Osteoblast Adhesion Dynamics: A Possible Role for ROS and LMW-PTP

• Published in: Journal of cellular biochemistry 2014-06, Vol.115 (6), p.1063-1069
• Main Authors: Fernandes, Gustavo V. O., Cavagis, Alexandre D. M., Ferreira, Carmen V., Olej, Beni, de Souza Leão, Maurício, Yano, Cláudia L., Peppelenbosch, Maikel, Granjeiro, José Mauro, Zambuzzi, Willian F.
• Format: Article
• Language: English
• Subjects: ADHESION; Animals; Catalase - metabolism; Cell Adhesion; Cell Line; FAK; Flow Cytometry; Focal Adhesion Kinase 1 - metabolism; Hydrogen Peroxide - metabolism; Immunoblotting; Kinetics; LMW-PTP; Mice; Microscopy, Confocal; OSTEOBLAST; Osteoblasts - cytology; Osteoblasts - metabolism; Phosphorylation; Protein Tyrosine Phosphatases - genetics; Protein Tyrosine Phosphatases - metabolism; Proto-Oncogene Proteins - genetics; Proto-Oncogene Proteins - metabolism; Proto-Oncogene Proteins pp60(c-src) - metabolism; Reactive Oxygen Species - metabolism; REDOX; RNA Interference; ROS; Superoxide Dismutase - metabolism; Time Factors; Tyrosine - metabolism
• ISSN: 0730-2312; 1097-4644
• DOI: 10.1002/jcb.24691

ABSTRACT Reactive oxygen species (ROS) modulate a variety of intracellular events, but their role in osteoblast adhesion and spreading remains unclear. ROS is a very‐known physiological modulators of Protein Tyrosine Phosphatases activities, mainly to low molecular weight protein tyrosine phosphatase (LMW‐PTP) activity. As this biological mechanism is not clear in osteoblast adhesion, we decided to investigate ROS levels and phosphorylations of FAK and Src, identifying these proteins as potential substrates to LMW‐PTP activity. Our results showed that during osteoblast adhesion/spreading (30 min and 2 h of seeding) the intracellular ROS content (hydrogen peroxide) is finely regulated by an effective anti‐oxidant system [catalase and Superoxide Dismutase (SOD) activities were evaluated]. During the first 30 min of adhesion, there was an increase in ROS production and a concomitant increase in focal adhesion kinase (FAK) activity after its phosphorylation at Tyrosine 397 (Y397). Moreover, after 2 h there was a decrease in ROS content and FAK phosphorylation. There was no significant change in LMW‐PTP expression at 30 min or 2 h. In order to validate our hypothesis that LMW‐PTP is able to control FAK activity by modulating its phosphorylation status, we decided to overexpress and silence LMW‐PTP in this context. Our results showed that FAK phosphorylation at Y397 was increased and decreased in osteoblasts with silenced or overexpressed LMW‐PTP, respectively. Together, these data show that ROS modulate FAK phosphorylation by an indirect way, suggesting that a LMW‐PTP/FAK supra‐molecular complex is involved in transient responses during osteoblast adhesion and spreading. J. Cell. Biochem. 115: 1063–1069, 2014. © 2013 Wiley Periodicals, Inc.