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P G Traber

Publications and source records attributed to P G Traber.

At least 19 recordsLinked to original sources
PubMed2001

Sucrase-isomaltase gene transcription requires the hepatocyte nuclear factor-1 (HNF-1) regulatory element and is regulated by the ratio of HNF-1 alpha to HNF-1 beta.

The mouse sucrase-isomaltase (SI) gene is an enterocyte-specific gene expressed in a complex developmental pattern. We previously reported that a short, evolutionarily conserved gene promoter regulates developmental expression of SI in mouse small intestine. Herein, we investigated the role of a hepatocyte nuclear factor-1 (HNF-1) cis-acting element to regulate SI gene expression in vivo. Transgenic SI gene constructs with a mutated HNF-1 element (SIF3) revealed a strong reduction in promoter activity in comparison with a wild-type construct in mice and during Caco-2 cell differentiation. Nuclear proteins isolated from enterocytes showed increased binding of the HNF-1 alpha complex with a concomitant decrease in the HNF-1 beta-containing complex to the SIF3 element both during the suckling-weaning developmental transition and Caco-2 cell differentiation. These changes coincided with a strong induction of SI gene transcription. In transfection experiments, HNF-1 alpha activated the SI promoter via the SIF3 element, and co-expression of HNF-1 beta impaired this transcriptional activation. These findings demonstrate the essential role of the HNF-1 regulatory element to support SI gene transcription in vivo and suggest that the ratio of HNF-1 alpha to HNF-1 beta plays a role in the transcriptional activity of this gene during intestinal development.

Animals↗
PubMed2001

Phosphorylation of the serine 60 residue within the Cdx2 activation domain mediates its transactivation capacity.

BACKGROUND & AIMS: Cdx2 is critical in intestinal proliferation and differentiation. Modulation of Cdx2 function in response to cellular signaling is to be elucidated. We hypothesize that phosphorylation of the Cdx2 activation domain can modulate its function. METHODS: The Cdx2 activation domain was delineated in transient transfections using different portions of Cdx2 fused to the Gal4-DNA binding domain. In vivo phosphorylation was studied by metabolic labeling with (32)P-orthophosphate. To study a potential phosphorylation site, polyclonal antibodies were generated: CNL was raised against amino acids 54-66 of Cdx2 and P-Cdx2-S60 against the same epitope in which serine 60 was phosphorylated. RESULTS: A critical region for transactivation resides within amino acids 60-70. Substitution of serine 60 with alanine reduces incorporation of (32)P-orthophosphate substantially. S60-phosphorylation decreases Cdx2 transactivation. Phosphorylation of serine 60 can be inhibited with the mitogen-activated protein kinase inhibitors PD98059 or UO126. P-Cdx2-S60 recognizes phosphorylated serine 60 mainly in proliferative compartment of the intestinal epithelial layer. In contrast, CNL recognizes Cdx2 predominantly in the differentiated compartment. CONCLUSIONS: The Cdx2 activation domain is phosphorylated at serine 60 via the mitogen-activated protein kinase pathway. S60-phosphorylated and S60-nonphosphorylated Cdx2 have different transcriptional activity, as well as different spatial expression patterns in the intestinal epithelium.

Amino Acid Sequence↗
PubMed2001

Clusterin gene transcription is activated by caudal-related homeobox genes in intestinal epithelium.

Caudal-related homeobox (Cdx) proteins play an important role in development and differentiation of the intestinal epithelium. Using cDNA differential display, we identified clusterin as a prominently induced gene in a Cdx2-regulated cellular model of intestinal differentiation. Transfection experiments and DNA-protein interaction assays showed that clusterin is an immediate downstream target gene for Cdx proteins. The distribution of clusterin protein in the intestine was assessed during development and in the adult epithelium using immunohistochemistry. In the adult mouse epithelium, clusterin protein was localized in both crypt and villus compartments but not in interstitial cells of the intestinal mucosa. Together, these data suggest that clusterin is a direct target gene for Cdx homeobox proteins, and the pattern of clusterin protein expression suggests that it is associated with the differentiated state in the intestinal epithelium.

