title: Osteoblast adhesion dynamics: a possible role for ROS and LMW-PTP.
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.
authors.affiliation: Antonio Pedro University Hospital, Fluminense Federal University, Niterói, RJ, Brazil.
authors.forename: Gustavo V O
authors.initials: GV
authors.lastname: Fernandes
authors.forename: Alexandre D M
authors.initials: AD
authors.lastname: Cavagis
authors.forename: Carmen V
authors.initials: CV
authors.lastname: Ferreira
authors.forename: Beni
authors.initials: B
authors.lastname: Olej
authors.forename: Maurício de Souza
authors.initials: Mde S
authors.lastname: Leão
authors.forename: Cláudia L
authors.initials: CL
authors.lastname: Yano
authors.forename: Maikel
authors.initials: M
authors.lastname: Peppelenbosch
authors.forename: José Mauro
authors.initials: JM
authors.lastname: Granjeiro
authors.forename: Willian F
authors.initials: WF
authors.lastname: Zambuzzi
mesh_terms: D000818:Animals; D002374:Catalase / Q000378:metabolism; D002448:Cell Adhesion; D002460:Cell Line; D005434:Flow Cytometry; D051417:Focal Adhesion Kinase 1 / Q000378:metabolism; D006861:Hydrogen Peroxide / Q000378:metabolism; D015151:Immunoblotting; D007700:Kinetics; D051379:Mice; D018613:Microscopy, Confocal; D010006:Osteoblasts / Q000166:cytology / Q000378:metabolism*; D010766:Phosphorylation; D017027:Protein Tyrosine Phosphatases / Q000235:genetics / Q000378:metabolism*; D011518:Proto-Oncogene Proteins / Q000235:genetics / Q000378:metabolism*; D016392:Proto-Oncogene Proteins pp60(c-src) / Q000378:metabolism; D034622:RNA Interference; D017382:Reactive Oxygen Species / Q000378:metabolism*; D013482:Superoxide Dismutase / Q000378:metabolism; D013997:Time Factors; D014443:Tyrosine / Q000378:metabolism
chemical_list: D011518:Proto-Oncogene Proteins; D017382:Reactive Oxygen Species; D014443:Tyrosine; D006861:Hydrogen Peroxide; D002374:Catalase; D013482:Superoxide Dismutase; D051417:Focal Adhesion Kinase 1; D016392:Proto-Oncogene Proteins pp60(c-src); C496051:Ptk2 protein, mouse; C490428:Acp1 protein, mouse; D017027:Protein Tyrosine Phosphatases
keywords: ADHESION; FAK; LMW-PTP; OSTEOBLAST; REDOX; ROS