The transcriptional repressor HIC1 regulates intestinal immune homeostasis.

The intestine is a unique immune environment that must respond to infectious organisms but remain tolerant to commensal microbes and food antigens. However, the molecular mechanisms that regulate immune cell function in the intestine remain unclear. Here we identify the POK/ZBTB family transcription factor hypermethylated in cancer 1 (HIC1, ZBTB29) as a central component of immunity and inflammation in the intestine. HIC1 is specifically expressed in immune cells in the intestinal lamina propria (LP) in the steady state and mice with a T-cell-specific deletion of HIC1 have reduced numbers of T cells in the LP. HIC1 expression is regulated by the Vitamin A metabolite retinoic acid, as mice raised on a Vitamin A-deficient diet lack HIC1-positive cells in the intestine. HIC1-deficient T cells overproduce IL-17A in vitro and in vivo, and fail to induce intestinal inflammation, identifying a critical role for HIC1 in the regulation of T-cell function in the intestinal microenvironment under both homeostatic and inflammatory conditions.

MMP-19 deficiency causes aggravation of colitis due to defects in innate immune cell function.

Matrix metalloproteinases (MMPs) are potential biomarkers for disease activity in inflammatory bowel disease (IBD). However, clinical trials targeting MMPs have not succeeded, likely due to poor understanding of the biological functions of individual MMPs. Here, we explore the role of MMP-19 in IBD pathology. Using a DSS-induced model of colitis, we show evidence for increased susceptibility of Mmp-19-deficient (Mmp-19(-/-)) mice to colitis. Absence of MMP-19 leads to significant disease progression, with reduced survival rates, severe tissue destruction, and elevated levels of pro-inflammatory modulators in the colon and plasma, and failure to resolve inflammation. There was a striking delay in neutrophil infiltration into the colon of Mmp-19(-/-) mice during the acute colitis, leading to persistent inflammation and poor recovery; this was rescued by reconstitution of irradiated Mmp-19(-/-) mice with wild-type bone marrow. Additionally, Mmp-19-deficient macrophages exhibited decreased migration in vivo and in vitro and the mucosal barrier appeared compromised. Finally, chemokine fractalkine (CX3CL1) was identified as a novel substrate of MMP-19, suggesting a link between insufficient processing of CX3CL1 and cell recruitment in the Mmp-19(-/-) mice. MMP-19 proves to be a critical factor in balanced host response to colonic pathogens, and for orchestrating appropriate innate immune response in colitis.

ADAM10/17-dependent release of soluble c-Met correlates with hepatocellular damage.

The signalling pathway elicited by hepatocyte growth factor (HGF) and its receptor c-Met is indispensable for liver development and regeneration. It has been described that c-Met is released from the cell surface by a disintegrin and metalloprotease 10 (ADAM10) resulting in a soluble c-Met form known as sMet. Using the human hepatocellular HepG2 and hepatic stellate cell LX2 lines we show that sMet is released from the cell surface of liver cells by both ADAM17 and ADAM10, with ADAM17 appearing to be the major proteinase. Moreover, using a mouse model of 3,5-diethoxycarbonyl- 1,4-dihydroxycollidine (DDC)-induced hepatobiliary obstruction we show that serum levels of sMet correlate well with the liver damage state and consecutive regeneration as well as with established markers of liver damage such as alanine aminotransferase (ALT), aspartate transaminase (AST), alkaline phosphatase (ALP), and total bilirubin. However, sMet exhibited remarkably better correlation with liver damage and inflammation than did serum tumour necrosis factor α (TNF-α), whose shedding is also mediated by ADAM proteolytic activity. Our results indicate that the proteolytic activity of ADAM10/17 is essential for regulating HGF/c-Met signalling during acute liver damage and following regeneration and that the differential serum levels of sMet together with expression of c-Met/HGF might be a useful indicator not only for damage, but also for ongoing liver regeneration.

Signal transduction pathways participating in homeostasis and malignant transformation of the intestinal tissue.

