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Transcriptional regulation of the ezrin gene during rat intestinal development and epithelial differentiation
Abstract
Polarized intestinal epithelial cells are characterized by the presence of a brush border at their apical surface. The brush border cytoskeleton is assembled during cell differentiation and is composed of parallel actin bundles, held together by specific actin-binding proteins. Using specific cDNA probes we have studied the expression of the mRNAs encoding ezrin and moesin, two members of a class of proteins that connect the microvillar cytoskeleton to the plasma membrane, during the process of enterocyte maturation that occurs both in the embryonic and in the adult small intestine, along the crypt-villus axis. The steady state levels of ezrin mRNA were found to increase in the fetal gut epithelium between day 15 and day 20 of gestation and during the first week after birth, in parallel with the morphogenetic process that leads to cell polarization and brush border assembly. On the contrary, moesin mRNA is expressed at very low levels in the mature small intestine, with a sudden drop in transcription occurring at birth. In the continuously renewing epithelium of adult animals, ezrin mRNA levels are higher in the differentiated villus cells of the distal portions of the gastrointestinal tract and very low in undifferentiated crypt cells. These data demonstrate that the expression of the ezrin gene is regulated at the level of mRNA abundance during development and differentiation of the intestinal epithelium.
Supplementary resource (1)
... Phosphorothioate antisense oligonucleotides used in this study were complementary to the position Ϫ 4 to 20 (relative to the translation initiation site) of rat ezrin mRNA (GenBank/EMBL/DDBJ, X67788; Barila et al., 1995). Oligonucleotides were purchased from Cornell University and Operon Technologies, Inc. Sense oligonucleotides corresponding to positions Ϫ 4 to 20 were used as controls. ...
... As immunoblots of RPE lysates obtained from rats of different ages did not detect the presence of moesin or radixin (Fig. 4), we can discard a possible additional role of these proteins in vivo. Ezrin has been identified as a minor component of apical brush borders of intestinal and renal epithelial cells and a previous report described an increase in ezrin mRNA as intestinal cells matured from an embryonic stage to a mature morphologically differentiated stage (Barila et al., 1995). However, in intestinal and kidney epithelia, potential morphogenetic roles of ezrin and other ERM proteins are overshadowed by the contribution of villin, a major component of microvilli that is a major player in the assembly of these structures. ...
Ezrin, a member of the ezrin/radixin/moesin (ERM) family, localizes to microvilli of epithelia in vivo, where it bridges actin filaments and plasma membrane proteins. Here, we demonstrate two specific morphogenetic roles of ezrin in the retinal pigment epithelium (RPE), i.e., the formation of very long apical microvilli and of elaborate basal infoldings typical of these cells, and characterize the role of ezrin in these processes using antisense and transfection approaches. In the adult rat RPE, only ezrin (no moesin or radixin) was detected at high levels by immunofluorescence and immunoelectron microscopy at microvilli and basal infoldings. At the time when these morphological differentiations develop, in the first two weeks after birth, ezrin levels increased fourfold to adult levels. Addition of ezrin antisense oligonucleotides to primary cultures of rat RPE drastically decreased both apical microvilli and basal infoldings. Transfection of ezrin cDNA into the RPE-J cell line, which has only trace amounts of ezrin and moesin, sparse and stubby apical microvilli, and no basal infoldings, induced maturation of microvilli and the formation of basal infoldings without changing moesin expression levels. Taken together, the results indicate that ezrin is a major determinant in the maturation of surface differentiations of RPE independently of other ERM family members.
... Primary and secondary antibodies were diluted in 1ϫ PBS ϩ , 2% BSA, and 0.05% Triton X-100. For tissue immunofluorescence, portions of proximal duodenum, jejunum, and distal ileum from 21-day-old rats were dissected, and longitudinal cryosections were prepared as described in Ref. 3. Sections were fixed and treated as described for cells. ...
... Our laboratory has been primarily concerned with the isolation and molecular characterization of genes that are upregulated during intestinal epithelial differentiation (2)(3)(4). Because the absorptive intestinal mucosa is the primary site of zinc uptake and we originally isolated the Dri 27 cDNA from this tissue on the basis of its upregulation during intestinal epithelial differentiation, we have sought to understand the role that its gene product might play in differentiated enterocytes through a detailed analysis of its intracellular localization and of its distinct functional domains. ...
We have identified the Dri 27 cDNA on the basis of its upregulated expression during rat intestinal development. It encodes a hydrophobic protein of 430 amino acids that shares significant homology with members of the mammalian zinc transporter family ZnT. The murine homologue of Dri 27 (named ZnT4) was recently associated with the mouse mutation "lethal milk." The primary sequence of Dri 27/ZnT4 displays features characteristic of polytopic membrane proteins. In this paper, we show that Dri 27/ZnT4 is localized in the membrane of intracellular vesicles, the majority of which concentrate in the basal cytoplasmic region of polarized enterocytes. A Dri 27/ZnT4 myc-tagged construct, transiently transfected in intestinal Caco-2 cells, partially colocalizes with the transferrin receptor and with the beta-subunits of the clathrin adaptor complexes AP-1 and AP-2 in a subpopulation of endosomal vesicles. By subcloning distinct portions of the protein in frame with glutathione-S-transferase, we also provide experimental evidence of their function as zinc-binding and protein-protein-interaction domains.
... Enterocytes are characterized by the existence of a brush border at their apical surfaces (Barila et al, 1995 andMeads andSchroer, 1995).. Snoeck et al (2005) described the enterocytes as hyperpolarized epithelial cells which are joined togather by tight junctions and their apices are covered by stiff, closely placed microvilli. In addition, the main function of these cells is to absorb nutrient materials. ...
The aim of this work was to explore the different types of carbohydrates
histochemically in the trachea of local Awase sheep and to compare it with that of
the local black goat. Ten tracheas from each sheep and goat were used for this
study. Different histochemical methods were used to explore types and locations
of carbohydrates in trachea’s wall of both animals. It was observed that the
amount of mucus secreted from the anterior third of trachea was comparatively
greater than that of the middle and posterior thirds due to its higher content oftracheal glands and goblet cells. The carbohydrate histochemistry of different
constituents of the trachea showed that there was no noticeable difference between
the two studied species. However, the glycogen was found in a greater amount in
goat's trachea in comparison to sheep. The goblet cells and the mucous secretory
units of the trachea showed a considerable amount of carboxylated glycoprotein
together with a little amount of other forms of carbohydrates. The mucus that
covers the lumen of the trachea contained almost all the carbohydrate substances
with the exception of neutral glycoproteins. Chondrocytes contain glycogen,
sulphated and neutral glycoproteins but didn't show any form of GAGs, whereas,
the territorial matrix of the cartilage contains a mixture of carboxylated and
sulphated GAGs with the predominance of the latter, but glycoproteins could not
be detected. The interterritorial matrix contained mainly carboxylated
glycosaminoglycans together with a little amount of glycoproteins. The columnar
cells and the basal surface epithelial cells showed no reaction to any form of
carbohydrates.
... Enterocytes are characterized by the existence of a brush border at their apical surfaces (Barila et al, 1995 andMeads andSchroer, 1995).. Snoeck et al (2005) described the enterocytes as hyperpolarized epithelial cells which are joined togather by tight junctions and their apices are covered by stiff, closely placed microvilli. In addition, the main function of these cells is to absorb nutrient materials. ...