Age Factors↗
PubMed2000

The caudal-related homeodomain protein Cdx1 inhibits proliferation of intestinal epithelial cells by down-regulation of D-type cyclins.

Cdx1 is a homeodomain transcription factor that regulates intestine-specific gene expression. Experimental evidence suggests that Cdx1 may be involved in cell cycle regulation, but its role is ill defined and the mechanisms have not been explored. We used stable transfection of inducible constructs and transient expression with a replication-deficient adenovirus to induce Cdx1 expression in rat IEC6 cells, a non-transformed intestinal epithelial cell line that does not express Cdx1 protein. Expression of Cdx1 markedly reduced proliferation of IEC6 cells with accumulation of cells in the G(0)/G(1) phase of the cell cycle. Cell cycle arrest was accompanied by an increase in the hypophosphorylated forms of the retinoblastoma protein (pRb) and the pRb-related p130 protein. Protein levels of multiple cyclin-dependent kinase inhibitors were either unchanged (p16, p18, p21, p27, and p57) or were not detected (p15 and p19). Most significantly, levels of cyclins D1 and D2 were markedly diminished with Cdx1 expression, but not cyclins D3, E, or the G(1) kinases. Additionally, cyclin-dependent kinase-4 activity was decreased in association with decreased cyclin D protein. We conclude that Cdx1 regulates intestinal epithelial cell proliferation by inhibiting progression through G(0)/G(1), most likely via modulation of cyclin D1 and D2 protein levels.

Adenoviridae↗
PubMed2000

Cdx1 and cdx2 expression during intestinal development.

BACKGROUND & AIMS: The intestine-specific transcription factors Cdx1 and Cdx2 are candidate genes for directing intestinal development, differentiation, and maintenance of the intestinal phenotype. This study focused on the complex patterns of expression of Cdx1 and Cdx2 during mouse gastrointestinal development. METHODS: Embryonic and postnatal mouse tissues were analyzed by immunohistochemistry to determine protein expression of Cdx1 and Cdx2 in the developing intestinal tract. RESULTS: Cdx2 protein expression was observed at 9. 5 postcoitum (pc), whereas weak expression of Cdx1 protein was first seen at 12.5 pc in the distal developing intestine (hindgut). Expression of Cdx1 increased from 13.5 to 14.5 pc during the endoderm/epithelial transition with predominately distal expression. In contrast to Cdx1, there was intense expression of Cdx2 in all but the distal portions of the developing intestine. Cdx2 expression remained low in the distal colon throughout postnatal development. A gradient of expression formed in the crypt-villus axis, with Cdx1 primarily in the crypt and Cdx2 primarily in the villus. CONCLUSIONS: Direct comparison of the patterns of Cdx1 and Cdx2 protein expression during development as performed in this study provides new insights into their potential functional roles. The relative expression of Cdx1 to Cdx2 protein may be important in the anterior to posterior patterning of the intestinal epithelium and in defining patterns of proliferation and differentiation along the crypt-villus axis.

3T3 Cells↗
PubMed1999

CREB-binding [corrected] protein interacts with the homeodomain protein Cdx2 and enhances transcriptional activity.

Cdx2 encodes for a homeodomain protein that is expressed in intestinal epithelial cells. The Cdx2 protein triggers intestinal differentiation in cell lines and is necessary for maintenance of the intestinal phenotype in mice. CBP (cAMP response element-binding protein) is a transcriptional co-activator that interacts with many transcription factors and components of the basal transcriptional machinery. In this study, we demonstrate that CBP is markedly induced upon differentiation of the Caco-2 intestinal cell line and augments Cdx2-dependent transcriptional activity. Cdx2 interacts with the amino-terminal domain of CBP, and the two proteins coexist in vivo within the same nuclear protein complex. Moreover, expression of the CBP domain that interacts with Cdx2 acts as a dominant-negative inhibitor of transcriptional activation by Cdx2. These findings demonstrate a direct interaction between an intestinal homeodomain protein and CBP and suggest that CBP participates in the network of transcriptional proteins that direct intestinal differentiation.

