FIGURE 4.

mTORC2 signaling mediates collagen I expression and lies upstream of mTORC1 in activated mesenchymal cells. A, Fib-MCs were transfected with Rictor siRNA (100 nm) or scrambled siRNA for 48 h followed by protein collection and Western blot analysis. Effect of Rictor silencing on baseline collagen I, P-S6K1 (Thr-389), P-4E-BP1 (Thr-37/46), P-AKT (Ser-473), P-TSC2 (Thr-462), and P-PKC (Ser-657) expression is shown (n = 6–8). B, Non-Fib MCs transfected with Rictor (100 nm) or scrambled siRNA were treated with or without 10 μm lysophosphatidic acid (LPA) for 6 h. Effect of rictor silencing in presence and absence of LPA on P-S6K1 (Thr-389), P-4E-BP1 (Thr-37/46), P-AKT (Ser-473), and collagen I expression is shown (n = 6–8). C, Fib-MCs were treated with AKT inhibitor MK-2206 (5um) or PKC inhibitor Go6796 (1um) for 24 h, and protein expression of mTORC substrates and collagen I was quantitated by Western blot analysis (n = 5). D, non-Fib MCs were transfected with pcDNA3 backbone, pcDNA3 HA-AKT or pcDNA3 HA-AKT S473D plasmids followed by protein collection at 24 h and Western blot analysis. (n = 6). E, Fib-MCs were infected with lentivirus containing pGIPZ Rictor shRNA for 72 h followed by transient transfection with HA-AKT plasmids and subsequent protein collection and Western blot analysis. Representative blots from all experiments are shown.