Abstract
The current study was conducted to investigate the histomorphological, histochemical and immunohistochemical changes that occurred in the small intestine and colon of the local rabbits at different postnatal ages. To reach this goal, specimens from duodenum, jejunum, ileum and colon were collected from 48 local rabbits at four different ages of 1, 10, 15, and 40 days which were set as newly born kits (P1), suckling puppies (P10), pre-weaned rabbits (P15) and post-weaned rabbits (P40), respectively. Routine histological stain (H&E), Masons trichrome stain, Mallory stain, PAS, Alcian blue pH2.5, Toluidine blue and Aldehyde fuchsine staining techniques in addition to special immunohistochemical marker (Lgr5) for intestinal stem cell identification were conducted to achieve current goals.
Microscopic findings revealed the presence of villi in the small intestinal epithelium in newly born kits of one day age and suckling puppies as finger-like projections covered with numerous vacuolated enterocytes and immature cells. In the next ages of 15 and 40 days, the vacuolated cells disappeared and the villi acquired its mature shape with wide bases and narrow tips. Crypts of Lieberkühn were absent in newly born kits, but with age progress they appeared and acquired lumen and be continuous with villi bases in pre-weaned 15 days aged rabbits, in this age crypt fission was clearly observed in all segments of the small intestine particularly in ileum. In the next age of 40 days, the crypts depth and number were increased.
Paneth cells were distinguished for the first time in the crypts of suckling puppies with ill-defined red stained granules with H&E and masons trichrome stains and appeared blue granules with Mallory stain. In the subsequent ages both their size and number was increased. The largest cell size and well developed granules was recorded in jejunum of pre-weaned rabbits, whereas the largest number of paneth cells was recorded in the post-weaned rabbits. Goblet cells were found in all segments ad all studied ages as pale large cells with H&E and magenta color with PAS and blue with Alcian blue stain. Its number increased along the proximo-distal axis of the intestine.
The applying of Lgr5 marker indicated the presence of intestinal stem cells as dark brown elongated cells at the bases of villi of duodenum, jejunum and ileum as well as in the crypts of colon in the newly born kits (P1) and at the crypt bases in other studied ages (P10, P15, P40). Few dividing stem cells or their mitotic figures were seen in the sections of newly born kits and suckling puppies. In the pre-weaned rabbits, mitotic figures increased dramatically simultaneously with maturation of paneth cells in the crypts. The highest number of mitotic figures was detected in both jejunum and ileum.
Peyer’s patches of ileum was absent in newly born kits but they were developed with age progress to reach its mature dome-shape in the pre-weaned and post-weaned rabbits. Follicle associated epithelium covered the dome-shaped lymphoid follicles possesses microfold (M) cells which were large, pale basolaterally located nuclei containing several lymphocytes within its pockets.
The 1st appearance of Brunner’s glands of duodenum was in suckling puppies (P10) and most of acini were of serous type. In the next age (P15), mucus acini appeared simultaneously with serous one to form mixed sero-mucous acini (serous demilune). In post-weaned rabbits (P40), the mucous acini increased in number and were the dominant type. Generally, epithelium height, length and width of the villi, crypt depth, apparent villus surface area and thickness of tunica muscularis were increased with age progress in all studied segments of the intestine.
Two types of enteric nerve plexuses were detected in small and large intestine of local rabbits, the 1st submucosal one (henle nerve plexus) was small and ill-defined located in deep part of submucosa adjacent to inner circular muscular layer of tunica muscularis. The second Auerbach’s nerve plexus found between the two layers of tunica muscularis as well developed structures containing neurons and glial cells. Auerbach’s plexuses found in all ages even in newly born kits and in all segments of intestine with an interesting, large and numerous plexuses in the colon.
Sacculus rotundus appeared as very developed lymphoid structure at the end of ileum even in the newly born kits. Numerous lymphoid follicles and its dome shaped structures were distributed along whole circumference of the wall of this organ with very distinctive follicular associated epithelium and M cells.
The surface epithelium of proximal colon possess villi-like structures and several lymphoid follicles within its lamina propria while the surface epithelium of distal colon was straight but its crypts were more packed than those of proximal part. The two parts of colon possess longitudinal folds in their wall with large number of goblet cells and vacuolated cell within their epithelium.
Carbohydrate profile of small intestine and colon revealed dominance of non sulfated (carboxylated) acidic mucin in the crypts and Brunner's glands whereas the neutral and sulfated mucins were dominant in the villi and surface epithelium. Glycosaminglycans was increased with age progress especially in ileum and colon.
... To check for the possibility that the lack of detection of galectins-4 and -6 in non-gastrointestinal tissues was due to loss of RNA, the blot was reprobed for GAPDH mRNA, known to have a relatively even distribution in most tissues (including the gastrointestinal tract) and commonly used as a marker of RNA integrity (e.g. Ref. 25). Except for lane b (tongue), the amount of RNA in the lanes from the non-gastrointestinal tissues was within the same order of magnitude as for lanes f-j, as estimated by the amount of GAPDH mRNA. ...
Galectins are a family of carbohydrate-binding proteins that share a conserved sequence and affinity for β-galactosides. Some,
such as galectin-1, are isolated as dimers and have a single carbohydrate recognition domain (CRD) in each monomer, whereas
others, such as galectin-4, are isolated as monomers and have two CRDs in a single polypeptide chain. In the course of studying
mouse colon mRNA for galectin-4, we detected a related mRNA that encodes a new galectin that also has two CRDs in a single
peptide chain. The new galectin, galectin-6, lacks a 24-amino acid stretch in the link region between the two CRDs that is
present in galectin-4. Otherwise, these two galectins have 83% amino acid identity. Expression of both galectin-4 and galectin-6
is confined to the epithelial cells of the embryonic and adult gastrointestinal tract. Galectin-4 is expressed at about equal
levels in colon and small intestine but much less in stomach, whereas galectin-6 is expressed at about equal levels throughout
the gastrointestinal tract.
... Demonstrating the validity of this approach, several proteins previously shown to be altered in expression along the crypt-villus axis were similarly identified in the present study. These included villin [21], ezrin [40], CRBPII [29] and FABP1 [29], each of which were maximally expressed in the villus compartment. Analyses to identify functionally enriched categories of proteins demonstrated that proteins with a role in lipid/fatty acid/steroid metabolism and cell structure were significantly enriched among the differentially expressed proteins identified. ...
Intestinal epithelial cells undergo progressive cell maturation as they migrate along the crypt-villus axis. To determine molecular signatures that define this process, proteins differentially expressed between the crypt and villus were identified by 2D-DIGE and MALDI-MS. Forty-six differentially expressed proteins were identified, several of which were validated by immunohistochemistry. Proteins upregulated in the villus were enriched for those involved in brush border assembly and lipid uptake, established features of differentiated intestinal epithelial cells. Multiple proteins involved in glycolysis were also upregulated in the villus, suggesting increased glycolysis is a feature of intestinal cell differentiation. Conversely, proteins involved in nucleotide metabolism, and protein processing and folding were increased in the crypt, consistent with functions associated with cell proliferation. Three novel paneth cell markers, AGR2, HSPA5 and RRBP1 were also identified. Notably, significant correlation was observed between overall proteomic changes and corresponding gene expression changes along the crypt-villus axis, indicating intestinal cell maturation is primarily regulated at the transcriptional level. This proteomic profiling analysis identified several novel proteins and functional processes differentially induced during intestinal cell maturation in vivo. Integration of proteomic, immunohistochemical, and parallel gene expression datasets demonstrate the coordinated manner in which intestinal cell maturation is regulated.
