Molecular Mechanism Of BNIP3in Cadmium-induced Neurotoxicity

BackgroundCadmium (Cd) is a highly toxic heavy metal, mainly originated from mining, metal smelting, metal clarifier, industrial waste and cigarette smoking. In addition to workers, residents are increasingly exposed population due to polluted water, air and soil by Cd. Cd can result in carcinogenesis, renal and reproductive dysfunction, but also neurological disorders. In children, Cd exposure elicits learning disabilities. In adult workers exposed to Cd, neurobehavioral defects such as slowing of psychomotor speed are found; meanwhile workers have increased complaints about equilibrium and ability to concentrate. The available findings indicate Cd is a possible etiological factor of neurodegenerative diseases.Cd induces cytotoxic effects in many cell types. Depending on the cell type, Cd results in two forms of cell death:apoptosis or necrosis. In neuronal cells both apoptosis and necrosis can be induced by Cd. Several studies report possible mechanism of cell death in Cd-exposed neuronal cells, which include:generation of reactive oxygen species, the activation of MAPKs (Mitogen-activated protein kinases) and mTOR. However, contribution of apoptosis or necrosis to Cd-induced neurotoxicity remains unclear. Moreover, the molecular mechanism accounting for caspase-independent necrosis in Cd-induced neurotoxicity is totally unknown.BNIP3protein (Bcl-2and adenovirus E1B-19kDa-interacting protein3), a member of the BH3-only subfamily of Bcl-2family proteins, plays a critical role in cell death. It is widely involved in many pathological processes, such as cardiomyocyte hypertrophy and atrophy, cancer progression and hypoxia/ischemia in nervous system. Recently, BNIP3has been reported to be closely related to HD (Huntington’s disease, a neurodegenerative disorder), which abnormally accumulates in muscle cells from HD patients and in brain tissues from HD model mice. Growing evidence show that BNIP3induces caspase-independent cell death. Furthermore, BNIP3can be induced by toxins including cyanide and cobalt (Co), which subsequently mediates caspase-independent cell death in response to these stimuli. However, it is unknown whether BNIP3plays a role in Cd-induced neurotoxicity and which form of cell death it mediates.MAPKs are important molecules involved in Cd-induced neurotoxicity. MAPKs are composed of a signaling cascade of serine-threonine kinases. The mammalian MAPK family consists of ERK (extracellular signal-regulated kinase), p38and JNK(c-Jun NH2-terminal kinase). Various observations suggest that MAPKs contribute to neuroinflammatory responses and neuronal death in neurodegenerative diseases. Although MAPKs appear to be important in Cd-induced neurotoxicity, currently the downstream effector of them is not clearly understood.ObjectiveBy combined use of several cell biology and molecular biology methods:RNA interference, real-time RT-PCR, western blot, trypan blue exclusion method, PI staining, etc, the effect and molecular mechanism of BNIP3pathway on cadmium-induced neurotoxicity. Method1. SH-SY5Y and PC12cells were chosen to study Cd-induced neuronal cells death. Treatment with different concentration of Cd, then detect the dead rate by using Trypan blue exclusion.2. Cells were treated with Cd for various times, expression of Caspase-3and PARP were examined by using western blot. Cells were pretreated with a pan-caspase inhibitor (zVAD-fink) or two necrosis inhibitors (Necrox-2and Necrox-5) respectively, before treatment with Cd for24h. To determine the cell dead rate by using Trypan blue exclusion and LDH release kit.3. To determine the gene and protein expression of BNIP3induced by Cd, real-time RT-PCR and western blot were performed.4. Chemically synthesized siRNAs against BNIP3were introduced into neuronal cells. After downregulating BNIP3protein expression induced by Cd, using trypan blue exclusion and percentage of Pi-positive cells to examined the cells death.5. HIF-la protein expression and VEGF (Vascular endothelial growth factor) gene expression were detected by using western blot and RT-PCR.6. Investigated activation of MAPKs after Cd treatment over different times via western blot.7. Cells were exposed to Cd for24h after pretreatment with MEK (upstream kinase of ERK) inhibitor U0126, JNK inhibitor SP600125, or p38inhibitor SB203580for30min. The expression of BNIP3and cleaved caspase-3were examined via western blot. The cell dead rate was detected by using trypan blue exclusion.8. Built up the Cd exposed mice model. Twenty male BALB/c mice were randomly assigned to control group and Cd treated group (lOmice/group). Two groups received one intraperitoneal (ip) injection of Cd solution