Anomalous apical plasma membrane phenotype in CK8-deficient mice indicates a novel role for intermediate filaments in the polarization of simple epithelia.

Previous results from our laboratory have indicated a requirement for CK intermediate filaments (IF) for the organization of the apical domain in polarized epithelial cells in culture. The results seemed to be challenged by the phenotype of cytokeratin (CK) 8-deficient mice, which comprises only colorectal hyperplasia, female sterility and a weaker hepatocyte integrity. In this work localization with anti-CK antibodies indicated that many Ck8-/- epithelia still form IF in CK8-deficient mice, perhaps because of the expression of the promiscuous CK7. In the small intestine, only villus enterocytes lacked IFs. These cells appeared to lose syntaxin 3, and three apical membrane proteins (alkaline phosphatase, sucrase isomaltase and cystic fibrosis transmembrane conductance regulator) as they progressed along the villus. At the distal third of the villi, gamma-tubulin was found scattered within the cytoplasm of enterocytes, in contrast to its normal sub-apical localization, and the microtubules were disorganized. These results could not be attributed to increased numbers of apoptotic or necrotic cells. The only other cell type we found without IFs in CK8 null mice, the hepatocyte, displayed increased basolateral levels of one apical marker (HA4), indicating a correlation between the lack of intermediate filaments and an apical domain phenotype. These data suggest a novel function for intermediate filaments organizing the apical pole of simple polarized epithelial cells.

Subcellular distribution of CFTR in rat intestine supports a physiologic role for CFTR regulation by vesicle traffic.

The cystic fibrosis transmembrane conductance regulator (CFTR) is a cAMP-activated chloride channel critical to intestinal anion secretion. In addition to phosphorylation, vesicle traffic regulates CFTR in some epithelial cells. Studies of cultured intestinal cells are conflicting regarding the role of cAMP-dependent vesicle traffic in regulating chloride transport. Whether CFTR is present in vesicular compartments within chloride secretory cells in the intestine is unknown and the role of cAMP-dependent vesicle insertion in regulating CFTR and intestinal fluid secretion remains unclear. The purpose of this study was to: (1) examine and quantify the subcellular distribution for CFTR in rat intestine, (2) further define the ultrastructure of the previously identified CFTR High Expresser (CHE) cell, and (3) examine the cellular distribution of CFTR following cAMP stimulation in vivo. Using the sensitive techniques of cryoimmunogold electron microscopy we identified CFTR in subapical vesicles and on the apical plasma membrane in crypt, Brunner glands, and CHE cells. cAMP stimulation in rat proximal small intestine produced a fluid secretory response and was associated with an apical redistribution of CFTR, supporting a physiologic role for cAMP-dependent CFTR vesicle insertion in regulating CFTR in the intestine.

Microvillus inclusion disease: a genetic defect affecting apical membrane protein traffic in intestinal epithelium.

The striking similarities between microvillus inclusions (MIs) in enterocytes in microvillus inclusion disease (MID) and vacuolar apical compartment in tissue culture epithelial cells, led us to analyze endoscopic biopsies of duodenal mucosa of a patient after the samples were used for diagnostic procedures. Samples from another patient with an unrelated disease were used as controls. The MID enterocytes showed a decrease in the thickness of the apical F-actin layer, and normal microtubules. The immunofluorescence analysis of the distribution of five apical membrane markers (sucrase isomaltase, alkaline phosphatase, NHE-3 Na+/H+ exchanger, cGMP-dependent protein kinase, and cystic fibrosis trans-membrane conductance regulator), showed low levels of these proteins in their standard localization at the apical membrane as compared with normal duodenal epithelium processed in parallel. Instead, four of these markers were found in a diffuse distribution in the apical cytoplasm, below the terminal web (as indicated by co-localization with F-actin and cytokeratin 19), and in MIs as well. The basolateral protein Na(+)-K+ATPase, in contrast, was normally localized. These results support the hypothesis that MID may represent the first genetic defect affecting apical membrane traffic, possibly in a late step of apical exocytosis.

CFTR channel insertion to the apical surface in rat duodenal villus epithelial cells is upregulated by VIP in vivo.

cAMP activated insertion of the cystic fibrosis transmembrane conductance regulator (CFTR) channels from endosomes to the apical plasma membrane has been hypothesized to regulate surface expression and CFTR function although the physiologic relevance of this remains unclear. We previously identified a subpopulation of small intestinal villus epithelial cells or CFTR high expressor (CHE) cells possessing very high levels of apical membrane CFTR in association with a prominent subapical vesicular pool of CFTR. We have examined the subcellular redistribution of CFTR in duodenal CHE cells in vivo in response to the cAMP activated secretagogue vasoactive intestinal peptide (VIP). Using anti-CFTR antibodies against the C terminus of rodent CFTR and indirect immunofluorescence, we show by quantitative confocal microscopy that CFTR rapidly redistributes from the cytoplasm to the apical surface upon cAMP stimulation by VIP and returns to the cytoplasm upon removal of VIP stimulation of intracellular cAMP levels. Using ultrastructural and confocal immunofluorescence examination in the presence or absence of cycloheximide, we also show that redistribution was not dependent on new protein synthesis, changes in endocytosis, or rearrangement of the apical cytoskeleton. These observations suggest that physiologic cAMP activated apical membrane insertion and recycling of CFTR channels in normal CFTR expressing epithelia contributes to the in vivo regulation of CFTR mediated anion transport.

