoxia/reoxygenation

HSP70, SERCA2a, SERCA2b and IP3R mRNA expression levels in PC12 cells were assessed by qRT-PCR after ischemia- hypoxia/reoxygenation for 8 hours. mRNA expression levels of HSP70, SERCA2a and SERCA2b were higher in the lentiviral infection group compared with the lentivirus control group or the non-infection group (P < 0.01). IP3R mRNA expression was lower in the lentiviral infection group compared with the lentivirus control group or the non-infection group (P < 0.01; [Table 2]).
Table 2: Effect of HSP70 overexpression on SERCA2a, SERCA2b and IP3R mRNA ((copies/mL)2) expression in PC12 cells after ischemia-hypoxia/reoxygenation

Click here to view


HSP70 overexpression upregulated SERCA protein levels and downregulated IP3R protein levels in PC12 cells after ischemia-hypoxia/reoxygenation

HSP70, SERCA and IP3R protein levels were detected by western blot assay after PC12 cells were exposed to ischemia-hypoxia/reoxygenation for 8 hours. HSP70 and SERCA protein levels were upregulated in the lentiviral infection group, while IP3R protein expression was downregulated, compared with the lentivirus control group or the non-infection group ([Figure 4]).
Figure 4: Effects of exogenous HSP70 on HSP70, SERCA and IP3R protein expression levels in PC12 cells exposed to ischemia-hypoxia/reoxygenation for 8 hours.
(A) Western blot assay was used to assess HSP70, SERCA and IP3R protein levels. Relative protein expression was calculated as the optical density ratio to that of β-actin. β-Actin was used as a loading control. (B) Statistical analysis. Data are presented as the ratio of HSP70, SERCA and IP3R to β-actin. Each bar represents the mean ± SEM, and data were analyzed by analysis of variance followed by the least significant difference test. **P < 0.01, vs. lentivirus control group; ##P < 0.01, vs. non-infection group. The experiment was performed in triplicate. Lentiviral infection group: Lentivirus-mediated HSP70 gene transfected PC12 cells; lentivirus control group: empty vector transfected PC12 cells; non-infection group: untransfected PC12 cells. Cells were exposed to ischemia-hypoxia/reoxygenation for 8 hours. HSP70: Heat shock protein 70; SERCA: sarcoplasmic/endoplasmic reticulum Ca2+-ATPase; IP3R: inositol 1,4,5-trisphosphate.


Click here to view


HSP70 overexpression decreased intracellular ROS production in PC12 cells after ischemia-hypoxia/ reoxygenation

ROS production was measured in PC12 cells for 8 hours. ROS levels were significantly lower in the lentiviral infection group than in the lentivirus control group or the non-infection group (P < 0.01). There was no difference between the lentivirus control and non-infection groups (P > 0.05; [Table 3]).
Table 3: Effect of HSP70 overexpression on cellular ROS levels (U) and intracellular Ca2+ concentration (U) in PC12 cells after ischemia-hypoxia/reoxygenation

Click here to view


HSP70 overexpression decreased intracellular Ca2+ concentration in PC12 cells after ischemia-hypoxia/ reoxygenation

Ca2+ concentration was measured in PC12 cells after ischemia-hypoxia/reoxygenation for 8 hours. Intracellular Ca2+ concentration was significantly lower in the lentiviral infection group than in the lentivirus control and non-infection groups (P < 0.01). There was no difference between the lentivirus control and non-infection groups (P > 0.05; [Table 3]).


