Secretagogue-stimulated pancreatic secretion is differentially regulated by constitutive NOS isoforms in mice

Nitric oxide (NO) and NO synthase (NOS) play controversial roles in pancreatic secretion. NOS inhibition reduces CCK-stimulated in vivo pancreatic secretion, but it is unclear which NOS isoform is responsible, because NOS inhibitors lack specificity and three NOS isoforms exist: neuronal (nNOS), endothelial (eNOS), and inducible (iNOS). Mice having individual NOS gene deletions were used to clarify the NOS species and cellular interactions influencing pancreatic secretion. In vivo secretion was performed in anesthetized mice by collecting extraduodenal pancreatic duct juice and measuring protein output. Nonselective NOS blockade was induced with N(omega)-nitro-L-arginine (L-NNA; 10 mg/kg). In vivo pancreatic secretion was maximal at 160 pmol.kg(-1).h(-1) CCK octapeptide (CCK-8) and was reduced by NOS blockade (45%) and eNOS deletion (44%). Secretion was unaffected by iNOS deletion but was increased by nNOS deletion (91%). To determine whether the influence of NOS on secretion involved nonacinar events, in vitro CCK-8-stimulated secretion of amylase from isolated acini was studied and found to be unaltered by NOS blockade and eNOS deletion. Influence of NOS on in vivo secretion was further examined with carbachol. Protein secretion, which was maximal at 100 nmol.kg(-1).h(-1) carbachol, was reduced by NOS blockade and eNOS deletion but unaffected by nNOS deletion. NOS blockade by L-NNA had no effect on carbachol-stimulated amylase secretion in vitro. Thus constitutive NOS isoforms can exert opposite effects on in vivo pancreatic secretion. eNOS likely plays a dominant role, because eNOS deletion mimics NOS blockade by inhibiting CCK-8 and carbachol-stimulated secretion, whereas nNOS deletion augments CCK-8 but not carbachol-stimulated secretion.     

Regulation of alternative 3' splice site selection by constitutive splicing factors.

We have devised an in vitro splicing assay in which the mutually exclusive exons 2 and 3 of alpha-tropomyosin act as competing 3' splice sites for joining to exon 1. Splicing in normal HeLa cell nuclear extracts results in almost exclusive joining of exons 1 and 3. Splicing in decreased nuclear extract concentrations and decreased ionic strength results in increased 1-2 splicing. We have used this assay to determine the role of three constitutive pre-mRNA splicing factors on alternative 3' splice site selection. Polypyrimidine tract binding protein (PTB) was found to inhibit the splicing of introns containing a strong binding site for this factor. However, the inhibitory effect of PTB could be partially reversed if pre-mRNAs were preincubated with U2 auxiliary factor (U2AF) prior to splicing in PTB-supplemented extracts. For alpha-tropomyosin, regulation of splicing by PTB and U2AF primarily affected the joining of exons 1-3 with no dramatic increases in 1-2 splicing being detected. Preincubation of pre-mRNAs with SR proteins led to small increases in 1-2 splicing. However, if pre-mRNAs were preincubated with SR proteins followed by splicing in PTB-supplemented extracts, there was a nearly complete reversal of the normal 1-2 to 1-3 splicing ratios. Thus, multiple pairwise, and sometimes antagonizing, interactions between constitutive pre-mRNA splicing factors and the pre-mRNA can regulate 3' splice site selection.     

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.     

Differentially expressed isoforms of the mouse retinoic acid receptor beta generated by usage of two promoters and alternative splicing.

