CaMKⅡ mediates cadmium induced apoptosis in rat primary osteoblasts through MAPK activation and endoplasmic reticulum stress

Cadmium triggers apoptosis of LLC-PK1 cells through induction of endoplasmic reticulum (ER) stress. We found that cadmium caused generation of reactive oxygen species (ROS) and that cadmium-induced ER stress was inhibited by antioxidants. In contrast, suppression of ER stress did not attenuate cadmium-triggered oxidative stress, suggesting that ER stress occurs downstream of oxidative stress. Exposure of the cells to either O(2)(*), H(2)O(2), or ONOO(-) caused apoptosis, whereas ER stress was induced only by O(2)(*) or ONOO(-). Transfection with manganese superoxide dismutase significantly attenuated cadmium-induced ER stress and apoptosis, whereas pharmacological inhibition of ONOO(-) was ineffective. Interestingly, transfection with catalase attenuated cadmium-induced apoptosis without affecting the level of ER stress. O(2)(*) caused activation of the activating transcription factor 6-CCAAT/enhancer-binding protein-homologous protein (CHOP) and the inositol-requiring ER-to-nucleus signal kinase 1-X-box-binding protein 1 (XBP1) proapoptotic cascades, and overexpression of manganese superoxide dismutase attenuated cadmium-triggered induction of both pathways. Furthermore, phosphorylation of proapoptotic c-Jun N-terminal kinase by O(2)(*) or cadmium was suppressed by dominant-negative inhibition of XBP1. These data elucidated 1) cadmium caused ER stress via generation of ROS, 2) O(2)(*) was selectively involved in cadmium-triggered, ER stress-mediated apoptosis through activation of the activating transcription factor 6-CHOP and inositol-requiring ER-to-nucleus signal kinase 1-XBP1 pathways, and 3) phosphorylation of JNK was caused by O(2)(*)-triggered activation of XBP1.

High glucose (HG) state is closely related to diabetic complications, and among these one main pathogenesis involves apoptosis of important functional cells. However, the mechanism of osteoblast (OB) apoptosis induced by HG state is not clear. Peroxynitrite (ONOO-), the strongest oxidant, can mediate apoptosis in various kinds of cells, including OB. Therefore, this study was aimed at investigating whether HG state could induce OB apoptosis through ONOO-. Cultured OB from rat calvariae explanted from E21 fetuses were treated with HG solution. The ratio of OB apoptosis and ONOOcontent was assayed by TUNEL, flow cytometry, and immunohistochemistry. The results showed that HG state could induce ONOO- overformation (p<0.01), and excessive OB apoptosis (p<0.001). However, the HG-induced OB apoptosis could be attenuated by peonol, a potent scavenger of ONOO-. In conclusion, HG state might trigger excessive ONOO- formation, mediating OB apoptosis.

The present study investigated the effects of fluoride on endoplasmic reticulum (ER) stress (ERS) and osteoblast apoptosis in vivo. Forty-eight Wistar rats were randomly divided into four groups (12/group) and exposed to 0, 50, 100, and 150 mg/L of fluoride in drinking water for 8 weeks, respectively. Peripheral blood samples and bilateral femurs were used to monitor the progression of fluorosis in the animals. Hematoxylin and eosin (H&E) staining of the bone tissues was used to determine the severity of osteofluorosis. The expression of ERS chaperones (glucose-regulated protein 78 (GRP78), X-box binding protein l (XBP1), cysteine aspartate specific protease-12 (caspase-12), and growth arrest and DNA damage-inducible gene 153 (Gadd153/CHOP) was analyzed by immunohistochemistry staining, and osteoblast apoptosis was determined by TUNEL staining and flow cytometry. Accumulation of fluoride in bone was associated with the severity of osteofluorosis. The expression of GRP78, XBP1, caspase-12, and CHOP was increased in a dose-dependent manner. Fluoride-induced apoptosis in osteoblasts was also dose-dependent. High concentrations of fluoride induced ERS and osteoblast apoptosis in vivo. The increased expression of GRP78 and XBP1 increased the adaptation of osteoblasts to ERS to a certain extent. Caspase-12 and CHOP activation was associated with ERS and osteoblast apoptosis.