Intestinal homeostasis is a complex and tightly regulated process governed by a variety of signalling pathways that balance cell proliferation and differentiation. As revealed by extensive use of defined mouse models, perturbations within the signalling circuitry trigger initial expansion of premalignant cells. In this review, we attempt to summarise recent advances in the knowledge of the cellular signalling mechanisms that drive tumorigenesis in the human and mouse intestine.

Developmentally regulated expression of the transmembrane adaptor protein trim in fetal and adult T cells.

TRIM is a recently identified transmembrane adaptor protein which is exclusively expressed in T cells and natural killer (NK) cells. In peripheral blood T cells TRIM has been reported to coprecipitate, comodulate, and cocap with the T-cell receptor (TCR), suggesting that it is an integral component of the TCR/CD3/zeta complex. Here we investigate the expression of TRIM mRNAs and proteins in developing thymocytes. Two splicing isoforms with open reading frames are observed, namely a full length (TRIM) and a truncated version (DeltaTM-TRIM). The latter lacks the extracellular and transmembrane domains as well as the first 10 cytoplasmic aminoacids and is significantly expressed only as mRNA in early fetal thymocytes. TRIM mRNA is detected in all mainstream thymocyte subsets in adult mice. TRIM protein, in contrast, first appears in the DN2 (CD44+ CD25+) subset of adult double negative (DN) cells. In fetal thymocyte development, TRIM mRNA is seen from dg 14.5 onwards whereas TRIM protein appears first on dg 16.5. In contrast to the adult, the TRIM protein was seen in a subset of fetal DN1 cells. In fetal and adult thymocytes, TRIM protein expression was highest in DN2, DN3 (CD44-25+) and in DP cells, compatible with a functional role at or around phases of thymic selection.

The transmembrane adaptor protein TRIM regulates T cell receptor (TCR) expression and TCR-mediated signaling via an association with the TCR zeta chain.

T cell receptor (TCR)-interacting molecule (TRIM) is a recently identified transmembrane adaptor protein, which is exclusively expressed in T cells. Here we demonstrate that in mature T cells, TRIM preferentially interacts with the TCR via the TCR-zeta chains and to a lesser extent via the CD3-straightepsilon/gamma heterodimer. Transient or stable overexpression of TRIM in Jurkat T cells results in enhancement of TCR expression on the cell surface and elevated induction of Ca(2+) mobilization after T cell activation. TRIM-mediated upregulation of TCR expression results from inhibition of spontaneous TCR internalization and stabilization of TCR complexes on the cell surface. Collectively, our data identify TRIM as a novel integral component of the TCR complex and suggest that one function of TRIM might be to modulate the strength of signals transduced through the TCR through regulation of TCR expression on the cell surface.

Phosphoprotein associated with glycosphingolipid-enriched microdomains (PAG), a novel ubiquitously expressed transmembrane adaptor protein, binds the protein tyrosine kinase csk and is involved in regulation of T cell activation.

According to a recently proposed hypothesis, initiation of signal transduction via immunoreceptors depends on interactions of the engaged immunoreceptor with glycosphingolipid-enriched membrane microdomains (GEMs). In this study, we describe a novel GEM-associated transmembrane adaptor protein, termed phosphoprotein associated with GEMs (PAG). PAG comprises a short extracellular domain of 16 amino acids and a 397-amino acid cytoplasmic tail containing ten tyrosine residues that are likely phosphorylated by Src family kinases. In lymphoid cell lines and in resting peripheral blood alpha/beta T cells, PAG is expressed as a constitutively tyrosine-phosphorylated protein and binds the major negative regulator of Src kinases, the tyrosine kinase Csk. After activation of peripheral blood alpha/beta T cells, PAG becomes rapidly dephosphorylated and dissociates from Csk. Expression of PAG in COS cells results in recruitment of endogenous Csk, altered Src kinase activity, and impaired phosphorylation of Src-specific substrates. Moreover, overexpression of PAG in Jurkat cells downregulates T cell receptor-mediated activation of the transcription factor nuclear factor of activated T cells. These findings collectively suggest that in the absence of external stimuli, the PAG-Csk complex transmits negative regulatory signals and thus may help to keep resting T cells in a quiescent state.

