Abstract
Wnts are morphogens with well recognized functions during embryogenesis. Aberrant Wnt signaling has been demonstrated to be important in colorectal carcinogenesis. However, the role of Wnt in regulating normal intestinal epithelial cell proliferation is not well established. Here we determine that Wnt11 is expressed throughout the mouse intestinal tract including the epithelial cells. Conditioned media from Wnt11-secreting cells stimulated proliferation and migration of IEC6 intestinal epithelial cells. Co-culture of Wnt11-secreting cells with IEC6 cells resulted in morphological transformation of the latter as evidenced by the formation of foci, a condition also accomplished by stable transfection of IEC6 with a Wnt11-expressing construct. Treatment of IEC6 cells with Wnt11 conditioned media failed to induce nuclear translocation of beta-catenin but led to increased activities of protein kinase C and Ca(2+)/calmodulin-dependent protein kinase II. Inhibition of protein kinase C resulted in a decreased ability of Wnt11 to induce foci formation in IEC6 cells. Finally, E-cadherin was redistributed in Wnt11-treated IEC6 cells, resulting in diminished E-cadherin-mediated cell-cell contact. We conclude that Wnt11 stimulates proliferation, migration, cytoskeletal rearrangement, and contact-independent growth of IEC6 cells by a beta-catenin-independent mechanism. These findings may help understand the molecular mechanisms that regulate proliferation and migration of intestinal epithelial cells.
Highlights
Wnt signaling pathways have established importance in determining tissue development, differentiation, cell fate, and embryonic patterning
We show that Wnt11 is one protein in this family that has a relatively compartmentalized pattern of expression in the gut, and that both exogenous treatment of intestinal epithelial cells IEC6 with Wnt11 and overexpression of Wnt11 in IEC6 result in accelerated proliferation, migration, and transformation in a manner that is dependent on protein kinase C (PKC) and/or calmodulin-independent kinase II (CamKII) but independent of -catenin
To demonstrate further that PKC is a mediator of the biological activity of Wnt11 in stimulating proliferation of IEC6 cells, we investigated the effect of a PKC-specific inhibitor, calphostin C, on the transforming activity of Wnt11 using the focus formation assay in co-cultured transwells
Summary
Cell Lines—The nontransformed rat small intestinal epithelial cell line, IEC6 [42, 43], was purchased from American Type Culture Collection (ATCC). A Wnt11-transfected quail cell line, W11ox, and its parental cell line, QCE6 [46], were cultured on fibronectin-coated plates (1 g/cm fibronectin in phosphate-buffered saline (PBS)) in media consisting of minimum essential medium with 292 mg/liter glutamine, 10% tryptose phosphate broth, 2% heat-inactivated chicken serum, 10% iron-supplemented calf serum, and 200 g/ml G418 for W11ox. IEC6 cells were transfected by electroporation (300 V, 1 millifarad) with pcDNA3-Wnt or the pCDNA3 vector control. Independent stable populations were tested for their ability to form foci in soft agar by plating 104 cells in 2.5% agar culture medium with antibiotic selection on plates with a 5% agar base. Cells were cultured for 3 weeks, and visible foci were selected and clonally expanded for further analysis (IEC6/W11 cells).
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