BackgroundCholesterol (CHO) is an essential component of the body. However, high CHO levels in the body can damage bone mass and promote osteoporosis. CHO accumulation can cause osteoblast apoptosis, which has a negative effect on bone formation. The pathogenesis of osteoporosis is a complicate process that includes oxidative stress, endoplasmic reticulum (ER) stress, and inflammation. Geniposide (GEN) is a natural compound with anti-osteoporotic effect. However, the roles of GEN in osteopathogenesis are still unclear. Our previous studies demonstrated that GEN could reduce the accumulation of CHO in osteoblasts and the activation of ER stress in osteoblasts. However, the molecular mechanism of GEN in inhibiting CHO-induced apoptosis in osteoblasts needs to be further investigated.MethodsMC3T3-E1 cells were treated with osteogenic induction medium (OIM). Ethanol-solubilized cholesterol (100 µM) was used as a stimulator, and 10 µM and 25 µM geniposide was added for treatment. The alterations of protein expression were detected by western blot, and the cell apoptosis was analyzed by a flow cytometer.ResultsCHO promoted osteoblast apoptosis by activating ER stress in osteoblasts, while GEN alleviated the activation of ER stress and reduced osteoblast apoptosis by activating the GLP-1R/ABCA1 pathway. Inhibition of ABCA1 or GLP-1R could eliminate the protective activity of GEN against CHO-induced ER stress and osteoblast apoptosis.ConclusionGEN alleviated CHO-induced ER stress and apoptosis in osteoblasts by mediating the GLP-1R/ABCA1 pathway.

Although total hip arthroplasty is considered to be an effective surgical procedure for treating hip joint diseases, it is hindered by implant wear debris, which induces aseptic loosening. Various cell types are involved in this pathogenesis; however, the interactions between wear debris and osteoblasts, which serve a crucial role in bone formation, have not been clearly illustrated. In the present study, minor metallic wear particles were collected from the interfacial membrane around loosened implants of patients, and the biological effects of these particles on rat primary osteoblasts were then explored. The results demonstrated that metallic wear debris was able to induce the apoptosis of treated cells in a concentration- and time-dependent manner. Furthermore, it was identified that reactive oxygen species (ROS) generation increased, the mitochondrial membrane potential collapsed, and the mitochondria-caspase-dependent and endoplasmic reticulum (ER) stress apoptotic pathways were activated following metallic wear debris application. In addition, apoptosis and associated pathways were inhibited by the use of N-acetyl-L-cysteine, an antioxidant that suppresses ROS production, indicating that the ROS generation triggered ER stress, mitochondrial dysfunction and downstream cascades that contributed to cell apoptosis. These findings suggest that metallic wear debris-induced ROS serve an important role in the apoptosis of osteoblasts. This provides a valuable insight, not only into understanding the mechanisms underlying the involvement of osteoblasts in osteolysis, but also into a potential novel therapeutic approach to treat implant aseptic loosening.

Molecular mechanism of nitric oxide-induced osteoblast apoptosis

Osteoporosis is an aging-associated disease that is attributed to excessive osteoblast apoptosis. It is known that the accumulation of advanced glycation end products (AGEs) in bone extracellular matrix deteriorates osteoblast functions. However, little is known about the interaction between intracellular AGE accumulation and the induction of osteoblast apoptosis. In this study, we investigated the effect of intracellular AGE accumulation on osteoblast apoptosis in vitro and in vivo. In vitro, murine osteoblastic MC3T3-E1 cells were treated with glycolaldehyde (GA), an AGE precursor. GA-induced intracellular AGE accumulation progressed in time- and dose-dependent manners, followed by apoptosis induction. Intracellular AGE formation also activated endoplasmic reticulum (ER) stress-related proteins (such as glucose-regulated protein 78, inositol-requiring protein-1α (IRE1α), and c-Jun N-terminal kinase) and induced apoptosis. In agreement, treatment with the ER stress inhibitor 4-phenylbutyric acid and knocking down IRE1α expression ameliorated osteoblast apoptosis. Furthermore, the ratio between AGE- and terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL)-positive osteoblasts in human vertebral bodies was significantly higher in an elderly group than in a younger group. A positive linear correlation between the ratio of AGE-positive and TUNEL-positive osteoblasts (r = 0.72) was also observed. Collectively, these results indicate that AGEs accumulated in osteoblasts with age and that intracellular AGE accumulation induces apoptosis via ER stress. These findings offer new insight into the mechanisms of osteoblast apoptosis and age-related osteoporosis. © 2020 American Society for Bone and Mineral Research.