Expression of CD44 in Apc and Tcf mutant mice implies regulation by the WNT pathway.

Overexpression of cell surface glycoproteins of the CD44 family is an early event in the colorectal adenoma-carcinoma sequence. This suggests a link with disruption of APC tumor suppressor protein-mediated regulation of beta-catenin/Tcf-4 signaling, which is crucial in initiating tumorigenesis. To explore this hypothesis, we analyzed CD44 expression in the intestinal mucosa of mice and humans with genetic defects in either APC or Tcf-4, leading to constitutive activation or blockade of the beta-catenin/Tcf-4 pathway, respectively. We show that CD44 expression in the non-neoplastic intestinal mucosa of Apc mutant mice is confined to the crypt epithelium but that CD44 is strongly overexpressed in adenomas as well as in invasive carcinomas. This overexpression includes the standard part of the CD44 (CD44s) as well as variant exons (CD44v). Interestingly, deregulated CD44 expression is already present in aberrant crypt foci with dysplasia (ACFs), the earliest detectable lesions of colorectal neoplasia. Like ACFs of Apc-mutant mice, ACFs of familial adenomatous polyposis (FAP) patients also overexpress CD44. In sharp contrast, Tcf-4 mutant mice show a complete absence of CD44 in the epithelium of the small intestine. This loss of CD44 concurs with loss of stem cell characteristics, shared with adenoma cells. Our results indicate that CD44 expression is part of a genetic program controlled by the beta-catenin/Tcf-4 signaling pathway and suggest a role for CD44 in the generation and turnover of epithelial cells.

Restricted high level expression of Tcf-4 protein in intestinal and mammary gland epithelium.

Tcf-4 is a member of the Tcf/Lef family of transcription factors that interact functionally with beta-catenin to mediate Wnt signaling in vertebrates. We have previously demonstrated that the tumor suppressor function of APC in the small intestine is mediated via regulation of Tcf-4/beta-catenin transcriptional activity. To gain further insight into the role of Tcf-4 in development and carcinogenesis we have generated several mouse monoclonal antibodies, one of which is specific for Tcf-4 and another of which recognizes both Tcf-3 and Tcf-4. Immunohistochemistry performed with the Tcf 4- specific monoclonal antibody revealed high levels of expression in normal intestinal and mammary epithelium and carcinomas derived therefrom. Additional sites of Tcf-3 expression, as revealed by staining with the Tcf-3/-4 antibody, occurred only within the stomach epithelium, hair follicles, and keratinocytes of the skin. A temporal Tcf-4 expression gradient was observed along the crypt-villus axis of human small intestinal epithelium: strong Tcf-4 expression was present within the crypts of early (week 16) human fetal small intestine, with the villi showing barely detectable Tcf-4 protein levels. Tcf-4 expression levels increased dramatically on the villi of more highly developed (week 22) fetal small intestine. We conclude that Tcf-4 exhibits a highly restricted expression pattern related to the developmental stage of the intestinal epithelium. The high levels of Tcf-4 expression in mammary epithelium and mammary carcinomas may also indicate a role in the development of this tissue and breast carcinoma.

Depletion of epithelial stem-cell compartments in the small intestine of mice lacking Tcf-4.

Mutations of the genes encoding APC or beta-catenin in colon carcinoma induce the constitutive formation of nuclear beta-catenin/Tcf-4 complexes, resulting in activated transcription of Tcf target genes. To study the physiological role of Tcf-4 (which is encoded by the Tcf7/2 gene), we disrupted Tcf7/2 by homologous recombination. Tcf7/2-/- mice die shortly after birth. A single histopathological abnormality was observed. An apparently normal transition of intestinal endoderm into epithelium occurred at approximately embryonic day (E) 14.5. However, no proliferative compartments were maintained in the prospective crypt regions between the villi. As a consequence, the neonatal epithelium was composed entirely of differentiated, non-dividing villus cells. We conclude that the genetic program controlled by Tcf-4 maintains the crypt stem cells of the small intestine. The constitutive activity of Tcf-4 in APC-deficient human epithelial cells may contribute to their malignant transformation by maintaining stem-cell characteristics.

