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A possible mechanism of low molecular weight protein tyrosine phosphatase (LMW-PTP) activity modulation by glutathione action during human osteoblast differentiation |
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| Authors: | Tatiana Salles de Souza Malaspina,Cé lio Xavier dos Santos,Francisco Rafael Martins Laurindo,José Mauro Granjeiro |
| Affiliation: | a Department of Biological Sciences, Bauru Dental School, University of São Paulo, Bauru, SP, Brazil b Department of Biochemistry, Biology Institute, University of Campinas (UNICAMP), Campinas, SP, Brazil c Heart Institute (INCOR), University of São Paulo, São Paulo, SP, Brazil d Chemistry Institute, University of São Paulo, São Paulo, SP, Brazil e Biology Institute, Fluminense Federal University, Niterói, RJ, Brazil |
| Abstract: | ObjectiveLow molecular weight protein tyrosine phosphatases (LMW-PTPs) are a family of enzymes strongly involved in the regulation of cell growth and differentiation. Since there is no information concerning the relationship between osteoblastic differentiation and LMW-PTP expression/activity, we investigated its involvement during human osteoblast-like cells (hFOB 1.19) differentiation. It is known that LMW-PTP is regulated by an elegant redox mechanism, so we also observed how the osteoblastic differentiation affected the reduced glutathione levels.DesignhFOB 1.19 cells were cultured in DMEM/F12 up to 35 days. The osteoblast phenotype acquisition was monitored by measuring alkaline phosphatase activity and mineralized nodule formation by Von Kossa staining. LMW-PTP activity and expression were measured using the p-nitrophenylphosphate as substrate and Western blotting respectively. Crystal violet assay determined the cell number in each experimental point. Glutathione level was determined by both HPLC and DNTB assays.ResultsLMW-PTP modulation was coincident with the osteoblastic differentiation biomarkers, such as alkaline phosphatase activity and presence of nodules of mineralization in vitro. Likewise LMW-PTP, the reduced glutathione-dependent microenvironment was modulated during osteoblastic differentiation. During this process, LMW-PTP expression/activity, as well as alkaline phosphatase and glutathione increased progressively up to the 21st day (p < 0.001) of culturing, decreasing thereafter.ConclusionsOur results clearly suggest that LMW-PTP expression/activity was rigorously modulated during osteoblastic differentiation, possibly in response to the redox status of the cells, since it seems to depend on suitable levels of reduced glutathione. In this way, we pointed out LMW-PTP as an important signaling molecule in osteoblast biology and bone formation. |
| Keywords: | LMW-PTP, low molecular weight protein tyrosine phosphatase GSH and GSSG, reduced glutathione and its oxidized form HPLC, high performance (pressure) liquid chromatograph ALP, alkaline phosphatase PBS, phosphate-buffered saline p-NPP, p-nitrophenylphosphate FBS, fetal bovine serum DTT, dithiothreitol SDS, sodium dodecyl sulfate PMSF, phenyl-methyl sulphonyl fluoride-serine-protease enzyme inhibitor PVDF, polyvinylidene fluoride ATCC, American Type Culture Collection TBS, Tris-buffered saline ECL, enhanced chemiluminescence ROS, reactive oxygen species |
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