Investigating the role of Ca+2/calmodulin dependent kinase pathways in osteoblasts exposed to cadmium Jacob A. Noeker, Matthew L. Goodwin, Thao T. Ha, Sara J. Heggland Department of Biology, The College of Idaho, Caldwell, Idaho 83605 CELL VIABILITY RESULTS ABSTRACT Cadmium, a heavy metal and known environmental contaminant, is linked to impaired human bone health. While bone is a target site for cadmium, the mechanism by which cadmium induces osteotoxicity is unclear. Our preliminary results demonstrate the pleotropic role of Ca+2/calmodulin dependent kinase (CAMK) pathways in cadmium-induced osteotoxicity within bone-forming osteoblasts. One possible regulatory target of the CAMK pathways is alkaline phosphatase (ALP), which is a known osteoblast marker. We hypothesize that exposure to cadmium inhibits ALP via CAMK pathways. MG-63 and Saos-2 cells were treated with 5μM CdCl2 alone or in co-treatment with 5 μM Calmodulin-dependent Phosphodiesterase (PDE) inhibitor CGS-9343β or 2.5 μM Calmodulin-dependent Kinase II (CAMKII) inhibitor KN-93 for 48 hours. MTT results showed that treatment with CGS-9343β protected against cadmium-induced toxicity, whereas KN-93 had no detectable effect on cadmium-induced toxicity. In contrast to the MTT results, co-treatment with CGS resulted in no effect on ALP activity whereas inhibition of the CAMKII pathway by KN-93 resulted in cadmium-induced decrease in ALP activity. Collectively, these results demonstrate a pleiotropic role for the CAMK pathways in cadmium-induced osteotoxicity. This research will help elucidate the mechanisms in which exposure to cadmium leads to the pathogenesis of bone diseases. ALP RESULTS Saos-2 MG-63 . * * Table 1. First ALP activity optimization experiment. Results: Saos-2 had more ALP activity compared to MG-63 cells. Further cell line specific assay optimization was required. * * Saos-2 MG-63 BACKGROUND Improperly disposed electronic waste is a growing issue throughout the world, causing serious harm to the environment and human health. Some of this toxicity is a result of heavy metals, such as cadmium, which is found  to have negative effects on the human body. Humans are exposed to cadmium primarily through consumption of contaminated water or food and inhalation of cigarette smoke. Once inside the body, cadmium is linked to the development of several degenerative bone diseases including osteoporosis. Compromised bone health may result from cadmium disrupting normal calcium-signaling in bone-forming  osteoblasts. The Ca+2/calmodulin dependent kinase (CAMK) pathways are one such mechanism we propose to be involved in cadmium-induced osteotoxicity. This is because cadmium has similar elemental properties as calcium, such as their divalent nature, which leads cadmium to disrupt calcium signaling. Although other studies identify CAMKII involvement in cadmium-induced toxicity, this study is unique in that it examines the pleiotropic role of multiple cadmium-dependent pathways. Figure 1. The effect of CdCl2 on cell viability using the MTT assay. Saos-2 or MG-63 cells were treated with 5µM CdCl2alone or in combination with 5µM CGS, 10µM STO, or 2.5µM KN-93 for 48 hrs.   Results: Cadmium reduced cell viability in both cell lines. Co-treatment with CGS resulted in partial recovery of cell viability, while co-treatment with STO exacerbated cadmium toxicity. Cadmium toxicity remained unchanged with KN-93 co-treatment. These results demonstrate that PDE promotes cadmium toxicity, CAMKK protects against toxicity, whereas CAMKII may not be involved in cadmium toxicity as it relates to cell viability. Next we chose to study the effect of PDE and CAMKII pathways on a specific bone marker, ALP. *Data still being collected for Saos-2 CAMKII pathway HYPOTHESIS We hypothesize that exposure to cadmium inhibits ALP via CAMK pathways. Figure 3. The effect of CdCl2 on ALP activity. Saos-2 or MG-63 cells were treated with 5µM CdCl2 alone or in combination with 5µM CGS, or 2.5µM KN-93 for 48 hrs. WORKING MODEL Results: Preliminary data indicates cadmium decreases ALP activity which does not involve the PDE pathway. In contrast CAMKII protects against cadmium’s inhibitory effect on ALP activity. METHODS Cell Culture and Treatment: MG-63 cells were plated in Eagles MEM medium while Saos-2 cells were plated in McCoy’s 5A medium, each supplemented with 10% FBS. After 24 hrs, treatment was initiated in Opti-MEM serum-free medium containing 5µM CdCl2 for 24 or 48 hrs. Inhibitor co-treatment studies for PDE, CaMKK, and CaMKII were completed using 5µM CGS, 5µM STO, or 2.5µM KN-93 respectively in Opti-MEM serum-free medium. Controls received 0.05% DMSO. Methylthiazol Tetrazolium (MTT) Assay: Cell viability was analyzed using the ATCC MTT Cell Proliferation Assay Kit and absorbance was read at 570 nm. Alkaline-phosphatase Assay: Cells were lysed using 1% Triton-X100 solution followed by two freeze/thaw cycles. Total protein determined with a Bradford assay. Assay conditions were optimized for substrate concentration, incubation and freeze/thaw time and cell density. Absorbance was read at 405 nm after 20 minute exposure to p-Nitrophenyl Phosphate substrate.     Statistical Analysis: Results are expressed as % control ± % SEM for the MTT. Significant differences from cadmium treatment are denoted with (*) for p < 0.05. MAJOR CONCLUSIONS CAMK pathways play a pleiotropic role in cadmium toxicity in bone: PDE activation facilitates cadmium-induced osteotoxicity whereas CAMKII appears not involved. CAMKII protects against cadmium-induced decrease in ALP activity whereas PDE appears not involved. This work contributes to the understanding of how cadmium exposure contributes to bone disease. ACKNOWLEDGEMENTS The project described was supported by an Institutional Development Award (IDeA) from the National Institute of General Medical Sciences of the National Institutes of Health under Grant #P20GM103408. Figure 2. Working model for cadmium-induced osteotoxicity and ALP activity.