... In normal intestinal epithelial cells, ribosomes are concentrated in the perinuclear region, away from the subapical cytoplasm (Kasai et al. 2003). More importantly, available in situ hybridization data on the distribution of ezrin mRNA in enterocytes suggests a diffuse, perhaps supranuclear distribution rather than apical localization of ezrin mRNA (Barilá et al., 1995). In tall, slender cells such as enterocytes, it means that newly synthesized ezrin must travel several microns to reach its final destination under the apical membrane. ...
Atypical protein kinase iota (PKCiota) is a key organizer of the apical domain in epithelial cells. Ezrin is a cytosolic protein that, upon activation by phosphorylation of T567, is localized under the apical membrane where it connects actin filaments to membrane proteins and recruits protein kinase A (PKA). To identify the kinase that phosphorylates ezrin T567 in simple epithelia, we analyzed the expression of active PKC and the appearance of T567-P during enterocyte differentiation in vivo. PKCiota phosphorylated ezrin on T567 in vitro, and in Sf9 cells that do not activate human ezrin. In CACO-2 human intestinal cells in culture, PKCiota co-immunoprecipitated with ezrin and was knocked down by shRNA expression. The resulting phenotype showed a modest decrease in total ezrin, but a steep decrease in T567 phosphorylation. The PKCiota-depleted cells showed fewer and shorter microvilli and redistribution of the PKA regulatory subunit. Expression of a dominant-negative form of PKCiota also decreased T567-P signal, and expression of a constitutively active PKCiota mutant showed depolarized distribution of T567-P. We conclude that, although other molecular mechanisms contribute to ezrin activation, apically localized phosphorylation by PKCiota is essential for the activation and normal distribution of ezrin at the early stages of intestinal epithelial cell differentiation.
Multidrug resistance-associated protein 2 (MRP2)/ATP-binding cassette protein C2 (ABCC2) and multidrug resistance protein 1 (MDR1)/ABCB1 are well-known efflux transporters located on the brush border membrane of the small intestinal epithelia, where they limit the absorption of a broad range of substrates. The expression patterns of MRP2/ABCC2 and MDR1/ABCB1 along the small intestinal tract are tightly regulated. Several reports have demonstrated the participation of ERM (ezrin/radixin/moesin) proteins in the post-translational modulation of MRP2/ABCC2 and MDR1/ABCB1, especially with regard to their membrane localization. The present study focused on the in vivo expression profiles of MRP2/ABCC2, MDR1/ABCB1, ezrin, and phosphorylated ezrin to further elucidate the relationship between the efflux transporters and the ERM proteins. The current results showed good correlation between the phosphorylation status of ezrin and Mrp2/Abcc2 expression along the gastrointestinal tract of rats, and between the expression profiles of both ezrin and Mdr1/Abcb1 in the small intestine. We also demonstrated the involvement of conventional protein kinase C isoforms in the regulation of ezrin phosphorylation. Furthermore, experiments conducted with wild-type (WT) ezrin and a T567A (Ala substituted Thr) dephosphorylated mutant showed a decrease in membrane surface-localized and total expressed MRP2/ABCC2 in T567A-expressing vs. WT ezrin-expressing Caco-2 cells. In contrast, T567A- and WT-expressing cells both showed an increase in membrane surface-localized and total expressed MDR1/ABCB1. These findings suggest that the phosphorylation status and the expression profile of ezrin differentially direct MRP2/ABCC2 and MDR1/ABCB1 expression, respectively, along the small intestinal tract.
We have previously reported that the induction of Vitamin A deficiency results in a threefold decrease in the hepatic expression of cellular retinol binding protein I (CRBP I) mRNA in vivo and that the treatment of intestinal cell lines in vitro with retinoids leads to the induction of CRBP I transcription. In the present paper we extend the analysis to retinoid-dependent gene expression in the testicular epithelium in vivo and in the intestinal cell line FRIC B. In rat testis excess Vitamin A results in the reduced production of mature spermatozoa and in the premature release of immature germ cells in the lumen, while Vitamin A deficiency leads to almost complete degeneration of the germinal epithelium. We show reduced level of expression of CRBP I mRNA in vitamin A deficient testis. Retinoid treatment of cultured intestinal cells, which induces a reorganization of the actin cytoskeleton, has no effect on the expression of the differentiation induced gene Dri 42. The results show that even though unable to trigger by themselves the differentiation process, retinoids exert a direct effect on the expression of specific genes.
Ezrin, which cross-links the cytoskeleton and plasma membrane, was involved in a wide variety of cellular processes. Here, to investigate the distribution of ezrin, tissue microarray technology was employed to perform immunohistochemical experiments on human embryos, fetuses at 4 to 22 weeks' gestation, and adult tissue specimens. Results showed that ezrin was widely expressed in the gastrointestinal tract throughout the human developmental stages studied. At 6 to 8 weeks' gestation, ezrin was found in epithelial cells, and this staining pattern was particularly pronounced in the brush border of mature absorptive cells lining the villus in later developmental stages and adult tissues. Throughout neural development, ezrin was only expressed in the neural tube at 4 weeks' gestation. Ezrin was also detected in the cortex and medulla of the adrenal gland at 8 to 12 weeks' gestation, whereas its immunoreactivity was increased from the zona glomerulosa through the zona reticularis and was essentially undetectable in the adrenal medulla of adult tissues. Significant expression of ezrin was seen throughout development in the kidney, spleen, lymph nodes, and cells of stratified squamous epithelia. However, ezrin was undetectable in lung, liver, heart, and blood vessels. These results demonstrated that the expression pattern of ezrin was highly time specific and tissue specific.
To understand the molecular mechanism underlying vigorous proliferative activity of hepatic stem-like (HSL) cells, we performed two-dimensional electrophoresis to identify the proteins statistically more abundant in rapidly growing undifferentiated HSL cells than in sodium butyrate-treated differentiated HSL cells. Matrix-assisted laser desorption/ionization-time of flight mass spectrometry and Mascot search identified 6 proteins including prohibitin, vimentin, ezrin, annexin A3, acidic ribosomal phosphoprotein P0 and Grp75. Prohibitin and vimentin control the mitogen-activated protein (MAP) kinase pathway. Ezrin is phosphorylated by various protein-tyrosine kinases and modulates interactions between cytoskeletal and membrane proteins. Annexin A3 has a role in DNA synthesis. Acidic ribosomal phosphoprotein P0 and Grp75 play in protein synthesis. These results suggest that the proteins related to the MAP kinase cascade had some role in continuous proliferation of HSL cells without differentiation.
A search for novel genes that are up-regulated during development and differentiation of the epithelial cells of the intestinal mucosa led us to the isolation of the Dri 42 cDNA clone (Dri, differentially expressed in rat intestine). The nucleotide sequence of the full-length cDNA has shown that it encodes a 35.5-kDa protein with one consensus sequence for N-linked glycosylation and alternating hydrophilic and hydrophobic domains. To determine the intracellular localization of Dri 42 we have raised polyclonal antibodies in hens against a bacterially produced Dri 42-glutathione S-transferase fusion protein. Immunofluorescence detection with these antibodies has shown specific staining of the endoplasmic reticulum (ER) in the relatively undifferentiated fetal rat intestinal cell line FRIC B and in sections of rat small intestine. ER membrane localization of Dri 42 was confirmed by laser confocal microscopy of polarized Madin-Darby canine kidney cells overexpressing a Dri 42-chloramphenicol acetyltransferase (CAT) fusion protein by transfection. Pulse labeling experiments on transiently transfected cells demonstrated that the protein does not acquire Golgi modifications up to 4 h after synthesis, thus indicating that Dri 42 is an ER resident protein. The transmembrane disposition of Dri 42 was studied using in vitro insertion of Dri 42-CAT fusion proteins into microsomal membranes. The fusion proteins consisted of several different lengths of truncated Dri 42 and a reporter protein, CAT, that was linked in-frame after each hydrophobic segment. We found that hydrophobic segments H1, H3, and H5 had a signal/anchor function, and that membrane insertion of Dri 42 was achieved co-translationally by the action of a series of alternating insertion signals and halt transfer signals, resulting in the exposure of both termini of the protein to the cytosolic side. The functional implications of the structure and localization of Dri 42, whose primary sequence does not share significant homology to any previously described protein, are discussed.