3T3 Cells↗
PubMed1999

Transcriptional regulation in intestinal development. Implications for colorectal cancer.

Deciphering the complex mechanisms of intestinal epithelial development will require multiple cell and molecular approaches in both in vitro and whole animal systems. Additionally, the use of model organisms such as D. melanogaster, C. elegans, and zebrafish will help describe paradigms that may be investigated in mammals as well as serve as test systems for findings from mammals. This manuscript reviewed only one approach to understanding intestinal development. However, the Cdx story and the information to be mined from an understanding of SI gene transcription is not at an end. As the other pieces of the transcriptional puzzle of the SI gene are assembled there will be new information to generate hypotheses on the relationship of transcriptional mechanisms to cancer pathogenesis.

Animals↗
PubMed1998

Control of gene expression in intestinal epithelial cells.

Coordination of gene transcription is a critical regulatory step in orchestrating developmental, differentiation and adaptation processes in the mammalian intestinal epithelium. Insight into these mechanisms has been gained by the study of transcriptional regulation of the sucrase-isomaltase gene. An understanding of the regulatory network of nuclear proteins that direct transcriptional initiation of intestinal genes such as sucrase-isomaltase will provide insight into the mechanisms of normal development and differentiation as well as disease processes such as neoplasia.

Animals↗
PubMed1997

Activation of enhancer elements by the homeobox gene Cdx2 is cell line specific.

Cdx2 is a caudal-related homeodomain transcription factor that is expressed in complex patterns during mouse development and at high levels in the intestinal epithelium of adult mice. Cdx2 activates transcription of intestinal gene promoters containing specific binding sites. Moreover, Cdx2 has been shown to induce intestinal differentiation in cell lines. In this study, we show that Cdx2 is able to bind to two well defined enhancer elements in the HoxC8 gene. We then demonstrate that Cdx2 is able to activate transcription of heterologous promoters when its DNA binding element is placed in an enhancer context. Furthermore, the ability to activate enhancer elements is cell-line dependent. When the Cdx2 activation domain was linked to the Gal4 DNA binding domain, the chimeric protein was able to activate Gal4 enhancer constructs in an intestinal cell line, but was unable to activate transcription in NIH3T3 cells. These data suggest that there are cell-specific factors that allow the Cdx2 activation domain to function in the activation of enhancer elements. We hypothesize that either a co-activator protein or differential phosphorylation of the activation domain may be the mechanism for intestinal cell line-specific function of Cdx2 and possibly in other tissues in early development.

3T3 Cells↗
PubMed1997

The mesenchymal winged helix transcription factor Fkh6 is required for the control of gastrointestinal proliferation and differentiation.

The winged helix transcription factor Fkh6 is expressed in the mesoderm of the gastrointestinal tract directly adjacent to the endoderm-derived epithelium. Homozygous null mice for Fkh6 showed postnatal growth retardation secondary to severe structural abnormalities of the stomach, duodenum, and jejunum. Dysregulation of epithelial cell proliferation in these organs resulted in an approximately fourfold increase in the number of dividing intestinal epithelial cells and marked expansion of the proliferative zone. As a consequence, the tissue architecture of the stomach and small intestine was distorted, with abnormal crypt structure, formation of mucin filled cysts, and lengthening of villi. Changes in the cellular phenotype and composition of the gastric and intestinal epithelia also suggests that epithelial cell-lineage allocation or differentiation may be affected by loss of Fkh6. From the analysis of a number of potential signaling molecules, we found Bmp2 and Bmp4 expression reduced in the gastrointestinal tract of Fkh6 mutant mice, suggesting that Fkh6 directs a signaling cascade that mediates communication between the mesenchyme and endoderm of the gut to regulate cell proliferation.

Animals↗
PubMed1997

Molecular pathogenesis of colorectal cancer.

The enormous progress made in the identification of genes that are involved in colon carcinogenesis has provided the foundation for further understanding the biology of both normal and cancer cells and for targeted therapeutic strategies. In one sense, the genes described in this review are only the building blocks of a larger puzzle that constitutes the integrated metabolic function of a cell. The current challenge is to understand the functional role of these genes in normal cellular physiology and make the connections between pathways that knit together integrated cellular homeostasis. A complete understanding of the regulatory pathways, and the synthesis and modifications of the proteins involved, will provide novel targets for therapeutic agents.

