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Carpi, Donatella
(2009).
DOI: https://doi.org/10.21954/ou.ro.0000f276
Abstract
2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) is an endocrine disrupting environmental pollutant that affects bone tissue, although the mechanistic basis of such effect is far from clear.
In this study a proteomic approach has been adopted to investigate the disturbance of the osteogenic process evoked by TCDD in an in vitro osteoblast differentiation model of rat mesenchymal stem cells. Stem cells were isolated from bone marrow of femurs and tibias of rats. Progress of osteoblastic differentiation was monitored by measuring mRNA expression levels of differentiation markers from control and TCDD-treated cells after 3, 7 and 10 days of culture in presence and absence of TCDD using quantitative RT-PCR.
Analysis of differential protein expression in the total cell lysate of untreated and TCDD-treated cells was carried out over the same time points, using two-dimensional gel electrophoresis, computerized gel image, univariate and multivariate statistical analysis, in-gel digestion and tandem mass spectrometry for protein identification. Expression of all measured markers characteristic for various stages of osteoblastic differentiation (Runx2, alkaline phosphatase and osteocalcin) was dramatically reduced only after 10 days of TCDD exposure, indicating that TCDD significantly inhibits osteoblast differentiation in vitro.
Similarly, the most significant rearrangement of the proteome during osteoblast differentiation was observed following the 10 days of TCDD exposure, at which time the full progress of osteoblast differentiation should have occurred. Overall, 18 individual proteins showed a statistically significant change in abundance as a result of 10 days of TCDD exposure.
These proteins were mostly involved in cytoskeleton organization and biogenesis, actin filament-based processes, protein transport and folding. The alteration in cell architecture and increase in cell adhesion were confirmed by confocal microscopy. The TCDD-induced decrease in the expression of calcium binding proteins may interfere with osteoblast calcium deposition, which was in fact reduced by TCDD. Evidence from proteomics and Western blot analysis indicated a decreased apoptotic capability in TCDD-treated osteoblasts.
This is the first study investigating, at the protein expression level, the effect evoked by TCDD during osteoblastic differentiation. Interestingly, MetaCore pathway analysis grouped the majority of these proteins around two principal nodes (c-fos and c-myc) suggesting that they may participate in the transcriptional activation of key pathways in TCDD-driven inhibition of osteoblast differentiation. These findings provide clues to the complex interplay between TCDD's action and the regulatory molecules that alter signal-transduction pathways regulating osteoblast differentiation and indicate new molecular players in the effects of TCDD on bone development.
CORE (COnnecting REpositories)
CORE (COnnecting REpositories)