Two members of the Tcf family implicated in Wnt/beta-catenin signaling during embryogenesis in the mouse.

Tcf transcription factors interact with beta-catenin and Armadillo to mediate Wnt/Wingless signaling. We now report the characterization of genes encoding two murine members of the Tcf family, mTcf-3 and mTcf-4. mTcf-3 mRNA is ubiquitously present in embryonic day 6.5 (E6.5) mouse embryos but gradually disappears over the next 3 to 4 days. mTcf-4 expression occurs first at E10.5 and is restricted to di- and mesencephalon and the intestinal epithelium during embryogenesis. The mTcf-3 and mTcf-4 proteins bind a canonical Tcf DNA motif and can complex with the transcriptional coactivator beta-catenin. Overexpression of Wnt-1 in a mammary epithelial cell line leads to the formation of a nuclear complex between beta-catenin and Tcf proteins and to Tcf reporter gene transcription. These data demonstrate a direct link between Wnt stimulation and beta-catenin/Tcf transcriptional activation and imply a role for mTcf-3 and -4 in early Wnt-driven developmental decisions in the mouse embryo.

Constitutive transcriptional activation by a beta-catenin-Tcf complex in APC-/- colon carcinoma.

The adenomatous polyposis coli (APC) tumor suppressor protein binds to beta-catenin, a protein recently shown to interact with Tcf and Lef transcription factors. The gene encoding hTcf-4, a Tcf family member that is expressed in colonic epithelium, was cloned and characterized. hTcf-4 transactivates transcription only when associated with beta-catenin. Nuclei of APC-/- colon carcinoma cells were found to contain a stable beta-catenin-hTcf-4 complex that was constitutively active, as measured by transcription of a Tcf reporter gene. Reintroduction of APC removed beta-catenin from hTcf-4 and abrogated the transcriptional transactivation. Constitutive transcription of Tcf target genes, caused by loss of APC function, may be a crucial event in the early transformation of colonic epithelium.

Activation of beta-catenin-Tcf signaling in colon cancer by mutations in beta-catenin or APC.

Inactivation of the adenomatous polyposis coli (APC) tumor suppressor gene initiates colorectal neoplasia. One of the biochemical activities associated with the APC protein is down-regulation of transcriptional activation mediated by beta-catenin and T cell transcription factor 4 (Tcf-4). The protein products of mutant APC genes present in colorectal tumors were found to be defective in this activity. Furthermore, colorectal tumors with intact APC genes were found to contain activating mutations of beta-catenin that altered functionally significant phosphorylation sites. These results indicate that regulation of beta-catenin is critical to APC's tumor suppressive effect and that this regulation can be circumvented by mutations in either APC or beta-catenin.

XTcf-3 transcription factor mediates beta-catenin-induced axis formation in Xenopus embryos.

XTcf-3 is a maternally expressed Xenopus homolog of the mammalian HMG box factors Tcf-1 and Lef-1. The N-terminus of XTcf-3 binds to beta-catenin. Microinjection of XTcf-3 mRNA in embryos results in nuclear translocation of beta-catenin. The beta-catenin-XTcf-3 complex activates transcription in a transient reporter gene assay, while XTcf-3 by itself is silent. N-terminal deletion of XTcf-3 (delta N) abrogates the interaction with beta-catenin, as well as the consequent transcription activation. This dominant-negative delta N mutant suppresses the induction of axis duplication by microinjected beta-catenin. It also suppresses endogenous axis specification upon injection into the dorsal blastomeres of a 4-cell-stage embryo. We propose that signaling by beta-catenin involves complex formation with XTcf-3, followed by nuclear translocation and activation of specific XTcf-3 target genes.

Extensive alternative splicing and dual promoter usage generate Tcf-1 protein isoforms with differential transcription control properties.

