Zanotto-Filho et al 2008 2009) The increase in RAGE immunocontent induced by retinol was not affected by the PKA inhibitor H89 (1 M) the pan-PKC inhibitor G6983 (10 M) the JNK inhibitor SP600125 (10 M) and the ERK1/2 inhibitor UO126 (10 M); however the p38 inhibitor SB203580 (10 M) and the Akt inhibitor LY294002 (10 M) inhibited the increase in RAGE immunocontent by retinol (Fig 1 C) The antioxidant Trolox (100 M) as previously observed also decreases the effect of retinol on RAGE These data indicated the KSP Inhibitors involvement of the protein kinases p38 and Akt on the effect of retinol upon RAGE upregulation Cell viability after 24 h was not affected by any of the protein kinase inhibitors tested except the PKA inhibitor H89 (data not shown) .

To confirm that the protein kinases p38 and Akt were activated by retinol we evaluated the phosphorylation state of these protein kinases by Western blot Phosphorylated forms (ie active) of p38 and Akt were detected within 60 min of incubation with retinol 7 M (Fig 2); p38 phosphorylation peaked at between 15 and 30 min while Akt phosphorylation peaked SB 216763 between 10 and 15 min of retinol incubation Time course evaluation of the phosphorylation state of p38 and Akt during 24 h shows that activation of these kinases during retinol treatment are transient (Fig 2B) The antioxidant Trolox (100 M) inhibited the effect of retinol on the phosphorylation of both kinases indicating that p38 and Akt phosphorylation are dependent on reactive species production (Fig 3) We know from previous works that incubation with these concentrations of Trolox blocks the increase in ROS production induced by retinol 7 M (Gelain et al 2008ab; Pasquali et al 2008).

Furthermore we confirmed that SB203580 and LY294002 were effective in the inhibition of p38 and Akt respectively Altogether these results indicate that the oxidant-dependent Hematomas up-regulation of RAGE by retinol is mediated by the activation of p38 and Akt p38 which are probably activated in response to oxidative stressAkt phosphorylation peaked earlier than p38 phosphorylation during retinol treatment suggesting Akt phosphorylation is an upstream event leading to p38 activation We then tested whether Akt and p38 phosphorylation were dependent on each other by analyzing the effect of Akt inhibition on p38 phosphorylation (Fig 4) Pre-incubation with LY294002 did not affect p38 phosphorylation; also the p38 inhibitor SB203580 did not exert any effect on Akt phosphorylation .