Identification of proteins that are differentially expressed in mammals that hibernate can provide insight into mechanisms that preserve cellular function at low temperatures. A candidate protein was identified in intestinal brush border membranes of 13-lined ground squirrels. Intestinal brush border membrane proteins were separated using SDS-PAGE and gels were stained with Coomassie blue. We observed a approximately 75-kDa band that was specifically increased in brush border membranes isolated from torpid squirrels compared with summer active squirrels. The 75-kDa band was cut from one-dimensional gels and sequenced. A 17 amino acid sequence was identified of which amino acids 2-17 matched exactly a portion of moesin, a membrane-cytoskeletal linking protein and member of the ERM (ezrin/radixin/moesin) family. The sequence results were confirmed using anti-moesin antibodies that detected strong bands at approximately 75 kDa on Western blots of brush border membranes in torpid squirrels (Tb approximately 7 degreesC) and only faint signals in summer squirrels (Tb approximately 37 degrees C) or aroused hibernators (Tb approximately 37 degrees C). In contrast, signals obtained using anti-ezrin antibodies were uniformly strong in all squirrels, regardless of activity state. Intestinal brush borders of mice and rats expressed ezrin but not moesin. These results provide evidence for the physiological induction of an ERM protein in intestinal epithelial cells of torpid hibernators and support the idea that hibernation involves differential expression of gene products that may facilitate viability of cells at low temperatures.
Stem cells in the intestinal epithelium give rise to enterocytes, goblet cells, enteroendocrine cells, and Paneth cells. Each of these cell lines plays a role in cytoprotection of the intestinal mucosa. In particular, it has been demonstrated that mature enterocytes can act as antigen presenting cells. Parenteral and enteral nutrition are used to nourish critically ill patients. However, these regimens are unfortunately associated with gut atrophy. Glutamine, the preferred intestinal nutrient, reverses this gut atrophy and plays a key role in maintaining the barrier function of the gut. Specific nutrients (putrescine, spermidine, spermine) have been used to modulate intestinal adaption. In addition, ornithine has been shown to act as a regulator of intestinal adaption. In this review, we discuss the relationship between the biology of enterocytes and failure of the gut barrier.
Cytochalasins are cytoskeleton disrupters, and cytochalasin E has been reported to increase intestinal paracellular permeability. In this study, the cytochalasin E effect on galactose transport has been investigated. Ussing-type chamber experiments show an inhibitory effect of 20 microM cytochalasin E on unidirectional mucosal to serosal flux of galactose. On the contrary, the opposite unidirectional flux is not modified by the inhibitor. Results using intestinal everted sacs and rings confirm that galactose uptake by the tissue is diminished by cytochalasin E. The effect appears already after 5 min incubation, depends on cytochalasin E concentration, and does not occur in the absence of Na+. The inhibition is accompanied by an increase in the apparent K(m) of the active sugar transport (11.5 vs.15.8 mM) without significant change in the VmaX (10.6 vs. 9.1 micromol x g(-1) wet weight x 5 min(-1)). Cytochalasin E does not modify either galactose uptake by brush border membrane vesicles or Na(+)-K(+) ATPase activity in the enterocytes, indicating that the inhibitory effect on the Na(+)-dependent sugar transport cannot be explained as a direct effect on SGLT1 activity or as an indirect effect through the Na(+)-K(+) ATPase. Thus, our results suggest that cytochalasin E decreases SGLTI activity indirectly through cytoskeleton disruption.
Organ transplantation has become routine in many centres throughout the world. Although success rates for intestinal transplantation are generally lower than for other organs, it is still high enough to warrant this form of therapy for patients with short bowel syndrome or other untreatable bowel diseases. Much of the success in transplantation is due to the impact of immunosuppressive drugs as well as the development of new surgical techniques and new methods of organ preservation. The intestine has been preserved successfully for about 24 hours, as judged by in vitro viability assessment1 and by survival after transplantation.2 However, problems associated with extended cold storage, ischaemia/reperfusion injury, and immunological rejection of grafts still limit the optimal use of intestinal transplantation. The benefit of hypothermic organ preservation is generally attributed to the reduction in oxygen and nutrient demands and in the rates of potentially damaging enzyme catalysed reactions. However, lack of oxygen in cold stored organs gradually leads to a time dependent loss of ATP, with rates of degradative reactions exceeding biosynthesis. In addition, prolonged hypothermia alters cell structure (for example, cytoskeletal proteins), reduces ion pump activity, and slows ATP synthesis. These changes, coupled with oxidative stress secondary to ischaemia-reperfusion injury and immunological factors, eventually lead to loss of metabolic control and critical membrane functions that compromise organ survival on reperfusion. The result for the cold stored transplanted bowel is too often mucosal destruction, loss of absorptive and barrier function, and eventual graft rejection.
Improvements in intestinal preservation could increase organ availability and perhaps more importantly the likelihood that a transplant is successful. To this end, novel insights may be found in animals that naturally experience extreme changes in physiology, particularly hypoxia and hypothermia. Mammalian hibernators offer the unique opportunity to examine what is essentially Nature's version of organ preservation. …
We find that ezrin, a cytoskeletal protein involved in anchoring actin to the cell membrane, is preferentially degraded and resynthesized after oxidative stress. Ezrin was identified using 2-dimensional gels and amino-terminal microsequencing as one of a select few [35S]methionine prelabeled proteins degraded in clone 9 rat liver cells exposed to hydrogen peroxide (H2O2). Metabolic labeling of cellular proteins with [35S]methionine after oxidative stress showed that resynthesis of ezrin rose dramatically but carboxyl terminus anti-ezrin monoclonal antibodies revealed constant intracellular ezrin levels; in other words, degradation and resynthesis were exactly matched. Ezrin degradation was blocked by selective inhibitors of the proteasome (lactacystin, NLVS, and epoxomycin) and by an antisense oligonucleotide directed against the proteasome C2 subunit. H2O2 also caused major changes in cell shape, including significant increases in cell diameter, which must require substantial cytoskeletal rearrangement. Peroxide-induced increases in cell diameter were, however, blocked by the selective proteasome inhibitor lactacystin. The degradation and resynthesis of ezrin may therefore be an underlying mechanism for overall cell shape changes observed during oxidative stress. Oxidative stress induces extensive protein oxidation and degradation and significant increases in cell blebbing, rounding-up, and overall size. Our results indicate that all these oxidant-induced changes may actually be catalyzed by the proteasome.
