Protein nitration is triggered by nanostructured TiO2 during PC12 differentiation

Introduction: Cells in their natural environment interact with extracellular matrix components in the nanometer scale and respond to nanoscale features when grown on synthetic substrates. Therefore, in studying neuronal proliferation and differentiation processes, nanoscale topography of synthetic materials has been receiving increasing attention because of its resemblance to in vivo surroundings. It has been recently proposed that the physical properties of the substrates can be considered as a new kind of stimulus based on the finding that surface energy distribution, through cell-substrate interactions, triggers neuritogenesis of PC12 cells in the absence of nerve growth factor (NGF) or other inducers. However the molecular mechanisms of the triggered signalling cascade has not been identified, yet. Methods: In order to detect nitrated proteins, cellular extracts were submitted to SDS-PAGE; gels were sliced and proteins reduced, carbamidated, digested with trypsin and analysed by nano-LC-MS/MS with an LTQ Orbitrap Velos (Thermo Scientific) mass spectrometer. Results: To investigate the potential role exerted by protein nitration, we studied the behaviour of PC12 cells on nanostructured TiO2 films of different thickness in the presence and in the absence of NGF while cells grown on PLL (poly-L-Lysine) coated glass were taken as the control. Since it has been demonstrated that NGF induces NO production by nitric oxide synthases (NOS) and that differentiation in PC12 cells grown on PLL-glass in the presence of NGF is associated to an increase in protein nitration, our investigation was aimed at detecting whether increased protein nitration is also observed during PC12 differentiation triggered by nanostructed TiO2 films in the absence of NGF. Conclusion: Altogether the data suggest that protein nitration is involved in the differentiation process induced by nanotopography similarly to what is described in PC12 cells differentiated upon NGF.