These authors contributed equally to this work.
CaMKII is involved in cadmium activation of MAPK and mTOR pathways leading to neuronal cell death
Version of Record online: 20 OCT 2011
© 2011 The Authors. Journal of Neurochemistry © 2011 International Society for Neurochemistry
Journal of Neurochemistry
Volume 119, Issue 5, pages 1108–1118, December 2011
How to Cite
Chen, S., Xu, Y., Xu, B., Guo, M., Zhang, Z., Liu, L., Ma, H., Chen, Z., Luo, Y., Huang, S. and Chen, L. (2011), CaMKII is involved in cadmium activation of MAPK and mTOR pathways leading to neuronal cell death. Journal of Neurochemistry, 119: 1108–1118. doi: 10.1111/j.1471-4159.2011.07493.x
- Issue online: 14 NOV 2011
- Version of Record online: 20 OCT 2011
- Accepted manuscript online: 20 SEP 2011 04:40PM EST
- Received August 1, 2011; revised manuscript received September 15, 2011; accepted September 15, 2011.
- calcium/calmodulin-dependent protein kinase II;
- calcium ion;
- mammalian target of rapamycin;
- mitogen-activated protein kinase
J. Neurochem. (2011) 119, 1108–1118.
Cadmium (Cd), a toxic environmental contaminant, induces neurodegenerative diseases. Recently, we have shown that Cd elevates intracellular free calcium ion ([Ca2+]i) level, leading to neuronal apoptosis partly by activating mitogen-activated protein kinases (MAPK) and mammalian target of rapamycin (mTOR) pathways. However, the underlying mechanism remains to be elucidated. In this study, we show that the effects of Cd-elevated [Ca2+]i on MAPK and mTOR network as well as neuronal cell death are through stimulating phosphorylation of calcium/calmodulin-dependent protein kinase II (CaMKII). This is supported by the findings that chelating intracellular Ca2+ with 1,2-bis(o-aminophenoxy) ethane-N,N,N′,N′-tetraacetic acid tetra(acetoxymethyl) ester or preventing Cd-induced [Ca2+]i elevation using 2-aminoethoxydiphenyl borate blocked Cd activation of CaMKII. Inhibiting CaMKII with KN93 or silencing CaMKII attenuated Cd activation of MAPK/mTOR pathways and cell death. Furthermore, inhibitors of mTOR (rapamycin), c-Jun N-terminal kinase (SP600125) and extracellular signal-regulated kinase 1/2 (U0126), but not of p38 (PD169316), prevented Cd-induced neuronal cell death in part through inhibition of [Ca2+]i elevation and CaMKII phosphorylation. The results indicate that Cd activates MAPK/mTOR network triggering neuronal cell death, by stimulating CaMKII. Our findings underscore a central role of CaMKII in the neurotoxicology of Cd, and suggest that manipulation of intracellular Ca2+ level or CaMKII activity may be exploited for prevention of Cd-induced neurodegenerative disorders.
eukaryotic initiation factor 4E-binding protein 1
1,2-bis(o-aminophenoxy) ethane-N,N,N′,N′-tetraacetic acid tetra(acetoxymethyl) ester
intracellular free calcium ion
Ca2+/Calmodulin-dependent protein kinase II
- CRAC channels
Ca2+release activated Ca2+channels
extracellular signal-regulated kinase 1/2
- GFP, green fluorescent protein; HRP
c-Jun N-terminal kinase
mitogen-activated protein kinase
mammalian target of rapamycin
S6 kinase 1
Cadmium (Cd) pollution in the environment may be accumulated in human organs through direct exposure or food chain, resulting in many diseases such as pulmonary edema, respiratory tract irritation, renal dysfunction, anemia, osteoporosis, and cancer in humans (Satarug et al. 2000; Kim et al. 2005; Lau et al. 2006; Chen et al. 2008a). Cd also severely affects the function of the nervous system (Lopez et al. 2003), with symptoms including headache and vertigo, olfactory dysfunction, parkinsonian-like symptoms, slowing of visuomotor functioning, peripheral neuropathy, decreased equilibrium, decreased ability to concentrate, and learning disabilities (Pihl and Parkes 1977; Kim et al. 2005; Monroe and Halvorsen 2006). A growing number of clinical investigations have pointed to Cd intoxication as a possible etiological factor of neurodegenerative diseases, including Parkinson’s disease, Alzheimer’s disease and Huntington’s disease (Okuda et al. 1997; Johnson 2001; Panayi et al. 2002). However, the exact mechanism(s) through which Cd elicits its neurotoxic effects is still unresolved.
