The Role Of CaMKⅡ In Cadmium-induced Endoplasmic Reticulum Apoptosis Pathway In Rat Osteoblasts

Cadmium, a ubiquitous heavy metal pollutants, enters cells through calcium ion channels or ion channel type receptor in the form of Cd2+, and then affects the distribution of intracellular calcium ion ([Ca2+]i). The change of [Ca2+]i concentration would alter the expression of CaMKⅡ and cause endoplasmic reticulum (ER) stress response. However, there is few study focusing on the role of CaMK II in osteoblast apoptosis and ER stress. In order to study the effects of cadmium-induced calcium homeostasis imbalance with ER stress, after rat primary osteoblasts exposed to cadmium or 2-APB, an ER calcium channel inhibitor, [Ca2+]i concentration was measured via flow cytometry, cell survival was detected by RTCA and Western Blot was performed for detection of UPR related protein expression. The results showed that after osteoblasts exposed to 2 μmol/L cadmium acetate,50 μmol/L 2-APB alone or co-treatment, compared with control group, cadmium treatment resulted in significantly decreased cell survival accompanied by significantly increased [Ca2+]i concentration (P<0.01); The [Ca2+]i concentration of osteoblasts in co-treatment group decreased significantly (P<0.01) when compared with cadmium treatment group; After osteoblasts exposed to different concentrations of cadmium (0,0.25,0.5,1,2,5,10 mol/L) for 6 h or 2 mol/L cadmium acetate for different time (0,0.5,1,5,10,2, or 30 min 4,6 h), compared with the control group,2 umol/L cadmium acetate treatment for 6 hours significantly caused the increased expression level of BiP, ATF4 and CHOP three proteins (P<0.05 or P<0.01). In order to study the impacts of cadmium on CaMK II expression in osteoblasts and the role of CaMK II in cadmium inducing ER stress, after osteoblasts exposed to cadmium,2-APB or KN93, a CaMKII inhibitor, cell survival was detected by RTCA, Western Blot was performed for detection of CaMK II, P-CaMKⅡ and UPR associated protein expression. Results showed that the phosphorylation level of CaMK Ⅱ in osteoblasts exposed to 2μmol/L cadmium acetate for 6 h was increased significantly, compared with the control group; the phosphorylation level of CaMK II in osteoblasts exposed to 2-APB with cadmium was significantly decreased, compared with the cadmium group and co-treatment with KN93 and cadmium significantly increased the cell survival and significantly reduced the ATF4 and CHOP protein expression (P<0.05 or P<0.01). In order to study the role of ER stress and CaMK II in cadmium-induced osteoblasts apoptosis, after osteoblasts exposed to cadmium,2-APB or KN93, the cell apoptosis was detected by flow cytometry, the bisbenzimide (Hoechst 33258) staining was performed for observation of nuclear morphology and Western blot for detection of caspase-12,3, Bax and bcl-2 protein expression. Results showed that after osteoblasts exposed to 2 μmol/L cadmium for 6 h, cell apoptosis rate, caspase-12,3 protein activation and Bax/Bcl-2 ratio was significantly higher than that of control group (P< 0.01); co-treatment with cadmium and 2-APB or KN93 caused significant decreases in the apoptosis rate, caspase-12,3 protein activation and Bax/Bcl-2 ratio when compared with cadmium group (P<0.01); result of Hoechst 33258 staining were consistent with showed above. In summary, cadmium could cause cytosolic calcium overload and induction of ER stress through inducing ER releasing calcium ions and finally result in rat primary osteoblasts apoptosis through the caspase-12,3 and BCL-2 family protein pathways; furthermore CaMK Ⅱ was involved in this process with its important role.