  Discussion Top


Our findings demonstrate that ischemia-hypoxia/reoxygenation for 8 hours increases the expression of HSP70. Compared to normal cells, cell morphology was altered. We infer that HSP70 may not rescue cells acutely. However, cells gradually recovered their normal morphology over time. Seven days after reoxygenation, cell viability gradually reached the level of normal cells. We found that HSP70 overexpression decreased ROS production and Ca2+ concentration in PC12 cells exposed to ischemia-hypoxia/reoxygenation for 8 hours. PC12 cells are significantly damaged by ischemia-hypoxia/reoxygenation for 8 hours (Hu et al., 2015). Moreover, the decreased Ca2+ concentration was related to the upregulation of SERCA and the downregulation of IP3R. Our results are consistent with a previous study (Amin et al., 1996), showing that overexpression of HSP70 protects cultured sensory neurons from nerve injury or ischemia. HSP70, in particular, has been demonstrated to play important roles in cerebrovascular disease (Zhang et al., 2009). It has been shown that HSP expression is correlated with ischemic vulnerability and neuronal survival (Nakka et al., 2010). The results of our study suggest that 8 hours of hypoxia/reoxygenation produces the most significant increase in HSP70 expression. Recent research has shown that Ca2+ plays a key role in cerebral ischemia (Kumar et al., 2014). Several proteins are responsible for cellular Ca2+ homeostasis. IP3R and RyR are Ca2+ release channels located on the sarcoplasmic reticulum in all cell types. SERCA is located in the sarcoplasmic reticulum within nerve cells. It is a Ca2+-ATPase that transfers Ca2+ from the cytosol to the lumen of the sarcoplasmic reticulum at the expense of ATP hydrolysis. NCX is an antiporter membrane protein that removes Ca2+ from cells. It has been reported that HSP70 may decrease Ca2+ overload in myocardial cells during myocardial ischemia (Chen et al., 2003). HSP70 may increase myocardial SERCA and RyR expression, enhance Ca2+ release from the endoplasmic reticulum into the cytosol by RyR, and increase cytosolic Ca2+ reuptake into the endoplasmic reticulum by SERCA. Furthermore, HSP70 increases NCX activity, suggesting that it may regulate Ca2+ homeostasis by affecting NCX function as well (Xu et al., 2009).

Ischemia-hypoxia/reoxygenation impacts Ca2+ flux and reduces reoxygenation injury. HSP70 decreases the tethering of the endoplasmic reticulum to mitochondria and prevents mitochondrial Ca2+ overload and reduces cell death after ischemia-hypoxia/reoxygenation (Dremina et al., 2012). Smaili et al. (2001) suggested that, in intact hepatocytes, cyclophilins play a role in Ca2+ cycling between the endoplasmic reticulum and mitochondria by showing that cyclosporine modifies IP3-dependent Ca2+ signals. The present data expand on this notion because both genetic and pharmacological inhibition of cyclophilin D leads to decreased Ca2+ transfer from the endoplasmic reticulum to mitochondria through IP3R, even in the in vivo cardiomyocyte model. Studies suggest that several isoforms of IP3R are enriched in the mitochondria-associated endoplasmic reticulum membrane (Mendes et al., 2005; Szabadkai et al., 2006; Hayashi et al., 2009). In the brain, the type-2 isoform of IP3R (IP3R2) is highly expressed in neurons (Vermassen et al., 2004). Most of the functions of IP3R are attributed to IP3R2, and our results show that HSP70 preferentially interacts with this isoform. Our results also demonstrate that HSP70 regulates endoplasmic reticulum Ca2+ in PC cells and that its absence from these cells can alter Ca2+ homeostasis. Our results also demonstrate that perturbed Ca2+ homeostasis plays an important pathophysiological role in neurons.

The essential role of HSP70 in Ca2+ homeostasis is unexpected given the other cell types studied thus far (Guo et al., 2004; Shahlaie et al., 2013). It is conceivable that synaptic transmission produces a moderate but persistent deficit in endoplasmic reticulum calcium concentration because of release through IP3R and RyR channels. Whereas primary Ca2+ signals in hematopoietic and other nonexcitable cells involve brief but massive Ca2+ release and require rapid refilling of depleted intracellular stores, higher HSP70 levels may be needed in neurons.

PC12 cells overexpressing HSP70 exhibited a significant increase in viability and ATPase activity, as well as decreased cellular ROS and intracellular Ca2+ concentration, after hypoxia/reoxygenation. HSP70 overexpression increased the mRNA and protein expression levels of SERCA, but it decreased the mRNA and protein levels of IP3R, thereby decreasing intracellular Ca2+ concentration after hypoxia/reoxygenation. These results suggest that HSP70 overexpression improves the ischemia-induced perturbation in Ca2+ homeostasis in neuronal cells.