Multiple studies have shown that the mitogen-activated protein kinases (MAPKs) signaling pathways, which comprise a highly conserved cascade of serine/threonine (Ser/Thr) kinases connecting cell surface receptors to regulatory targets in response to various stimuli, play a critical role in neuronal apoptosis (Kyriakis and Avruch 2001; Pearson et al. 2001; Li et al. 2004). There exist at least three distinct groups of MAPKs, including extracellular signal-regulated protein kinase 1/2 (Erk1/2), c-Jun N-terminal kinase (JNK), and p38 MAPK, in mammalian cells. Erk1/2 can be activated by growth factors, and is involved in cellular proliferation, differentiation and development, whereas JNK and p38 signaling cascades can be activated by environmental stress and inflammatory cytokines, and have been shown to promote neuronal cell death (Rockwell et al. 2004). Mammalian target of rapamycin (mTOR), a Ser/Thr kinase, lies downstream of protein kinase B (Akt/PKB) (Kim et al. 2000; Huang and Houghton 2003), and senses mitogenic stimuli, nutrient conditions (Fang et al. 2001; Hara et al. 2002; Kim et al. 2002) and ATP (Dennis et al. 2001), regulating cell proliferation, growth and survival (Bjornsti and Houghton 2004). Akt may positively regulate mTOR, leading to increased phosphorylation of ribosomal p70 S6 kinase (S6K1) and eukaryotic initiation factor 4E-binding protein 1 (4E-BP1), the two best characterized downstream effector molecules of mTOR (Bjornsti and Houghton 2004). We have demonstrated that all three MAPK members can be activated by Cd in neuronal (PC12 and SH-SY5Y) cells, and identified that Cd-induced neuronal apoptosis is partially associated with activation of JNK, Erk1/2, but not by p38 signaling (Chen et al. 2008b). Especially, we have pinpointed that Cd induces apoptosis of neuronal cells in part by activation of Akt/mTOR signaling pathway (Chen et al. 2008b). However, it is still not well understood how Cd activates MAPKs and mTOR signaling pathways in the neuronal cells.
Other studies have implicated that Cd-induced cell apoptosis involves a sustained elevation of intracellular free Ca2+([Ca2+]i) (Shen et al. 2001; Li et al. 2003; Lemarie et al. 2004; Liu et al. 2007; Biagioli et al. 2008; Wang et al. 2008). Prolonged change in calcium distribution in cells triggers various cascades that lead to apoptosis (Hajnoczky et al. 2003). Recently, we have shown that Cd-induced [Ca2+]i elevation activates MAPK and mTOR pathways, thereby leading to apoptosis of neuronal cells (Xu et al. 2011). However, the underlying mechanism remains elusive. Calcium/calmodulin-dependent protein kinase II (CaMKII), a Ser/Thr-specific protein kinase, is a general integrator of Ca2+ signaling (Colbran and Brown 2004; Liu and Templeton 2007). CaMKII is activated in the presence of Ca2+and calmodulin (CaM), which leads to autophosphorylation, generating a Ca2+/CaM-independent form of the enzyme (Schworer et al. 1986). In addition, CaMKII transduces signals to MAPKs involved in cell proliferation or apoptosis (Wright et al. 1997; Yamanaka et al. 2007; Kim et al. 2008a,b; Lin et al. 2008; Liu and Templeton 2008; Olofsson et al. 2008). This prompted us to study whether Cd activates MAPK and mTOR pathways triggering neuronal apoptosis, through CaMKII activation by elevated [Ca2+]i.
