The significant inhibitory effect of the PI3K inhibitor LY294002 on IGF1-induced, HIF1α-dependent VEGF secretion is consistent with a major role of PI3K/AKT in mediating IGF1 signaling in cholangiocytes. In agreement with the aforementioned data, IGF1 stimulated cell proliferation in PC2-defective cells (Fig. 5), and this effect was significantly inhibited by rapamycin. However, IGF1 stimulates secretion of VEGF, also a strong mitogen for cystic cholangiocytes. As shown in Fig. 5, IGF1-induced cell proliferation in cystic cholangiocytes was significantly inhibited by the VEGFR2

inhibitor SU5416. find more SU5416 did not affect the phosphorylation of the IGF1 receptor, in contrast to the specific IGF1R inhibitor AG102429 used as a positive control, and this

indicates that the effects of SU5416 on VEGFR2 are specific (Supporting Fig. 5). These data strongly argue for the presence of an autocrine loop in which IGF1 stimulates PI3/AKT/mTOR and mTOR stimulates HIF1a-dependent secretion of VEGF, which in turn, interacting with its receptor VEGFR2, activates an MEK/ERK1/2-dependent proliferative effect. In agreement with this interpretation, both rapamycin and SU5416 inhibited the increase in pERK expression A-769662 purchase induced by IGF1 in cystic cholangiocytes. A further indication of this autocrine loop involving IGF, mTOR, and VEGF secretion is the strong reduction in pericystic microvascular density in mice treated with rapamycin (Fig. 3A). An open question is the mechanistic relationships between the aforementioned mechanisms and the polycystin defect. Shillingford et al.11 and Distefano et al.19 proposed that PC1 acts as an inhibitor selleck compound of TSC2; this mechanism would be lost in ADPKD, and increased activity of mTOR would result. These data represent an important clue; however, they have been generated by overexpression of PC1. In our study,

we instead used a strategy involving the loss of function of PC2. In addition to its functional relationships with PC1, PC2 participates in the cellular and ER homeostasis of calcium.3, 4 Lower cellular and ER calcium stimulates PKA and ERK phosphorylation.30-33 We have recently shown that in PC2-defective cholangiocytes, the increase in pERK1/2 is PKA-dependent.7 We here provide evidence that baseline p-mTOR activation in PC2-defective cholangiocytes (as judged by its downstream kinase P70S6) is PKA-dependent and ERK-dependent and is thus linked to the altered calcium homeostasis. We can only speculate about why the cystic epithelium produces this vast array of growth factors. The mechanisms are unclear, but this is akin to what happens to WT biliary epithelium during liver repair. Thus, cystic cholangiocytes resemble activated cholangiocytes in terms of their ability to generate autocrine and paracrine growth factors.