Creb1 regulates late stage mammalian lung development via respiratory epithelial and mesenchymal-independent mechanisms.

During mammalian lung development, the morphological transition from respiratory tree branching morphogenesis to a predominantly saccular architecture, capable of air-breathing at birth, is dependent on physical forces as well as molecular signaling by a range of transcription factors including the cAMP response element binding protein 1 (Creb1). Creb1(-/-) mutant mice exhibit complete neonatal lethality consistent with a lack of lung maturation beyond the branching phase. To further define its role in the developing mouse lung, we deleted Creb1 separately in the respiratory epithelium and mesenchyme. Surprisingly, we found no evidence of a morphological lung defect nor compromised neonatal survival in either conditional Creb1 mutant. Interestingly however, loss of mesenchymal Creb1 on a genetic background lacking the related Crem protein showed normal lung development but poor neonatal survival. To investigate the underlying requirement for Creb1 for normal lung development, Creb1(-/-) mice were re-examined for defects in both respiratory muscles and glucocorticoid hormone signaling, which are also required for late stage lung maturation. However, these systems appeared normal in Creb1(-/-) mice. Together our results suggest that the requirement of Creb1 for normal mammalian lung morphogenesis is not dependent upon its expression in lung epithelium or mesenchyme, nor its role in musculoskeletal development.

Intestinal-specific activatable Myb initiates colon tumorigenesis in mice.

Transcription factor Myb is overexpressed in most colorectal cancers (CRC). Patients with CRC expressing the highest Myb are more likely to relapse. We previously showed that mono-allelic loss of Myb in an Adenomatous polyposis coli (APC)-driven CRC mouse model (Apc(Min/+)) significantly improves survival. Here we directly investigated the association of Myb with poor prognosis and how Myb co-operates with tumor suppressor genes (TSGs) (Apc) and cell cycle regulator, p27. Here we generated the first intestinal-specific, inducible transgenic model; a MybER transgene encoding a tamoxifen-inducible fusion protein between Myb and the estrogen receptor-α ligand-binding domain driven by the intestinal-specific promoter, Gpa33. This was to mimic human CRC with constitutive Myb activity in a highly tractable mouse model. We confirmed that the transgene was faithfully expressed and inducible in intestinal stem cells (ISCs) before embarking on carcinogenesis studies. Activation of the MybER did not change colon homeostasis unless one p27 allele was lost. We then established that MybER activation during CRC initiation using a pro-carcinogen treatment, azoxymethane (AOM), augmented most measured aspects of ISC gene expression and function and accelerated tumorigenesis in mice. CRC-associated symptoms of patients including intestinal bleeding and anaemia were faithfully mimicked in AOM-treated MybER transgenic mice and implicated hypoxia and vessel leakage identifying an additional pathogenic role for Myb. Collectively, the results suggest that Myb expands the ISC pool within which CRC is initiated while co-operating with TSG loss. Myb further exacerbates CRC pathology partly explaining why high MYB is a predictor of worse patient outcome.

Selective CREB-dependent cyclin expression mediated by the PI3K and MAPK pathways supports glioma cell proliferation.

The cyclic-AMP response element binding (CREB) protein has been shown to have a pivotal role in cell survival and cell proliferation. Transgenic rodent models have revealed a role for CREB in higher-order brain functions, such as memory and drug addiction behaviors. CREB overexpression in transgenic animals imparts oncogenic properties on cells in various tissues, and aberrant CREB expression is associated with tumours. It is the central position of CREB, downstream from key developmental and growth signalling pathways, which gives CREB this ability to influence a spectrum of cellular activities, such as cell survival, growth and differentiation, in both normal and cancer cells. We show that CREB is highly expressed and constitutively activated in patient glioma tissue and that this activation closely correlates with tumour grade. The mechanism by which CREB regulates glioblastoma (GBM) tumour cell proliferation involves activities downstream from both the mitogen-activated protein kinase and phosphoinositide 3-kinase (PI3K) pathways that then modulate the expression of three key cell cycle factors, cyclin B, D and proliferating cell nuclear antigen (PCNA). Cyclin D1 is highly CREB-dependent, whereas cyclin B1 and PCNA are co-regulated by both CREB-dependent and -independent mechanisms. The precise regulatory network involved appears to differ depending on the tumour-suppressor phosphatase and tensin homolog status of the GBM cells, which in turn allows CREB to regulate the activity of the PI3K itself. Given that CREB sits at the hub of key cancer cell signalling pathways, understanding the role of glioma-specific CREB function may lead to improved novel combinatorial anti-tumour therapies, which can complement existing PI3K-specific drugs undergoing early phase clinical trials.

