Jacqueline L. M. Vermeulen

Patterns of Expression of Sarcoplasmic Reticulum Ca2+-ATPase and Phospholamban mRNAs During Rat Heart Development

This study reports the clonal analysis and sequence of rat phospholamban (PLB) cDNA clones and the temporal appearance and patterns of distribution of the mRNAs encoding sarcoplasmic/endoplasmic reticulum Ca(2+)-ATPase (SERCA2) and PLB in the developing rat heart determined by in situ hybridization. Both proteins play a critical role in the contraction-relaxation cycle of the heart. SERCA2 mRNA is already abundantly present in the first stage studied, in the cardiogenic plate of the 9-day-old presomite embryo, before the occurrence of the first contractions. This very early expression makes it an excellent marker for the study of early heart development. Subsequently, SERCA2 mRNA becomes expressed in a craniocaudal gradient, being highest at the venous pole and decreasing in concentration toward the arterial pole of the heart. PLB mRNA can be detected in hearts from 12 days of development onward in a virtually opposite gradient. In essence, these patterns do not change during further development. PLB mRNA levels remain highest in the ventricle and outflow tract, whereas SERCA2 mRNA prevails in the inflow tract and atrium, although the difference between atrium and ventricle becomes less pronounced. These observations are compatible with a model in which the upstream part of the heart (inflow tract and atrium) would have a greater capacity to clear calcium and hence would have a longer duration of the diastole than the downstream compartments (atrioventricular canal, ventricle, and outflow tract), similar to the observed pattern of contraction of the embryonic heart. The sinoatrial and atrioventricular nodes do not reveal an expression pattern of SERCA2 and PLB mRNA that allows one to distinguish them from the surrounding atrial working myocardium. However, the ventricular part of the conduction system, comprising atrioventricular bundle and bundle branches, are almost devoid of SERCA2 mRNA.

Identification of the leukocyte cell‐derived chemotaxin 2 as a direct target gene of β‐catenin in the liver

To clarify molecular mechanisms underlying liver carcinogenesis induced by aberrant activation of Wnt pathway, we isolated the target genes of β‐catenin from mice exhibiting constitutive activated β‐catenin in the liver. Adenovirus‐mediated expression of oncogenic β‐catenin was used to isolate early targets of β‐catenin in the liver. Suppression subtractive hybridization was used to identify the leukocyte cell‐derived chemotaxin 2 (LECT2) gene as a direct target of β‐catenin. Northern blot and immunohistochemical analyses demonstrated that LECT2 expression is specifically induced in different mouse models that express activated β‐catenin in the liver. LECT2 expression was not activated in livers in which hepatocyte proliferation was induced by a β‐catenin–independent signal. We characterized by mutagenesis the LEF/TCF site, which is crucial for LECT2 activation by β‐catenin. We further characterized the chemotactic property of LECT2 for human neutrophils. Finally, we have shown an up‐regulation of LECT2 in human liver tumors that expressed aberrant activation of β‐catenin signaling; these tumors constituted a subset of hepatocellular carcinomas (HCC) and most of the hepatoblastomas that were studied. In conclusion, our results show that LECT2, which encodes a protein with chemotactic properties for human neutrophils, is a direct target gene of Wnt/β‐catenin signaling in the liver. Since HCC develops mainly in patients with chronic hepatitis or cirrhosis induced by viral or inflammatory factors, understanding the role of LECT2 in liver carcinogenesis is of interest and may lead to new therapeutic perspectives. (HEPATOLOGY 2004;40:167–176.)

Hepatic HNF4α deficiency induces periportal expression of glutamine synthetase and other pericentral enzymes