In summary, lentivirus-mediated HSP70 overexpression protects PC12 cells against ischemic/hypoxic injury by maintaining cellular Ca2+ homeostasis. Our findings suggest that lentivirus-mediated exogenous HSP70 overexpression may have clinical potential for the prevention and treatment of cerebral ischemia-hypoxia/reoxygenation.[33]

 
  References Top

1.
Amin V, Cumming DV, Latchman DS (1996) Over-expression of heat shock protein 70 protects neuronal cells against both thermal and ischaemic stress but with different efficiencies. Neurosci Lett 206:45-48.  Back to cited text no. 1
    
2.
Bedirli A, Sakrak O, Muhtaroglu S, Soyuer I, Guler I, Riza Erdogan A, Sozuer EM (2004) Ergothioneine pretreatment protects the liver from ischemia-reperfusion injury caused by increasing hepatic heat shock protein 70. J Surg Res 122:96-102.  Back to cited text no. 2
    
3.
Brustovetsky T, Bolshakov A, Brustovetsky N (2010) Calpain activation and Na+/Ca2+ exchanger degradation occur downstream of calcium deregulation in hippocampal neurons exposed to excitotoxic glutamate. J Neurosci Res 88:1317-1328.  Back to cited text no. 3
    
4.
Chen M, Zhou JJ, Kam KW, Qi JS, Yan WY, Wu S, Wong TM (2003) Roles of KATP channels in delayed cardioprotection and intracellular Ca(2+) in the rat heart as revealed by kappa-opioid receptor stimulation with U50488H. Br J Pharmacol 140:750-758.  Back to cited text no. 4
    
5.
Dijkmans TF, van Hooijdonk LW, Schouten TG, Kamphorst JT, Vellinga AC, Meerman JH, Fitzsimons CP, de Kloet ER, Vreugdenhil E (2008) Temporal and functional dynamics of the transcriptome during nerve growth factor-induced differentiation. J Neurochem 105:2388-2403.  Back to cited text no. 5
    
6.
Dremina ES, Sharov VS, Schöneich C (2012) Heat shock proteins attenuate SERCA inactivation by the anti-apoptotic protein Bcl-2: possible implications for the ER Ca(2+) mediated apoptosis. Biochem J 444:127-139.  Back to cited text no. 6
    
7.
Fei G, Guo C, Sun HS, Feng ZP (2008) HSP70 reduces chronic hypoxia/reoxygenation-induced neural suppression via regulating expression of syntaxin. Adv Exp Med Biol 605:35-40.  Back to cited text no. 7
    
8.
Galán-Cobo A, Sánchez-Silva R, Serna A, Abreu-Rodríguez I, Muñoz-Cabello AM, Echevarría M (2013) Cellular overexpression of Aquaporins slows down the natural HIF-2α degradation during prolonged hypoxia/reoxygenation. Gene 522:18-26.  Back to cited text no. 8
    
9.
Guo J, Meng F, Fu X, Song B, Yan X, Zhang G (2004) N-methyl-D-aspartate receptor and L-type voltage-gated Ca2+ channel activation mediate proline-rich tyrosine kinase 2 phosphorylation during cerebral ischemia in rats. Neurosci Lett 355:177-180.  Back to cited text no. 9
    
10.
Hayashi T, Rizzuto R, Hajnoczky G, Su TP (2009) MAM: more than just a housekeeper. Trends Cell Biol 19:81-88.  Back to cited text no. 10
    
11.
Honisch S, Yu W, Liu G, Alesutan I, Towhid ST, Tsapara A, Schleicher S, Handgretinger R, Stournaras C, Lang F (2015) Chorein addiction in VPS13A overexpressing rhabdomyosarcoma cells. Oncotarget 6:10309-10319.  Back to cited text no. 11
    
12.
Hu Y, Li Q, Li Z, Hu D, Qu Y (2015) Effects of lentivirus-mediated heat shock protein 70 gene on calcium homeostasis in PC12 cells undergone ischemia and hypoxia/reoxygenation. Zhonghua Wei Zhong Bing Ji Jiu Yi Xue 27:295-299.  Back to cited text no. 12
    