Materials and methods
Cadmium chloride (Sigma, St Louis, MO, USA) was dissolved in sterile distilled water to prepare the stock solutions (0–120 μM), filtered through a 0.22 μm pore size membrane, aliquoted, and stored at 20–25°C. Rapamycin (ALEXIS, San Diego, CA, USA) was dissolved in dimethylsulfoxide to prepare a 100 μg/mL stock solution and was stored at −20°C. Fluo-3/AM was from Fluka (Buchs, SG, Switzerland). Dulbecco’s Modified Eagle Medium, 0.05% Trypsin–EDTA, NEUROBASALTM Media, and B27 Supplement were purchased from Invitrogen (Grand Island, NY, USA), whereas horse serum and fetal bovine serum were supplied by Minhai Corp. (Lanzhou, China). Enhanced chemiluminescence solution was from Millipore (Billerica, MA, USA). 1,2-Bis(o-aminophenoxy) ethane-N,N,N′,N′-tetraacetic acid tetra(acetoxymethyl) ester (BAPTA/AM) and 2-aminoethoxydiphenyl borane (2-APB) were purchased from Calbiochem (San Diego, CA, USA). KN93 was from ALEXIS, whereas SP600125, U0126, and PD169316 were form Sigma. The following antibodies were used: CaMKII, phospho-CaMKII (Thr286), phospho-Akt (Ser473), phospho-S6K1 (Thr389), phospho-Erk1/2 (Thr202/Tyr204), phospho-p38 (Thr180/Tyr182), phospho-4E-BP1 (Thr70), 4E-BP1 (all from Cell Signaling Technology, Beverly, MA, USA), Akt, S6K1, Erk2, JNK1, phospho-JNK (Thr183/Tyr185), c-Jun, phospho-c-Jun (Ser63), p38 (all from Santa Cruz Biotechnology, Santa Cruz, CA, USA), b-tubulin (Sigma), goat anti-rabbit IgG-horseradish peroxidase (HRP), goat anti-mouse IgG-HRP, and rabbit anti-goat IgG-HRP (Pierce, Rockford, IL, USA). Poly-d-lysine (PDL) and 4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) were purchased from Sigma. Other chemicals were purchased from local commercial sources and were of analytical grade quality.
Rat pheochromocytoma (PC12) cell line was from American Type Culture Collection (ATCC) (Manassas, VA, USA). PC12 cells were used for no more than 10 passages, and cultured in antibiotic-free Dulbecco’s Modified Eagle’s Medium supplemented with 10% horse serum and 5% fetal bovine serum. Cells were maintained at 37°C in a humidified incubator containing 5% CO2. Primary cortical neurons were obtained from fetal mice at 16–18 days of gestation and cultured as described (Chen et al. 2010).
Lentiviral shRNA cloning, production, and infection
To generate lentiviral shRNA to CaMKII, oligonucleotides containing the target sequences were synthesized, annealed and inserted into FSIPPW lentiviral vector via the EcoR1/BamH1 restriction enzyme site, as described previously (Liu et al. 2008). Oligonucleiotides used were: sense: 5′-AATTCCCCTGATTGAAGCCATCAACATGCAAGAGATGTTGATGGCTTCAATCAGTTTTTG-3′, anti-sense: 5′-GATCCAAAAACTGATTGAAGCCATCAACATCTCT TGCATGTTGATGGCTTCAATCAGGGG-3′. Lentiviral shRNA targeting green fluorescent protein (GFP) for control was described (Liu et al. 2008). To produce lentivirus with CaMKII shRNA, above constructs were transfected to 293TD cells, then the lentivirus-containing supernatant was used to infect targeting cells following the procedure described (Chen et al. 2010).