In liver, most genes are expressed with a porto‐central gradient. The transcription factor hepatic nuclear‐factor4α (HNF4α) is associated with 12% of the genes in adult liver, but its involvement in zonation of gene expression has not been investigated. A putative HNF4α‐response element in the upstream enhancer of glutamine synthetase (GS), an exclusively pericentral enzyme, was protected against DNase‐I and interacted with a protein that is recognized by HNF4α‐specific antiserum. Chromatin‐immunoprecipitation assays of HNF4α‐deficient (H4LivKO) and control (H4Flox) livers with HNF4α antiserum precipitated the GS upstream enhancer DNA only from H4Flox liver. Identical results were obtained with a histone‐deacetylase1 (HDAC1) antibody, but antibodies against HDAC3, SMRT and SHP did not precipitate the GS upstream enhancer. In H4Flox liver, GS, ornithine aminotransferase (OAT) and thyroid hormone‐receptor β1 (TRβ1) were exclusively expressed in pericentral hepatocytes. In H4LivKO liver, this pericentral expression remained unaffected, but the genes were additionally expressed in the periportal hepatocytes, albeit at a lower level. The expression of the periportal enzyme phosphoenolpyruvate carboxykinase had declined in HNF4α‐deficient hepatocytes. GS‐negative cells, which were present as single, large hepatocytes or as groups of small cells near portal veins, did express HNF4α. Clusters of very small GS‐ and HNF4α‐negative, and PCNA‐ and OV6‐positive cells near portal veins were contiguous with streaks of brightly HNF4α‐positive, OV6‐, PCNA‐, and PEPCK‐dim cells. Conclusion: Our findings show that HNF4α suppresses the expression of pericentral proteins in periportal hepatocytes, possibly via a HDAC1‐mediated mechanism. Furthermore, we show that HNF4α deficiency induces foci of regenerating hepatocytes. (HEPATOLOGY 2007;45:433–444.)

Glutamine synthetase is essential in early mouse embryogenesis

Glutamine synthetase (GS) is expressed in a tissue‐specific and developmentally controlled manner, and functions to remove ammonia or glutamate. Furthermore, it is the only enzyme that can synthesize glutamine de novo. Since congenital deficiency of GS has not been reported, we investigated its role in early development. Because GS is expressed in embryonic stem (ES) cells, we generated a null mutant by replacing one GS allele in‐frame with a β‐galactosidase‐neomycine fusion gene. GS+/LacZ mice have no phenotype, but GSLacZ/LacZ mice die at ED3.5, demonstrating GS is essential in early embryogenesis. Although cells from ED2.5 GSLacZ/LacZ embryos and GSGFP/LacZ ES cells survive in vitro in glutamine‐containing medium, these GS‐deficient cells show a reduced fitness in chimera analysis and fail to survive in tetraploid‐complementation assays. The survival of heavily (>90%) chimeric mice up to at least ED16.5 indicates that GS deficiency does not entail cell‐autonomous effects and that, after implantation, GS activity is not essential until at least the fetal period. We hypothesize that GS‐deficient embryos die when they move from the uterine tube to the harsher uterine environment, where the embryo has to catabolize amino acids to generate energy and, hence, has to detoxify ammonia, which requires GS activity. Developmental Dynamics 236:1865–1875, 2007.

Glutamine synthetase in muscle is required for glutamine production during fasting and extrahepatic ammonia detoxification.

The main endogenous source of glutamine is de novo synthesis in striated muscle via the enzyme glutamine synthetase (GS). The mice in which GS is selectively but completely eliminated from striated muscle with the Cre-loxP strategy (GS-KO/M mice) are, nevertheless, healthy and fertile. Compared with controls, the circulating concentration and net production of glutamine across the hindquarter were not different in fed GS-KO/M mice. Only a ∼3-fold higher escape of ammonia revealed the absence of GS in muscle. However, after 20 h of fasting, GS-KO/M mice were not able to mount the ∼4-fold increase in glutamine production across the hindquarter that was observed in control mice. Instead, muscle ammonia production was ∼5-fold higher than in control mice. The fasting-induced metabolic changes were transient and had returned to fed levels at 36 h of fasting. Glucose consumption and lactate and ketone-body production were similar in GS-KO/M and control mice. Challenging GS-KO/M and control mice with intravenous ammonia in stepwise increments revealed that normal muscle can detoxify ∼2.5 μmol ammonia/g muscle·h in a muscle GS-dependent manner, with simultaneous accumulation of urea, whereas GS-KO/M mice responded with accumulation of glutamine and other amino acids but not urea. These findings demonstrate that GS in muscle is dispensable in fed mice but plays a key role in mounting the adaptive response to fasting by transiently facilitating the production of glutamine. Furthermore, muscle GS contributes to ammonia detoxification and urea synthesis. These functions are apparently not vital as long as other organs function normally.

