p27(Kip1) is an inducer of intestinal epithelial cell differentiation.

Constant renewal of the intestinal epithelium is a highly coordinated process that has been subject to intense investigation, but its regulatory mechanisms are still essentially unknown. In this study, we have demonstrated that forced expression of the cyclin-dependent kinase inhibitors (CKIs) p27(Kip1) and p21(Cip1/WAF1) in human intestinal epithelial cells led to expression of differentiation markers at both the mRNA and protein levels. Cell differentiation was temporally dissociated from inhibition of retinoblastoma protein phosphorylation and growth arrest, already established 1 day after infection with recombinant adenoviruses. p27(Kip1) proved significantly more efficient than p21(Cip1/WAF1) in induction of cell differentiation. In contrast, forced expression of p16(INK4a) resulted in growth arrest without induction of differentiation markers. These results implicate both p27(Kip1) and p21(Cip1/WAF1) in the differentiation-timing process, but p21(Cip1/WAF1) may act indirectly by increasing p27(Kip1) levels. These results also suggest that induction of intestinal epithelial cell differentiation by CKIs is not related to their effects on the cell cycle and may involve interactions with cellular components other than cyclins and cyclin-dependent kinases.

Contribution of villous atrophy to reduced intestinal maltase in infants with malnutrition.

BACKGROUND: It has been known for many years that small intestinal maltase activities are reduced in malnourished infants and in other patients with villous atrophy. The recent availability of human maltase-glucoamylase cDNA provides the opportunity to test the hypothesis that villous atrophy accounts for the reduced maltase enzyme activity in malnourished infants. METHODS: Mucosal biopsy specimens obtained for clinical evaluation of malnourished infants with poor responses to refeeding were examined by quantitative methods for enzyme activity and mRNA levels. RESULTS: Maltase activity and maltase-glucoamylase mRNA were reduced (approximately 45% of normal). When maltase-glucoamylase message was normalized to villin message, a structural protein expressed only in enterocytes, a preservation of maltase messages in surviving enterocytes was documented. The luminal glucose transporter-villin message was also preserved. CONCLUSIONS: The loss of maltase-glucoamylase message paralleled the reduction in villin message and degree of villous atrophy. The reduced maltase-glucoamylase message also paralleled sucrase-isomaltase message, previously found to be decreased in proportion to villous atrophy of malnourished infants. The data directly demonstrate, for the first time, that the terminal steps of starch 1-4 starch digestion and sucrase-isomaltase 1-6 starch digestion are decreased in malnourished infants, secondary to villous atrophy. These data in prior and present reports suggest that mechanisms underlying the chronic villous atrophy of malnutrition should be a priority for investigations in malnourished infants with slower than expected weight gain during refeeding.

Glucocorticoids have pleiotropic effects on small intestinal crypt cells.

Glucocorticoids have long been known to accelerate maturation of the intestinal tract, but the molecular mechanisms that account for their physiological function in the epithelium remain poorly characterized. Using rat intestinal epithelial cell lines (IEC-6, IEC-17, and IEC-18) as models, we have characterized glucocorticoid receptors in crypt cells and documented striking morphological, ultrastructural, and functional alterations induced by these hormones in intestinal cells. They include arrest of growth, formation of tight junctions, appearance of long, slender microvilli, reorganization of the endoplasmic reticulum and trans-Golgi network, and downregulation of the cell cycle regulatory proteins cyclin-dependent kinase 6 and p27(Kip1). These effects are consistent with the activation or modulation of multiple genes important in the physiological function of absorptive villous cells but are probably not directly involved in the induction of cell differentiation.

Involvement of p21(WAF1/Cip1) and p27(Kip1) in intestinal epithelial cell differentiation.

Using the conditionally immortalized human cell line tsFHI, we have investigated the role of cyclin-dependent kinase inhibitors (CKIs) in intestinal epithelial cell differentiation. Expression of cyclins, cyclin-dependent kinases (Cdk), and CKIs was examined under conditions promoting growth, growth arrest, or expression of differentiated traits. Formation of complexes among cell cycle regulatory proteins and their kinase activities were also investigated. The tsFHI cells express three CKIs: p16, p21, and p27. With differentiation, p21 and p27 were strongly induced, but with different kinetics: the p21 increase was rapid but transient and the p27 increase was delayed but sustained. Our results suggest that the function of p16 is primarily to inhibit cyclin D-associated kinases, making tsFHI cells dependent on cyclin E-Cdk2 for pRb phosphorylation and G1/S progression. Furthermore, they indicate that p21 is the main CKI involved in irreversible growth arrest during the early stages of cell differentiation in association with D-type cyclins, cyclin E, and Cdk2, whereas p27 may induce or stabilize expression of differentiated traits acting independently of cyclin-Cdk function.