daily (1mg/kg bodyweight in saline) or equivalent saline, respectively for2weeks. Brain tissues were collected to examine the Cd level and protein expression of BNIP3and MAPKs via western blot.Result1. Trypan blue exclusion shows that treatment with Cd for24h caused a concentration-dependent increase of neuronal cells death.2. Cd modestly activated caspase-3and PARP (poly (ADP-ribose) polymerase) in neuronal cells. Consistently, pretreatment cells with100μM zVAD-fmk only slightly inhibited Cd-induced neuronal cells death, though zVAD-fink obviously prevented cleavage of caspase-3. Furthermore,0.3uM Necrox-2or0.1μM Necrox-5, two necrosis inhibitors, did not prevent cleavage of caspase-3, but suppressed cell death more significantly compared with zVAD-fink (p<0.01). Moreover, Cd-induced LDH release due to necrosis was more markedly inhibited by Necrox-2or5compared with zVAD-fmk (p<0.05).3. Cd induced BNIP3gene expression in a time-and concentration-dependent manner. In response to20μM Cd treatment, BNIP3gene expression increased at12h, further elevated over48h observation period. Consistent with gene expression, BNIP3protein level drastically increased in a concentration-and time-dependent manner after Cd exposure. Moreover, Co (Cobalt, a known BNIP3protein inducer) led to a similar protein bands pattern.4. Four siRNAs were screened and western blot showed siRNA-1,2or3had little effects on downregulating BNIP3protein expression induced by Cd. However, BNIP3protein in cells transfected with siRNA-4were reduced to4.5%, of that found in cultures transfected with NC siRNA in Cd-exposed SH-SY5Y cells. Furthermore, inhibitory effect of siRNA-4on BNIP3protein expression was dose dependent. Transfection with siRNA-4also reduced Cd-induced cell death in a dose-dependent manner. Consistent with the cell death data, release of LDH into the culture medium and percentage of PI-positive cells, two characteristic of necrotic cells due to rupture of the cell membrane, were also greatly inhibited by siRNA. Moreover, transfection of siRNA against BNIP3did not change cleavage of caspase-3induced by Cd in neuronal cells.5. Co (a positive control) increased strongly HIF-la protein expression over time in SH-SY5Y cells. However, protein bands corresponding to HIF-1α could not be detected at any time after Cd exposure. Moreover, VEGF, a well-known target gene of HIF-1, was upregulated significantly by Co. However, the similar effects were not observed in Cd-exposed neuronal cells.6. Activation of MAPKs after Cd treatment, ERK, p38and JNK were activated at different time by Cd. ERK was activated, peaked at1h and returned to basal level within8h. P38was activated progressively and remained elevated for8h. Phosphorylation of JNK increased until4h, but rapidly reached peak at4h.7. Pretreatment with ERK inhibitor U0126or JNK inhibitor SP600125suppressed BNIP3upregulation and following cell death. However, pretreatment with p38inhibitor SB203580did not change the BNIP3expression at any time after Cd exposure, but augmented Cd-induced cell death. Pretreatment with U0126obviously inhibited cleaved caspase-3induced by Cd. In contrast, pretreatment with SB203580augmented cleavage of caspase-3in Cd-exposed neuronal cells. Pretreatment with SP600125had little effects on cleaved caspase-3.8. Cd treatment increased the levels of Cd in mouse brain tissues. Importantly, thesame treatment induced BNIP3expression in mouse brain suchas cortex and hippocampus, and Cd treatment activatedMAPKs in mouse brain. 9. Results were expressed as Mean±SD. A Student’s t-test was used to determine the statistical significance of difference in values between two groups. One-way ANOVA was used for statistical analysis of difference in values among multiple groups. A value of p<0.05was considered significant.Conclusion1. Cd induces both apoptosis and necrosis in neuronal cells and among the two types of cell death; necrosis is a more prevalent pathway.2. Cd exposure strongly induces BNIP3expression in transcriptional and translational level in vivo and in vitro.3. BNIP3is a critical mediator for Cd-induced necrosis, but not apoptosis.4. Regulation of BNIP3expression is not related to HIF-1pathway in Cd-treated cells.5. Cd induces the activation of MAPKs in vivo and in vitro. And activation of ERK and JNK participate in BNIP3upregulation and following cell death. In contrast, p38promotes cell survival by inhibiting activation of caspase-3.In summary, the present study identified that BNIP3, upregulated by activation of ERK and JNK, mediates Cd-induced caspase-independent necrosis in neuronal cells. Our results suggest ERK/BNIP3and JNK/BNIP3pathway might be promising pathway for new study of Cd-induced neurotoxicity.