The PI 3‐kinase and mTOR signaling pathways are important modulators of epithelial tubule formation

Using MDCK cells as a model system, evidence is presented demonstrating that the signaling pathways mammalian target of rapamycin (mTOR) and phosphoinositide 3‐kinase (PI 3‐kinase) play important roles in the regulation of epithelial tubule formation. Incubation of cells with collagen gel overlays induced early (4–8 h) reorganization of cells (epithelial remodeling) into three‐dimensional multicellular tubular structures over 24 h. An MDCK cell line stably expressing the PH domain of Akt, a PI 3‐kinase downstream effector, coupled to green fluorescent protein (GFP‐Akt‐PH) was used to determine the distribution of phosphatidyl inositol‐3,4,5‐P 3 (PIP 3 ), a product of PI 3‐kinase. GFP‐Akt‐PH was associated with lateral membranes in control cells. After incubation with collagen gel overlays, GFP‐Akt‐PH redistributed into the lamellipodia of migrating cells suggesting that PIP 3 plays a role in epithelial remodeling. Using the small molecule inhibitor LY‐294002 that inhibits both mTOR and PI 3‐kinase, we demonstrated that kinase activity was required for epithelial remodeling, disruption of cell junctions and subsequent modulation of tubule formation. Since the mTOR signaling pathway is downstream of PI 3‐kinase, the effects of rapamycin, a specific mTOR inhibitor, on tubule formation were assessed. Rapamycin did not affect epithelial remodeling or GFP‐Akt‐PH redistribution but inhibited elongated tubule formation that occurred later (24 h) in morphogenesis. These results were further supported by using RNA interference to down‐regulate mTOR and inhibit tubule formation. Our studies demonstrate that PI 3‐kinase regulates early epithelial remodeling stages while mTOR modulates latter stages of tubule development. J. Cell. Physiol. 216: 469–479, 2008. © 2008 Wiley‐Liss, Inc.