Jianchun Bian

Cadmium-induced apoptosis in primary rat cerebral cortical neurons culture is mediated by a calcium signaling pathway.

Cadmium (Cd) is an extremely toxic metal, capable of severely damaging several organs, including the brain. Studies have shown that Cd disrupts intracellular free calcium ([Ca2+]i) homeostasis, leading to apoptosis in a variety of cells including primary murine neurons. Calcium is a ubiquitous intracellular ion which acts as a signaling mediator in numerous cellular processes including cell proliferation, differentiation, and survival/death. However, little is known about the role of calcium signaling in Cd-induced apoptosis in neuronal cells. Thus we investigated the role of calcium signaling in Cd-induced apoptosis in primary rat cerebral cortical neurons. Consistent with known toxic properties of Cd, exposure of cerebral cortical neurons to Cd caused morphological changes indicative of apoptosis and cell death. It also induced elevation of [Ca2+]i and inhibition of Na+/K+-ATPase and Ca2+/Mg2+-ATPase activities. This Cd-induced elevation of [Ca2+]i was suppressed by an IP3R inhibitor, 2-APB, suggesting that ER-regulated Ca2+ is involved. In addition, we observed elevation of reactive oxygen species (ROS) levels, dysfunction of cytochrome oxidase subunits (COX-I/II/III), depletion of mitochondrial membrane potential (ΔΨm), and cleavage of caspase-9, caspase-3 and poly (ADP-ribose) polymerase (PARP) during Cd exposure. Z-VAD-fmk, a pan caspase inhibitor, partially prevented Cd-induced apoptosis and cell death. Interestingly, apoptosis, cell death and these cellular events induced by Cd were blocked by BAPTA-AM, a specific intracellular Ca2+ chelator. Furthermore, western blot analysis revealed an up-regulated expression of Bcl-2 and down-regulated expression of Bax. However, these were not blocked by BAPTA-AM. Thus Cd toxicity is in part due to its disruption of intracellular Ca2+ homeostasis, by compromising ATPases activities and ER-regulated Ca2+, and this elevation in Ca2+ triggers the activation of the Ca2+-mitochondria apoptotic signaling pathway. This study clarifies the signaling events underlying Cd neurotoxicity, and suggests that regulation of Cd-disrupted [Ca2+]i homeostasis may be a new strategy for prevention of Cd-induced neurodegenerative diseases.

Zearalenone induces apoptosis and cytoprotective autophagy in primary Leydig cells.

Zearalenone (ZEA) is a nonsteroidal estrogenic mycotoxin found in several food commodities worldwide. ZEA causes reproductive disorders, genotoxicity, and testicular toxicity in animals. However, little is known about the functions of apoptosis and autophagy after exposure to ZEA in Leydig cells. This study investigated the effects of ZEA on rat Leydig cells. Results showed that ZEA at different doses significantly inhibited the growth of Leydig cells by inducing apoptosis. ZEA treatment upregulated Bax expression, promoted cytochrome c release into the cytosol, and triggered mitochondria-mediated apoptosis. Consequently, caspase-9 and downstream effector caspase-3 were activated, followed by the cleavage of poly(ADP-ribose) polymerase (PARP), resulting in Leydig cell apoptosis. ZEA treatment also upregulated LC3-II and Beclin-1 expression, suggesting that ZEA induced a high level of autophagy. Pretreatment with chloroquine (an autophagy inhibitor) and rapamycin (an autophagy inducer) increased and decreased the rate of apoptosis, respectively, in contrast to other ZEA-treated groups. Autophagy delayed apoptosis in the ZEA-treated Leydig cells. Therefore, autophagy may prevent cells from undergoing apoptosis by reducing ZEA-induced cytotoxicity.

Induction of cytoprotective autophagy in PC-12 cells by cadmium.

Laboratory data have demonstrated that cadmium (Cd) may induce neuronal apoptosis. However, little is known about the role of autophagy in neurons. In this study, cell viability decreased in a dose- and time-dependent manner after treatment with Cd in PC-12 cells. As cells were exposed to Cd, the levels of LC3-II proteins became elevated, specific punctate distribution of endogenous LC3-II increased, and numerous autophagosomes appeared, which suggest that Cd induced a high level of autophagy. In the late stages of autophagy, an increase in the apoptosis ratio was observed. Likewise, pre-treatment with chloroquine (an autophagic inhibitor) and rapamycin (an autophagic inducer) resulted in an increased and decreased percentage of apoptosis in contrast to other Cd-treated groups, respectively. The results indicate that autophagy delayed apoptosis in Cd-treated PC-12 cells. Furthermore, co-treatment of cells with chloroquine reduced autophagy and cell activity. However, rapamycin had an opposite effect on autophagy and cell activity. Moreover, class III PI3 K/beclin-1/Bcl-2 signaling pathways served a function in Cd-induced autophagy. The findings suggest that Cd can induce cytoprotective autophagy by activating class III PI3 K/beclin-1/Bcl-2 signaling pathways. In sum, this study strongly suggests that autophagy may serve a positive function in the reduction of Cd-induced cytotoxicity.