A delta F508 mutation in mouse cystic fibrosis transmembrane conductance regulator results in a temperature-sensitive processing defect in vivo.

The most prevalent mutation (delta F508) in cystic fibrosis patients inhibits maturation and transfer to the plasma membrane of the mutant cystic fibrosis transmembrane conductance regulator (CFTR). We have analyzed the properties of a delta F508 CFTR mouse model, which we described recently. We show that the mRNA levels of mutant CFTR are normal in all tissues examined. Therefore the reduced mRNA levels reported in two similar models may be related to their intronic transcription units. Maturation of mutant CFTR was greatly reduced in freshly excised oviduct, compared with normal. Accumulation of mutant CFTR antigen in the apical region of jejunum crypt enterocytes was not observed, in contrast to normal mice. In cultured gallbladder epithelial cells from delta F508 mice, CFTR chloride channel activity could be detected at only two percent of the normal frequency. However, in mutant cells that were grown at reduced temperature the channel frequency increased to over sixteen percent of the normal level at that temperature. The biophysical characteristics of the mutant channel were not significantly different from normal. In homozygous delta F508 mice we did not observe a significant effect of genetic background on the level of residual chloride channel activity, as determined by the size of the forskolin response in Ussing chamber experiments. Our data show that like its human homologue, mouse delta F508-CFTR is a temperature sensitive processing mutant. The delta F508 mouse is therefore a valid in vivo model of human delta F508-CFTR. It may help us to elucidate the processing pathways of complex membrane proteins. Moreover, it may facilitate the discovery of new approaches towards therapy of cystic fibrosis.

A unique subset of rat and human intestinal villus cells express the cystic fibrosis transmembrane conductance regulator.

BACKGROUND/AIMS: In the intestine, the cystic fibrosis transmembrane conductance regulator (CFTR) has been localized to the apical pole of crypt epithelial cells. Recent data indicate that some villus cells may also express CFTR, although the identity of these cells has not been established. The aim of the current study was to characterize the distribution, morphology, and surface marker expression of CFTR-expressing villus cells. METHODS: Immunofluorescence and immunoelectron microscopy was performed using anti-CFTR and enzyme marker antibodies. RESULTS: In the rat and human proximal small intestine, a subpopulation of scattered villus and superficial crypt epithelial cells label brightly with anti-CFTR antibodies. The fluorescent signal is detected throughout the cells with its greatest concentration apically. At the ultrastructural level, labeling involves the brush border and a prominent subapical vesicular compartment. The cells resemble adjacent villus enterocytes in their abundance of mitochondria and expression of basolateral Na(+)-K(+)-adenosine triphosphatase yet differ in their absence of brush-border sucrase and lactase expression. CONCLUSIONS: A previously uncharacterized subpopulation of villus cells with high levels of intracellular CFTR expression exists in the proximal small intestine. Morphological and cytochemical studies suggest that this subset of villus cells has a unique transport function.

Localization of the cystic fibrosis transmembrane conductance regulator to a distinct sub-population of enterocytes in the small intestine - abstract

Immunocytochemical studies have localized the cystic fibrosis transmembrane regulator (CFTR) to the apical domain of a variety of epithelial cell types. This distribution is consistent with an established function of CFTR as an apical membrane chloride channel. In the intestine, several studies have identified the apical domain of crypt epithelial cells as the predominant site of CFTR expression. In addition to crypt epithelial cells, we have identified a distinct sub-population of small intestinal epithelial cells with high levels of CFTR immunoreactivity. Cryosections of human and rat intestine were stained with an affinity-purified anti-CFTR antibody (q-1468) and examined by indirect immunofluorescent microscopy. In both species, intense staining of a sub-population of isolated intestinal mucosal epithelial cells was observed. This staining pattern is eliminated by preincubation of a-1468 with the C-terminal peptide against which it was raised. In the rat, these cells are most abundant in the villi of the proximal small intestine and comprise less than 2 percent of the total epithelial cell population. They are not seen in the terminal ileum or colon. A similar sub-population of CFTR-expressing cells was identified in the human small intestine. Within these cells from both species, staining is punctate and present throughout the cytoplasm, with the greatest intensity near the apical (luminal) pole. We have confirmed the specificity of this staining, using two other anti-CFTR antibodies. Using in-situ hybridization, other investigators have identified a similar subset of intestinal epithelial cells that exhibit high levels of CFTR mRNA expression. We performed immunoelectron microscopy to determine the morphological characteristics of these cells. The CFTR-labelled villus cells possess morphological features ofenterocytes. Within these enterocytes, CFTR labelling was detected in the apical brush border and in several small vesicles scattered throughout the cytoplasm. Thus, in the small intestine of the rat and human, CFTR is present in two mucosal cell populations. Apical staining of contiguous cells, characteristic of CFTR, is present in the crypts in both the small and the large intestine. In addition, a distinct sub-population of enterocytes with an apparent subcellular distribution for CFTR is present, predominantly in the villi of proximal small intestine. The relative contribution of these CFTR-expressing enterocytes to the pathogenesis of CF remains to be elucidated (AU)