Dissociation between growth arrest and differentiation in Caco-2 subclone expressing high levels of sucrase.

Growth arrest and cell differentiation are generally considered temporally and functionally linked phenomena in small intestinal crypt cells and colon tumor cell lines (Caco-2, HT-29). We have derived a Caco-2 subclone (NGI3) that deviates from such a paradigm. In striking contrast with the parental cells, proliferative and subconfluent NGI3 cells were found to express sucrase-isomaltase (SI) mRNA and to synthesize relatively high levels of SI, dipeptidyl peptidase IV, and aminopeptidase N (APN). In postconfluent cells, little difference was seen in SI mRNA levels between Caco-2 and NGI3 cells, but the latter still expressed much higher levels of SI that could be attributed to higher rates of translation. APN expression was also greatly enhanced in NGI3 cells. To determine whether high levels of brush-border enzymes correlated with expression of cell-cycle regulatory proteins, we investigated their relative cellular levels in growing and growth-arrested cells. The results showed that the cyclin-dependent kinase inhibitors (p21 and p27) and D-type cyclins (D1 and D3) were all induced in postconfluent cells, but NGI3 cells expressed much higher levels of p21. This study demonstrated that cell growth and expression of differentiated traits are not mutually exclusive in intestinal epithelial cells and provided evidence indicating that posttranscriptional events play an important role in regulation of SI expression.

Cytocentrin is a Ral-binding protein involved in the assembly and function of the mitotic apparatus.

Cytocentrin is a cytosolic protein that transiently associates with the mitotic spindle poles in early prophase, and dissociates from them after completion of mitosis. Cloning of its cDNA demonstrated a high degree of homology with three proteins known to specifically interact with an activated form of Ral. Herein we demonstrate that overexpression of cytocentrin inhibits assembly of the mitotic spindle without affecting polymerization or distribution of interphase microtubules. Conversely, loss of cytocentrin expression leads to formation of monopolar spindles. These results indicate that association of cytocentrin with the centrosome may be essential for a timely separation of the diplosomes. They also implicate Ral GTPases and their related pathways in the assembly and function of the mitotic apparatus.

Human small intestinal maltase-glucoamylase cDNA cloning. Homology to sucrase-isomaltase.

It has been hypothesized that human mucosal glucoamylase (EC 3.2.1. 20 and 3.2.1.3) activity serves as an alternate pathway for starch digestion when luminal alpha-amylase activity is reduced because of immaturity or malnutrition and that maltase-glucoamylase plays a unique role in the digestion of malted dietary oligosaccharides used in food manufacturing. As a first step toward the testing of this hypothesis, we have cloned human small intestinal maltase-glucoamylase cDNA to permit study of the individual catalytic and binding sites for maltose and starch enzyme hydrolase activities in subsequent expression experiments. Human maltase-glucoamylase was purified by immunoisolation and partially sequenced. Maltase-glucoamylase cDNA was amplified from human intestinal RNA using degenerate and gene-specific primers with the reverse transcription-polymerase chain reaction. The 6,513-base pair cDNA contains an open reading frame that encodes a 1,857-amino acid protein (molecular mass 209,702 Da). Maltase-glucoamylase has two catalytic sites identical to those of sucrase-isomaltase, but the proteins are only 59% homologous. Both are members of glycosyl hydrolase family 31, which has a variety of substrate specificities. Our findings suggest that divergences in the carbohydrate binding sequences must determine the substrate specificities for the four different enzyme activities that share a conserved catalytic site.

Cell dynamics and differentiation of conditionally immortalized human intestinal epithelial cells.