Using anchored PCR, three different cDNA isoforms of the mouse retinoic acid receptor beta [mRAR-beta 1, mRAR-beta 2 (formerly mRAR-beta 0) and mRAR-beta 3], generated from the same gene by differential promoter usage and alternative splicing, were isolated. These three isoforms encode RAR proteins with different N-terminal A regions and identical B - F regions. The sequence encoding the first 59 amino acids of the mRAR-beta 3 A region is identical with the entire A region of mRAR-beta 1. However, the sequence of mRAR-beta 3 region A differs from that of mRAR-beta 1 by an additional 27 C-terminal amino acids encoded in an 81 nucleotide-long putative exon which is spliced in between the exons encoding the A and B regions of mRAR-beta 1. Both mRAR-beta 1 and beta 3 cDNAs differ entirely from mRAR-beta 2 in their 5'-untranslated (5'-UTR) and A region coding sequences. This N-terminal variability, in a region which was shown to be important for cell-type specific differential target gene trans-activation by other nuclear receptors, suggests that the three mRAR-beta isoforms may be functionally distinct. The conservation of RAR-beta isoform sequences from mouse to human, as seen by cross-hybridization on Southern blots or DNA sequence analysis, as well as their differential patterns of expression in various mouse tissues, corroborates this view. Additionally, the mRNA analysis data suggest that mRAR-beta 2, whose expression predominates in RA-treated embryonal carcinoma (EC) and embryonic stem (ES) cells, may be important during early stages of development. mRAR-beta 1 and beta 3, on the other hand, which are predominantly expressed in fetal and adult brain, may play some specific role in the development of the central nervous system.     

A two-locus model for experience-conditioned direction of paw usage in the mouse is suggested by dominant and recessive constitutive paw usage behaviours.

Left-right direction of paw usage in the mouse depends on the genotype and the directional nature of the test. There are two phenotypic classes; in some strains, direction of paw usage is learned or conditioned by the direction of the initial test chamber and the experience of reaching and, in other strains, paw usage is a constitutive behaviour not affected by previous experience. We report the evidence for locus heterogeneity in the cause of constitutive versus experience-conditioned paw usage from a phenotypic analysis of F1 hybrid generations from the experience-conditioned C57BL/6J, C3H/HeHa, and SWV strains and the constitutive CDS/Lay and DBA/2J strains. The F1 hybrids between strains of different phenotypic classes provide evidence of locus heterogeneity. Constitutive paw usage in CDS/Lay is phenotypically dominant to experience-conditioned behaviour in both C57BL/6J and SWV. However, constitutive paw usage in DBA/2J is phenotypically recessive to experience-conditioned behaviour in C57BL/6J and dominant to experience-conditioned behaviour in SWV. Among the experience-conditioned strains, C57BL/6J is highly lateralized but SWV is only weakly lateralized. Our data suggest a model in which C57BL/6J may have a "strong" allele that identifies a functional difference between the constitutive paw usage of CDS/Lay and DBA/2J. DBA/2J may have a loss-of-function mutation at the same locus that is recessive to the strong C57BL/6J allele. SWV may have a "weak" allele and the (SWV x D2)F1 compound heterozygote may be below a threshold for detectability of experience-conditioned behaviour, making the constitutive behaviour of DBA/2J appear to be dominant to the experience-conditioned behaviour of SWV. CDS/Lay may have a dominant allele at a second locus that suppresses experience-conditioned behaviour in all F1 hybrids.     

Expression of mouse Fbxw7 isoforms is regulated in a cell cycle- or p53-dependent manner

Fbxw7 is the F-box protein component of an SCF-type ubiquitin ligase that contributes to the ubiquitin-dependent degradation of cell cycle activators and oncoproteins. Three isoforms (alpha, beta, and gamma) of Fbxw7 are produced from mRNAs with distinct 5' exons. We have now investigated regulation of Fbxw7 expression in mouse tissues. Fbxw7alpha mRNA was present in all tissues examined, whereas Fbxw7beta mRNA was detected only in brain and testis, and Fbxw7gamma mRNA in heart and skeletal muscle. The amount of Fbxw7alpha mRNA was high during quiescence (G0 phase) in mouse embryonic fibroblasts (MEFs) and T cells, but it decreased markedly as these cells entered the cell cycle. The abundance of Fbxw7alpha mRNA was unaffected by cell irradiation or p53 status. In contrast, X-irradiation increased the amount of Fbxw7beta mRNA in wild-type MEFs but not in those from p53-deficient mice, suggesting that radiation-induced up-regulation of p53 leads to production of Fbxw7beta mRNA. Our results thus indicate that expression of Fbxw7 isoforms is differentially regulated in a cell cycle- or p53-dependent manner.     