Geniposide ameliorated dexamethasone-induced endoplasmic reticulum stress and mitochondrial apoptosis in osteoblasts

Introduction: Markers of hypoxia (HIF-1α) and Endoplasmic Reticulum (ER) stress are present in alveolar epithelial cells (AEC) of patients with Idiopathic Pulmonary Fibrosis (IPF). However, the origin and specific contribution of ER stress remain unclear in this disease. Aims: To investigate the link between hypoxic and ER stress pathways in the context of alveolar cell dysfunction. Methods: Impact of hypoxia on cell dysfunction was evaluated in a rat hypoxic model. ER stress modulators were used to understand the relationship between ER stress, apoptosis, and epithelial-mesenchymal transition (EMT) induction in primary rat AEC exposed to hypoxia. The role of HIF-1α on the unfolded protein response pathways and Chop promoter activation was documented through the overexpression of HIF-1α in AEC. Then, crosstalk between the hypoxic and ER stress pathways was analysed by co-immunoprecipitation on hypoxic-AEC, or by proximity ligation assay in pathologic lungs. Results:In vivo, 24h-hypoxia exposure induced ER stress and apoptosis markers specifically in AEC. Loss of epithelial phenotype was observed at 48h. In vitro, ER stress inhibitors limited apoptosis and EMT induction by hypoxia whereas calcium chelation limits only hypoxia-induced EMT. Overexpression of HIF-1α was able to induce ATF4, ATF6α-XBP1s transactivation activity and to enhance Chop promoter activity. Finally, the co-expression of CHOP and HIF-1α was observed in mice with bleomycin-induced pulmonary fibrosis and in IPF lungs. Conclusions: Our results suggest that hypoxic microenvironment and expression of HIF-1α could trigger ER stress in AEC, which may promote the development of the fibrosing process.

OBJECTIVE—Endoplasmic reticulum (ER) stress has been implicated in the pathogenesis of diabetes, but the roles of specific ER Ca2+ release channels in the ER stress–associated apoptosis pathway remain unknown. Here, we examined the effects of stimulating or inhibiting the ER-resident inositol trisphosphate receptors (IP3Rs) and the ryanodine receptors (RyRs) on the induction of β-cell ER stress and apoptosis.RESEARCH DESIGN AND METHODS—Kinetics of β-cell death were tracked by imaging propidium iodide incorporation and caspase-3 activity in real time. ER stress and apoptosis were assessed by Western blot. Mitochondrial membrane potential was monitored by flow cytometry. Cytosolic Ca2+ was imaged using fura-2, and genetically encoded fluorescence resonance energy transfer (FRET)–based probes were used to measure Ca2+ in ER and mitochondria.RESULTS—Neither RyR nor IP3R inhibition, alone or in combination, caused robust death within 24 h. In contrast, blocking sarco/endoplasmic reticulum ATPase (SERCA) pumps depleted ER Ca2+ and induced marked phosphorylation of PKR-like ER kinase (PERK) and eukaryotic initiation factor-2α (eIF2α), C/EBP homologous protein (CHOP)–associated ER stress, caspase-3 activation, and death. Notably, ER stress following SERCA inhibition was attenuated by blocking IP3Rs and RyRs. Conversely, stimulation of ER Ca2+ release channels accelerated thapsigargin-induced ER depletion and apoptosis. SERCA block also activated caspase-9 and induced perturbations of the mitochondrial membrane potential, resulting eventually in the loss of mitochondrial polarization.CONCLUSIONS—This study demonstrates that the activity of ER Ca2+ channels regulates the susceptibility of β-cells to ER stress resulting from impaired SERCA function. Our results also suggest the involvement of mitochondria in β-cell apoptosis associated with dysfunctional β-cell ER Ca2+ homeostasis and ER stress.

