Lead Inhibits Skeletal Wnt Signaling and Manifests in Osteoporosis

Abstract

Thesis (Ph.D.)--University of Rochester. School of Medicine & Dentistry. Dept. of Environmental Medicine & Toxicology, 2013.Purpose Exposure to lead (Pb) from environmental sources remains an overlooked, but serious public health risk. We believe that intoxication from Pb, starting during childhood, results in decreased attainment of peak bone mass and disruption of bone homeostasis, and thus disposes an individual to osteoporosis. Elucidating the effect of various doses and duration of Pb exposure and subsequent mechanisms of Pb on bone processes (formation, resorption) is therefore critical for understanding risk of bonerelated diseases. We may have found a novel target of Pb toxicity in osteoblasts, the cells responsible for bone formation. We hypothesize that suppression of Wnt signaling in osteoblasts by sclerostin (Scl) is a major component to the Pb-induced decrease in bone mass. Methods We used rodent models to mimic plausible Pb exposures and determine its effect on the growing and adult skeleton. First, we used a model of low-level Pb exposure in Long-Evans rats over a lifespan of 18 months. Second, we used low, moderate, and high Pb exposure in C57BL/6 mice and determined bone quality over 13 months. Lastly, we used a mouse devoid of the sclerostin gene (SOST-/-), treated them with Pb, and conducted tests to measure bone quantity, bone quality, and bone strength. Histological assessment was performed to delineate the effect of Pb on various bone processes (formation, resorption, or fatty marrow accrual) and changes in signaling pathway components. We then used Wnt pathway reporter mice (TOPgal) to assess β-catenin signaling in the context of Pb using a luminescent substrate and X-gal staining of the β- galactosidase protein. Next, we injected mice with PTH to induce ectopic bone formation in the calvaria to assay the influence of Pb on new bone growth. Finally we used various cell models to elucidate the direct effect of Pb on osteoblastogenesis, adipogenesis, and osteoclast activity. We did this using Western blotting, quantitative PCR, cellular staining, and luciferase reporter assays. Results Using DXA and micro-CT, we found that low-level Pb decreased bone mass in rats and mice. It also resulted in bones that were more susceptible to tensile and compression forces. Pb decreased osteoblastic cell numbers and osteoblast activity with no change in osteoclasts, with an accompanying elevation in Scl protein levels in the skeleton, and correspondingly reduced levels of β-catenin and Runx2 in stromal precursor cells. Pb also increased expression of peroxisome proliferator-activated receptor-γ (PPAR-γ). These findings were substantiated in vitro, where Pb directly inhibited bone nodule formation and enhanced lipid formation. High-level Pb, however, had the opposite phenotype, increased bone mass that was more resistant to flexure testing. Osteoclast numbers were increased, however, osteoclast activity was significantly reduced. In vitro, high-level Pb osteoclast activity was decreased when added in the media or within bone wafers. We found that depression of Wnt signaling was associated with increased Scl levels in vitro and in vivo. Activation of β-catenin downstream showed amelioration of Pb-inhibition in signaling activity in the TOPgal mouse. Ultimately, removal of the sclerostin gene did not prevent against Pb-induced trabecular bone loss in long bones, but did in vertebral bone. Bone strength was ameliorated with increased Wnt signaling. Bone marrow cells from the KO mouse showed improved bone formation potential and colony formation under exposure of Pb. Conclusions Low-level Pb decreased bone mass, which was found most significant during the first 3 months of life. Pb was able to disrupt mesenchymal cell signaling, by inhibiting bone formation and promoting adipogenesis, which can be explained by depression of the Wnt/β-catenin pathway. These findings also indicate that Pb inhibition of Wnt signaling is upstream of β-catenin activation. The increased bone mass caused by gain-of-function Wnt signaling (SOST-/-) was partially blunted by Pb exposure. This reveals that SOST may not be the only target of Pb effects on the Wnt signaling pathway, yet removal of SOST did improve the Pb-induced skeletal deficits in bone quality. In all, these findings may provide a molecular basis for novel therapeutic strategies to combat Pb-induced bone pathologies