Mammalian hibernators undergo a remarkable phenotypic switch that involves profound changes in physiology, morphology, and behavior in response to periods of unfavorable environmental conditions. The ability to hibernate is found throughout the class Mammalia and appears to involve differential expression of genes common to all mammals, rather than the induction of novel gene products unique to the hibernating state. The hibernation season is characterized by extended bouts of torpor, during which minimal body temperature (Tb) can fall as low as -2.9 degrees C and metabolism can be reduced to 1% of euthermic rates. Many global biochemical and physiological processes exploit low temperatures to lower reaction rates but retain the ability to resume full activity upon rewarming. Other critical functions must continue at physiologically relevant levels during torpor and be precisely regulated even at Tb values near 0 degrees C. Research using new tools of molecular and cellular biology is beginning to reveal how hibernators survive repeated cycles of torpor and arousal during the hibernation season. Comprehensive approaches that exploit advances in genomic and proteomic technologies are needed to further define the differentially expressed genes that distinguish the summer euthermic from winter hibernating states. Detailed understanding of hibernation from the molecular to organismal levels should enable the translation of this information to the development of a variety of hypothermic and hypometabolic strategies to improve outcomes for human and animal health.
Because most potential molecular markers and targets are proteins, proteomic profiling is expected to yield more direct answers to functional and pharmacological questions than does transcriptional profiling. To aid in such studies, we have developed a protocol for making reverse-phase protein lysate microarrays with larger numbers of spots than previously feasible. Our first application of these arrays was to profiling of the 60 human cancer cell lines (NCI-60) used by the National Cancer Institute to screen compounds for anticancer activity. Each glass slide microarray included 648 lysate spots representing the NCI-60 cell lines plus controls, each at 10 two-fold serial dilutions to provide a wide dynamic range. Mouse monoclonal antibodies and the catalyzed signal amplification system were used for immunoquantitation. The signal levels from the >30,000 data points for our first 52 antibodies were analyzed by using p-scan and a quantitative dose interpolation method. Clustered image maps revealed biologically interpretable patterns of protein expression. Among the principal early findings from these arrays were two promising pathological markers for distinguishing colon from ovarian adenocarcinomas. When we compared the patterns of protein expression with those we had obtained for the same genes at the mRNA level by using both cDNA and oligonucleotide arrays, a striking regularity appeared: cell-structure-related proteins almost invariably showed a high correlation between mRNA and protein levels across the NCI-60 cell lines, whereas non-cell-structure-related proteins showed poor correlation.
Protein 4.1 family proteins are thought to interact with membrane proteins and also membrane skeletons. In this study, immunohistochemical studies by light and electron microscopy were performed with a specific antibody against protein 4.1B. Specific protein 4.1B immunolabeling was observed in simple columnar epithelium in the adult rat large intestine, small intestine and stomach. Protein 4.1B immunolabeling was localized along the membranes facing the adjacent cells (lateral portion) and also facing the extracellular matrix (basal portion). Moreover, a spatial protein 4.1B expression gradient was observed along the crypt-villus axis of the rat small and large intestinal epithelium: strong protein 4.1B expression was present within the villus, with the crypt showing barely any detectable protein 4.1B. The expression of protein 4.1B was not detected in the stratified squamous epithelium in the forestomach or the esophagus. By immunoelectron microscopy, the immunolabeling of the cells was observed to be restricted to the cytoplasmic side just beneath the plasma membrane, including the membranes adjacent to the next cells, except for the tight junctions. We conclude that the protein 4.1B expression pattern is related to the maturation of simple columnar epithelium in the rat digestive system, probably by the effect of adhesion.
To evaluate the utility of transcript profiling for prediction of protein expression levels, we compared profiles across the NCI-60 cancer cell panel, which represents nine tissues of origin. For that analysis, we present here two new NCI-60 transcript profile data sets (A based on Affymetrix HG-U95 and HG-U133A chips; Affymetrix, Santa Clara, CA) and one new protein profile data set (based on reverse-phase protein lysate arrays). The data sets are available online at http://discover.nci.nih.gov in the CellMiner program package. Using the new transcript data in combination with our previously published cDNA array and Affymetrix HU6800 data sets, we first developed a "consensus set" of transcript profiles based on the four different microarray platforms. Using that set, we found that 65% of the genes showed statistically significant transcript-protein correlation, and the correlations were generally higher than those reported previously for panels of mammalian cells. Using the predictive analysis of microarray nearest shrunken centroid algorithm for functional prediction of tissue of origin, we then found that (a) the consensus mRNA set did better than did data from any of the individual mRNA platforms and (b) the protein data seemed to do somewhat better (P = 0.027) on a gene-for-gene basis in this particular study than did the consensus mRNA data, but both did well. Analysis based on the Gene Ontology showed protein levels of structure-related genes to be well predicted by mRNA levels (mean r = 0.71). Because the transcript-based technologies are more mature and are currently able to assess larger numbers of genes at one time, they continue to be useful, even when the ultimate aim is information about proteins.
The ontogeny of alpha-fetoprotein (AFP) gene expression has been examined in the fetal and adult mouse gastrointestinal tract. AFP mRNA constitutes approximately 0.1% of total mRNA in the fetal gut. The transcripts were localized by in situ hybridization to the epithelial cells lining the villi of the fetal gut. At birth, AFP mRNA declines rapidly to achieve low adult basal levels, which are not affected by different alleles of raf, a gene that determines the adult basal level of AFP mRNA in the liver. The basal level in the adult gut is the consequence of continued AFP transcription in a small number of enteroendocrine cells that are distributed infrequently on the villi. These cells were identified by double antibody staining with antibodies to chromogranin A, an enteroendocrine cell marker and AFP. Previous studies resulted in the generation of a line of transgenic mice containing an internally deleted AFP gene that was greatly overexpressed in the fetal gut. The basis for the inappropriately high level expression of the transgene was shown to be the consequence of very high levels of transcription in the epithelial cells of the villi rather than to expression in inappropriate cell types. The cis-acting DNA sequences required for expression of the AFP gene in the gut were investigated using Caco-2 cells, a human colon adenocarcinoma cell line. These experiments indicated that, with one exception, the regulatory elements required in both the promoter and enhancer regions of the gene coincided with those that are necessary for high level expression in the liver. The one exception was enhancer II, located 5 kbp of DNA upstream of the gene, which exhibited no activity in Caco-2 cells.
To examine the functions of ERM family members (ezrin, radixin, and moesin), mouse epithelial cells (MTD-1A cells) and thymoma cells (L5178Y), which coexpress all of them, were cultured in the presence of antisense phosphorothioate oligonucleotides (PONs) complementary to ERM sequences. Immunoblotting revealed that the antisense PONs selectively suppressed the expression of each member. Immunofluorescence microscopy of these ezrin, radixin, or moesin "single-suppressed" MTD-1A cells revealed that the ERM family members are colocalized at cell-cell adhesion sites, microvilli, and cleavage furrows, where actin filaments are densely associated with plasma membranes. The ezrin/radixin/moesin antisense PONs mixture induced the destruction of both cell-cell and cell-substrate adhesion, as well as the disappearance of microvilli. Ezrin or radixin antisense PONs individually affected the initial step of the formation of both cell-cell and cell-substrate adhesion, but did not affect the microvilli structures. In sharp contrast, moesin antisense PONs did not singly affect cell-cell and cell-substrate adhesion, whereas it partly affected the microvilli structures. These data indicate that ezrin and radixin can be functionally substituted, that moesin has some synergetic functional interaction with ezrin and radixin, and that these ERM family members are involved in cell-cell and cell-substrate adhesion, as well as microvilli formation.