BACKGROUND & AIMS: Intestinal epithelial cell lines capable of differentiating in monolayer culture have been difficult to obtain, prompting the use of novel approaches including immortalization with oncogenes or tumor viruses. The aim of this study was to obtain conditionally immortalized human intestinal epithelial cells (temperature-sensitive fetal human intestinal [tsFHI] cells) and study their growth and differentiation capabilities. METHODS: Intestinal cells from human fetuses were transformed with a temperature-sensitive simian virus 40 large tumor antigen, cloned, and screened for expression of intestinal markers. RESULTS: Establishment of tsFHI cells was crucially dependent on a growth medium based on OptiMEM. At 32 degrees C, the tsFHI cells proliferate and display crypt cell markers. Only a few scattered differentiated cells are observed together with cells displaying the morphological signs of apoptosis. A shift to 39 degrees C results in irreversible growth arrest and acquisition of an enterocyte-like phenotype. Expression of the cyclin-dependent kinase inhibitor p21/WAF1/Cip1 is induced within hours of a shift to 39 degrees C 1-2 days before corresponding increases in brush border enzymes, suggesting a role in the induction rather than in the maintenance of a differentiated phenotype. CONCLUSIONS: The tsFHI cells recapitulate key aspects of intestinal cell dynamics in vitro and seem particularly suited for the study of early events in cell differentiation.

Effects of malnutrition on expression and activity of lactase in children.

BACKGROUND & AIMS: Many malnourished infants have reduced lactase specific activity in the small intestine. The aim of this study was to test the hypothesis that the hypolactasia of malnourished infants results from transcriptional suppression of lactase expression. METHODS: Biopsy specimens were studied from two groups of infants: 29 with malnutrition and 10 normally nourished controls with normal morphology and lactase activity. RESULTS: In malnourished infants, lactase messenger RNA (mRNA) was reduced to 32% and sucrase to 61% of normal. Lactase and sucrase enzyme proteins and activities were lower in malnourished infants, and partial villus atrophy was present. The genotype of adult hypolactasia was not present. CONCLUSIONS: Because the hypolactasia of malnourished children was associated with much lower lactase than sucrase mRNA abundance and because the epigenetic suppression, which accounted for the reduction of sucrase mRNA, was inadequate to explain the greater reduction of lactase mRNA, this study concludes that malnutrition suppresses lactase gene transcription or mRNA stability in infants. The reductions of lactase mRNA, distinct from those found in adults with genetic hypolactasia, explain the low lactase activities commonly found in malnourished infants.

Lactase phlorhizin hydrolase turnover in vivo in water-fed and colostrum-fed newborn pigs.

We have estimated the synthesis rates in vivo of precursor and brush-border (BB) polypeptides of lactase phlorhizin hydrolase (LPH) in newborn pigs fed with water or colostrum for 24h post partum. At the end of the feeding period, piglets were anaesthetized and infused intravenously for 3h with L-[4-3H]- phenylalanine. Blood and jejunal samples were collected at timed intervals. The precursor and BB forms of LPH were isolated from jejunal mucosa by immunoprecipitation followed by SDS/PAGE, and their specific radioactivity in Phe determined. The kinetics of precursor and BB LPH labelling were analysed by using a linear compartmental model. Immunoisolated LPH protein consisted of five polypeptides [high-mannose LPH precursor (proLPHh), complex glycosylated LPH precursor (proLPHe), intermediate complex glycosylated LPH precursor (proLPH1i) and two forms of BB LPH]. The fractional synthesis rate (Ks) of proLPHh and proLPHc (approx. 5%/min) were the same in the two groups but the absolute synthesis rate (in arbitrary units, min-1) of proLPHh in the colostrum-fed animals was twice that of the water-fed animals. The Ks values of proLPHi polypeptides were significantly different (water-fed, 3.89%/min; colostrum-fed, 1.6%/min), but the absolute synthesis rates did not differ. The Ks of BB LPH was not different between experimental treatment groups (on average 0.037%/min). However, the proportion of newly synthesized proLPHh processed to BB LPH was 48% lower in colostrum-fed than in water-fed animals. We conclude that in neonatal pigs, the ingestion of colostrum stimulates the synthesis of proLPHh but, at least temporarily, disrupts the processing of proLPH polypeptides to the BB enzyme.

Identification of a 102 kDa protein (cytocentrin) immunologically related to keratin 19, which is a cytoplasmically derived component of the mitotic spindle pole.