Previously, we reported the isolation of cDNA clones representing four alternative splice forms of TCF-1, a T-cell-specific transcription factor. In the present study, Western blotting (immunoblotting) yielded a multitude of TCF-1 proteins ranging from 25-55 kDa, a pattern not simply explained from the known splice alternatives. Subsequent cDNA cloning, PCR amplification, and analysis by rapid amplification of 5' cDNA ends revealed (i) the presence of an alternative upstream promoter, which extended the known N terminus by 116 amino acids, (ii) the presence of four alternative exons, and (iii) the existence of a second reading frame in the last exon encoding an extended C terminus. Inclusion of the extended N terminus into the originally reported protein resulted in a striking similarity to the lymphoid factor Lef-1. Several of the TCF-1 isoforms, although less potent, mimicked Lef-1 in transactivating transcription through the T-cell receptor alpha-chain (TCR-alpha) enhancer. These data provide a molecular basis for the complexity of the expressed TCF-1 proteins and establish the existence of functional differences between these isoforms. Furthermore, the functional redundancy between Tcf-1 and Lef-1 explains the apparently normal TCR-alpha expression in single Tcf-1 or Lef-1 knockout mice despite the firm in vitro evidence for the importance of the Tcf/Lef site in the TCR-alpha enhancer.

Genomic structure of the human CD53 gene.

The genomic structure of the gene encoding human pan-leukocyte surface glycoprotein CD53 (a member of the "tetraspan family" of membrane proteins) was determined. The gene consists of eight exons encoding all sequences found in cDNA and is spread over more than 26 kilobases of genomic DNA. The exon-intron organization of the CD53 gene is strikingly similar to the CD63 and TAPA-1 genes, which suggests a close evolutionary relationship between these genes. The 5' end of the gene upstream of the first exon contains at least three close transcription start points (approximately 20 base pairs 5' of the 5' end of the published cDNA). The region upstream of the transcription initiation sites is not G+C rich; it contains potential binding sites for several transcriptional factors but no TATA or CCAAT boxes.

The human leucocyte antigen CD48 (MEM-102) is closely related to the activation marker Blast-1.

The glycosyl phosphatidylinositol (GPI)-linked antigen recognized by monoclonal antibody (mAb) MEM-102 is expressed on all peripheral blood lymphocytes, both resting and activated. Its properties are very similar to a previously described activation antigen, Blast-1. The amino acid sequence deduced from the structure of cloned cDNA is identical to that of the Blast-1 antigen except for a single amino acid residue. There are several other minor differences in the nucleotide sequence of the Blast-1 and MEM-102 cDNAs that do not affect the predicted structure of the polypeptide product. The amino acid sequence of the first 15 N-terminal residues of the antigen purified from Raji cells is found in the deduced sequence close to the presumed boundary between the leader peptide and mature polypeptide. Properties of the recombinant product expressed in COS cells are similar to the antigen isolated from peripheral blood mononuclear cells (PBMNCs) or B-and T-cells lines. The antigen purified on immobilized mAb MEM-102 is recognized by all six known CD48 mAbs under western blotting conditions. COS cells transfected with MEM-102 cDNA react with all the CD48 mAbs. It is concluded that mAb MEM-102 is directed against the as yet poorly characterized antigen CD48, which is therefore structurally closely related to Blast-1. Several possibilities are discussed that might account for the apparent discrepancy between the broad pan-leucocyte expression of the between the broad pan-leucocyte expression of the MEM-102/CD48 antigen and much more restricted expression of the epitope recognized by the previously described mAb defining the Blast-1 antigen.

Expression of hepatitis B virus large envelope protein in Escherichia coli and Saccharomyces cerevisiae.

The gene coding for hepatitis B large envelope protein was cloned under the lac promoter in bacterial vector pUC-8 and under the ADH1 promoter in yeast expression shuttle vector pVT103-U, and expression of HBsAg in bacteria and yeast was determined. The strongest expression of large envelope protein was obtained after transformation of the protease-deficient yeast strain BJ1991. The recombinant large envelope protein did not form complex 22-nm particles and was not secreted into medium.