Intestinal dysplasia and adenoma in transgenic mice after overexpression of an activated beta-catenin.

Mutations in the adenomatous polyposis coli gene or activating mutations in the beta-catenin gene itself are thought to be responsible for the excessive beta-catenin signaling involved in intestinal carcinogenesis. We generated transgenic mice that expressed large amounts of a NH2-terminally truncated mutant beta-catenin (deltaN131beta-catenin) in the intestine. These mice had multifocal dysplastic lesions in the small intestine, reminiscent of the early lesions observed in the mouse models of familial adenomatous polyposis. The number of apoptotic cells in the villi of these transgenic mice was 3-4-fold higher than in nontransgenic mice. Expression of the truncated beta-catenin mutant in the kidney led to the development of severe polycystic kidney disease. Our findings support the concept that deregulation of the beta-catenin signaling pathway is the major oncogenic consequence of adenomatous polyposis coli mutations in intestinal neoplasia.

Characterization of the aldolase B intronic enhancer.

The aldolase B gene is transcribed at a high level in the liver, kidney, and small intestine. This high level of gene expression results from cooperation between a weak but liver-specific promoter and an intronic activator. A deletional study of this activator present in the first intron allowed us to ascribe the maximal enhancer function to a 400-base pair (bp) fragment (+1916 to + 2329). This enhancer is highly liver-specific and enhances the activity of heterologous minimal promoters in a position and distance-independent fashion in transiently transfected Hep G2 hepatoma cells. The aldolase B enhancer is composed of two domains, a 200-bp module (Ba) inactive by itself but which synergizes with another 200-bp module (Bb) that alone retains 25% of the total enhancer activity. The Bb sequence is 76% homologous between human and rat genes and contains several binding sites for liver-enriched nuclear factors. By electrophoretic mobility shift assays, we demonstrated that elements 5 and 7 bind hepatic nuclear factor 1 (HNF1), whereas element 2 binds hepatic nuclear factor 4 (HNF4). A functional analysis of the enhancer whose elements have been mutated demonstrated that mutation of any of the HNF1 sites totally suppressed enhancer activity, whereas mutation of the HNF4-binding site reduced it by 80%.

An intronic enhancer essential for tissue-specific expression of the aldolase B transgenes.

Expression in mice of transgenes directed by regulatory regions of the rat aldolase B gene requires the presence of a B element located in the first intron, while constructs devoid of this intronic enhancer are silent. Histo- and immunochemical staining of transgenic tissue sections showed that the longer transgene was expressed in the proximal tubular cells of the kidney, enterocytes located in small intestine villi and liver parenchymal cells. In the liver, a maximal expression was observed in perivenous hepatocytes, while the transgene was weakly active in periportal hepatocytes, which reproduced the pattern of functional zonation already reported for other glycolytic and gluconeogenic genes in the liver. We also established that the transgene retained the necessary elements for a correct chronological expression during development but was lacking elements necessary for activation by high carbohydrate diet. Instead, transgene expression was paradoxically stimulated in fasted animals, suggesting that the endogenous gene, which must be active under both glycolytic and gluconeogenic conditions, could possess distinct elements activating it in fasted as well as in carbohydrate-fed animals; the former element might be conserved in the transgene and the latter one might be lost.

Activity of the rat liver-specific aldolase B promoter is restrained by HNF3.

Although it contains binding sites for HNF1, NFY and C/EBP/DBP, the proximal promoter of the aldolase B gene is surprisingly weak when tested by transient transfection in differentiated hepatoma cells. This low activity could be due to overlapping between HNF1 and HNF3 binding sites in element PAB, from -127 to -103 bp with respect to the cap site. Replacement of the PAB region by a consensus HNF1 binding site unable to bind HNF3, results in a 30 fold activation of the promoter, in accordance with the hypothesis that activity of the wild-type promoter is normally restrained by HNF3 binding to PAB competitively with HNF1. Consistently, transactivation of the wild-type promoter by excess HNF1 is very high, most likely due to the displacement of HNF3, while the construct with the exclusive HNF1 binding site is weakly transactivated by HNF1. The inhibitory effect of HNF3 on HNF1-dependent transactivation is clearly due to competition between these two factors for binding to mutually exclusive, overlapping sites; indeed, when HNF1 and HNF3 sites are contiguous and not overlapping, the resulting promoter is as active as the one containing an exclusive HNF1 binding site. A construct in which PAB has been replaced by an exclusive HNF3 binding site is weakly expressed and is insensitive to HNF3 hyperexpression. DBP-dependent transactivation, finally, is independent of the nature of the element present in the PAB region.

