J Vet Sci.  2015 Sep;16(3):297-306. 10.4142/jvs.2015.16.3.297.

Cadmium induces apoptosis in primary rat osteoblasts through caspase and mitogen-activated protein kinase pathways

• Zhao H
• Liu W
• Wang Y
• Dai N
• Gu J
• Yuan Y
• Liu X
• Bian J
• Liu ZP

• KMID: 2344302
• DOI: http://doi.org/10.4142/jvs.2015.16.3.297

Abstract

Exposure to cadmium (Cd) induces apoptosis in osteoblasts (OBs); however, little information is available regarding the specific mechanisms of Cd-induced primary rat OB apoptosis. In this study, Cd reduced cell viability, damaged cell membranes and induced apoptosis in OBs. We observed decreased mitochondrial transmembrane potentials, ultrastructure collapse, enhanced caspase-3 activity, and increased concentrations of cleaved PARP, cleaved caspase-9 and cleaved caspase-3 following Cd treatment. Cd also increased the phosphorylation of p38-mitogen-activated protein kinase (MAPK), extracellular signal-regulated kinases (ERK)1/2 and c-jun N-terminal kinase (JNK) in OBs. Pretreatment with the caspase inhibitor, N-benzyloxycarbonyl-Val-Ala-Asp-fluoromethylketone, ERK1/2 inhibitor (U0126), p38 inhibitor (SB203580) and JNK inhibitor (SP600125) abrogated Cd-induced cell apoptosis. Furthermore, Cd-treated OBs exhibited signs of oxidative stress protection, including increased antioxidant enzymes superoxide dismutase and glutathione reductase levels and decreased formation of reactive oxygen species. Taken together, the results of our study clarified that Cd has direct cytotoxic effects on OBs, which are mediated by caspase- and MAPK pathways in Cd-induced apoptosis of OBs.

Keyword

apoptosis; cadmium; caspase; mitogen-activated protein kinase; osteoblasts

MeSH Terms

Animals
Apoptosis/*drug effects
Cadmium/*toxicity
Caspases/metabolism
Environmental Pollutants/*toxicity
Osteoblasts/*drug effects/metabolism
Oxidative Stress/drug effects
Rats
Rats, Sprague-Dawley
p38 Mitogen-Activated Protein Kinases/metabolism
Cadmium
Caspases
Environmental Pollutants
p38 Mitogen-Activated Protein Kinases

Figure

• 

  Fig. 1 Cadmium (Cd) altered cell viability, lactate dehydrogenase (LDH), antioxidant enzymatic activity, reactive oxygen species (ROS), mitochondrial membrane potential and ultrastructure in osteoblasts (OBs). (A) OBs were treated with 0-20 µM Cd, and cell viability was assessed at 3 to 48 h. (B and C) OBs were treated with 0-5 µM Cd for 24 h, and LDH, SOD, and glutathione reductase (GR) activities were determined. (D and E) Cells were exposed to 0-5 µM Cd for 0.75 to 24 h. Intracellular ROS production and mitochondrial transmembrane potential (MMP) were measured, and the results are presented as the mean fluorescence intensity (MFI) relative to the control group (100%). Data are presented as the mean and SDs of three independent experiments, each performed in triplicate. Significant differences are indicated by *p < 0.05 and **p < 0.01 relative to the control. (F) Ultrastructural morphology of OBs (5,200× magnification) after treatment with 0, 1, 2, and 5 µM Cd (F1-4) for 24 h.

• 

  Fig. 2 Cd triggers apoptosis in OBs. (A and B) Cell nuclei as observed under fluorescence microscopy. Cells were exposed to 0 (A1 and B1), 1 (A2 and B2), 2 (A3 and B3), and 5 (A4 and B4) µM Cd for 12 h (A) and 24 h (B). Scale bars = 20 µm (A and B). (C) Flow cytometric analysis of cells showed an increased apoptotic rate in a dose-dependent manner. (D) The expression levels of Bax and Bcl-2 were assessed by western blot analysis, while β-actin was probed as a protein loading control. (E) Densitometry analysis of Bax, Bcl-2 and the Bax/Bcl-2 ratio. All data were expressed as the mean and SDs (n = 3). **p < 0.01 compared with the control group.

• 

  Fig. 3 Caspase activation is partially associated with Cd-induced apoptosis. (A) Time course of Cd-induced (2 µM) changes in caspase-3 activity. Data are expressed as the x-fold change over control values estimated in untreated cells. (B) Cd (0, 1, 2, 5 µM) stimulated caspase-3 activity. Data are expressed as the x-fold change over control values as estimated in untreated cells. (C) Cells were exposed to the indicated concentrations of Cd in the presence or absence of 50 µM z-VAD-fmk for 12 and 24 h, and cell viability was analyzed. (D) Morphological changes in OB nuclei after 2 µM Cd treatment in the presence or absence of 50 µM z-VAD-fmk for 24 h. Scale bar = 20 µm. (E) Cd increased the protein expression levels of cleaved PARP, cleaved caspase-9 and cleaved caspase-3. The intensity of each band was quantified by densitometry. The protein expression from the control group was designated as 1, while that of the other groups were expressed as fold changes compared to the control. Data are presented as the means and SDs of three independent experiments, each performed in triplicate. Significant differences are indicated by ap < 0.05 and bp < 0.01 relative to the control, and cp < 0.05 and dp < 0.01 compared with the Cd group.

• 

  Fig. 4 The involvement of MAPK signaling in Cd-induced apoptosis. Cell lysates analyzed by western blot with the indicated antibodies. The β-actin protein level was used as a loading control. (A) Cells were cultured with 2 µM Cd for 1 to 6 h. (B) OBs were pre-incubated with 10 µM SB203580, SP600125, and U0126 for 30 min, followed by incubation with 2 µM Cd for 12 h. (C and D) The amount of phosphorylated protein was quantified by densitometry and corrected for sample loading based on the density of the β-actin band. The results are expressed as the fold changes relative to the control lane. Each blot is representative of at least three replicate experiments. ap < 0.05, bp < 0.01 relative to the control and cp < 0.01 relative to the Cd-treated group.

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