The Myb-p300-CREB axis modulates intestine homeostasis, radiosensitivity and tumorigenesis.

The gastrointestinal (GI) epithelium is constantly renewing, depending upon the intestinal stem cells (ISC) regulated by a spectrum of transcription factors (TFs), including Myb. We noted previously in mice with a p300 mutation (plt6) within the Myb-interaction-domain phenocopied Myb hypomorphic mutant mice with regard to thrombopoiesis, and here, changes in GI homeostasis. p300 is a transcriptional coactivator for many TFs, most prominently cyclic-AMP response element-binding protein (CREB), and also Myb. Studies have highlighted the importance of CREB in proliferation and radiosensitivity, but not in the GI. This prompted us to directly investigate the p300-Myb-CREB axis in the GI. Here, the role of CREB has been defined by generating GI-specific inducible creb knockout (KO) mice. KO mice show efficient and specific deletion of CREB, with no evident compensation by CREM and ATF1. Despite complete KO, only modest effects on proliferation, radiosensitivity and differentiation in the GI under homeostatic or stress conditions were evident, even though CREB target gene pcna (proliferating cell nuclear antigen) was downregulated. creb and p300 mutant lines show increased goblet cells, whereas a reduction in enteroendocrine cells was apparent only in the p300 line, further resembling the Myb hypomorphs. When propagated in vitro, crebKO ISC were defective in organoid formation, suggesting that the GI stroma compensates for CREB loss in vivo, unlike in MybKO studies. Thus, it appears that p300 regulates GI differentiation primarily through Myb, rather than CREB. Finally, active pCREB is elevated in colorectal cancer (CRC) cells and adenomas, and is required for the expression of drug transporter, MRP2, associated with resistance to Oxaliplatin as well as several chromatin cohesion protein that are relevant to CRC therapy. These data raise the prospect that CREB may have a role in GI malignancy as it does in other cancer types, but unlike Myb, is not critical for GI homeostasis.

Intestinal adenoma formation and MYC activation are regulated by cooperation between MYB and Wnt signaling.

Aberrant Wnt signaling mediated by mutations affecting APC (adenomatous polyposis coli) or beta-catenin initiates the majority of human colorectal cancers (CRC) and drives tumorigenesis through the activation of specific genes such as MYC. We report here a novel association whereby another oncogenic transcription factor, MYB/c-Myb, is necessary for intestinal adenoma development directed by activated Wnt signaling. APC(Min/+) mice in which c-myb is haploinsufficient survive longer than wild-type APC(Min/+) animals due to a delay in adenoma formation. Intestinal adenomas from APC(Min/+) mice were assessed and found to have high levels of c-myc gene expression. We explored the relationship between activated Wnt signaling and MYB in regulating MYC and found activated beta-catenin in combination with MYB induces robust upregulation of MYC promoter activity, as well as endogenous MYC mRNA and protein expression, in human cells. This cooperation occurred through independent binding of MYB and beta-catenin to the MYC promoter. These data highlight a cooperative function for MYB in the context of activated Wnt signaling and provide a molecular basis for the expression of MYC in CRC.

Licensing regulators Geminin and Cdt1 identify progenitor cells of the mouse CNS in a specific phase of the cell cycle.