Adenoma↗
PubMed1997

CDX1 protein expression in normal, metaplastic, and neoplastic human alimentary tract epithelium.

BACKGROUND & AIMS: CDX1 is an intestine-specific transcription factor expressed early in intestinal development that may be involved in regulation of proliferation and differentiation of intestinal epithelial cells. We examined the pattern of CDX1 protein expression in metaplastic and neoplastic tissue to provide insight into its possible role in abnormal differentiation. METHODS: Tissue samples were stained by immunohistochemistry using an affinity-purified, polyclonal antibody against a peptide epitope of CDX1. RESULTS: Specific nuclear staining was found in epithelial cells of the small intestine and colon. Esophagus and stomach did not express CDX1 protein; however, adjacent areas of intestinal metaplastic tissue intensely stained for CDX1. Adenocarcinomas of the stomach and esophagus had both positive and negative nuclear staining for CDX1. Colonic epithelial cells in adenomatous polyps and adenocarcinomas had a decreased intensity of staining compared with normal colonic crypts in the same specimen. CONCLUSIONS: CDX1 may be important in the transition from normal gastric and esophageal epithelium to intestinal-type metaplasia. The variability in expression of CDX1 in gastric and esophageal adenocarcinomas suggests more than one pathway in the development of these carcinomas. The decrease of CDX1 in colonic adenocarcinomas may indicate a role for CDX1 in growth regulation and in the maintenance of the differentiated phenotype.

3T3 Cells↗
PubMed1997

Comparison of intestinal phospholipase A/lysophospholipase and sucrase-isomaltase genes suggest a common structure for enterocyte-specific promoters.

Intestinal phospholipase A/lysophospholipase (IPAL) is an intestine-specific brush-border enzyme expressed during development and along the intestinal crypt-villus axis in a pattern similar to another well characterized brush-border enzyme, sucrase-isomaltase (SI). A tissue-specific DNase I hypersensitive site was identified in chromatin from intestinal nuclei immediately upstream from the transcriptional start site of the IPAL gene. Footprinting analysis showed that two DNA elements within the IPAL promoter were protected by intestinal nuclear proteins. The IPAL-FP1 element was shown to be a monomer binding site for Cdx1 and Cdx2, intestine-specific homeobox proteins. Moreover, this site was important for transcriptional activation of the promoter in intestinal cell lines via interaction with Cdx proteins. Nuclear proteins from both liver and intestine interacted with the IPAL-FP2 element, forming a complex consistent with binding to HNF1. Cdx and HNF1 binding sites have also been shown to be the two major regulatory elements responsible for transcriptional activation of the SI gene promoter, which directs intestine-specific transcription in transgenic mice. These findings suggest that enterocyte genes that are expressed in similar developmental patterns may be regulated by the interaction of common DNA elements and their associated transcription factors.

Animals↗
PubMed1997

Developmental expression of SI is regulated in transgenic mice by an evolutionarily conserved promoter.

Developmental expression of the sucrase-isomaltase (SI) gene in the mouse intestine involves two major transitions that correspond to critical developmental events. Low levels of SI mRNA were first identified in day 16.5 fetal mouse intestine, immediately after the transition from stratified endoderm to a columnar epithelium organized in nascent villi. Low levels were maintained until the third week of life, when induction of SI mRNA to adult levels was observed coincident with the time of weaning. The mechanism of this pattern of SI gene expression was studied in transgenic mice using a reporter gene construct containing an SI gene promoter that is evolutionarily conserved between mouse and human (nucleotides -201 to +54 of the mouse SI gene). This promoter included the necessary regulatory information to direct transcription to enterocytes in developmental and differentiation-dependent patterns that recapitulated the expression of the endogenous SI gene. However, transgenes lacked the ability to direct induction of precocious expression in suckling animals after administration of corticosteroids. These findings define a short SI gene promoter that contains cis-acting elements that are responsible for developmental and differentiation-dependent transcriptional regulation.