Zearalenone inhibits testosterone biosynthesis in mouse Leydig cells via the crosstalk of estrogen receptor signaling and orphan nuclear receptor Nur77 expression

Zearalenone (ZEA) directly inhibits testosterone biosynthesis in Leydig cells, although the mechanisms involved remains unclear. Various experiments were performed to elucidate the molecular pathway of ZEA-mediated androgen inhibition. Leydig cells were isolated from 6 week-old male ICR mice and subjected to ZEA pre-treatment. The levels of testosterone and a series of influncing factors were measured. The results showed that ZEA caused a concentration- and time-dependent inhibition of testosterone stimulated both by hCG and cAMP (P<0.05). Exposure to ZEA did not affect the LHR binding activity nor the protein expression (P>0.05). However, ZEA exposure significantly elevated the cellular cAMP levels (P<0.05) in low concentrations (5 μg/ml) or for long time periods (24 h), significantly reduce the mitochondrial membrane potential (P<0.05). The expression of P450scc, 17β-HSD, and P450c17 at the mRNA level were significantly decreased (P<0.05). The steroidogenic acute regulatory (StAR) and 3β-HSD expression was significantly increased (P<0.05). Furthermore, the ERα protein expression was not affected by ZEA, but Nur77 expression was significantly inhibited (P<0.05). These observations imply that ZEA activity interferes with testosterone biosynthesis in mouse Leydig cells via the crosstalk of estrogen receptor signaling and Nur77 expression.

Calcium–calmodulin signaling elicits mitochondrial dysfunction and the release of cytochrome c during cadmium-induced apoptosis in primary osteoblasts

Cadmium (Cd) is a toxic heavy metal used in industry and is associated with adverse effects on human health following long- or short-term environmental exposure. Although Cd is known to induce apoptosis in many human organ systems, the mechanism that underlies its toxicity in primary osteoblasts (OBs) is not yet established. In the present study, we confirmed that Cd induced apoptosis in OBs isolated from the craniums of fetal Sprague-Dawley rats. We then showed that exposure to Cd transiently increased intracellular calcium ([Ca(2+)]i) levels for up to 1.5h, after which the levels returned to normal. Pretreatment with the calcium chelator BAPTA-AM was able to prevent Cd-induced apoptosis by reversing Cd-induced changes in the mitochondrial transmembrane potential (ΔΨm). In addition, we found that the antagonist of calcium-dependent calmodulin (CaM), W-7, inhibited the conformational change of calmodulin induced by Cd. Furthermore, Cd-induced apoptosis could be inhibited by W-7 through the suppression of the mitochondrial release of cytochrome c to the cytosol and the reversal of Cd-activation of caspase-3. These data indicate that activated Ca(2+)/CaM might transmit apoptotic signals to the mitochondria during Cd-induced apoptosis. Our findings provide new insights into the mechanisms underlying apoptosis in OBs following exposure to Cd.

The role of mitogen-activated protein kinase in cadmium-induced primary rat cerebral cortical neurons apoptosis via a mitochondrial apoptotic pathway.

Cadmium (Cd) is an extremely toxic metal capable of severely damaging several organs, including the brain. Studies have shown that Cd induces neuronal apoptosis partially by activating the mitogen-activated protein kinase (MAPK) pathways. However, the underlying mechanism of MAPK involving the mitochondrial apoptotic pathway in neurons remains unclear. In this study, primary rat cerebral cortical neurons were exposed to Cd, which significantly decreased cell viability and the B-cell lymphoma 2/Bcl-2 associate X protein (Bcl-2/Bax) ratio and increased the percentage of apoptotic cells, release of cytochrome c, cleavages of caspase-3 and poly (ADP-ribose) polymerase (PARP), and nuclear translocation of apoptosis-inducing factor (AIF). In addition, Cd induced phosphorylation of extracellular signal-regulated kinase (ERK), c-Jun N-terminal kinase (JNK) and p38 MAPK. Inhibition of ERK and JNK, but not p38 MAPK, partially protected the cells from Cd-induced apoptosis. ERK and JNK inhibition also blocked alteration of the Bcl-2/Bax ratio, release of cytochrome c, cleavages of caspase-3 and PARP, and nuclear translocation of AIF. Taken together, these data suggest that the ERK- and JNK-mediated mitochondrial apoptotic pathways play important roles in Cd-induced neuronal apoptosis.