The mAb RK7, previously shown to recognize keratin 19, was also found to cross-react with a biologically unrelated 102 kDa protein, which becomes associated with the poles of the mitotic apparatus. This newly identified protein, called cytocentrin, is a stable cellular component, may be at least in part phosphorylated, and displays a cell cycle-dependent cellular localization. In interphase cells, it is diffusely distributed in the cytosol and shows no affinity for cytoplasmic microtubules. It becomes localized to the centrosome in early prophase, prior to nuclear envelope breakdown, separation of replicated centrosomes, and nucleation of mitotic apparatus microtubules. During metaphase, cytocentrin is located predominately at the mitotic poles, often appearing as an aggregate of small globular sub-components; it also associates with some polar microtubules. In late anaphase/early telophase cytocentrin dissociates entirely from the mitotic apparatus and becomes temporarily localized with microtubules in the midbody, from which it disappears by late telophase. In taxol-treated cells cytocentrin was associated with the center of the miniasters but also showed affinity for some cytoplasmic microtubules. Studies employing G2-synchronized cells and nocodazole demonstrated that cytocentrin can become associated with mitotic centrosomes independently of tubulin polymerization and that microtubules regrow from antigen-containing foci. We interpret these results to suggest that cytocentrin is a cytoplasmic protein that becomes specifically activated or modified at the onset of mitosis so that it can affiliate with the mitotic poles where it may provide a link between the pericentriolar material and other components of the mitotic apparatus.

Conditionally immortalized intestinal epithelial cells: novel approach for study of differentiated enterocytes.

Clonal cell lines have been established from primary fetal rat intestinal epithelial cells by stable transfection with plasmids containing either the simian virus 40 (SV40) large T-antigen gene under the control of the heavy metal inducible metallothionein promoter (pMTWt) or the thermolabile SV40 T-antigen gene under the control of the SV40 early promoter (pZipSVtsa58). pMTWt-transfected cells produced sufficient T-antigen to allow them to proliferate both when the metallothionein promoter was induced and uninduced. No differences were observed in the pattern of intestinal epithelial markers expressed when the cells were cultured in the presence or absence of inducing agent (zinc). In contrast, fetal rat intestinal epithelial cells transfected with pZipSVtsa58 were immortalized conditionally; cells proliferated at 32 degrees C but ceased to proliferate between 48 and 72 h of culture at 39 degrees C. Four of these cell lines were characterized in detail; they showed microvilli and tight junctions as well as dome formation and expressed functional and biochemical markers of intestinal epithelial cells, including keratins 8, 19, and 21, aminopeptidase N, and dipeptidyl peptidase IV. One cell line, 2/4/A1, expressed in addition a low level of lactase and sucrase-isomaltase. The amount and/or activity of some of these markers changed during the switch from the proliferative to the nonproliferative state (switch from culture at 32 to 39 degrees C), resulting in a more differentiated phenotype and mimicking similar changes taking place during intestinal epithelial cell differentiation in vivo.

Differential localization by in situ hybridization of distinct keratin mRNA species during intestinal epithelial cell development and differentiation.

The distribution of the major keratin mRNAs expressed during terminal differentiation and fetal development of the rat intestinal epithelium has been examined by in situ hybridization. We have obtained and characterized a partial cDNA clone encoding human keratin 20 whose sequence spans from the coil la region through the 3' poly(A) tail. Sequence data and immunoblot analysis demonstrated that keratin 20 is the human homologue of the rat keratin 21, suggesting the existence of a single type I keratin specifically expressed by differentiated intestinal epithelial cells. Four cRNA probes, specific for keratins 8, 18, 19, and 20 respectively, were prepared and found to specifically hybridize with their respective mRNA species from total intestinal RNA preparations. Analysis of frozen tissue sections by in situ hybridization revealed that, in the adult intestine, keratin 18 and 19 mRNAs are restricted to the region of the crypts, keratin 8 mRNA is found along the entire crypt-villus axis, and keratin 20 mRNA is expressed only by the differentiated villus cells. This pattern is established late during fetal rat intestinal development: in the undifferentiated stratified epithelium present at 16 days gestation (16dg) mRNAs coding for keratins 8, 18, and 19 are expressed by all epithelial cells and keratin 20 mRNA is absent. Upon completion of villus formation at 20dg (2 days before birth) keratin 18 and 19 mRNAs become strictly confined to cells at the base of the nascent villi and we observed the appearance of keratin 20 mRNA which, like keratin 8 mRNA, is expressed by the entire epithelium. These results strongly suggest that transcriptional regulation of keratin genes in the intestinal epithelium occurs at the level of both immature and terminally differentiated epithelial cells, and is tightly regulated during both fetal development and crypt-to-villus differentiation of the intestinal epithelium.

