| Lead Induces Chondrogenesis and Alters Transforming Growth Factor-β and Bone Morphogenetic Protein Signaling in Mesenchymal Cell Populations Michael J. Zuscik,* Lin Ma,* Taylor Buckley, J. Edward Puzas, Hicham Drissi, Edward M. Schwarz, and Regis J. O'Keefe Center for Musculoskeletal Research, University of Rochester School of Medicine and Dentistry, Rochester, New York, USA Abstract
Background: It has been established that skeletal growth is stunted in lead-exposed children. Because chondrogenesis is a seminal step during skeletal development, elucidating the impact of Pb on this process is the first step toward understanding the mechanism of Pb toxicity in the skeleton. Objectives: The aim of this study was to test the hypothesis that Pb alters chondrogenic commitment of mesenchymal cells and to assess the effects of Pb on various signaling pathways. Methods: We assessed the influence of Pb on chondrogenesis in murine limb bud mesenchymal cells (MSCs) using nodule formation assays and gene analyses. The effects of Pb on transforming growth factor-β (TGF-β) and bone morphogenetic protein (BMP) signaling was studied using luciferase-based reporters and Western analyses, and luciferase-based assays were used to study cyclic adenosine monophosphate response element binding protein (CREB) , β-catenin, AP-1, and nuclear factor-kappa B (NF-κB) signaling. We also used an ectopic bone formation assay to determine how Pb affects chondrogenesis in vivo. Results: Pb-exposed MSCs showed enhanced basal and TGF-β/BMP induction of chondrogenesis, evidenced by enhanced nodule formation and up-regulation of Sox-9, type 2 collagen, and aggrecan, all key markers of chondrogenesis. We observed enhanced chondrogenesis during ectopic bone formation in mice preexposed to Pb via drinking water. In MSCs, Pb enhanced TGF-β but inhibited BMP-2 signaling, as measured by luciferase reporter assays and Western analyses of Smad phosphorylation. Although Pb had no effect on basal CREB or Wnt/β-catenin pathway activity, it induced NFκB signaling and inhibited AP-1 signaling. Conclusions: The in vitro and in vivo induction of chondrogenesis by Pb likely involves modulation and integration of multiple signaling pathways including TGF-β, BMP, AP-1, and NFκB. Key words: BMP signaling, chondrogenesis, lead, mesenchymal stem cells, TGF-β signaling. Environ Health Perspect 115:1276–1282 (2007) . doi:10.1289/ehp.10028 available via http://dx.doi.org/ [Online 3 July 2007]
Address correspondence to R.J. O'Keefe, Center for Musculoskeletal Research, University of Rochester Medical Center, 601 Elmwood Ave., Box 665, Rochester, NY 14642 USA. Telephone: (585) 273-1261. Fax: (585) 275-1121. E-mail: regis_okeefe@urmc.rochester.edu *These authors contributed equally to this work. This work was supported by grant PO1 ES011854 from the National Institutes of Health (J.E.P. and R.J.O.) . The authors declare they have no competing financial interests. Received 21 December 2006 ; accepted 2 July 2007.
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