Structural basis of natural promoter recognition by a unique nuclear receptor, HNF4alpha. Diabetes gene product

HNF4alpha (hepatocyte nuclear factor 4alpha) plays an essential role in the development and function of vertebrate organs, including hepatocytes and pancreatic beta-cells by regulating expression of multiple genes involved in organ development, nutrient transport, and diverse metabolic pathways. As such, HNF4alpha is a culprit gene product for a monogenic and dominantly inherited form of diabetes, known as maturity onset diabetes of the young (MODY). As a unique member of the nuclear receptor superfamily, HNF4alpha recognizes target genes containing two hexanucleotide direct repeat DNA-response elements separated by one base pair (DR1) by exclusively forming a cooperative homodimer. We describe here the 2.0 angstroms crystal structure of human HNF4alpha DNA binding domain in complex with a high affinity promoter element of another MODY gene, HNF1alpha, which reveals the molecular basis of unique target gene selection/recognition, DNA binding cooperativity, and dysfunction caused by diabetes-causing mutations. The predicted effects of MODY mutations have been tested by a set of biochemical and functional studies, which show that, in contrast to other MODY gene products, the subtle disruption of HNF4alpha molecular function can cause significant effects in afflicted MODY patients.     

Diurnal expression of Dnmt3b mRNA in mouse liver is regulated by feeding and hepatic clockwork

DNA methyltransferase 3B (DNMT3B) is critically involved in de novo DNA methylation and genomic stability, while the regulatory mechanism in liver is largely unknown. We previously reported that diurnal variation occurs in the mRNA expression of Dnmt3b in adult mouse liver. The aim of this study was to determine the mechanism underlying the diurnal expression pattern. The highest level and the lowest level of Dnmt3b mRNA expression were confirmed to occur at dawn and in the afternoon, respectively, and the expression pattern of Dnmt3b closely coincided with that of Bmal1. Since the diurnal pattern of Dnmt3b mRNA expression developed at weaning and scheduled feeding to separate the feeding cycle from the light/dark cycle led to a phase-shift in the expression, it could be assumed that feeding plays a critical role as an entrainment signal. In liver-specific Bmal1 knockout (L-Bmal1 KO) mice, L-Bmal1 deficiency resulted in significantly higher levels of Dnmt3b at all measured time points, and the time when the expression was the lowest in wild-type mice was shifted to earlier. Investigation of global DNA methylation revealed a temporal decrease of 5-methyl-cytosine percentage in the genome of wild-type mice in late afternoon. By contrast, no such decrease in 5-methyl-cytosine percentage was detected in L-Bmal1 KO mice, suggesting that altered Dnmt3b expression affects the DNA methylation state. Taken together, the results suggest that the feeding and hepatic clockwork generated by the clock genes, including Bmal1, regulate the diurnal variation in Dnmt3b mRNA expression and the consequent dynamic changes in global DNA methylation.     

Diurnal expression of Dnmt3b mRNA in mouse liver is regulated by feeding and hepatic clockwork

DNA methyltransferase 3B (DNMT3B) is critically involved in de novo DNA methylation and genomic stability, while the regulatory mechanism in liver is largely unknown. We previously reported that diurnal variation occurs in the mRNA expression of Dnmt3b in adult mouse liver. The aim of this study was to determine the mechanism underlying the diurnal expression pattern. The highest level and the lowest level of Dnmt3b mRNA expression were confirmed to occur at dawn and in the afternoon, respectively, and the expression pattern of Dnmt3b closely coincided with that of Bmal1. Since the diurnal pattern of Dnmt3b mRNA expression developed at weaning and scheduled feeding to separate the feeding cycle from the light/dark cycle led to a phase-shift in the expression, it could be assumed that feeding plays a critical role as an entrainment signal. In liver-specific Bmal1 knockout (L-Bmal1 KO) mice, L-Bmal1 deficiency resulted in significantly higher levels of Dnmt3b at all measured time points, and the time when the expression was the lowest in wild-type mice was shifted to earlier. Investigation of global DNA methylation revealed a temporal decrease of 5-methyl-cytosine percentage in the genome of wild-type mice in late afternoon. By contrast, no such decrease in 5-methyl-cytosine percentage was detected in L-Bmal1 KO mice, suggesting that altered Dnmt3b expression affects the DNA methylation state. Taken together, the results suggest that the feeding and hepatic clockwork generated by the clock genes, including Bmal1, regulate the diurnal variation in Dnmt3b mRNA expression and the consequent dynamic changes in global DNA methylation.