The results, summarized in Figure 8, clearly show that when cells are grown on ns TiO2 in NGF free media ERK is thing phosphor ylated to the same extent as in cell grown on glass or on flat TiO2 upon stimulation by NGF. In the latter two substrates the activation of ERK is almost undetectable in the absence of NGF. To further confirm the involvement of the ERK signa ling cascade in the process, we tested the effect of an inhibitor of MEK kinase, the enzyme responsible for ERK activation in the signaling cascade. As shown in Figure 9, cells treated with the inhibitor display a significant sup pression of neurite outgrowth compared to control condi tions, both on PLL plus NGF and on ns TiO2, and present a behavior similar to unstimulated cells.
Accordingly, differentiation induced by NGF on PLL glass and by ns TiO2 is prevented by MEK kinase inhibitor to a similar extent, suggesting that the same pathway is in volved in differentiation Inhibitors,Modulators,Libraries process started by the two dif ferent stimuli. Our data are in extremely good agreement with previ ous findings by Foley et al. who described the in volvement of ERK in the differentiation Inhibitors,Modulators,Libraries of PC12 cells cultured on synthetic substrates whose topographical features act to modulate neuritogenesis under sub optimal concentration of NGF. Since NGF treatment has been Inhibitors,Modulators,Libraries shown to up regulate 1B1 integrin molecules in PC12 cells and integrin mediated FAK activation augments EGF ERK signaling, they suggested that the formation and organization of focal adhesions on nanoscale features may cooperate with NGF to promote neuritogenesis when the concentration of the chemical inducer is low while it is ineffective at 50 ng mL NGF when the signaling cascade is already at its maximum.
This is in accordance with our finding that nanotopography mimics the effect of NGF but it does not act cooperatively with NGF to promote neuritogenesis. Based on our finding, we propose that the perturbation of the actin cytoskeleton caused by the surface nanoroughness, shown in the immu nostaining results reported in Figure 3B, increases NOS expression Inhibitors,Modulators,Libraries and the NO signaling cascade activation as well as ERK activation therefore explaining the cell behavior observed on nanostructured TiO2. One question arises from this picture how nano topography may increase NOS expression in order to produce NO.
Many data suggest that NOS activity may be regulated by cytoskeleton at transcriptional, post transcriptional and post translational level and that the cytoskeletal reorganization induced by extracellular stimuli such as shear stress, hypoxia and drugs play an important role in regulating NOS expression and ac tivity. iNOS gene Inhibitors,Modulators,Libraries transcription is regulated selleck by changes in the actin cytoskeleton in alveolar epithelial cells, glomerular mesangial cells and vascular smooth muscle cells.