Aging↗
PubMed1997

Epithelial cell growth and differentiation. V. Transcriptional regulation, development, and neoplasia of the intestinal epithelium.

Coordination of gene transcription is a critical regulatory step in orchestrating developmental, differentiation, and adaptation processes in the mammalian intestinal epithelium. An understanding of the regulatory network of nuclear proteins that direct transcriptional initiation of intestinal genes will provide insight into the mechanisms of normal development and differentiation as well as disease processes such as neoplasia.

Animals↗
PubMed1996

cis-Acting elements and transcription factors involved in the intestinal specific expression of the rat calbindin-D9K gene: binding of the intestine-specific transcription factor Cdx-2 to the TATA box.

The calbindin-D9K (CaBP9k) gene is mainly expressed in differentiated duodenal epithelial cells and is used as a model for studying the molecular mechanisms of intestine-specific transcription. The gene has been cloned, two major DNase-I-hypersensitive sites in the duodenum have been described, and a vitamin-D-response element has been identified. We have now analysed the transcription factors and regulatory sequences involved in the transcription of the CaBP9k gene in the intestine in ex vivo and in vitro experiments. Transfection experiments in intestinal (CaCo-2) and non-intestinal (HeLa) cell lines defined two regions in the 5'-flanking sequences of the rat CaBP9k gene. A minimal proximal region (-117 to +20) promoted transcription in both intestinal expressing and non-expressing cell lines. Tissue specificity was conferred by the sequences situated further upstream, which are responsible for complete repression in the non-intestinal cells. Intestinal transcription was specified by the proximal region, containing a specialized TATA box, and a distal region, which contains a previously described intestinal DNase-I-hypersensitive site. In vitro DNase I footprinting, electrophoretic mobility shift assays and antibody supershift assays were used to examine the factors bound to the proximal promoter region (-800 to +80 bp). Rat duodenal nuclear extracts protected 12 sites. Some of them appear to be binding sites for ubiquitous (nuclear factor 1) or hepatic-enriched sites (hepatocyte nuclear factors 1 and 4, enhancer binding protein alpha and beta factors. DNA binding studies and transfection experiments indicated that an intestine-specific transcription factor, caudal homeobox-2, binds to the TATA box of the rat CaBP9k gene. These data contribute to our understanding of the control of the intestinal transcription of the CaBP9k gene and demonstrate that several trans-acting factors, other than the vitamin D receptor, may be factors for intestine-specific CaBP9k gene expression.

Animals↗
PubMed1996

An intestine-specific homeobox gene regulates proliferation and differentiation.

Precise regulation of cellular proliferation, differentiation, and senescence results in the continuous renewal of the intestinal epithelium with maintenance of a highly ordered tissue architecture. Here we show that an intestine-specific homeobox gene, Cdx2, is a transcription factor that regulates both proliferation and differentiation in intestinal epithelial cells. Conditional expression of Cdx2 in IEC-6 cells, an undifferentiated intestinal cell line, led to arrest of proliferation for several days followed by a period of growth resulting in multicellular structures containing a well-formed columnar layer of cells. The columnar cells had multiple morphological characteristics of intestinal epithelial cells. Enterocyte-like cells were polarized with tight junctions, lateral membrane interdigitations, and well-organized microvilli with associated glycocalyx located at the apical pole. Remarkably, there were also cells with a goblet cell-like ultrastructure, suggesting that two of the four intestinal epithelial cell lineages may arise from IEC-6 cells. Molecular evidence for differentiation was shown by demonstrating that cells expressing high levels of Cdx2 expressed sucrase-isomaltase, an enterocyte-specific gene which is a well-defined target for the Cdx2 protein. Taken together, our data suggest that Cdx2 may play a role in directing early processes in intestinal cell morphogenesis and in the maintenance of the differentiated phenotype by supporting transcription of differentiated gene products. We propose that Cdx2 is part of a regulatory network that orchestrates a developmental program of proliferation, morphogenesis, and gene expression in the intestinal epithelium.

Animals↗