In response to epidermal growth factor (EGF) the microvillar core protein ezrin is phosphorylated transiently to a high level on tyrosine residues in human epidermoid carcinoma A431 cells. Here we report the identification of the tyrosine phosphorylation sites in ezrin using bacterially expressed protein as a substrate for in vitro phosphorylation with the EGF receptor. The two major phosphotyrosine-containing peptides observed in vivo were also phosphorylated in vitro. By secondary digestions and site-directed mutagenesis tyrosines 145 and 353 were identified as the sites of phosphorylation. One of the sites, Tyr145, lies in the N-terminal region of homology that is common to the band 4.1-talin-ezrin protein family. This tyrosine residue and its vicinal amino acids are conserved throughout the family members, including radixin, moesin, and the two phosphotyrosine phosphatases, PTP H1 and PTP MEG, but not in band 4.1 or talin. Tyr353 is localized within the alpha-helical domain of ezrin and comparison of the protein sequences reveals that this site is unique to ezrin.
The ontogeny of alpha-fetoprotein (AFP) gene expression has been examined in the fetal and adult mouse gastrointestinal tract. AFP mRNA constitutes approximately 0.1% of total mRNA in the fetal gut. The transcripts were localized by in situ hybridization to the epithelial cells lining the villi of the fetal gut. At birth, AFP mRNA declines rapidly to achieve low adult basal levels, which are not affected by different alleles of raf, a gene that determines the adult basal level of AFP mRNA in the liver. The basal level in the adult gut is the consequence of continued AFP transcription in a small number of enteroendocrine cells that are distributed infrequently on the villi. These cells were identified by double antibody staining with antibodies to chromogranin A, an enteroendocrine cell marker and AFP. Previous studies resulted in the generation of a line of transgenic mice containing an internally deleted AFP gene that was greatly overexpressed in the fetal gut. The basis for the inappropriately high level expression of the transgene was shown to be the consequence of very high levels of transcription in the epithelial cells of the villi rather than to expression in inappropriate cell types. The cis-acting DNA sequences required for expression of the AFP gene in the gut were investigated using Caco-2 cells, a human colon adenocarcinoma cell line. These experiments indicated that, with one exception, the regulatory elements required in both the promoter and enhancer regions of the gene coincided with those that are necessary for high level expression in the liver. The one exception was enhancer II, located 5 kbp of DNA upstream of the gene, which exhibited no activity in Caco-2 cells.
Moesin (membrane-organizing extension spike protein, pronounced mó ez in) has previously been isolated from bovine uterus and characterized as a possible receptor protein for heparan sulfate. We now have cloned and sequenced its complete cDNA, which represents a single 4.2-kilobase mRNA encoding a protein of 577 amino acids. It contains no apparent signal peptide or transmembrane domain. In addition, the protein shows significant sequence identity (72%) to ezrin (cytovillin, p81), as well as similarity to protein 4.1 and talin. All of the latter proteins have been postulated to serve as structural links between the plasma membrane and the cytoskeleton. A similar role for moesin is implied by structure and domain predictions derived from the cDNA-deduced peptide sequence. Furthermore, our data indicate that moesin is identical to the 77-kDa band that copurifies with ezrin in its isolation from human placenta [Bretscher, A. (1989) J. Cell Biol. 108, 921-930].
Radixin is an actin barbed-end capping protein which is highly concentrated in the undercoat of the cell-to-cell adherens junction and the cleavage furrow in the interphase and mitotic phase, respectively (Tsukita, Sa., Y. Hieda, and Sh. Tsukita. 1989 a.J. Cell Biol. 108:2369-2382; Sato, N., S. Yonemura, T. Obinata, Sa. Tsukita, and Sh. Tsukita. 1991. J. Cell Biol. 113:321-330). To further understand the structure and functions of the radixin molecule, we isolated and sequenced the cDNA clones encoding mouse radixin. Direct peptide sequencing of radixin and immunological analysis with antiserum to a fusion protein were performed to confirm that the protein encoded by these clones is identical to radixin. The composite cDNA is 4,241 nucleotides long and codes for a 583-amino acid polypeptide with a calculated molecular mass of 68.5 kD. Sequence analysis has demonstrated that mouse radixin shares 75.3% identity with human ezrin, which was reported to be a member of the band 4.1 family. We then isolated the cDNA encoding mouse ezrin. Sequence analysis and Northern blot analysis revealed that radixin and ezrin are similar but distinct (74.9% identity), leading us to conclude that radixin is a novel member of the band 4.1 family. In erythrocytes the band 4.1 protein acts as a key protein in the association of short actin filaments with a plasma membrane protein (glycophorin), together with spectrin. Therefore, the sequence similarity between radixin and band 4.1 protein described in this study favors the idea that radixin plays a crucial role in the association of the barbed ends of actin filaments with the plasma membrane in the cell-to-cell adherens junction and the cleavage furrow.
Villin is an evolutionarily well conserved, Ca2+ regulated actin-binding protein, and a major structural component of the brush border of specialized absorptive cells. Using paraffin sections and an affinity purified polyclonal anti-villin antibody, we have investigated the early expression of villin during mouse embryogenesis. Villin is first detectable at the early post-implantation stage in visceral endodermal cells at the periphery of the egg cylinder. In this extra embryonic layer, the expression of villin increases and then persists until full term gestation. In the embryo, villin first appears in gut anlage during the axial rotation. Using the same methodology, villin expression is also demonstrated in differentiating embryoid bodies from a teratocarcinoma. Both in extra embryonic and embryonic extracts, villin expression is confirmed by immunoblot and Northern blot analysis which reveal, respectively, a single polypeptide of 93 kd and an mRNA of 3.4 kb in length, two well defined parameters for adult mouse villin gene expression. The results presented here show that paraffin sections allow very sensitive and highly resolutive detection of antigens in early embryogenesis. They provide a detailed developmental profile of villin expression and demonstrate the usefulness of villin as a marker for epithelial cells involved in absorptive processes.
Ezrin is a component of the microvilli of intestinal epithelial cells and serves as a major cytoplasmic substrate for certain protein-tyrosine kinases. We have cloned and sequenced a human ezrin cDNA and report here the entire protein sequence derived from the nucleotide sequence of the cDNA as well as from partial direct protein sequencing. The deduced protein sequence indicates that ezrin is a highly charged protein with an overall pI of 6.1 and a calculated molecular mass of 69,000. The cDNA clone was used to survey the distribution of the ezrin transcript, and the 3.2 kb ezrin mRNA was found to be expressed in the same tissues that are known to express the protein and at the same relative levels. Highest expression was found in intestine, kidney and lung. The cDNA clone hybridized to DNAs from widely divergent organisms indicating that its sequence is highly conserved throughout evolution. The amino acid sequence of ezrin revealed a high degree of similarity within its N-terminal domain to the erythrocyte cytoskeletal protein, band 4.1 and secondary structure predictions indicate that a second region of ezrin contains a long alpha-helix, a feature also common to band 4.1. The structural similarity of ezrin to band 4.1 suggests a mechanism for the observed localization to the membrane, and a role for ezrin in modulating the association of the cortical cytoskeleton with the plasma membrane.