Cell viability assay and morphological analysis
Primary neurons, PC12 cells, or PC12 cells infected with lentiviral shRNA to CaMKII or green fluorescent protein (GFP) for control, respectively, were seeded at a density of 1 × 104 cells/well in a flat-bottomed 96-well plate, pre-coated with PDL (0.2 μg/mL). Next day, cells were treated with 0–120 μM Cd for 24 h, with 20 μM Cd for 0–24 h, or with/without 10 and 20 μM Cd for indicated time following pre-incubation with/without 100 μM 2-APB or 10 μM KN93 for 1 h with five replicates of each treatment. Subsequently, each well was added 0.01 mL (5 mg/mL) of MTT reagent and incubated for 4 h. After the incubation, the incubation precipitates were dissolved with 0.1 mL of sodium dodecyl sulfate. Cell viability was determined by measuring the optical density at 570 nm using an ELx800 Microplate Reader (Bio-Tek Instruments, Inc. Winooski, VT, USA). In addition, the cells were also seeded at a density of 5 × 105 cells/well in a PDL-coated six-well plate. Next day, cells were exposed to Cd (10 and 20 μM) following pre-incubation with/without 20 μM BAPTA/AM, 2-APB, or KN93 for 1 h. After incubation for 24 h, the images were taken with a Nikon Eclipse TE2000-U inverted phase-contrast microscope (Nikon Instruments Inc., Tokyo, Japan) (200×) equipped with a digital camera.
Flow cytometry for [Ca2+]i staining
PC12 cells were seeded in 60-mm dishes, pre-coated with PDL, at a density of 1 × 106 cells/dish in the complete growth medium. Next day, cells were exposed to Cd (0–20 μM) for 4 and 24 h with triplicate of each treatment, followed by harvesting cells, washing three times with phosphate-buffered saline (PBS). Subsequently, cell suspensions (100 μL) for [Ca2+]i staining were loaded with 5 μM Fluo-3/AM for 30 min at 37°C in the dark, and then washed once with PBS to remove the extracellular Fluo-3/AM. Fluo-3/AM was replaced by PBS as a negative control. All samples were analyzed to detect the status of [Ca2+]i under a fluorescence-activated cell sorter Vantage SE flow cytometer (Becton Dickinson Co., San Jose, CA, USA) using the CellQuest software.
Analysis for [Ca2+]i fluorescence intensity
PC12 cells were seeded at a density of 5 × 105 cells/well in the complete growth medium in a PDL-coated six-well plate. Next day, cells were treated with/without Cd (10 and 20 μM) for 4 and 24 h following pre-incubation with/without rapamycin (Rap, 0.2 μg/mL) for 48 h, or with/without SP600125 (20 μM), U0126 (5 μM), PD169316 (20 μM), BAPTA/AM (20 μM), or 2-APB (100 μM) for 1 h, followed by harvesting cells and washing with PBS. Subsequently, cell suspensions (100 μL) for [Ca2+]i analysis were loaded with Fluo-3/AM as described above. Afterwards, [Ca2+]i fluorescent intensity was detected as described (Chen et al. 2011).
Western blot analysis
Western blotting was performed as described (Chen et al. 2011).
Results were expressed as mean values ± standard error (mean ± SE). Statistical analysis was performed by Student’s t-test (STATISTICA; Statsoft Inc, Tulsa, OK, USA). A level of p < 0.05 was considered to be significant.
Cd-induced CaMKII phosphorylation in neuronal cells
Activation of CaMKII is pro-apoptotic or anti-apoptotic depending on cell types or experimental conditions (Wright et al. 1997; Fladmark et al. 2002; Liu and Templeton 2007; Rokhlin et al. 2007; Vila-Petroff et al. 2007). To determine the role of CaMKII activity in Cd-induced neuronal apoptosis, PC12 cells and primary neurons, respectively, were exposed to 0–20 μM Cd for 12 h, or to 20 μM Cd for different time (0–12 h). We found that exposure of neuronal cells to Cd resulted in phospho-CaMKII increase in a time- and concentration-dependent manner (Fig. 1a and b), and a dramatically increased phosphorylation of CaMKII was seen at 12 h of exposure to 10 and 20 μM Cd (Fig. 1a) and at 8–12 h post-exposure to 20 μM Cd (Fig. 1b), respectively. Furthermore, Cd-elevated phospho-CaMKII level was consistent with decreased cell viability (Fig.1c and d) or increased apoptosis in PC12 cells and/or primary neurons (Chen et al. 2008b), suggesting that Cd-induced neuronal apoptosis might be associated with its induction of CaMKII phosphorylation.
