Nucleotide exchange factor ECT2 regulates epithelial cell polarity.
Cell polarity regulates diverse biological events such as localization of embryonic determinants and establishment of tissue and organ architecture. Epithelial cell polarity is regulated by the polarity complex Par6/Par3/atypical protein kinase C (aPKC). We previously found that the nucleotide exchange factor ECT2 associates with this polarity complex and regulates aPKC activity, but the role of ECT2 in cell polarity is still unclear. Here we show that expression of a dominant negative (ECT2-N2) or constitutively active (ECT2-DeltaN5) form of ECT2 inhibits normal cyst formation of MDCK cells in 3-dimensional collagen gels. Central lumens were not observed in cysts formed by cells expressing either ECT2-DeltaN5 or ECT2-N2. Apical localization of ZO-1 and basolateral localization of beta-catenin were no longer observed in these cells. Interestingly, cells expressing ECT2-N2 did form normal cysts when cultured in the basement membrane matrix Matrigel instead of collagen gels. Addition of a major Matrigel component, laminin, partially rescued the normal cyst formation inhibited by ECT2-N2 in 3-dimensional collagen gels. Thus, signaling through laminin might override the defects of signaling through collagen and ECT2. Whereas ECT2-N2 inhibited the lumen formation of MDCK cysts, caspase-3, which is reportedly involved in lumen formation through apoptosis, was activated at various locations of cells in the cysts. It is likely that perturbation of ECT2 signaling inhibits the establishment of epithelial cell polarity leading to the inhibition of selected elimination of cells at the center of cysts. Thus, ECT2 appears to play a critical role in epithelial cell polarity.
Laboratory of Cell Biology, National Cancer Institute, Bethesda, Maryland 20892-4256, USA.
Xiu Fen
XF
Liu
Shigeo
S
Ohno
Toru
T
Miki
D000818:Animals; D001485:Basement Membrane / Q000378:metabolism; D002118:Calcium / Q000378:metabolism; D002352:Carrier Proteins / Q000378:metabolism; D053148:Caspase 3; D020169:Caspases / Q000378:metabolism; D016764:Cell Polarity; D002478:Cells, Cultured; D003094:Collagen / Q000378:metabolism; D003560:Cysts / Q000378:metabolism; D004285:Dogs; D004789:Enzyme Activation; D004847:Epithelial Cells / Q000166:cytology*; D005109:Extracellular Matrix / Q000378:metabolism; D005799:Genes, Dominant / Q000235:genetics; D020662:Guanine Nucleotide Exchange Factors / Q000378:metabolism*; D050505:Mutant Proteins / Q000378:metabolism; D011493:Protein Kinase C / Q000378:metabolism; D021381:Protein Transport; D019108:Tight Junctions / Q000378:metabolism
D002352:Carrier Proteins; D020662:Guanine Nucleotide Exchange Factors; D050505:Mutant Proteins; D003094:Collagen; C115015:PKC-3 protein; D011493:Protein Kinase C; D053148:Caspase 3; D020169:Caspases; D002118:Calcium