Addition of EGF to human carcinoma A-431 cells is known to induce membrane ruffling after approximately 2 min (Chinkers, M., J. A. McKanna, and S. Cohen. 1979. J. Cell Biol. 83:260-265) and the phosphorylation of a protein referred to as p81, a known substrate for various protein-tyrosine kinases (Cooper, J. A., D. F. Bowen-Pope, E. Raines, R. Ross, and T. Hunter. 1982. Cell. 31:263-273). Ezrin, a Mr approximately 80,000 cytoskeletal protein of the isolated chicken microvillar core, is present in actin-containing cell surface structures of a wide variety of cells (Bretscher, A. 1983. J. Cell Biol. 97:425-432). Ezrin was then found to be homologous to p81 and to be phosphorylated on tyrosine in response to EGF (Gould, K. L., J. A. Cooper, A. Bretscher, and T. Hunter. 1986. J. Cell Biol. 102:660-669). Here, the purification of ezrin from human placenta is described. Antibodies to human ezrin, together with antibodies to other microfilament-associated proteins, were used to follow the distribution and phosphorylation of these proteins in A-431 cells after EGF treatment. EGF induces the formation of microvillar-like surface structures on these cells within 30 s and these give way to membrane ruffles at approximately 2-5 min after EGF addition; the cells then round up after approximately 10-20 min. Ezrin is recruited into the microvillar-like structures and the membrane ruffles, and is phosphorylated on tyrosine and serine in a time course that parallels the formation and disappearance of these surface structures. Spectrin is recruited into the membrane ruffles and shows a similar rapid kinetics of phosphorylation, but only on serine residues, and remains phosphorylated through the rounding up of the cells. The microvillar-like structures and membrane ruffles are also enriched in fimbrin and alpha-actinin. Myosin becomes rapidly reorganized into a striated pattern that is consistent with it playing a role in cell rounding. These results show that two cortical proteins, ezrin and spectrin, become phosphorylated in a time course coincident with remodeling of the cell surface. The results are consistent with the notion that ezrin phosphorylation may play a role in the formation of cell surface projections whereas spectrin phosphorylation may be involved in remodelling of more planar areas of the cell surface.
The mammalian intestinal epithelium represents a unique model system for studying cellular differentiation since it undergoes continuous and rapid renewal. Perpetual differentiation results in the generation of its four principal cell types, each with a unique phenotype. The processes of proliferation and cellular differentiation are topologically very well organized. Gradients in gene expression are established and maintained in several spatial dimensions within this epithelium. Recent experiments involving mouse aggregation chimeras and transgenic mice have provided insights about the origins of intestinal stem cells, the migration pathways followed during cellular differentiation as well as the molecular mechanisms which produce complex geographic differences in gut gene expression. The purpose of this mini-review is to emphasize the usefulness of these powerful methods for examining questions related to enteric epithelial biology.
Villin, a Ca2+-regulated actin-binding protein is a major component of microvilli of intestinal epithelial cells and kidney proximal tubule cells. Villin expression during assembly of the brush border can be investigated using a human colon adenocarcinoma cell line HT29-18. This cell line is able to differentiate under nutritional control and develops an enterocyte-like phenotype. A cDNA library from a subclone HT29-18-C1 was constructed in an expression vector and a cDNA specific for human villin was isolated. This cDNA codes for the 110 carboxy-terminal residues of villin. Within that region, the 76 carboxy-terminal residues present 65% homology with the chicken villin 'head piece'. We show that two mRNA species 4.0 kb and 3.2 kb long hybridize with this cDNA probe in humans, whereas in rat and chicken only one mRNA species can be detected. The two villin mRNA species are co-expressed in normal human small and large intestinal mucosa and tumoral HT29-18 cells as well as in normal kidney. No villin mRNAs were detected in other normal or malignant epithelial cell types. Finally, we observed an accumulation of the two mRNA species coding for villin when HT29-18 cells become differentiated, suggesting that control of villin expression during terminal differentiation can occur at the transcription level or by RNA stabilization.
To examine the functions of ERM family members (ezrin, radixin, and moesin), mouse epithelial cells (MTD-1A cells) and thymoma cells (L5178Y), which coexpress all of them, were cultured in the presence of antisense phosphorothioate oligonucleotides (PONs) complementary to ERM sequences. Immunoblotting revealed that the antisense PONs selectively suppressed the expression of each member. Immunofluorescence microscopy of these ezrin, radixin, or moesin "single-suppressed" MTD-1A cells revealed that the ERM family members are colocalized at cell-cell adhesion sites, microvilli, and cleavage furrows, where actin filaments are densely associated with plasma membranes. The ezrin/radixin/moesin antisense PONs mixture induced the destruction of both cell-cell and cell-substrate adhesion, as well as the disappearance of microvilli. Ezrin or radixin antisense PONs individually affected the initial step of the formation of both cell-cell and cell-substrate adhesion, but did not affect the microvilli structures. In sharp contrast, moesin antisense PONs did not singly affect cell-cell and cell-substrate adhesion, whereas it partly affected the microvilli structures. These data indicate that ezrin and radixin can be functionally substituted, that moesin has some synergetic functional interaction with ezrin and radixin, and that these ERM family members are involved in cell-cell and cell-substrate adhesion, as well as microvilli formation.
Ezrin, a widespread protein present in actin-containing cell-surface structures, is a substrate of some protein tyrosine kinases. Based on its primary and secondary structure similarities with talin and band 4.1 it has been suggested that this protein could play a role in linking the cytoskeleton to the plasma membrane (Gould, K.L., A. Bretscher, F.S. Esch, and T. Hunter. 1989. EMBO (Eur. Mol. Biol. Organ.), J. 8:4133-4142; Turunen, O., R. Winqvist, R. Pakkanen, K.-H. Grzeschik, T. Wahlström, and A. Vaheri. 1989. J. Biol. Chem. 264:16727-16732). To test this hypothesis, we transiently expressed the complete human ezrin cDNA, or truncated cDNAs encoding the amino- and carboxy-terminal domains of the protein, in CV-1 cells. Protein epitope tagging was used to unambiguously determine the subcellular distribution of the protein encoded by the transfected cDNA. We show that this protein is concentrated underneath the dorsal plasma membrane in all actin-containing structures and is partially detergent insoluble. The amino-terminal domain displays the same localization but is readily extractable by nonionic detergent. The carboxy-terminal domain colocalizes with microvillar actin filaments as well as with stress fibers and remains associated with actin filaments after detergent extraction, and with disorganized actin structures after cytochalasin D treatment. Our results clearly demonstrate that ezrin interacts with membrane-associated components via its amino-terminal domain, and with the cytoskeleton via its carboxy-terminal domain. The amino-terminal domain could include the main determinant that restricts the entire protein to the cortical cytoskeleton in contact with the dorsal plasma membrane and its specialized microdomains such as microvilli, microspikes and lamellipodia.
Radixin is an actin barbed-end capping protein which is highly concentrated in the undercoat of the cell-to-cell adherens junction and the cleavage furrow in the interphase and mitotic phase, respectively (Tsukita, Sa., Y. Hieda, and Sh. Tsukita. 1989 a.J. Cell Biol. 108:2369-2382; Sato, N., S. Yonemura, T. Obinata, Sa. Tsukita, and Sh. Tsukita. 1991. J. Cell Biol. 113:321-330). To further understand the structure and functions of the radixin molecule, we isolated and sequenced the cDNA clones encoding mouse radixin. Direct peptide sequencing of radixin and immunological analysis with antiserum to a fusion protein were performed to confirm that the protein encoded by these clones is identical to radixin. The composite cDNA is 4,241 nucleotides long and codes for a 583-amino acid polypeptide with a calculated molecular mass of 68.5 kD. Sequence analysis has demonstrated that mouse radixin shares 75.3% identity with human ezrin, which was reported to be a member of the band 4.1 family. We then isolated the cDNA encoding mouse ezrin. Sequence analysis and Northern blot analysis revealed that radixin and ezrin are similar but distinct (74.9% identity), leading us to conclude that radixin is a novel member of the band 4.1 family. In erythrocytes the band 4.1 protein acts as a key protein in the association of short actin filaments with a plasma membrane protein (glycophorin), together with spectrin. Therefore, the sequence similarity between radixin and band 4.1 protein described in this study favors the idea that radixin plays a crucial role in the association of the barbed ends of actin filaments with the plasma membrane in the cell-to-cell adherens junction and the cleavage furrow.