Cd-induced phosphorylation of CaMKII was attributed to [Ca2+]i elevation in neuronal cells
CaMKII is a general integrator of Ca2+ signaling (Liu and Templeton 2007), which is activated in the presence of Ca2+ and calmodulin, resulting in autophosphorylation and generation of a Ca2+/CaM-independent form of the enzyme (Schworer et al. 1986). Our recent studies have demonstrated that Cd-induced neuronal apoptosis is associated with its induction of [Ca2+]i elevation, and unveiled that Cd-elevated [Ca2+]i activates MAPK/mTOR pathways and apoptosis in neuronal cells through calcium-binding protein CaM (Xu et al. 2011). Therefore, we proposed that Cd induction of [Ca2+]i elevation may activate MAPK/mTOR pathways and neuronal apoptosis by induction of CaMKII phosphorylation. To test the hypothesis, first of all, we examined whether Cd-elevated [Ca2+]i really activates CaMKII in neuronal cells. Consistent with our previous findings (Xu et al. 2011), treatment with Cd for 4 and 24 h resulted in a concentration-dependent increase of [Ca2+]i at concentrations of 0–20 μM in PC12 cells (Fig. 2a and b). Pre-treatment for 1 h with 20 μM BAPTA/AM, an intracellular Ca2+chelator, significantly attenuated [Ca2+]i elevation induced by Cd (10 and 20 μM) exposure for 4 and 24 h in PC12 cells (Fig. 2c) or primary neurons (data not shown). Interestingly, chelating intracellular Ca2+ with BAPTA/AM obviously inhibited Cd-induced CaMKII phosphorylation in PC12 cells and primary neurons (Fig. 2d).
2-APB, at concentrations of ≥ 50 μM, functions not only as a membrane-permeable inhibitor of inositol 1,4,5-trisphosphate (IP3) receptors on endoplasmic reticulum (ER), but also as an inhibitor of extracellular Ca2+ influx through the Ca2+ release activated Ca2+(CRAC) channels on plasma membrane (Prakriya and Lewis 2001; Bilmen et al. 2002; Braun et al. 2003). Next, 2-APB was employed to prevent Cd-induced [Ca2+]i elevation. As expected, pre-treatment with 2-APB (100 μM) markedly attenuated Cd-induced [Ca2+]i elevation (Fig. 3a). Like BAPTA/AM, 2-APB also partially blocked Cd activation of CaMKII in PC12 cells or primary neurons (Fig. 3b). Taken together, the results indicate Cd induction of [Ca2+]i elevation stimulates CaMKII phosphoryaltion.
Cd elicited CaMKII phosphorylation leading to activation of MAPK and mTOR pathways and neuronal apoptosis
To unravel whether Cd induction of CaMKII phosphorylation is correlated with its activation of MAPK and mTOR signaling pathways as well as apoptosis in neuronal cells, PC12 cells were exposed to Cd (10 and 20 μM) for 12 h after pre-treatment with CaMKII inhibitor KN93 (10 μM) for 1 h. We found that Cd-induced phosphorylation of CaMKII was obviously attenuated by KN93 in PC12 cells or primary neurons (Fig. 4a). Morphological analysis (Fig. 4b) revealed that KN93 itself did not alter cell shape. Cd alone (10 and 20 μM) induced cell roundup and shrinkage. However, KN93 partially prevented Cd-induced morphological change. Results from the MTT assay (Fig. 4c) further demonstrated that KN93 significantly suppressed Cd-induced loss of cell viability in PC12 cells and primary neurons. Of importance, KN93 partially blocked Cd-induced phosphorylation of Erk1/2, JNK, and p38 MAPK in PC12 cells and primary neurons (Fig. 4d). Cd-activated phosphorylation of Akt, S6K and 4E-BP1 in PC12 cells and primary neurons were also markedly reduced by KN93 (Fig. 4e).