Nervous system formation integrates control of cellular proliferation and differentiation and is mediated by multipotent neural progenitor cells that become progressively restricted in their developmental potential before they give rise to differentiated neurons and glial cells. Evidence from different experimental systems suggests that Geminin is a candidate molecule linking proliferation and differentiation during nervous system development. We show here that Geminin and its binding partner Cdt1 are expressed abundantly by neural progenitor cells during early mouse neurogenesis. Their expression levels decline at late developmental stages and become undetectable upon differentiation. Geminin and Cdt1 expressing cells also express Sox2 while no overlap is detected with cells expressing markers of a differentiated neuronal phenotype. A fraction of radial glial cells expressing RC2 and Pax6 are also immunoreactive for Geminin and Cdt1. The majority of the Geminin and Cdt1 expressing cell populations appears to be distinct from fate-restricted precursor cells expressing Mash1 or Neurogenin2. Bromo-deoxy-uridine (BrdU) incorporation experiments reveal a cell cycle specific expression in neural progenitor cells, with Geminin being present from S to M phase, while Cdt1 expression characterizes progenitor cells in G1 phase. Furthermore, in vitro differentiation of adult neurosphere cultures shows downregulation of Geminin/Cdt1 in the differentiated state, in line with our data showing that Geminin is present in neural progenitor cells of the CNS during mouse embryogenesis and adulthood and becomes downregulated upon cell fate specification and differentiation. This suggests a role for Geminin in the formation and maintenance of the neural progenitor cells.

Collagen loss and impaired wound healing is associated with c-Myb deficiency.

Collagen type I serves as an abundant structural and signalling component of skin. It is also an established target gene of the transcription factor, c-Myb. When c-myb-/- embryos were examined it was observed that their skin was markedly thinner than normal. Importantly, immunohistochemical investigation showed complete absence of collagen type I. Although these homozygous knock-out embryos fail to develop beyond day 15, fibroblasts established from these embryos (mouse embryonic fibroblasts [MEFs]) show defective proliferative responses. Furthermore, in vitro scratch wound assays demonstrated that these c-myb-/- MEFs also exhibit slower closure than their wild-type counterparts. Embryonic lethality has meant that examination of the role of c-Myb in adult mouse skin has not been reported to date. However, in view of the abundance of collagen type I in normal skin, its role in skin integrity and the in vitro data showing proliferative and migration defects in c-myb-/- MEFs, we investigated the consequences of heterozygous c-myb loss in adult mice on the complex process of skin repair in response to injury. Our studies clearly demonstrate that heterozygous c-myb deficiency has a functional effect on wound repair, collagen type I levels and, in response to wounding, transforming growth factor-beta1 (an important collagen stimulating factor) induction expression is aberrantly high. Manipulation of c-Myb may therefore provide new therapeutic opportunities for improving wound repair while uncontrolled expression may underpin some fibrotic disorders.

Transcriptional regulation of cyclo-oxygenase expression: three pillars of control.

Blocking cyclo-oxygenase (COX) isoform activities with non-steroidal anti-inflammatory drugs (NSAIDS) is widely employed in the treatment of arthritis. These agents also hold great promise in the context of pre and post-neoplastic diseases such as colorectal cancer (CRC). Nevertheless, issues of isoform specificity and delivery necessitate the exploration of other strategies to specifically block expression of the COX genes. Approaches that target gene transcription may complement enzyme inhibition. Thus, understanding the regulation of COX isoform transcription may improve the specific inhibition of expression. Three tiers of transcriptional regulation are evident: initiation, alternative splicing and messenger RNA stability. Transcription factors that activate COX-2 expression are elevated in certain disease states and emergency responses such as infection and are therefore potential targets. These factors include C/EBP-beta, phospho- CREB, NF-IL6, AP1, NFkB, and TCF-4/LEF-1. In this review we highlight another factor, c-MYB as a key COX-2 regulator in CRC. Alternative exon usage is another tier of regulation that has not received much attention. For instance, COX-1 splice variants (also known as COX-3 and PCOX-1a) may broaden the spectrum of COX activities in disease. Similarly, whilst mRNA stability is clearly modulated by steroids in the case of COX-2, the wider implications of targeting mRNA stability have not been afforded the same attention. Finally, it seems that some NSAIDS exert part of their action directly on COX-2 transcriptional regulation explaining why such agents display greater effects on this isoform than enzyme inhibition data would suggest.