Competition between transcription factors HNF1 and HNF3, and alternative cell-specific activation by DBP and C/EBP contribute to the regulation of the liver-specific aldolase B promoter.

The aldolase B proximal promoter is controlled by at least five elements spanning from -190 to -103 bp with respect to the start site of transcription. From 5' to 3', we found: a negative DE element, an activating C/EBP-DBP binding site, a CCAAT box binding NFY that seems to play a negative role, and an activating element consisting of two overlapping binding sites for HNF-1 and HNF-3. Contransfection experiments of aldolase B/CAT constructs and of expression vectors for different transcription factors were carried out in human hepatoma Hep G2 cells. We found that DBP and HNF-1 are strong transactivators of the aldolase B promoter while C/EBP and vHNF-1 are only weak activators and HNF-3 alone does not modify such activity. Deletion of the distal negative element results in a similar transactivation by C/EBP and DBP, enhanced for the former and reduced for the latter. In hepatocytes in primary culture, the strong transactivator is C/EBP while DBP is essentially inactive. This tissue-specificity of C/EBP and DBP action could depend on interaction with tissue-specific proteins bound to a neighbouring site, probably DE. Finally, HNF3 behaves as a very strong anti-activator of the aldolase B promoter. It competitively antagonizes transactivation by HNF-1 and non-competitively transactivation by DBP. This negative effect of HNF-3 and tissue-specificity of the transactivation potential of DBP and C/EBP are unique features of the aldolase B promoter.

Interplay of an original combination of factors: C/EBP, NFY, HNF3, and HNF1 in the rat aldolase B gene promoter.

The rat aldolase B 5' flanking region (nucleotides - 194 to +41) contains sufficient information for liver-specific expression. A detailed investigation of factors binding to the rat aldolase B 5' flanking region has allowed us to identify three distinct factors that filled different sites of this region (A, B, C). The liver-enriched C/EBP or related factors bind to box C, as demonstrated by the specific interaction with bacterially expressed C/EBP protein. Box B bearing the CCAAT sequence binds the ubiquitous factor NFY. Surprisingly, Box A is able to bind two liver enriched factors, namely HNF1 and HNF3. However, in the context of the intact promoter, as shown by footprinting competition experiments, HNF3 binds solely to this sequence. HNF3, but not HNF1 is a transcriptional activator as demonstrated in the in vitro transcription assay.

Expression of the rat aldolase B gene: a liver-specific proximal promoter and an intronic activator.

The nature and location of the cis-acting DNA sequences regulating expression of the rat aldolase B gene has been investigated. Two liver-specific DNAse I hypersensitive sites were detected, one located just upstream from the cap site, the second in the middle of the first, 4.8-kbp-long, intron. A fragment of 190 bp 5' to the cap site behaved as a tissue-specific but weak core promoter: it directed a detectable reporter gene expression in the Hep G2 cells and hepatocytes, but not in fibroblasts. The tissue-specific expression was stimulated at least 16 fold when constructs contained the entire first intron. The intronic activating sequences could be ascribed to an inner 2 kbp fragment in which the downstream liver-specific DNAse I hypersensitive site was located.

Glucose-dependent and -independent effect of insulin on gene expression.

Insulin action on gene expression can be glucose-dependent or -independent. Accumulation of aldolase B mRNA and of an unidentified 5.4-kilobase mRNA as well as accumulation of L-type pyruvate kinase mRNAs (Decaux, J.F., Antoine, B., and Kahn, A. (1989) J. Biol. Chem. 264, 11584-11590) in cultured hepatocytes isolated from fasted rats require the presence of both glucose and insulin, these agents not being effective individually. In contrast, maintaining the amount of albumin and transferrin mRNAs in these hepatocytes requires the presence of insulin alone, glucose having no effect by itself. Transcription of the albumin gene, investigated by run-on assay, is active in the presence of insulin alone, with or without glucose, whereas transcription of the aldolase B gene is stimulated by glucose and insulin together, but not by insulin or glucose alone. In addition, the stability of the albumin and aldolase B mRNAs in cultured hepatocytes is lowered in the absence of glucose and insulin together as compared to the stability in the presence of one or both agents. These results confirm that transduction of the insulin signal occurs via distinct pathways; one of these pathways could involve a secondary insulin-dependent modification of metabolite concentration, whereas other pathways could be more directly related to the activity(ies) of the occupied insulin receptor.

