A possible mechanism of low molecular weight protein tyrosine phosphatase (LMW-PTP) activity modulation by glutathione action during human osteoblast differentiation.
OBJECTIVE
Low 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.


METHODS
hFOB 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.


RESULTS
LMW-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.


CONCLUSIONS
Our 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.
Department of Biological Sciences, Bauru Dental School, University of São Paulo, Bauru, SP, Brazil.
Tatiana Salles
TS
de Souza Malaspina
Willian Fernando
WF
Zambuzzi
Célio Xavier
CX
dos Santos
Ana Paula
AP
Campanelli
Francisco Rafael Martins
FR
Laurindo
Mari Cleide
MC
Sogayar
José Mauro
JM
Granjeiro
D000469:Alkaline Phosphatase / Q000032:analysis; D015415:Biomarkers / Q000032:analysis; D015153:Blotting, Western; D002113:Calcification, Physiologic / Q000502:physiology; D002130:Calcium Phosphates / Q000032:analysis; D002452:Cell Count; D002454:Cell Differentiation / Q000502:physiology; D002460:Cell Line; D049109:Cell Proliferation; D002851:Chromatography, High Pressure Liquid; D004136:Dinitrobenzenes; D005978:Glutathione / Q000032:analysis / Q000378:metabolism*; D006241:Haptens; D006801:Humans; D009596:Nitrophenols; D009943:Organophosphorus Compounds; D010006:Osteoblasts / Q000502:physiology*; D010084:Oxidation-Reduction; D018384:Oxidative Stress / Q000502:physiology; D010641:Phenotype; D017027:Protein Tyrosine Phosphatases / Q000032:analysis / Q000378:metabolism*; D011518:Proto-Oncogene Proteins / Q000032:analysis / Q000378:metabolism*; D013997:Time Factors
D015415:Biomarkers; D002130:Calcium Phosphates; D004136:Dinitrobenzenes; D006241:Haptens; D009596:Nitrophenols; D009943:Organophosphorus Compounds; D011518:Proto-Oncogene Proteins; C008644:nitrophenylphosphate; D000469:Alkaline Phosphatase; C490429:ACP1 protein, human; D017027:Protein Tyrosine Phosphatases; D005978:Glutathione; C010936:2,4-dinitrothiocyanatobenzene