EGF activates p38 Kinase, Jnk and p70S6 Kinase via PI three K and mTOR dependent mechanisms in HC11 mammary epithelial cells The two Akt1 and p38MapK are actually identified like a poten tial downstream targets of EGF signaling in mammary epi thelial cells. Also, Akt stimulates activation of mTOR. The effect of blocking PI 3 kinase pathway, including mTOR as well as anxiety kinase pathways, on EGF induced inhibition of lactogenic differentiation was deter MP-470 ic50 mined in HC11 luci cells. Inhibitors of Mek, PI three kinase and p38 kinase at the same time as Rapamycin, an mTOR inhibitor, were extra to HC11 luci cells in DIP induction media in the presence of EGF. Luciferase action was measured 48 hrs post induction and normalized to protein concen tration.
As expected the addition of EGF for the DIP induction media resulted in inhibition of luciferase exercise, and each selelck kinase inhibitor inhibitor alone appreciably restored the casein promotor action compared to DIP plus EGF. In mixture analyses it appeared that PD98059, the Mek Erk inhibitor, made synergistic effects with LY294002 and Rapamycin. On the other hand, combinations of LY294002 with Rapamycin and SB203580 produced additive or significantly less than additive responses. This was also the situation to get a blend of Rapamycin with SB203580. These results demonstrate that the EGF induced disrup tion of lactogenic differentiation proceeds by blocking both the Ras Raf Mek Erk pathway plus the PI 3 kinase pathway. Moreover, the results recommend that EGF induced activation of mTOR and p38 are each dependent on PI three kinase signaling in HC11 cells.
It ought to be mentioned that the enhance in luciferase activity detected in inhibitor taken care of cells is distinct to recovery of action blocked by EGF. The treatment of HC11 luci cells with large amounts of PI three kinase or mTOR inhibitors while in the absence of EGF decreased cell viability and thereby decreased lactogenic differentiation. HC11 cells had been examined to a lot more entirely characterize the result of PI 3 kinase and mTOR inhibitors on a number of sig nal transduction pathways induced by EGF. HC11 cells have been serum starved in the absence of EGF and incubated for 4 hours with LY294002 or Rapamycin before stimu lation with EGF. The cell lysates had been harvested following EGF stimulation and analyzed by western blotting for expression and phosphorylation of Akt, Gsk3?, p70S6 kinase and also the Map kinases Erk, Jnk and p38. The PI three kinase inhibitor completely blocked the phosphorylation and subsequent activation of Akt on serine 473 and p70S6 kinase on threonine 389 and partially blocked the phos phorylation and activation of p38 and Jnk. The mTOR inhibitor Rapapmycin wholly blocked the activation of p38, Jnk and p70S6 kinase. How ever, neither inhibitor blocked the activation of Erk1.