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Poster No. 885 • 54th Annual Meeting of the Orthopaedic Research Society ENVIRONMENTAL TOXICANTS SUCH AS LEAD MAY INHIBIT BONE FORMATION THROUGH A SCLEROSTIN DEPEN- DENT MECHANISM Resika Ubayawardena, Debbie Dao, Taylor Buckley, Edward J. Puzas Orthopaedic, University of Rochester, School of Medicine and Dentistry, Rochester, NY [email protected] Introduction: Lead is an environmental toxicant that was used in the manu- facture of paints, gasoline and plumbing materials for decades. Prolonged exposure to this heavy metal has a major impact on the health of both adults and children. Especially in children, this is known to have an adverse effect on skeletal metabo- lism . At the cellular level, lead adversely affects osteoblastic bone formation, ulti- mately leading to a decrease in bone density and bone strength [1]. In order to define a mechanism for this effect we investigated the effects of lead on sclerostin gene and protein expression. We found that lead significantly increases the levels of both. Sclerostin is a secretory glycoprotein and a product of the SOST gene [2]. It was first detected in osteocytes and found to be widespread in the lacuno- canalicular network [3]. Previous studies have shown that sclerostin negatively reg- ulates the formation of bone by repressing the differentiation and/or function of osteoblasts through several mechanisms [4,5,6]. Thus, in this study we hypothesize that one mechanism by which lead represses the formation of new bone is by up regulation of sclerostin in osteoblasts. Materials and Methods: Primary osteoblasts were isolated from rat calvaria and used to evaluate markers of bone formation (i.e. alkaline phosphatase, type 1 colla- gen, osteocalcin) and sclerostin expression [7]. Cells were exposed to lead for 24- 48 hours. The in vivo effects of lead were also determined using immunohisto- chemistry in 8 week old mice (C57/B6). The mice were exposed to lead in the drinking water at 2 different concentrations (0 and 230 ppm) for a period of 8 weeks [8]. Results: A 24-h and 48-h lead exposure results in decreased alkaline phos- phatase activity in osteoblasts in a dose dependent manner. Also, after 24 hours of lead exposure alkaline phosphatase, type 1 collagen and osteocalcin mRNA levels are significantly depressed. These effects are paralleled in both time and dose by a significant increase in sclerostin mRNA and protein levels. Additionally, immunohistochemical evaluation of osteoblasts, osteo- cytes and subchondral articular chondrocytes in mice exposed to lead showed a strong up regulation of sclerostin protein expression after 8 weeks. (See Figure 1) Discussion: Our investigations into the effects of lead exposure on bone for- mation have identified a number of potential pathways by which lead may have an adverse effect on the skeleton. The data presented in this abstract show that yet another mechanism may be operant for the toxicological manifestations of this heavy metal. Lead, at physiologically relevant concentrations, induces sclerostin gene and protein levels in osteoblasts, osteocytes and some chondrocytes. Sclerostin is a potent inhibitor of bone formation that is thought to work, in part, by blocking signaling through the beta-catenin pathway. These results indicate that the adverse effects of lead on bone formation are not due to a toxic effect on cell viability, but rather may relate to the induction of a specific inhibitor of osteoblast function. References: 1. Holz J et al, Birth defects Research 2007, 81, 41-50; 2. Brunkow M et al, 2001, Am. J. Hum. Genet., 68:577-589; 3. Kenneth E. S. Poole FASEB J., 2005 Nov;19(13):1842-4; 4. Winkler D et.al The EMBO, 2003, 22(23):6267- 6276; 5. Naoki Kusu et.al JBC, 2003, 278(26):24113-24117; 6. Xiaofeng Li et.al, JBC, 2005, 280(20):19883-19887; 7. Zuscik MJ et al J Cell Biochem., 2004 May 15, 92(2):316-331; 8. Carmouche et al Environ Health Perspect., 2005 June;113(6):749. Acknowledgements: Dr TJ. Sheu, D. Metz and Dr V. Jamburuthugoda are gratefully acknowledged for their invaluable assistance. Supported by NIH grant P01 ES 11854. Figure 1. Lead in the drinking water increases the expression of sclerostin protein in osteocytes, osteoblasts and sub-chondral chondrocytes. A & B; Control animals. C&D; Animals consum- ing water at 230 ppm lead. (Magnification :20x)
