Gap junction blockage promotes cadmium-induced apoptosis in BRL 3A derived from Buffalo rat liver cells

Gap junctions mediate direct communication between cells; however, toxicological cascade triggered by nonessential metals can abrogate cellular signaling mediated by gap junctions. Although cadmium (Cd) is known to induce apoptosis in organs and tissues, the mechanisms that underlie gap junction activity in Cd-induced apoptosis in BRL 3A rat liver cells has yet to be established. In this study, we showed that Cd treatment decreased the cell index (a measure of cellular electrical impedance) in BRL 3A cells. Mechanistically, we found that Cd exposure decreased expression of connexin 43 (Cx43), increased expression of p-Cx43 and elevated intracellular free Ca2+ concentration, corresponding to a decrease in gap junctional intercellular communication. Gap junction blockage pretreatment with 18β-glycyrrhizic acid (GA) promoted Cd-induced apoptosis, involving changes in expression of Bax, Bcl-2, caspase-3 and the mitochondrial transmembrane electrical potential (Δψm). Additionally, GA was found to enhance ERK and p38 activation during Cd-induced activation of mitogen-activated protein kinases, but had no significant effect on JNK activation. Our results indicated the apoptosis-related proteins and the ERK and p38 signaling pathways may participate in gap junction blockage promoting Cd-induced apoptosis in BRL 3A cells.

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

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.

Involvement of the Ca2+ signaling pathway in osteoprotegerin inhibition of osteoclast differentiation and maturation

The purpose of this study was to determine whether the Ca2+ signaling pathway is involved in the ability of osteoprotegerin (OPG) to inhibit osteoclast differentiation and maturation. RAW264.7 cells were incubated with macrophage colony-stimulating factor (M-CSF) + receptor activator of nuclear factor-kappaB ligand (RANKL) to stimulate osteoclastogenesis and then treated with different concentrations of OPG, an inhibitor of osteoclast differentiation. The intracellular Ca2+ concentration [Ca2+]i and phosphorylation of Ca2+/calmodulin-dependent protein kinase II (CaMKII) in the different treatment groups were measured by flow cytometry and Western blotting, respectively. The results confirmed that M-CSF + RANKL significantly increased [Ca2+]i and CaMKII phosphorylation in osteoclasts (p < 0.01), and that these effects were subsequently decreased by OPG treatment. Exposure to specific inhibitors of the Ca2+ signaling pathway revealed that these changes varied between the different OPG treatment groups. Findings from the present study indicated that the Ca2+ signaling pathway is involved in both the regulation of osteoclastogenesis as well as inhibition of osteoclast differentiation and activation by OPG.

Oxidative stress and mitogen-activated protein kinase pathways involved in cadmium-induced BRL 3A cell apoptosis.

In this study, BRL 3A cells were treated with different Cd concentrations (0, 10, 20, and 40 µmol/L) for 12 h and preincubated with or without N-acetyl-L-cysteine (NAC) (2 mmol/L) for 30 min, and cells were treated with Cd (0 and 20 µmol/L), pretreated with p38 inhibitor (SB203580), JNK (c-Jun NH2-terminal kinases) inhibitor (SP600125), and extracellular signal-regulated kinase (ERK) inhibitor (U0126) for 30 min, and then treated with 20 µmol/L Cd for 12 h. Cd decreased cell viability, SOD, and GSH-Px activity in a concentration-dependent manner. Increased MDA level, ROS generation, nuclear condensation, shrinkage, and fragmentation in cell morphology were inhibited by NAC. Cd-induced apoptosis was attenuated by pretreatment with SB203580, SP600125, and U0126. The results of western blot showed that NAC preincubation affected Cd-activated MAPK pathways, p38 and ERK phosphorylation. Cd treatment elevated the mRNA levels of Bax and decreased the mRNA levels of Bcl-2, respectively. The same effect was found in their protein expression levels. These results suggest that oxidative stress and MAPK pathways participate in Cd-induced apoptosis and that the balance between pro- and antiapoptotic genes (Bax and Bcl-2) is important in Cd-induced apoptosis.

EFFECT OF 1?,25-DIHYDROXYVITAMIN D_3 ON CYTOSKELETON,GAP JUNCTION INTERCELLULAR COMMUNICATION AND [Ca~(2+)]_i OF OSTEOBLASTS / 营养学报

Objective To study the effect of 1?,25-dihydroxyvitamin D3[1,25-(OH)2D3] on cytoskeleton,gap junction intercellular communication (GJIC) and intracellular Ca2+ ([Ca2+]i) in osteoblasts (OB) in vitro. Method OB were isolated from calvaria bone. After 20 min and after 24 h treated by 1,25-(OH)2 VD3 (0,10-9,10-8,10-7 mol/L),[Ca2+]i was evaluated. F-actin and GJIC were observed after 24 and 48 h incubation later. Results Compared with the control group,[Ca2+]i in all 1,25-(OH)2 D3 groups was increased significantly at 20 min. [Ca2+]i in 10-9 mol/L 1,25-(OH)2D3 group was the lowest at 24 h after treatment. OB in 10-8 and 10-7 mol/L 1,25-(OH)2D3 groups were flat,and stress fibers were formed. The expression of F-actin in control group and 10-9 mol/L 1,25-(OH)2D3 group was reduced at 48 h after treatment. Compared with the control group,GJIC was weakened very significantly after treated with 10-9 mol/L 1,25-(OH)2D3 at 48 h,but enhanced very significantly in the group with 10-8 and 10-7 mol/L. Conclusion Higher dosage of 1,25-(OH)2D3 can maintain the morphology of OB and stimulate the communication among OB,but lower dosage can inhibit it.