Vertical inhibition of PI3K/Akt/mTOR signaling demonstrates in vitro and in vivo anti-fibrotic activity

•BEZ235 leads to superior inhibitory effect to rapamycin for dermal fibrosis in two complementary systemic sclerosis mouse models.•BEZ235 attenuates dermal fibrosis by vertical inhibiting PI3K/Akt/mTOR signaling via suppression of fibroblast activation.•Feedback induction of phosphorylation of the Akt on Ser473 by single mTOR inhibitor rapamycin might hamper its anti-fibrotic effect.•The strategy with BEZ235 overcomes the limitation of rapamycin in feedback activation of p-Akt on Ser473 following TGF-β treatment.

BackgroundThe mammalian target of rapamycin (mTOR) regulates cellular activity in many diseases, but the complex interplay with PI3K/Akt pathway may hampers its function.ObjectiveThis study was undertaken to determine the activity of PI3K/Akt/mTOR signaling in the fibroblasts from systemic sclerosis (SSc) patients, and compare the effects of vertical inhibiting PI3K/Akt/mTOR by BEZ235 and inhibiting mTOR alone by rapamycin in fibroblast activation and in two complementary established mouse model of SSc.MethodsPharmaceutical specific inhibitors BEZ235 and rapamycin were used to vertical inhibit PI3K/Akt/mTOR signaling and mTOR signaling alone in cultured fibroblasts and in mice. SSc mouse model was established by daily injecting bleomycin subcutaneously or by overexpression of constitutively active type I TGF-β receptor (TβRIca). To delineate the mechanisms underlying the antifibrotic effects of BEZ235 and rapamycin, activity of PI3K/Akt/mTOR signaling was analyzed by determining the expressions of phosphorylated Akt, GSK-3β, mTOR and S6 ribosomal protein (S6).ResultsPrimary dermal fibroblasts demonstrated hyperactivity of PI3K/Akt and mTOR signaling. mTOR inhibitor rapamycin failed to inhibit dermal fibrosis in an established SSc mouse model. However, administration of a dual inhibitor for PI3K/Akt and mTOR signaling BEZ235 attenuated dermal fibrosis by reversing increased dermal thickness and collagen deposition in two SSc mouse models. Furthermore, BEZ235 showed superior inhibitory effect on fibroblast activation relative to rapamycin in vitro. Also both BEZ235 and rapamycin could prevent the phosphorylation of mTOR and S6 completely. BEZ235 also blocked the activation of Akt and GSK-3β dramatically, whereas rapamycin has been shown to increase further upregulation of phosphorylated Akt on Ser473 both in vitro and in vivo.ConclusionThese data show that blocking PI3K/Akt/mTOR with BEZ235 leads to superior inhibitory effect for dermal fibrosis, suggesting that vertical inhibition of PI3K/Akt/mTOR signaling may have therapeutic potential for SSc.