Glutamine Synthetase Deficiency in Murine Astrocytes Results in Neonatal Death

Glutamine synthetase (GS) is a key enzyme in the “glutamine‐glutamate cycle” between astrocytes and neurons, but its function in vivo was thus far tested only pharmacologically. Crossing GSfl/lacZ or GSfl/fl mice with hGFAP‐Cre mice resulted in prenatal excision of the GSfl allele in astrocytes. “GS‐KO/A” mice were born without malformations, did not suffer from seizures, had a suckling reflex, and did drink immediately after birth, but then gradually failed to feed and died on postnatal day 3. Artificial feeding relieved hypoglycemia and prolonged life, identifying starvation as the immediate cause of death. Neuronal morphology and brain energy levels did not differ from controls. Within control brains, amino acid concentrations varied in a coordinate way by postnatal day 2, implying an integrated metabolic network had developed. GS deficiency caused a 14‐fold decline in cortical glutamine and a sevenfold decline in cortical alanine concentration, but the rising glutamate levels were unaffected and glycine was twofold increased. Only these amino acids were uncoupled from the metabolic network. Cortical ammonia levels increased only 1.6‐fold, probably reflecting reduced glutaminolysis in neurons and detoxification of ammonia to glycine. These findings identify the dramatic decrease in (cortical) glutamine concentration as the primary cause of brain dysfunction in GS‐KO/A mice. The temporal dissociation between GSfl elimination and death, and the reciprocal changes in the cortical concentration of glutamine and alanine in GS‐deficient and control neonates indicate that the phenotype of GS deficiency in the brain emerges coincidentally with the neonatal activation of the glutamine‐glutamate and the associated alanine‐lactate cycles.

Interorgan Coordination of the Murine Adaptive Response to Fasting

Starvation elicits a complex adaptive response in an organism. No information on transcriptional regulation of metabolic adaptations is available. We, therefore, studied the gene expression profiles of brain, small intestine, kidney, liver, and skeletal muscle in mice that were subjected to 0–72 h of fasting. Functional-category enrichment, text mining, and network analyses were employed to scrutinize the overall adaptation, aiming to identify responsive pathways, processes, and networks, and their regulation. The observed transcriptomics response did not follow the accepted “carbohydrate-lipid-protein” succession of expenditure of energy substrates. Instead, these processes were activated simultaneously in different organs during the entire period. The most prominent changes occurred in lipid and steroid metabolism, especially in the liver and kidney. They were accompanied by suppression of the immune response and cell turnover, particularly in the small intestine, and by increased proteolysis in the muscle. The brain was extremely well protected from the sequels of starvation. 60% of the identified overconnected transcription factors were organ-specific, 6% were common for 4 organs, with nuclear receptors as protagonists, accounting for almost 40% of all transcriptional regulators during fasting. The common transcription factors were PPARα, HNF4α, GCRα, AR (androgen receptor), SREBP1 and -2, FOXOs, EGR1, c-JUN, c-MYC, SP1, YY1, and ETS1. Our data strongly suggest that the control of metabolism in four metabolically active organs is exerted by transcription factors that are activated by nutrient signals and serves, at least partly, to prevent irreversible brain damage.

The human neonatal small intestine has the potential for arginine synthesis; developmental changes in the expression of arginine-synthesizing and -catabolizing enzymes

BackgroundMilk contains too little arginine for normal growth, but its precursors proline and glutamine are abundant; the small intestine of rodents and piglets produces arginine from proline during the suckling period; and parenterally fed premature human neonates frequently suffer from hypoargininemia. These findings raise the question whether the neonatal human small intestine also expresses the enzymes that enable the synthesis of arginine from proline and/or glutamine. Carbamoylphosphate synthetase (CPS), ornithine aminotransferase (OAT), argininosuccinate synthetase (ASS), arginase-1 (ARG1), arginase-2 (ARG2), and nitric-oxide synthase (NOS) were visualized by semiquantitative immunohistochemistry in 89 small-intestinal specimens.ResultsBetween 23 weeks of gestation and 3 years after birth, CPS- and ASS-protein content in enterocytes was high and then declined to reach adult levels at 5 years. OAT levels declined more gradually, whereas ARG-1 was not expressed. ARG-2 expression increased neonatally to adult levels. Neurons in the enteric plexus strongly expressed ASS, OAT, NOS1 and ARG2, while varicose nerve fibers in the circular layer of the muscularis propria stained for ASS and NOS1 only. The endothelium of small arterioles expressed ASS and NOS3, while their smooth-muscle layer expressed OAT and ARG2.ConclusionThe human small intestine acquires the potential to produce arginine well before fetuses become viable outside the uterus. The perinatal human intestine therefore resembles that of rodents and pigs. Enteral ASS behaves as a typical suckling enzyme because its expression all but disappears in the putative weaning period of human infants.

