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.
Walid
Shereaf
S
Department of Anatomy and Cell Biology, SUNY Downstate Medical Center, Brooklyn, New York 11203, USA.
Eisen
Randi
R
Ratcliffe
Don R
DR
Dai
Kezhi
K
Hussain
M Mahmood
MM
Ojakian
George K
GK
D000818:Animals; D015415:Biomarkers / Q000378:metabolism; D002460:Cell Line; D002867:Chromones / Q000378:metabolism; D004285:Dogs; D004791:Enzyme Inhibitors / Q000378:metabolism; D004848:Epithelium / Q000033:anatomy & histology / Q000196:embryology; D007365:Intercellular Junctions / Q000378:metabolism; D009024:Morphogenesis; D009025:Morpholines / Q000378:metabolism; D019869:Phosphatidylinositol 3-Kinases / Q000235:genetics / Q000378:metabolism*; D011494:Protein Kinases / Q000235:genetics / Q000378:metabolism*; D034622:RNA Interference; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D015398:Signal Transduction / Q000502:physiology*; D020123:Sirolimus / Q000378:metabolism; D058570:TOR Serine-Threonine Kinases
D015415:Biomarkers; D002867:Chromones; D004791:Enzyme Inhibitors; D009025:Morpholines; D019869:Phosphatidylinositol 3-Kinases; D011494:Protein Kinases; D011993:Recombinant Fusion Proteins; D020123:Sirolimus; D058570:TOR Serine-Threonine Kinases; C085911:2-(4-morpholinyl)-8-phenyl-4H-1-benzopyran-4-one