To further substantiate the role of CaMKII in Cd activation of MAPK/mTOR pathways and neuronal cell death, expression of CaMKIIα was silenced by RNA interference. As shown in Fig. 5a, lentiviral shRNA to CaMKIIα, but not to GFP, down-regulated CaMKII expression by ∼90% in PC12 cells. Down-regulation of CaMKII in part prevented Cd-induced cell death in PC12 cells (Fig. 5b and c). Furthermore, silencing CaMKII obviously attenuated Cd-induced phosphorylation of CaMKII (Fig. 5d). Consistently, down-regulation of CaMKII conferred partial resistance to Cd activation of MAPKs and mTOR signaling pathways (Fig. 5d and e). The results indicate that Cd activates MAPK/mTOR signaling pathways and neuronal apoptosis by inducing CaMKII phosphorylation.
Inhibitors of mTOR, JNK and Erk1/2, but not p38, partially inhibited Cd-induced [Ca2+]i elevation and CaMKII phosphorylation, preventing neuronal cell death
Recently, we have demonstrated that rapamycin, a specific inhibitor of mTOR (Huang and Houghton 2003), may exert a beneficial effect against Cd-induced neuronal cell death by blocking mTOR-mediated phosphorylation of S6K1 and 4E-BP1 (Chen et al. 2008b). We have also shown that inhibitors of JNK and Erk1/2 partially prevent Cd-induced neuronal apoptosis by inhibiting phosphorylation of JNK and Erk1/2, respectively (Chen et al. 2008b). Cd induced [Ca2+]i elevation activates MAPKs and mTOR network (Xu et al. 2011). To investigate whether MAPKs and mTOR pathways reversely regulate Cd-induced intracellular Ca2+ elevation and CaMKII phosphorylation, PC12 cells and/or primary neurons were exposed to Cd (10 and 20 μM) for 4 and 24, or 12 h after pre-treatment with rapamycin, JNK inhibitor SP600125, MEK1/2 (upstream of Erk1/2) inhibitor U0126, or p38 inhibitor PD169316 for indicated time, respectively. As shown in Fig. 6a, pre-treatment with rapamycin obviously reduced Cd-induced [Ca2+]i elevation in PC12 cells. Consistently, we found that rapamycin dramatically blocked activation of mTOR-mediated S6K1 and 4E-BP1 with a concomitant reduction of CaMKII phosphorylaiton in Cd-exposed PC12 cells and primary neurons (Fig. 6b). Similar effects of 20 μM SP600125 (Fig. 6c and d) and 5 μM U0126 (Fig. 6e and f), but not 20 μM PD169316 (Fig. 6g and h), on Cd-induced [Ca2+]i elevation and CaMKII activation were seen in PC12 cells and/or primary neurons. The findings suggest that inhibitors of mTOR, JNK and Erk1/2 may prevent Cd-induced neuronal cell death in part through inhibition of [Ca2+]i elevation and CaMKII phosphorylation.
Calcium ion (Ca2+), as a second messenger, mediates a variety of physiological responses of neurons to neurotransmitters and neurotrophic factors (Cheng et al. 2003). However, deregulated cellular Ca2+ homeostasis contributes to neuronal cell death, which is implicated in neurodegenerative disorders, such as Alzheimer’s disease and Parkinson’s disease (Gibbons et al. 1993; Kawahara and Kuroda 2000; Mattson 2007; Marambaud et al. 2009). Cd, a highly toxic heavy metal, occurs frequently in the polluted environment and has a long biological half-life (15–20 years) in humans (Jin et al. 1998). Its targets of toxicity including liver, lung, kidney, testis, bone, etc. resulting in many diseases such as pulmonary edema, respiratory tract irritation, renal dysfunction, anemia, osteoporosis, and cancer in humans (Satarug et al. 2000; Kim et al. 2005; Lau et al. 2006; Chen et al. 2008a). Cd also severely affects the function of the nervous system by its induction of neuronal apoptosis, and thereby contributes to neurodegenerative diseases (Lopez et al. 2003). We previously have demonstrated that Cd induces apoptosis of neuronal (PC12 and SH-SY5Y) cells via activation of MAPK and mTOR signaling network (Chen et al. 2008b). Recently, our further studies revealed that Cd elevated [Ca2+]i level, leading to neuronal apoptosis in part through activation of MAPKs and mTOR pathways (Xu et al. 2011). Here, we provide evidence that the effects of Cd-elevated [Ca2+]i on MAPKs and mTOR network as well as neuronal cell death are through stimulating phosphorylation of CaMKII. The data indicate that CaMKII, as a key signaling protein, plays a bridging role between Cd induction of [Ca2+]i elevation and activation of MAPK and mTOR network, leading to neuronal cell death.
