In particular, it was observed that IGF I can induce Akt activation and phosphorylation of Ser 473 located in the C terminal regulator domain of the protein and this effect is selleck compound totally dependent on PI 3K activation since it was completed inhibited by wortman nin or LY294002. Phosphorylation at this site results in the binding of Bad to 14 3 3t protein, thus inhibiting Bad binding to Bcl 2 and Bcl Xl. Of note, IGF I induced Bad phosphorylation was not completely reversed by PI 3 K inhibitors. This could be due to the fact that other IGF I activated proteins able to phosphorylate Bad are not acti vated by PI 3K. In this context we could exclude the involvement of either ERK or PKA activation in Bad phos phorylation.

In addition, exposure to IGF I for 24 hours induced an increased expression of the anti apoptotic protein Bcl Xl, an anti apoptotic protein that binds Bad. Taken together, these data indicate that IGF I could protect cells from apoptosis acting both on anti apoptotic signalling and the expression of anti apoptotic proteins. We then evaluated the involvement of GSK3? in IGF I induced PI 3K activation. GSK3? was initially identified as an enzyme that regulates glycogen synthesis in response to insulin. GSK3? is a ubiquitously expressed serine/threonine protein kinase that phosphorylates and inactivates glycogen synthase. GSK3? has been shown to regulate cyclin D1 proteolysis and subcellular localisation. GSK3? knock out mice show accelerated wound clo sure and fibrogenesis, thus suggesting an inhibitory role of this kinase.

In our experimental Cilengitide setting, IGF I induced the phosphorylation of GSK3? after 15 minutes of incubation, and this effect was PI 3K dependent. This observation provides additional molecular insights into the pro survival action of IGF I and reinforces its role in the fibrogenic process. Other downstream targets of Akt are the FOXO family of transcription factors. Phosphorylation of FKHR family members by Akt promotes cell survival and regulates the cell cycle. Phosphorylation of FKHR protein regulates their nuclear translocation and target gene transcription. Our data indicate that IGF I induces the phosphor ylation of Fox 1 and Fox 4 of the Forkhead family and this phosphorylation is strongly reduced by pre incubation with WMN, thus confirming a predominant anti apop totic action of this growth factor through the activation of PI 3K and related downstream pathways.

Finally, a dedicated set of experiments confirmed the apoptosis resistant phenotype of this activated human HSC. Numerous factors were used to induce human HSC apoptosis but only with selleck screening library high doses of FasL cyclohex ymide, were caspase 3 and PARP cleavage observed. In support of the survival action of IGF I, incubation with this growth factor resulted in a partial reversion of this effect.