Characterization and metabolic regulation of a liver-specific 5.4-kilobase mRNA whose synthesis is transcriptionally induced by carbohydrates and repressed by glucagon and cyclic AMP.

Four clones derived from a carbohydrate-induced rat liver cDNA library were found to hybridize with a 5.4-kilobase mRNA species encoding a 36 kDa protein. This mRNA was abundant in the liver, barely detectable in adipocytes and kidney, and absent from the other tissues tested. In the liver, the mRNA was fully induced by a carbohydrate-rich diet, but was undetectable during both starvation and feeding with a protein-rich or lipid-rich diet. Adrenalectomized, thyroidectomized and diabetic animals did not express the mRNA in their liver when re-fed with the carbohydrate-rich diet. When these animals were given the missing hormone, the amount of hybridizable RNA returned to normal values, but administration of the hormone alone failed to induce mRNA synthesis in starved animals. Both glucagon and its second messenger, cyclic AMP, abolished the induction of the mRNA in re-fed animals. Exogenous insulin, whatever the dose, did not reverse the inhibitory action of glucagon. In an isolated nuclei transcription system, no detectable RNA transcripts were found in starved animals, whereas feeding the animals with the carbohydrate-rich diet led to a maximum rate of gene transcription. Although unidentified, this mRNA proves to be a remarkable marker of dietary and hormonal control of gene expression in vivo. It will provide a useful model for further analysis of the role of cyclic AMP in regulating the transcription of eukaryotic genes.

Cyclic 3':5'-nucleotide phosphodiesterase in human myometrium at the end of pregnancy: partial purification and characterization of the different soluble isoenzymes.

Most of the cyclic nucleotide phosphodiesterase (PDE) activity of pregnant human myometrium was found in the soluble fraction. Chromatography of this fraction on DEAE-cellulose resolved two peaks of PDE activities. Peak I hydrolyzed both cAMP and cGMP and was activated by the Ca2+-calmodulin complex. Peak II, insensitive to this complex, hydrolyzed specifically cAMP. Sucrose gradient centrifugation of the soluble fraction resolved three peaks (A, B, C) of cAMP PDE activities, and only the first two peaks (A, B) were active towards cGMP. A subsequent sucrose gradient centrifugation of peak I, previously determined by DEAE-cellulose, allowed us to restore two peaks A and B identical to those directly obtained from the soluble fraction: peak A hydrolyzes both substrates, while peak B is specific to cGMP hydrolysis. For peak II, a single large cAMP PDE activity peak is generated. Considering Ca2+ and cAMP as intracellular messengers in the control of uterine motility, the characterization of the different forms of PDE in pregnant human myometrium will be of importance in developing improved tocolytic therapy.

Cyclic AMP as a transcriptional inhibitor of upper eukaryotic gene transcription.

Recently, glucagon and its second messenger, cyclic AMP, have been shown to stimulate the transcription rate of several upper eukaryotic genes (1-5). We show here that glucagon can also block gene transcription. Both glucagon and cyclic AMP were found to inhibit the transcription of the genes encoding three liver glycolytic enzymes, including L-type pyruvate kinase and aldolase B. Thus, cyclic AMP proves to be not only an activator but also an inhibitor of gene transcription in eukaryotes.

Endogenous proteolysis of membrane-bound red cell cytochrome-b5 reductase in adults and newborns: its possible relevance to the generation of the soluble "methemoglobin reductase".

The problem of the low activity of so-called methemoglobin reductase in red cells from newborns was reinvestigated in view of our current knowledge of this enzyme, i.e., (1) its being cytochrome-b5 reductase and (2) its presence in two forms: soluble and membrane-bound. We found that red cells from cord blood and newborns exhibited a 50% decrease of soluble cytochrome-b5 reductase activity, whereas membrane-bound activity was in the adult range. Ghosts from these cells possessed diminished ability to solubilize membrane-bound cytochrome-b5 reductase in the course of in vitro auto-incubation. This autosolubilizing ability increased with age and reached adult level concomitantly with soluble cytochrome-b5 reductase activity at 6 mo. We conclude that the relative deficiency of soluble cytochrome-b5 reductase observed at birth is due to diminished post-translational processing of the membrane-bound enzyme during erythropoiesis of fetal cells. This processing is calcium-dependent related to calmodulin.