CaMKII, a multifunctional Ser/Thr kinase ubiquitously expressed in all neuronal compartments, is prominent among the Ca2+-sensitive processes (Colbran and Brown 2004). It is activated upon binding of Ca2+-containing CaM, which undergoes autophosphorylation, and then takes on autonomous activity independent of calmodulin binding (Hudmon and Schulman 2002). Based on the unique regulatory properties of CaMKII and our recent findings that Cd induces [Ca2+]i elevation with a concomitant increase of CaM function contributing to neuronal apoptosis, we speculated that CaMKII is an ‘interpreter’ of Cd induction of Ca2+ signaling, leading to apoptosis of neuronal cells. In the present study, we observed that exposure of neuronal cells (PC12 cells and primary neurons) to Cd resulted in phospho-CaMKII increase in a time- and concentration-dependent manner, which was consistent with decreased cell viability (Fig.1c and d) or increased apoptosis of PC12 cells (Chen et al. 2008b). Chelation of intracellular Ca2+ with BAPTA/AM blocked Cd activation of CaMKII. We have recently noticed that BAPTA/AM attenuated Cd activation of MAPK and mTOR signaling pathways as well as cell death in PC12 cells or primary neurons (Xu et al. 2011). These findings suggest that Cd-elevated [Ca2+]i activates CaMKII, which might be associated with activation of MAPK and mTOR signaling pathways, leading to neuronal apoptosis.
Many cell death stimuli are known to alter the concentration of Ca2+ in the cytosol and the storage of Ca2+ in the intracellular organelles (Baffy et al. 1993; Bian et al. 1997). [Ca2+]i increase is usually attribute to Ca2+ mobilization from intracellular stores and/or Ca2+ entry from the extracellular space (Jan et al. 2001). The ER is one of the major calcium storage units in cells, and blockers of the ER calcium channel, such as IP3 receptors, can effectively prevent Ca2+ release induced by various stimuli including Cd (Wang et al. 2007, 2008). Influx of Ca2+ from the extracellular environment, following internal Ca2+ store depletion, provides the elevated and sustained [Ca2+]i levels, which is associated with the activation of the CRAC channels on the plasma membrane (Prakriya and Lewis 2001; Lewis et al. 2008). Studies have demonstrated that 2-APB (≥ 50 μM) inhibits both IP3 receptors and CRAC channels, blocking ER Ca2+ release and extracellular Ca2+ influx (Prakriya and Lewis 2001; Bilmen et al. 2002; Braun et al. 2003). Therefore, 2-APB (100 μM) was employed to prevent Cd-induced [Ca2+]i elevation. As expected, 2-APB did block Cd-induced [Ca2+]i elevation. Importantly, like BAPTA/AM, 2-APB remarkably attenuated Cd-induced phosphorylation of CaMKII. Furthermore, we found that Cd-activated MAPK/mTOR pathways were partially inhibited by 2-APB as well (data not shown). This is in line with our recent findings that pre-treatment with ethylene glycol tetra-acetic acid (EGTA), an extracellular Ca2+ chelator, which renders the inaccessibility of extracellular Ca2+ to the cells, dramatically prevented Cd-induced [Ca2+]i elevation and activation of MAPK and mTOR pathways, as well as cell death (Xu et al. 2011). Taken together, our results suggest that Cd may induce ER Ca2+ release via activation of IP3 receptors on the ER and extracellular Ca2+ influx by activation of CRAC channels on plasma membrane, resulting in [Ca2+]i elevation contributing to CaMKII phosphorylation.