Gene expression profiling of osteoblasts subjected to dexamethasone-induced apoptosis with/without GSK3β-shRNA

The endoplasmic reticulum represents the quality control site of the cell for folding and assembly of cargo proteins. A variety of conditions can alter the ability of the endoplasmic reticulum (ER) to properly fold proteins, thus resulting in ER stress. Cells respond to ER stress by activating different signal transduction pathways leading to increased transcription of chaperone genes, decreased protein synthesis, and eventually to apoptosis. In the present paper we analyzed the role that the adaptor protein tumor necrosis factor-receptor associated factor 2 (TRAF2) plays in regulating cellular responses to apoptotic stimuli from the endoplasmic reticulum. Mouse embryonic fibroblasts derived from TRAF2-/- mice were more susceptible to apoptosis induced by ER stress than the wild type counterpart. This increased susceptibility to ER stress-induced apoptosis was because of an increased accumulation of reactive oxygen species following ER stress, and was abolished by the use of antioxidant. In addition, we demonstrated that the NF-kappaB pathway protects cells from ER stress-induced apoptosis, controlling ROS accumulation. Our results underscore the involvement of TRAF2 in regulating ER stress responses and the role of NF-kappaB in protecting cells from ER stress-induced apoptosis.

Particle-induced osteolysis is mediated by endoplasmic reticulum stress-associated osteoblast apoptosis

Palmitic acid (PA) upregulates oxidized LDL receptor-1 (LOX-1), a scavenger receptor responsible for uptake of oxidized LDL (oxLDL), and enhances oxLDL uptake in macrophages. However, the precise underlying mechanism remains to be elucidated. PA is known to induce endoplasmic reticulum (ER) stress in various cell types. Therefore, we investigated whether ER stress is involved in PA-induced LOX-1 upregulation. PA induced ER stress, as determined by phosphorylation of PERK, eIF2α, and JNK, as well as induction of CHOP in macrophage-like THP-1 cells. Inhibitors [4-phenylbutyric acid (PBA), sodium tauroursodeoxycholate (TUDCA), and salubrinal] and small interfering RNA (siRNA) for the ER stress response decreased PA-induced LOX-1 upregulation. Thapsigargin, an ER stress inducer, upregulated LOX-1, which was decreased by PBA and TUDCA. We next examined whether unsaturated FAs could counteract the effect of PA. Both oleic acid (OA) and linoleic acid (LA) suppressed PA-induced LOX-1. Activation of the ER stress response observed in the PA-treated cells was markedly attenuated when the cells were cotreated with OA or LA. In addition, OA and LA suppressed thapsigargin-induced LOX-1 upregulation with reduced activation of ER stress markers. Our results indicate that activation of ER stress is involved in PA-induced LOX-1 upregulation in macrophages, and that OA and LA inhibit LOX-1 induction through suppression of ER stress.

Idiopathic scoliosis (IS) is a spinal 3-dimensional deformity with an unknown cause. Melatonin is secreted by the pineal body and contributes to the occurrence and progression of IS. In our previous preliminary study, it was reported that high concentrations of melatonin can induce osteoblast apoptosis, thus acting as an IS treatment, but the mechanism of action is unknown. Therefore, the present study was performed to further investigate the possible mechanism underlying the efficacy of melatonin as a treatment for IS. The present results indicated that high concentrations of melatonin mediate endoplasmic reticulum stress (ERS)-induced apoptosis in hFOB 1.19 cells, and this resulted in a significant and dose-dependent increase in the expression of Septin4, as well as the expression levels of glucose-regulated protein (GRP)78, GRP94 and cleaved caspase-3. Furthermore, osteoblasts were overexpressed with Septin4 and the mechanism via which melatonin induces osteoblast ERS was demonstrated to be via the regulation of Septin4. In addition, it was indicated that cytoskeleton destruction, cell morphology changes and the decrease in the number of cells were aggravated after osteoblasts were overexpressed with Septin4, as indicated by phalloidin and DAPI staining. Collectively, the present results suggest that the Septin4 protein may be a target of ERS in melatonin-induced osteoblast apoptosis, which is involved in bone metabolism diseases, thus providing novel evidence for clinical melatonin treatment of IS.