A CamKIIalpha iCre BAC allows brain-specific gene inactivation.

We describe the generation of transgenic mouse lines expressing the Cre recombinase enzyme in brain under control of the CamKIIalpha gene present in a BAC expression vector. The CamKIIalpha BAC transgene gave a faithful expression pattern resembling the pattern of the endogenous CamKIIalpha gene. Specifically, high levels of CamKIIalpha Cre were detected in hippocampus, cortex, and amygdala, and lower levels were detected in striatum, thalamus, and hypothalamus. As expected, no expression was detected in the cerebellum or outside of the brain. The expression level of the BAC CamKIIalpha driven Cre was shown to be copy number dependent. To test the activity of the Cre recombinase, the transgenic mice were crossed with mice harbouring the CREB (cAMP response element binding protein) allele with the 10th exon flanked by two loxP sites, and recombination was monitored by the disappearance of the CREB protein. Finally, evaluation of the developmental postnatal expression of the CamKIIalpha Cre BAC revealed the expression of the Cre recombinase as early as P3.

Primary structure, chromosomal mapping, expression and transcriptional activity of murine hepatocyte nuclear factor 4gamma.

We demonstrate the presence of a new member of the orphan nuclear receptor hepatocyte nuclear factor 4 (HNF4) subfamily in mouse which is genetically distinct from the previously characterized mouse HNF4alpha gene. The new member of the HNF4 subfamily shows highest amino acid identity, similar tissue distribution and syntenous chromosomal localization to the recently described human HNF4gamma (NR2A2), we therefore classify it as mouse HNF4gamma (mHNF4gamma). A combination of RT-PCR and immunohistochemical analysis showed expression of mHNF4gamma mRNA and protein in the endocrine pancreas, testes, kidney and gut. By co-transfection experiments, we show that mHNF4gamma is able to activate transcription, acting through binding sites that have been previously characterized as HNF4alpha binding sites. The presence of HNFgamma in human and mouse implies that a complex transcriptional network exists in higher vertebrates involving a number of HNF4 members with overlapping yet distinct function and tissue distribution.

Expression of gastrin/CCK-B receptors does not lead to a mitogenic response to gastrin in two colon cell lines.

To clarify the impact of the classic gastrin/CCK-B receptor on the growth of benign and malignant colonic cells, two permanent cell lines expressing this receptor have been established. The conditionally immortalized nonmalignant colonic cell line YAMC and the colonic carcinoma cell line SW 403 were stably transfected with a plasmid encoding the gastrin/CCK-B receptor (GR), or with plasmid alone (V). Expression of the gastrin/CCK-B receptor in the transfected YAMC-GR and SW 403-GR cells was demonstrated by gastrin binding experiments. The YAMC-GR cell line did not respond mitogenically to pentagastrin or gastrin(17) in vitro and was not tumorigenic. The SW 403-GR cell line was stimulated by gastrin(17) in vitro, but the growth patterns of SW 403-GR and SW 403-V were the same in nude mice with cells injected either subcutaneously or into the spleen. These results provide further evidence that the gastrin/CCK-B receptor is not responsible for gastrin-stimulated growth in colonic tumors.

Glycine-extended gastrin acts as an autocrine growth factor in a nontransformed colon cell line.

BACKGROUND & AIMS: The hypothesis that progastrin-derived peptides act as autocrine growth factors for colorectal carcinomas has generated considerable interest. However, the influence of autocrine gastrins on nontumorigenic colonic cells has not been investigated. This study tested the above hypothesis in the nontumorigenic, conditionally immortalized mouse colon cell line YAMC. METHODS: The effects of expression of antisense or sense gastrin messenger RNA, treatment with antibodies against progastrin-derived peptides, or treatment with gastrin receptor antagonists on YAMC cell proliferation were measured. RESULTS: YAMC clones expressing antisense gastrin messenger RNA had reduced levels of immunoreactive progastrin-derived peptides and a reduced rate of proliferation, relative to vector only-transfected cells. Glycine-extended gastrin17, but not amidated gastrin17, reversed the antisense-induced inhibition of proliferation and stimulated the proliferation of sense- or vector only-transfected cells. YAMC cells bound 125I-glycine-extended gastrin17 (Kd, 0.36 nmol/L, 1810 sites/cell), but not 125I-amidated gastrin17, and binding was unaffected by gastrin receptor antagonists including benzotript. Proliferation of all YAMC clones was partially inhibited either by an antibody selective for glycine-extended gastrin or by preincubation with benzotript, and the inhibitory effects were additive. CONCLUSIONS: YAMC cells use nonamidated progastrin-derived peptides as autocrine growth factors, partly through binding to an extracellular receptor selective for glycine-extended gastrin, and partly through an intracellular mechanism.