To demonstrate that CaMKII is essential for Cd activation of MAPK and mTOR pathways as well as neuronal cell death, pharmacological inhibition or genetic manipulation of CaMKII activity was utilized. Pre-treatment with KN93, a specific inhibitor of CaMKII (Choi et al. 2006), blocked Cd-induced CaMKII phosphorylation, and partially prevented Cd-induced death in PC12 cells and primary neurons (Fig. 4a–c). Consistently, Cd activation of MAPK and mTOR pathways was obviously blocked by KN93 in PC12 cells and primary neurons as well (Fig.4d and e). Furthermore, down-regulation of CaMKII by RNA interference also attenuated Cd activation of MAPK and mTOR pathways as well as neuronal cell death. These findings strongly suggest that Cd activates MAPK and mTOR signaling pathways by CaMKII phosphorylation, leading to neuronal cell death.
Recently, we have shown that a selective mTOR inhibitor, rapamycin, may dramatically rescue Cd-induced neuronal cell death by blocking mTOR-mediated phosphorylation of S6K1 and 4E-BP1 (Chen et al. 2008b). Consistently, in the study, we noted that rapamycin was able to attenuate Cd-induced [Ca2+]i elevation and CaMKII phosphorylaiton in PC12 cells and/or primary neurons. In addition, we also found that SP600125 (a specific JNK inhibitor) and U0126 (a specific inhibitor of MEK1/2, upstream kinases of Erk1/2), but not PD169136 (a specific p38 inhibitor), could prevent Cd-induced [Ca2+]i elevation and CaMKII activation in PC12 cells and/or primary neurons. The results are consistent with our previous findings that inhibition of JNK or Erk1/2 by specific inhibitors or RNA interference partially prevented Cd-induced neuronal apoptosis (Chen et al. 2008b). Our data suggest that JNK, Erk1/2 and mTOR are involved in controlling the cellular Ca2+ homeostasis, and their inhibitors prevent neuronal cell death, not only by directly inhibiting the activities of the kinases, but also by inhibiting Cd induction of [Ca2+]i elevation, which, in turn, results in less activation of CaMKII-MAPK/mTOR signaling network.
Four isoforms of p38 (-α, -β, -γ, and -δ) have been identified and especially, various isoforms of p38 have unique cellular functions (Wang et al. 1998; Enslen et al. 2000; Zhang et al. 2011). In this study, an antibody to phospho-p38 (Thr180/Tyr182) (Cat.No. 9215; Cell Signaling) was used, which cannot differentiate isoforms of p38-α, -β, -γ, and -δ. Therefore, currently we do not know what isoforms of p38 MAPK is activated by Cd-activated CaMKII. As activation of p38 MAPK is not involved in Cd-induced neuronal cell death (Chen et al. 2008b), we did not further study what isoforms of p38 was activated.
In conclusion, we have shown that Cd-elevated [Ca2+]i activates CaMKII in neuronal cells, which elicits MAPK and mTOR network, leading to neuronal cell death. Our findings implicate an important role of CaMKII in Cd neurotoxicology, and suggest that manipulation of intracellular Ca2+ level or CaMKII activity may be exploited for prevention of Cd-induced neurodegenerative disorders.
This work was supported in part by the grants from the National Natural Science Foundation of China (No. 30971486; LC), the Scientific Research Foundation of the State Education Ministry of China (SEMR20091341, LC), the Project for the Priority Academic Program Development and the Natural Science Foundation of Jiangsu Higher Education Institutions of China (10KJA180027; LC), NIH (CA115414; SH), American Cancer Society (RSG-08-135-01-CNE; SH), and Louisiana Board of Regents (NSF-2009-PFUND-144; SH). We thank Dr Ling Wang for technical assistance in flow cytometry analysis.
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