The ERM family members, ezrin, radixin, and moesin, localizing just beneath the plasma membranes, are thought to be involved in the actin filament/plasma membrane association. To identify the integral membrane protein directly associated with ERM family members, we performed immunoprecipitation studies using antimoesin mAb and cultured baby hamster kidney (BHK) cells metabolically labeled with [35S]methionine or surface-labeled with biotin. The results indicated that moesin is directly associated with a 140-kD integral membrane protein. Using BHK cells as antigens, we obtained a mAb that recognized the 140-kD membrane protein. We next cloned a cDNA encoding the 140-kD membrane protein and identified it as CD44, a broadly distributed cell surface glycoprotein. Immunoprecipitation with various anti-CD44 mAbs showed that ezrin and radixin, as well as moesin, are associated with CD44, not only in BHK cells, but also in mouse L fibroblasts. Furthermore, immunofluorescence microscopy revealed that in both BHK and L cells, the Triton X-100-insoluble CD44 is precisely colocalized with ERM family members. We concluded that ERM family members work as molecular linkers between the cytoplasmic domain of CD44 and actin-based cytoskeletons.
Heterologous cDNA clones were used as hybridization probes to define the temporal expression of intestinal functions during fetal and postnatal development in the pig. Northern hybridization analysis revealed the presence of the mRNAs for the cellular retinol binding protein CRBP II, for the digestive enzyme aminopeptidase N, and for the microvillar proteins villin and ezrin in the small intestine of both weaned and 40-day fetal pigs. The presence of these mRNAs suggests that at the end of the first third of gestation the pig fetal intestine is already exhibiting some characteristics of a differentiated epithelium. The mRNAs for the two fatty acid-binding proteins I-FABP and L-FAPB, both involved in the metabolism of long chain fatty acids, were detected only in the intestinal mRNA extracted from weaned animals, while that for the cellular retinol-binding protein CRBP I was expressed only in the fetal tissue. The temporal limits of expression of intestinal genes in the pig epithelium seem therefore more easily defined than in other experimental animals with shorter times of fetal development. To isolate pig genes expressed at different developmental stages during intestinal epithelial cell differentiation, a cDNA library was constructed from extracted from mature pig intestine. This library was employed in the isolation of clones encoding CRBP II and L-FABP. The nucleotide sequence of the two pig cDNA clones was determined, and the sequences of the deduced proteins compared with their homologues from other species. The results of this analysis showed that the two pig clones share a high level of homology with human and rat homologues both at the DNA and at the protein level.
Intact ribonucleic acid (RNA) has been prepared from tissues rich in ribonuclease such as the rat pancreas by efficient homogenization in a 4 M solution of the potent protein denaturant guanidinium thiocyanate plus 0.1 M 2-mercaptoethanol to break protein disulfide bonds. The RNA was isolated free of protein by ethanol precipitation or by sedimentation through cesium chloride. Rat pancreas RNA obtained by these means has been used as a source for the purification of alpha-amylase messenger ribonucleic acid.
The distribution of the mRNA encoding for villin, the major actin-binding protein of intestinal brush border, was studied during the differentiation of mouse intestinal epithelial cells and compared to the distribution of the protein. In situ hybridization using a cRNA clone specific for villin indicated that the distribution of the mRNA did not fully parallel that of the protein, although the overall labelling pattern for mRNA and protein along the crypt-villus axis was similar. While villin was present in equal amounts in all cells along the villi, villin-specific mRNA was mainly accumulated in the cells at the villus base, the area of the epithelium where terminal differentiation takes place and where the brush border is formed.
A chemically modified phage T7 DNA polymerase has three properties that make it ideal for DNA sequencing by the chain-termination method. The enzyme is highly processive, catalyzing the polymerization of thousands of nucleotides without dissociating. By virtue of the modification the 3' to 5' exonuclease activity is eliminated. The modified polymerase efficiently uses nucleotide analogs that increase the electrophoretic resolution of bands in gels. Consequently, dideoxynucleotide-terminated fragments have highly uniform radioactive intensity throughout the range of a few to thousands of nucleotides in length. There is virtually no background due to terminations at pause sites or secondary-structure impediments. Processive synthesis with dITP in place of dGTP eliminates band compressions, making possible the unambiguous determination of sequences from a single orientation.
Intestinal genes whose expression is regulated during development and differentiation were identified and cloned from a rat villi cDNA library using a subtracted cDNA probe. The isolated clones are transcribed in the fully differentiated intestinal epithelium 21 days after birth and absent or poorly expressed in the fetal gut at 15 days of gestation. Two of the DRI (differentially-expressed in rat intestine) genes are novel, while the others encode the microvillar protein ezrin and intracellular carrier proteins for retinol and fatty acids. Expression of the newly isolated DRI27 and DRI42 clones parallels epithelial differentiation during development and it is more pronounced in the distal portions of the small intestine. In situ hybridization experiments indicate that the DRI mRNAs are expressed in the differentiated cell types of the gut epithelium. Moreover, the expression of DRI27 and DRI42 is strongly related to the stage of epithelial differentiation during gut development. This relationship holds true also for the expression of DRI42 in other tissues. These clones will be a valuable tool to identify regulatory sequences and factors responsible for confining gene expression to the differentiated epithelial cell types in mammalian small intestine.
Recent studies have greatly expanded our understanding of actin-bundling proteins. A new group of actin-bundling proteins, the fascins, has been recognized. An actin-bundling protein inhibits actin depolymerization even under conditions in which it cannot produce a gel, which suggests that bundling proteins may affect actin filament dynamics. A villin-like protein is present in Dictyostelium, shedding doubt on current ideas on the evolution of villin. Domain mapping continues to be a major thrust of research into most groups of bundling proteins.
This study was undertaken with the aim of investigating the effects of retinoids on the expression of differentiated traits in intestinal cell models. The cell lines used included epithelial cells isolated from fetal rat intestines (FRIC), displaying a relatively undifferentiated phenotype, and the human colon adenocarcinoma cell lines Caco 2 and HT29, which express some of the traits of the mature enterocytes under defined culture conditions. The effects of retinoids were also studied in organ cultures of fetal rat intestine, where the epithelial-mesenchymal interactions are preserved. All-trans-retinol and all-trans-retinoic acid treatments were compared in their ability to regulate the expression of genes coding for proteins involved in retinoid metabolism and for cytoskeletal proteins. The results have shown that the effects of the two retinoids were qualitatively similar. A specific induction of the cellular retinol-binding protein CRBP I mRNA was observed following retinoid treatment in one of the two FRIC lines examined (FRIC B) and in organ culture. The expression of the retinoic acid receptors RAR alpha and gamma was not affected by treatment in any of the cultures examined, while RAR beta was expressed only by the organ cultures and was transcriptionally induced by retinoic acid treatment. The retinoids also induced a reorganization of the actin cytoskeleton in the FRIC B cell line, accompanied by a decrease in the expression of two components of the microvillar cytoskeleton, ezrin and villin. The results obtained in both cell and organ cultures suggest that retinoids alone are not able to trigger the differentiation program in the intestinal epithelial cell, irrespective of the level of differentiation already achieved at the time of treatment.
- Berryman
- Sato