Gastrin and gastrin receptor antagonists bind to both N- and C-terminal halves of the 78 kDa gastrin-binding protein.

A 78 kDa gastrin-binding protein (GBP) has previously been identified as the target of the anti-proliferative effects of non-selective gastrin/cholecystokinin receptor antagonists on colorectal carcinoma cell lines. The GBP was related in sequence to a family of fatty acid oxidation enzymes possessing enoyl CoA hydratase and 3-hydroxyacyl CoA dehydrogenase activity. This study aims to define the binding site for gastrin and gastrin antagonists in greater detail. The N- and C-terminal halves of the porcine GBP were expressed independently as glutathione S-transferase fusion proteins in E. coli. Affinities of gastrin and gastrin antagonists for the fusion proteins were measured by competition for 125I-[Nle15]-gastrin binding in a covalent cross-linking assay. The N- and C-terminal fusion proteins bound gastrin with affinities of 9.9 +/- 6.1 and 71 +/- 48 microM, respectively (n = 3). These values were 40-fold and 300-fold lower than the affinity of the full-length GBP for gastrin (0.23 +/- 0.15 microM). In contrast, the affinities of the N- and C-terminal halves for the antagonists proglumide (22 +/- 13 and 10 +/- 4 mM, respectively) and benzotript (350 +/- 90 and 400 +/- 160 micro M, respectively) were similar to each other and to the affinities of proglumide and benzotript for the full-length GBP (5.1 +/- 3.6 mM and 200 +/- 120 microM, respectively). It is concluded that proglumide and benzotript bind independently to both the hydratase and dehydrogenase active sites of the GBP, while a single molecule of gastrin may bind simultaneously to both active sites. A model is proposed which is consistent with these data, and which will assist in the development of more potent and selective GBP antagonists.

Expression of gastrin, gastrin/CCK-B and gastrin/CCK-C receptors in human colorectal carcinomas.

To investigate further the presence of an autocrine proliferative loop involving gastrin in colorectal carcinomas and to clarify the receptor responsible, 102 human colorectal carcinomas and 10 hepatic metastases were investigated for the expression of the genes encoding gastrin, the gastrin/CCK-B receptor and the gastrin/CCK-C receptor. Levels of RNA expression were assayed by RNase protection assay. In addition, gastrin/CCK receptors on crude membranes of tumour tissue were assayed by radioligand binding. High-affinity gastrin/CCK-B receptors were not detected in any of the carcinomas investigated, whereas in 36% low-affinity binding was observed, consistent with the expression of the gastrin/CCK-C receptor. RNase protection assay detected the RNA for the gastrin/CCK-B receptor in 11% of the carcinomas investigated, whereas the RNA for the gastrin/CCK-C receptor was demonstrated in 75% and the RNA for gastrin in 86% of the carcinomas investigated. These results confirm the recent demonstration of progastrin fragments in colorectal carcinomas. One possible explanation for progastrin expression is that such progastrin fragments may participate in an autocrine proliferative loop. The receptor involved in this loop is more likely to be the low-affinity gastrin/CCK-C receptor rather than the gastrin/CCK-B receptor, which is rarely expressed in colorectal carcinomas.

The seventh transmembrane domain of gastrin/CCK receptors contributes to non-peptide antagonist binding.