Elevated NAD(P) glycohydrolase activity: a possible enzymatic marker for malignancy in Burkitt's lymphoma cells.

A comparative analysis of enzymatic activities has been performed on 47 human continuous lymphoid lines: 22 tumors derived from Burkitt's lymphoma lines, 6 other lymphomatous long-term cultures, and 19 nonmalignant ties determined on the cell extracts. 4 showed no significant differences between the various lines. They included adenosine diphosphoribose incorporation, glucose-6-phosphate dehydrogenase, cyclic-AMP phosphodiesterase, and glutathione reductase. However, striking differences of activity were found for the enzyme, NAD(P) glycohydrolase (EC 3.2.2.6). Activity levels were, as a mean, four times higher in Burkitt's lymphoma-derived cell lines than in nonmalignant control lines, and the difference was highly significant (p less than 0.02). All Burkitt cell lines containing translocations of chromosome 8 with either chromosomes 2, 14 or 22 showed an increased activity. The specificity and significance of this possible enzymatic marker of Burkitt's lymphoma cells is discussed.

3',5' Cyclic nucleotide phosphodiesterase from human platelets: effect of heat upon the multiple forms and their interconversion.

A 33,000 g supernatant from human platelets showed a biphasic heat inactivation curve at 45, 50 and 55 degrees C of the cAMP and cGMP phosphodiesterase. This could suggest the presence of two differently heat sensitive phosphodiesterases. However, a preparation heated for 30 min at 55 degrees C, where only the apparently thermostable form of the enzyme remained, still displayed the same characteristics as the starting material, i.e. two apparent Km values for cAMP, a cAMP specific activity lower at low protein concentration (less than 50 micrograms/ml) than at high protein concentration(greater than 100 micrograms/ml), and three peaks of activity upon linear sucrose density gradient. Moreover, a biphasic inactivation curve was again observed after a second heat treatment. These results demonstrated that the heat effect is not a simple protein denaturation of one of two independent species. A study at different temperatures of the profile of the cAMP phosphodiesterase upon sucrose gradient demonstrated that the dissociated form was predominant at high temperature whereas lower temperature favored the associated form. During heat treatment, the dissociated form is at first denatured and this leads to a shift in the equilibrium between the associated and dissociated forms of the phosphodiesterase in favor of the dissociated form. From the overall results, one can draw a model for phosphodiesterase regulation by dissociation-reassociation.

Calmodulin ligands. The interaction of muscle phosphorylase kinase with phosphodiesterase. Comparison of calmodulin ligands in muscle extracts from normal and phosphorylase kinase-deficient mice.

Interactions between phosphorylase kinase (ATP:phosphorylase-b phosphotransferase, EC 2.7.1.38) and calmodulin were studied with pure preparations of muscle phosphorylase kinase, and with crude extracts from muscles of control (C57 Black) and deficient (ICR/IAn) mice, which lack muscle phosphorylase kinase activity. Calmodulin was determined by its ability to stimulate a calmodulin-dependent phosphodiesterase. The amount of calmodulin bound to phosphorylase kinase in muscle extract was estimated to a maximum of 30% of the total amount of calmodulin. In the muscle of the deficient strain a decrease of 35% in the total amount of calmodulin was observed. This correlates with the absence of the calmodulin fraction specifically bound to phosphorylase kinase. From sucrose gradient studies we demonstrated that in the presence of Ca2+ the amount of calmodulin bound to phosphorylase kinase was enhanced, compared to the control in the presence of EGTA. This observation was made both in crude extracts and in pure phosphorylase kinase preparations. Sucrose gradient also showed that muscle phosphorylase kinase can be dissociated to low molecular species when extracts are made in the presence of Ca+; this dissociation was found to be related to a Ca2+-dependent proteolytic effect.

Cyclic 3':5'-nucleotide phosphodiesterase. Stimulation of bovine brain cytoplasmic enzyme by lysophosphatidylcholine.