13.
Kumar VS, Gopalakrishnan A, Naziroglu M, Rajanikant GK (2014) Calcium ion--the key player in cerebral ischemia. Curr Med Chem 21:2065-2075.  Back to cited text no. 13
    
14.
Liu J, Kam KW, Zhou JJ, Yan WY, Chen M, Wu S, Wong TM (2004) Effects of heat shock protein 70 activation by metabolic inhibition preconditioning or kappa-opioid receptor stimulation on Ca2+ homeostasis in rat ventricular myocytes subjected to ischemic insults. J Pharmacol Exp Ther 310:606-613.  Back to cited text no. 14
    
15.
Liu JC, Wan L, He M, Cheng XS (2007) Protection of myocardiocytes against anoxia-reoxygeneration injury by heat shock protein 70 gene transfection: experiment with rats. Zhonghua Yi Xue Za Zhi 87:3436-3439.  Back to cited text no. 15
    
16.
Mendes CC, Gomes DA, Thompson M, Souto NC, Goes TS, Goes AM, Rodrigues MA, Gomez MV, Nathanson MH, Leite MF (2005) The type III inositol 1,4,5-trisphosphate receptor preferentially transmits apoptotic Ca2+ signals into mitochondria. J Biol Chem 280:40892-40900.  Back to cited text no. 16
    
17.
Nakka VP, Gusain A, Raghubir R (2010) Endoplasmic reticulum stress plays critical role in brain damage after cerebral ischemia/reperfusion in rats. Neurotox Res 17:189-202.  Back to cited text no. 17
    
18.
Orsenigo MN, Porta C, Sironi C, Laforenza U, Meyer G, Tosco M (2012) Effects of creatine in a rat intestinal model of ischemia/reperfusion injury. Eur J Nutr 51:375-384.  Back to cited text no. 18
    
19.
Oyake J, Otaka M, Matsuhashi T, Jin M, Odashima M, Komatsu K, Wada I, Horikawa Y, Ohba R, Hatakeyama N, Itoh H, Watanabe S (2006) Over-expression of 70-kDa heat shock protein confers protection against monochloramine-induced gastric mucosal cell injury. Life Sci 79:300-305.  Back to cited text no. 19
    
20.
Pignataro L, Miller AN, Ma L, Midha S, Protiva P, Herrera DG, Harrison NL (2007) Alcohol regulates gene expression in neurons via activation of heat shock factor 1. J Neurosci 27:12957-12966.  Back to cited text no. 20
    
21.
Radons J, Multhoff G (2005) Immunostimulatory functions of membrane-bound and exported heat shock protein 70. Exerc Immunol Rev 11:17-33.  Back to cited text no. 21
    
22.
Ramaglia V, Buck LT (2004) Time-dependent expression of heat shock proteins 70 and 90 in tissues of the anoxic western painted turtle. J Exp Biol 207:3775-3784.  Back to cited text no. 22
    
23.
Riezzo I, Neri M, De Stefano F, Fulcheri E, Ventura F, Pomara C, Rabozzi R, Turillazzi E, Fineschi V (2010) The timing of perinatal hypoxia/ischemia events in term neonates: a retrospective autopsy study. HSPs, ORP-150 and COX2 are reliable markers to classify acute, perinatal events. Diagn Pathol 5:49.  Back to cited text no. 23
    
24.
Sazontova TG, Zhukova AG, Anchishkina NA, Arkhipenko IuV (2007) Dynamic changes in transcription factor HIF-1alpha, rapid response protein, and membrane structure resistance following acute hypoxia/reoxygenation. Vestn Ross Akad Med Nauk:17-25.  Back to cited text no. 24
    
25.
Shahlaie K, Gurkoff GG, Lyeth BG, Muizelaar JP, Berman RF (2013) Neuroprotective effects of SNX-185 in an in vitro model of TBI with a second insult. Restor Neurol Neurosci 31:141-153.  Back to cited text no. 25
    
26.
Smaili SS, Stellato KA, Burnett P, Thomas AP, Gaspers LD (2001) Cyclosporin A inhibits inositol 1,4,5-trisphosphate-dependent Ca2+ signals by enhancing Ca2+ uptake into the endoplasmic reticulum and mitochondria. J Biol Chem 276:23329-23340.  Back to cited text no. 26
    