The related rat cholecystokinin (CCK)-A and gastrin/CCK-B receptors can be selectively blocked by the antagonists L364718 and L365260, respectively. In order to determine receptor domains which are important in conferring specificity for L365260 and L364718 we constructed by overlap-PCR a rat gastrin/CCK-B receptor chimaera which contained the seventh transmembrane domain of the rat CCK-A receptor. Receptor binding assays on transiently transfected COS cells demonstrated a selective reduction in the affinity of the chimaeric receptor for L364718, so that the L365260 and L364718 affinities were of a similar order. Since the chimaera differs from the wild-type gastrin/CCK-B receptor by only six amino acids we conclude that one or more of these six amino acids contributes to L364718 binding and that the affinity determinants of L365260 and L364718 must, at least in part, be different. Furthermore, the affinity of the chimaera for gastrin is essentially the same as the gastrin/CCK-B receptor, indicating that the six different amino acids probably do not contribute to peptide agonist binding.

Nucleotide sequence encoding a novel member of the hydratase/dehydrogenase family.

The nucleotide sequence encoding a novel member of the family of fatty acid oxidation enzymes has been determined. Clones were generated from porcine gastric corpus cDNA by application of the polymerase chain reaction (PCR) using oligodeoxyribonucleotides based on the amino acid sequence of a 78 kDa gastrin-binding protein isolated from porcine gastric mucosal membranes (Baldwin et al., J. Biol. Chem. 261 (1986) 12252-12257). Clones encoding the 3' and 5' ends of the cDNA were then isolated by conventional screening of a porcine liver cDNA library, and by application of anchored PCR, respectively. The composite cDNA of 2744 nucleotides encoded a protein of 763 amino acids, the sequence of which was related to a rat peroxisomal trifunctional enzyme, delta 3, delta 2-enoyl-CoA isomerase/enoyl-CoA hydratase/3-hydroxyacyl-CoA dehydrogenase, and to other members of the same enzyme family. Northern blots indicated that the 3-kb mRNA encoding the novel protein was abundant in gastric corpus and liver, with lower amounts also present in gastric antrum and forestomach.

Structure-function studies of human interferons-alpha: enhanced activity on human and murine cells.

To identify functionally important regions of the human interferon (IFN)-alpha molecule, mutagenesis in vitro of human IFN-a genes was used to create analogs with deletions or specific amino acid replacements. These analogs were expressed in vitro using SP6 RNA polymerase and a rabbit reticulocyte lysate protein synthesis system. Deletion of 7 highly conserved hydrophilic amino acids from the C-terminus of human IFN-alpha 4 reduced, but did not abolish, antiviral activity on human cells. However, analogs with deletions of 15 or 25 amino acids from the C-terminus, or 28 amino acids from the N-terminus, had no measurable antiviral activity. The antiviral activity of human IFN-alpha 4 was increased by substitution of cysteine for serine at position 86, and lysine for arginine at position 121. However, other amino acid substitutions at positions 121, 122 or 123 reduced antiviral activity. The size of the side chain of the amino acid residue at position 130 was shown to be important. Replacement of the absolutely conserved leucine residue at position 131 with glutamine had little effect on antiviral activity. However, the introduction of a proline residue at this position abolished antiviral activity, probably due to the formation of a beta turn in the polypeptide chain. The antiviral activity of human IFN-alpha 4 on murine cells was increased by substitutions at positions 86, 121 and 133. This study illustrates the utility of the in vitro mutagenesis and rabbit reticulocyte lysate systems for the investigation of structure-function relationships, and extends our knowledge of the biologically active regions and species specificity of the human IFN-alpha molecule.

PCR cloning and sequence of gastrin mRNA from carcinoma cell lines.

The hypothesis that a gastrin-like peptide is acting as an autocrine growth factor in gastric and colonic carcinoma cell lines requires that the cells should synthesize a gastrin-like mRNA. Although no gastrin mRNA was observed in the gastric line Okajima or the colonic lines HCT 116 or LIM 1215 by Northern blotting, gastrin mRNA was detected by application of the polymerase chain reaction. Two products were observed corresponding to mRNA with and without a 130 bp intron. The sequences of both products were identical to the sequences predicted from the normal human gastrin gene.