Cadmium Induces PC12 Cells Apoptosis via an Extracellular Signal-Regulated Kinase and c-Jun N-Terminal Kinase-Mediated Mitochondrial Apoptotic Pathway

To investigate the role of mitogen-activated protein kinase (MAPK) and downstream events in cadmium (Cd)-induced neuronal apoptosis executed via the mitochondrial apoptotic pathway, this study used the PC-12 cell line as a neuronal model. The result showed that Cd significantly decreased cell viability and the Bcl-2 / Bax ratio and increased the percentage of apoptotic cells, release of cytochrome c, caspase-3, and poly(ADP-ribose) polymerase cleavage, and nuclear translocation of apoptosis-inducing factor (AIF) and endonuclease G. In addition, exposure to Cd-induced phosphorylation of extracellular signal-regulated kinase (ERK), c-Jun N-terminal kinase (JNK), and p38 MAPK. Inhibition of ERK and JNK, but not p38 MAPK, partially protected the cells from Cd-induced apoptosis. ERK and JNK inhibition also blocked alteration of the Bcl-2 / Bax ratio and cytochrome c release and suppressed caspase-3 and poly(ADP-ribose) polymerase cleavage and AIF and endonuclease G nuclear translocation. Taken together, these data suggest that the ERK- and JNK-mediated mitochondrial apoptotic pathway played an important role in Cd-induced PC12 cells apoptosis.

Osteoprotegerin Induces Apoptosis of Osteoclasts and Osteoclast Precursor Cells via the Fas/Fas Ligand Pathway.

Osteoprotegerin (OPG) is known to inhibit differentiation and activation of osteoclasts (OCs) by functioning as a decoy receptor blocking interactions between RANK and RANKL. However, the exact role of OPG in the survival/apoptosis of OCs remains unclear. OPG caused increased rates of apoptosis of both OCs and osteoclast precursor cells (OPCs). The expression of Fas and activated caspase-8 was increased by both 20 ng/mL and 40 ng/mL of OPG, but was markedly decreased at 80 ng/mL. Interestingly, we noted that while levels of Fas ligand (FasL) increased with increasing doses of OPG, the soluble form of FasL in the supernatant decreased. The results of a co-immunoprecipitation assay suggested that the decrease of sFasL might be caused by the binding of OPG. This would block the inhibition of the apoptosis of OCs and OPCs. Furthermore, changes in expression levels of Bax/Bcl-2, cleaved-caspase-9, cleaved-caspased-3 and the translocation of cytochrome c, illustrated that OPG induced apoptosis of OCs and OPCs via the classic Fas/FasL apoptosis pathway, and was mediated by mitochondria. Altogether, our results demonstrate that OPG induces OCs and OPCs apoptosis partly by the Fas/FasL signaling pathway.

Autophagy Plays a Cytoprotective Role During Cadmium-Induced Oxidative Damage in Primary Neuronal Cultures.

Cadmium (Cd) induces significant oxidative damage in cells. Recently, it was reported that autophagy could be induced by Cd in neurons. However, little is known about the role of reactive oxygen species (ROS) during Cd-induced autophagy. In our study, we examined the cross-talk between ROS and autophagy by using N-acetyl cysteine (NAC, an antioxidant) and chloroquine (CQ, a pharmacological inhibitor of autophagy) in a primary rat neuronal cell cultures. We observed accumulation of acidic vesicular organelles and the increased expression of endogenous protein light chain 3 (LC3) in Cd-treated neurons, revealing that Cd induced a high level of autophagy. Moreover, increased levels of ROS were observed in neurons treated with Cd, showing that ROS accumulation was closely associated with neuron’s exposure to Cd. Furthermore, we found that autophagy was inhibited by using CQ and/or NAC with further aggravation of mitochondrial damage, lactate dehydrogenase (LDH) leakage and hypoploid apoptotic cell number in Cd-treated neurons. These results proved that autophagy has a cytoprotective role during Cd-induced toxicity in neurons, and it can prevent the oxidative damage. These findings may enable the development of novel therapeutic strategies for neurological diseases.

Effects of 1α,25-(OH)2D3 on the formation and activity of osteoclasts in RAW264.7 cells.

The hormonally active form of vitamin D3, 1α,25-(OH)2D3, has an important role in bone metabolism. This study examined the effects of 1α,25-(OH)2D3 on the ability of two cytokines, receptor activator of nuclear factor-κB ligand (RANKL) and macrophage-colony stimulating factor (M-CSF), to induce RAW 264.7 cells to form osteoclasts. A TRAP histochemical staining assay and bone resorption analysis were used to identify the rate of formation and activity of osteoclasts. The numbers of osteoclasts formed, and their bone resorption activity, was enhanced by the addition of 1α,25-(OH)2D3. The expression levels of osteoclast-specific proteins that are essential for bone resorption, integrin β3, V-ATPase, CAII, CTSK, TRAP and MMP-9, were detected by western blotting. During 48 h, the expression levels of all these proteins significantly increased. Quantitative real-time polymerase chain reaction was used to determine the expression levels of the transcription factors, c-Fos and NFATcl. The expression levels of c-Fos and NFATc1 also increased 24h after treatment with 1α,25-(OH)2D3. These results suggest that 1α,25-(OH)2D3 can regulate bone metabolism by directly enhancing the formation and maturation of osteoclasts.