27.
Szabadkai G, Bianchi K, Várnai P, De Stefani D, Wieckowski MR, Cavagna D, Nagy AI, Balla T, Rizzuto R (2006) Chaperone-mediated coupling of endoplasmic reticulum and mitochondrial Ca(2+) channels. J Cell Biol 175:901-911.  Back to cited text no. 27
    
28.
Vermassen E, Parys JB, Mauger JP (2004) Subcellular distribution of the inositol 1,4,5-trisphosphate receptors: functional relevance and molecular determinants. Biol Cell 96:3-17.  Back to cited text no. 28
    
29.
Xu L, Voloboueva LA, Ouyang Y, Emery JF, Giffard RG (2009) Overexpression of mitochondrial Hsp70/Hsp75 in rat brain protects mitochondria, reduces oxidative stress, and protects from focal ischemia. J Cereb Blood Flow Metab 29:365-374.  Back to cited text no. 29
    
30.
Yan JQ, Ma YJ, Sun JC, Bai SF, Huang LN (2014) Neuroprotective effect of lovastatin by inhibiting NMDA receptor1 in 6-hydroxydopamine treated PC12 cells. Int J Clin Exp Med 7:3313-3319.  Back to cited text no. 30
    
31.
Yuan G, Nanduri J, Bhasker CR, Semenza GL, Prabhakar NR (2005) Ca2+/calmodulin kinase-dependent activation of hypoxia/reoxygenation inducible factor 1 transcriptional activity in cells subjected to intermittent hypoxia/reoxygenation. J Biol Chem 280:4321-4328.  Back to cited text no. 31
    
32.
Yuan ZQ, Li XL, Peng YZ, Wang P, Huang YS, Yang ZC (2008) Influence of HSP70 on function and energy metabolism of mitochondria in intestinal epithelial cells after hypoxia/reoxygenation. Zhonghua Shao Shang Za Zhi 24:203-206.  Back to cited text no. 32
    
33.
Zhang K, Zhao T, Huang X, Liu ZH, Xiong L, Li MM, Wu LY, Zhao YQ, Zhu LL, Fan M (2009) Preinduction of HSP70 promotes hypoxic tolerance and facilitates acclimatization to acute hypobaric hypoxia/reoxygenation in mouse brain. Cell Stress Chaperones 14:407-415.  Back to cited text no. 33
    

Author contributions: YQ and DH conceived and designed the study. YL and QSL performed the experiments. YL wrote the paper. XCW, SRH and ZL reviewed and edited the paper. All authors approved the final version of the paper.
Conflicts of interest: None declared.
Plagiarism check: This paper was screened twice using CrossCheck to verify originality before publication.
Peer review: This paper was double-blinded and stringently reviewed by international expert reviewers.


    Figures

  [Figure 1], [Figure 2], [Figure 3], [Figure 4]
 
 
    Tables

  [Table 1], [Table 2], [Table 3]


This article has been cited by
1 Quinacrine pretreatment reduces microwave-induced neuronal damage by stabilizing the cell membrane
Xue-feng Ding,Yan Wu,Wen-rui Qu,Ming Fan,Yong-qi Zhao
Neural Regeneration Research. 2018; 13(3): 449
[Pubmed] | [DOI]
2 A Controversial Medicolegal Issue: Timing the Onset of Perinatal Hypoxic-Ischemic Brain Injury
Vittorio Fineschi,Rocco Valerio Viola,Raffaele La Russa,Alessandro Santurro,Paola Frati
Mediators of Inflammation. 2017; 2017: 1
[Pubmed] | [DOI]



 

Top
 
 
  Search
 
Similar in PUBMED
   Search Pubmed for
   Search in Google Scholar for
 Related articles
Access Statistics
Email Alert *
Add to My List *
* Registration required (free)

 
  In this article
Abstract
Introduction
Materials and Me...
Results
Discussion
References
Article Figures
Article Tables

 Article Access Statistics
    Viewed587    
    Printed0    
    Emailed0    
    PDF Downloaded159    
    Comments [Add]    
    Cited by others 2    

Recommend this journal


[TAG2]
[TAG3]
[TAG4]