Intracellular degradation and reduced cell-surface expression of sucrase-isomaltase in heat-shocked Caco-2 cells.

To investigate the role of post-translational events in intestinal cell differentiation we have studied the effects of heat shock on processing and cell surface delivery of sucrase-isomaltase (SI), dipeptidylpeptidase IV (DPPIV) and aminopeptidase N (APN) in Caco-2 cells. In cells cultured at 42.5 degrees C there was a rapid decline in sucrase activity, while DPPIV and APN were unaffected over a 3-day period. Immunofluorescence staining confirmed the selective disappearance of SI from the surface membrane after only 1 day of culture at 42.5 degrees C. Cell-surface biotinylation of cells metabolically labelled with [35S]methionine 4 h after a switch from 37 degrees C to 42.5 degrees C demonstrated that newly synthesized APN and DPPIV were associated with the surface membrane, while SI was almost completely retained intracellularly. Pulse-chase experiments confirmed that, in these cells, DPPIV and APN were normally processed and vectorially delivered to the cell surface; in contrast, conversion between the two conformationally distinct high-mannose precursor forms of SI (hmP1 and hmP2) was markedly inhibited, a significant fraction of newly synthesized enzyme was degraded, probably in the ER, and an immature form of complex-glycosylated SI precursor (cP) was produced and mostly retained intracellularly. Double labelling of Caco-2 cells for SI and cathepsin D excluded an accumulation of SI in the lysosomes, suggesting that this organelle was not involved in the degradation of SI. These results indicate that the ER may play an important role in intestinal cell differentiation by regulating the conformational maturation, degradation and eventual cellular localization of some digestive enzymes.

In vivo sucrase-isomaltase and lactase-phlorizin hydrolase turnover in the fed adult rat.

We estimated in vivo turnover rates of sucrase-isomaltase and lactase-phlorizin hydrolase in adult rats. Fed animals received a primed continuous infusion of phenylalanine (300 microCi, 150 mumol Phe/100 g of body weight for 30 s, then 7.5 microCi, 3.75 mumol Phe/min for 10 to 140 min). Sucrase-isomaltase and lactase-phlorizin hydrolase were immunoprecipitated from jejunal mucosal membranes; isoforms were separated by SDS-polyacrylamide gel electrophoresis. Endoglycosidase H digestions and (for lactase-phlorizin hydrolase) N-terminal amino acid sequencing were performed on all isoforms. Specific radioactivity of prosucrase-isomaltase and prolactase-phlorizin hydrolase isoforms reached isotopic equilibrium by 60 and 90 min, respectively. Specific radioactivity of brush border sucrase and lactase did not reach steady state. The isotope kinetic, N-terminal amino acid sequencing, and endoglycosidase H digestion data suggested that one of the high molecular weight lactase isoforms is a dimer of mature lactase. Compartmental modeling of specific radioactivity demonstrated that mean intracellular residence time is 59 min for prosucrase-isomaltase isoforms and 68 min for prolactase-phlorizin hydrolase isoforms. Mean residence time in the brush border was 5.8 h for sucrase and 7.8 h for lactase. Fractional synthesis rates were 414%/day for sucrase and 307%/day for lactase. Thus, in vivo brush border sucrase and lactase turn over at similar rates in the adult rat.

GPI-anchored proteins associate to form microdomains during their intracellular transport in Caco-2 cells.