Brain cytoplasmic cyclic 3':5'-nucleotide phosphodiesterase (EC 3.1.4.17) requires an endogenous Ca2+-binding protein for ful activity. We now show that lysophosphatidylcholine also effectively enhances activator-deficient phosphodiesterase activity. Stimulation by both ligands was immediate and reversible; both rendered the enzyme more thermally labile, decreased the energy of activation, and increased the Vmax of phosphodiesterase without affecting its apparent Km for adenosine 3'5'-monophosphate. However, the cofactor requirements of the two ligands were different. Although the protein activator gave a greater stimulation than lysophosphatidylcholine, the simultaneous presence of the two gave a stimulation comparable to lysophosphatidylcholine, suggesting that the effect of the latter was predominant. Phosphodiesterase was also stimulated by oleic acid, cardiolipin, and phosphatidylinositol, albeit to a less extent.

Cyclid 3':5'-nucleotide phosphodiesterase. Interconvertible multiple forms and their effects on enzyme activity and kinetics.

An extract of rat liver or human platelet displayed three cyclic 3':5'-nucleotide phosphodiesterase activity peaks (I, II, and III) in a continuous sucrose density gradient when assayed with millimolar adenosine 3':5'-monophosphate (cAMP) or guanosine 3':5'-monophosphate (cGMP). The three fractions obtained from each nucleotide were not superimposable. The molecular weights corresponding to the three activity peaks of cAMP phosphodiesterase in rat liver were approximately: I, 22,000; II, 75,000; and III, 140,000. In both tissues, fraction I was barely detectable when assayed with micromolar concentrations of either nucleotide, presumably because fraction I has low affinity for cAMP and cGMP. Any one of the three forms upon recentrifugation on the gradient generated the others, indicating that they were interconvertible. The multiple forms appear to represent different aggregated states of the enzyme. The ratio of the three forms of cAMP phosphodiesterase in the platelet was shifted by dibutyryl cAMP (B2cAMP) and by the enzyme concentration. B2cAMP enhanced the formation of fraction I. Low enzyme concentration favored the equilibrium towards fraction I, while high enzyme concentration favored fraction III. When phosphodiesterase activities in the extract of rat liver, human platelets, or bovine brain were examined as a function of enzyme concentration, rectilinear rates were observed with micromolar, but not with millimolar cAMP or cGMP. The specific activity with millimolar cAMP was higher with low than with high protein concentrations, suggesting that the dissociated form catalyzed the hydrolysis of cAMP faster than that of the associated form. In contrast, the specific activity with millimolar cGMP was lower with low than with high protein concentrations. Supplementing the reaction mixture with bovine serum albumin to a final constant protein concentration did not affect the activity, suggesting that the concentration of the enzyme rather than that of extraneous proteins affected the enzyme activity. A change in enzyme concentration affected the kinetic properties of phosphodiesterase. A low enzyme concentration of cAMP phosphodiesterase yielded a linear Lineweaver-Burk plot, and a Km of 1.2 X 10(-4) M (bovine), 3 X 10(-5) M (platelet), or 5 X 10(-4) M (liver), while a high enzyme concentration yielded a nonlinear plot, and apparent Km values of 1.4 X 10(-4) M and 2 X 10(-5) M (brain), 4 X 10(-5) M and 3 X 10(-6) M (platelet), or 4 X 10(-5) M and 3 X 10(-6) (liver). Since a low enzyme concentration favored fraction I, the dissociated form, whereas a high enzyme concentration favored fraction III, the associated form, these kinetic constants suggest that the dissociated form exhibits a high Km and the associated form exhibits a low Km. In contrast, a high enzyme concentration gave a linear kinetic plot for cGMP phosphodiesterase, while a low enzyme concentration gave a nonlinear plot...

[Interconvertibility of molecular forms of 3'5' cyclic AMP phosphodiesterase of human platelets]. / Interconvertibilité des formes moléculaires de l'AMP 3'-5' cyclique phosphodiestérase des plaquettes humaines

Three forms of cyclic AMP phosphodiesterase from human platelets are resolved upon sucrose gradient centrifugation : 2.5S (monomer), 4.8S (dimer) and 7S (tetramer). They are interconvertible and form an association-dissociation equilibrium depending on the concentration of enzyme. The dissociated form has a high Km for cyclic AMP (Km : 3-5.10(-4) M) whereas the associated form has a low Km (Km : 3-5.10(-5) M).