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