In this study, we have investigated the possibility that glycosyl-phosphatidylinositol (GPI)-anchored proteins form insoluble membrane complexes in Caco-2 cells and that transmembrane proteins are associated with these complexes. GPI-anchored proteins were mainly resistant to Triton X-100 (TX-100) extraction at 4 degrees C but fully soluble in n-octyl-glucoside. Resistance to Triton X-100 extraction was not observed in the endoplasmic reticulum but appeared during transport through the Golgi complex. It was not dependent upon N-glycosylation processing, or pH variation from 6.5 to 8.5, and was not affected by sterol-binding agents. Other apical or basolateral transmembrane proteins were well solubilized in TX-100, with the exception of sucrase-isomaltase, which was partly insoluble. We isolated a membrane fraction from Caco-2 cells that contained GPI-anchored proteins and sucrase-isomaltase but no antigen 525, a basolateral marker, or dipeptidylpeptidase IV, an apical one. These data suggest that GPI-anchored proteins cluster to form membrane microdomains together with an apical transmembrane protein, providing a possible apical sorting mechanism for intestinal cells in vitro that might be related to apical sorting in MDCK cells, and that other mechanisms might exist to sort proteins to the apical membrane.

Patchy expression of lactase protein in adult rabbit and rat intestine.

Enzymatic and immunohistological analyses of lactase were performed at different stages of development and within different regions of the small intestine of the rabbit and rat. As previously reported, there seems to be a sharp decline of lactase activity on weaning but variable and higher levels of activity are seen in adult animals. Two monoclonal antibodies to rat lactase were available to study the protein in rats. Four monoclonal antibodies to human lactase were shown to cross-react with rabbit lactase and used for the rabbit studies. Immunohistological analysis of small intestine of adult rabbits and rats showed residual lactase protein within the enterocytes throughout the small intestine. In the middle of the small intestine (lower jejunum, upper ileum), uniform staining of the brush border was observed. In the proximal and distal regions, a patchy pattern of staining was observed. This pattern, which resembles that observed in adult hypolactasic humans, indicates an underlying heterogeneity of enterocyte differentiation.

Changes in keratin expression during fetal and postnatal development of intestinal epithelial cells.

We have investigated keratin expression in fetal, newborn and adult rat intestines by immunofluorescence staining, immunoblotting of two-dimensional gels and Northern blot analysis of total cellular RNAs. Keratin-type intermediate filaments, composed predominantly of keratin no. 19, were observed already in the undifferentiated stratified epithelium present at 15-16 days of gestation. The marked maturation and differentiation of the epithelium taking place at 18-19 days of gestation was characterized by the appearance of the differentiation-specific keratin no. 21 and by a significant increase in the relative amount of keratin no. 8. The keratin pattern typical of adult villus cells became established at the time of birth, and was marked by a considerable increase in the complexity of the keratin-related polypeptides detected on two-dimensional gels, indicative of extensive post-translational modification of all keratins. Starting at 20 days of gestation there was a major increase in the relative abundance of mRNAs coding for keratin nos. 8, 19 and 21; in contrast, the relative amount of keratin no. 18 mRNA reached a peak shortly after birth and declined to very low levels in adult intestine. These results demonstrated marked changes in keratin expression and post-translational processing taking place at key stages of intestinal development. The appearance of keratin no. 21 in coincidence with the formation of an adult-type brush border and terminal web would be consistent with it having an important role in the organization of the intermediate filament network in the apical cytoplasm of the differentiated intestinal cells.

Modulation of transcytotic and direct targeting pathways in a polarized thyroid cell line.

Two biosynthetic pathways exist for delivery of membrane proteins to the apical surface of epithelial cells, direct transport from the trans-Golgi network (TGN) and transcytosis from the basolateral membrane. Different epithelial cells vary in the expression of these mechanisms. Two extremes are MDCK cells, that use predominantly the direct route and hepatocytes, which deliver all apical proteins via the basolateral membrane. To determine how epithelial cells establish a particular targeting phenotype, we studied the apical delivery of endogenous dipeptidyl peptidase IV (DPPIV) at early and late stages in the development of monolayers of a highly polarized epithelial cell line derived from Fischer rat thyroid (FRT). In 1 day old monolayers, surface delivery of DPPIV from the TGN was unpolarized (50%/50%) but a large basal to apical transcytotic component resulted in a polarized apical distribution. In contrast, after 7 days of culture, delivery of DPPIV was mainly direct (85%) with no transcytosis of the missorted component. A basolateral marker, Ag 35/40 kD, on the other hand, was directly targeted (90-98%) at all times. These results indicate that the sorting machinery for apical proteins develops independently from the sorting machinery for basolateral proteins and that the sorting site relocates progressively from the basal membrane to the TGN during development of the epithelium. The transient expression of the transcytotic pathway may serve as a salvage pathway for missorted apical proteins when the polarized phenotype is being established.