Developmental changes in the expression of the liver-enriched transcription factors LF-B1, C/EBP, DBP and LAP/LIP in relation to the expression of albumin, α-fetoprotein, carbamoylphosphate synthase and lactase mRNA

SummaryExpression of α-fetoprotein, carbamoylphosphate synthase and albumin, that are generally accepted markers for the hepatic phenotype, require a distinct set of transcription factors. We investigated by in situ hybridization whether this set of transcription factors, LF-B1, C/EBP, DBP and LAP/LIP, is expressed coordinately in the liver during embryonic development and to what extent they are also expressed elsewhere. Our results demonstrate that mRNA levels of all transcription factors tested are significantly above background in the whole embryo and are either reduced or enhanced in expression during subsequent development. Interestingly, cardiac mesoderm, which induces prehepatic endoderm to liver formation, is temporarily permissive to its own signals, showing enhanced expression of these transcription factors and, as a result, the hepatocyte-specific genes α-fetoprotein and carbamoylphosphate synthase. In addition, these transcription factors and many liver-specific structural genes rise concomitantly in intestine and kidney just before birth, suggesting the expression of hepatogenic factors in these tissues as well. Despite the extrahepatic expression of these transcription factors, expression of albumin remains confined to the liver at all developmental stages.

Stromal Indian Hedgehog Signaling Is Required for Intestinal Adenoma Formation in Mice

BACKGROUND & AIMS Indian hedgehog (IHH) is an epithelial-derived signal in the intestinal stroma, inducing factors that restrict epithelial proliferation and suppress activation of the immune system. In addition to these rapid effects of IHH signaling, IHH is required to maintain a stromal phenotype in which myofibroblasts and smooth muscle cells predominate. We investigated the role of IHH signaling during development of intestinal neoplasia in mice. METHODS Glioma-associated oncogene (Gli1)-CreERT2 and Patched (Ptch)-lacZ reporter mice were crossed with Apc(Min) mice to generate Gli1CreERT2-Rosa26-ZSGreen-Apc(Min) and Ptch-lacZ-Apc(Min) mice, which were used to identify hedgehog-responsive cells. Cyp1a1Cre-Apc (Apc(HET)) mice, which develop adenomas after administration of β-naphthoflavone, were crossed with mice with conditional disruption of Ihh in the small intestine epithelium. Apc(Min) mice were crossed with mice in which sonic hedgehog (SHH) was overexpressed specifically in the intestinal epithelium. Intestinal tissues were collected and analyzed histologically and by immunohistochemistry and quantitative reverse-transcription polymerase chain reaction. We also analyzed levels of IHH messenger RNA and expression of IHH gene targets in intestinal tissues from patients with familial adenomatous polyposis (n = 18) or sessile serrated adenomas (n = 15) and normal colonic tissue from control patients (n = 12). RESULTS Expression of IHH messenger RNA and its targets were increased in intestinal adenomas from patients and mice compared with control colon tissues. In mice, IHH signaling was exclusively paracrine, from the epithelium to the stroma. Loss of IHH from Apc(HET) mice almost completely blocked adenoma development, and overexpression of SHH increased the number and size of adenomas that developed. Loss of IHH from Apc(HET) mice changed the composition of the adenoma stroma; cells that expressed α-smooth muscle actin or desmin were lost, along with expression of cyclooxygenase-2, and the number of vimentin-positive cells increased. CONCLUSIONS Apc mutant epithelial cells secrete IHH to maintain an intestinal stromal phenotype that is required for adenoma development in mice.