Transcriptional Modulation of Genes Encoding Structural Characteristics of Differentiating Enterocytes During Development of a Polarized Epithelium In Vitro

Abstract

Although there is considerable evidence implicating posttranslational mechanisms in the development of epithelial cell polarity, little is known about the patterns of gene expression and transcriptional regulation during this process. We characterized the temporal program of gene expression during cell-cell adhesion-initiated polarization of human Caco-2 cells in tissue culture, which develop structural and functional polarity similar to that of enterocytes in vivo. A distinctive switch in gene expression patterns occurred upon formation of cell-cell contacts between neighboring cells. Expression of genes involved in cell proliferation was down-regulated concomitant with induction of genes necessary for functional specialization of polarized epithelial cells. Transcriptional up-regulation of these latter genes correlated with formation of important structural and functional features in enterocyte differentiation and establishment of structural and functional cell polarity; components of the apical microvilli were induced as the brush border formed during polarization; as barrier function was established, expression of tight junction transmembrane proteins peaked; transcripts encoding components of the apical, but not the basal-lateral trafficking machinery were increased during polarization. Coordinated expression of genes encoding components of functional cell structures were often observed indicating temporal control of expression and assembly of multiprotein complexes. INTRODUCTIONThe human intestinal epithelium has a distinctive organization in which a small population of stem cells in the crypt gives rise to postmitotic cells that differentiate as they migrate away from the crypt into the villus (Clatworthy and Subramanian, 2001;Vidrich et al., 2003;Pinto and Clevers, 2005). The major polarized cell type, the enterocyte, forms a tight epithelial monolayer along the crypt-villus axis of the intestine, serving as a permeability barrier between luminal contents and the blood supply and as a conduit for nutrient absorption and enzyme secretion (Clatworthy and Subramanian, 2001).Enterocyte polarization requires the formation of functionally and structurally distinct plasma membrane domains, termed apical and basal-lateral. The apical surface faces the lumen and consists of a dense array of microvilli, the "brush border," which increases the absorptive surface area and contains enzymes and transporters to facilitate uptake of nutrients. The basal-lateral surface, which is separated from the apical surface by the tight junction, faces neighboring cells and the extracellular matrix (ECM). Basallateral membranes contain intercellular junctional complexes, ECM receptors, and channels and transporters that regulate ion/solute transfer from the intestinal lumen to the interstitium (Yeaman et al., 1999b).Differentiation and polarization of epithelial cells involves the reorganization of cytoskeletal structures to initiate changes in cell shape and correct orientation of vesicle trafficking machineries to generate and ma...

A Flippase-Independent Function of Atp8b1, the Protein Affected in Familial Intrahepatic Cholestasis Type 1, Is Required for Apical Protein Expression and Microvillus Formation in Polarized Epithelial Cells

“…Disruption of cytoskeletal linkages between the actin bundle and the microvillar membrane results in a profound loss of SI. 37 These and other data 24 suggest that microvilli formation is tightly connected to apical protein biosynthesis. As microvilli dramatically increase the surface area of the apical membrane, this coupling may serve to prevent overloading of the apical membrane with protein.…”

“…and villin 24 indicated that all clonal cell lines retained the ability to undergo normal epithelial differentiation (Fig. 2D).…”

“…The perturbed expression of apical membrane proteins in ATP8B1-depleted cells suggested a possible defect in the assembly of the apical brush border. 24,27 Contrary to differentiated control cells (EV1, untransfected), ATP8B1-depleted clones (A5, A3, B3) generally lacked the patchy apical staining pattern indicative of microvilli (Fig. 8A).…”

Polar Effects on Ion Transport and Cell Proliferation Induced by GC-C Ligands in Intestinal Epithelial Cells

“…However, ERK1/2 inhibition did not affect cGMP level, which suggests that cGMP is not the target of the homeostatic mechanisms involved in serosal ST-induced chlo- ride secretion. The Caco-2 cell line that we used to investigate the polarization of GC-C activation is considered a reliable model of human intestinal epithelium (30).…”

Mophogenesis and Dynamics of Post-Golgi Transport Carriers

“…This might happen because the target patch for the PGC fusion forms at the lateral surface of epithelial cells earlier then at the apical targeting patch (Mostov et al, 2003;Nelson, 2003;Rodriguez-Boulan et al, 2005). Indeed, Sec6 undergoes recruitment to the sites of cell-to-cell junctions as soon as subconfluent cells start to contact each other (Yeaman et al, 2004), while expression of the apical sorting machinery components occurs later during the process of cell polarization (Halbleib et al, 2007). Thus, during the early stages of polarization, most of PGCs fuse near junctional complexes at the lateral domain of the PM , which contributes to fast vertical elongation of a cell within the epithelial sheet (Mostov et al, 2003;Nelson, 2003;Rodriguez-Boulan et al, 2005).…”