Mutually dependent response elements in the cis-regulatory region of the neurotensin/neuromedin N gene integrate environmental stimuli in PC12 cells

The expression of the gene encoding the neuroendocrine peptides neurotensin (NT) and neuromedin N is strictly dependent on simultaneous exposure to multiple inducers in PC12 pheochromocytoma cells. NT peptide and NT/N mRNA levels are synergistically induced by combinations of NGF, dexamethasone, activators of adenylate cyclase, and lithium ion. We have used transient transfection assays to delineate the rat NT/N gene sequences necessary for this complex regulation. Progressive deletions of the 5\u27 flanking region revealed that sequences between -216 and +56 are sufficient to confer the full spectrum of responses exhibited by the endogenous gene to a reporter gene. Detailed mutational analysis of this region indicates that it is composed of an array of inducible cis-regulatory sequences, including AP-1, cAMP response, and glucocorticoid response elements. Specific mutation of either the AP-1 site or each of two cAMP response elements indicates that they are functionally interdependent. This array of response elements serves to integrate multiple environmental stimuli into a unified transcriptional response

Synergistic induction of neurotensin gene transcription in PC12 cells parallels changes in AP-1 activity

A consensus AP-1 site in the promoter of the rat neurotensin/neuromedin N (NT/N) gene is a critical regulatory element required for synergistic regulation by combinations of nerve growth factor (NGF), lithium, glucocorticoids, and adenylate cyclase activators. A rapid RNase protection assay was developed to examine the kinetics of NT/N gene activation and to determine whether activation requires newly synthesized proteins. Either NGF or lithium in combination with dexamethasone and forskolin transiently activated NT/N gene expression, but with distinct kinetics. Protein synthesis was not required for activation when NGF was used as the permissive inducer, but was required for the rapid down-regulation of the response. In contrast, lithium responses were attenuated in the absence of protein synthesis, consistent with a requirement for newly synthesized AP-1 complexes in activation. In all cases, increases in NT/N gene expression closely paralleled increases in AP-1 binding activity. Lithium in combination with other inducers caused delayed increases in both AP-1 binding activity and c-jun, c-fos and fra-1 gene expression. These results indicate that NGF and lithium exert their effects on NT/N gene expression through distinct pathways. The lithium pathway is active in neuronally-differentiated PC12 cells and could potentially be involved in the regulation of NT/N gene expression in the nervous system

Induction of the neurotensin (NT) gene in PC12 cells gives rise to NT precursor (approximately 88%), NT(3-13)-like peptide (approximately 10%), and NT (approximately 2%)

Neurotensin (NT) is coexpressed with catecholamines in sympathetic neurons and adrenal chromaffin cells. A pheochromocytoma PC12 cell line can also be induced to express the NT gene and produce immunoreactive NT. In the present study, NT mRNA was quantified under various hormonal conditions and NT precursor synthesis rates were determined by pulse labeling and immunoprecipitation. In addition, NT precursor and NT-related products were measured using RIA and were characterized using HPLC and Sephadex chromatography. Neurotensin mRNA, NT precursor synthesis, and NT precursor/product levels were correlated. Surprisingly, NT appeared to be a minor product, both in cells and media: NT precursor (approximately 88%), NT(3-13)-like peptide (approximately 10%), and NT (approximately 2%). Neurotensin added to cultures was not converted to NT(3-13). Treatment of cells with 60 mM KCl or various secretagogues induced Ca(2+)-dependent release of NT precursor, NT(3-13), and NT in proportion to their cellular contents. These results suggest a) that NT precursor processing in induced PC12 cells was much slower than NT precursor synthesis, b) that NT(3-13) was a major product and NT a minor one, and c) that NT precursor and its products were stored within secretory vesicles

Endogenous neurotensin facilitates enterohepatic bile acid circulation by enhancing intestinal uptake in rats

Initial studies on the digestive hormone neurotensin (NT) showing that intestinal NT mRNA expression and blood levels were altered in rats fed chow containing bile acid (BA) and the BA chelator cholestyramine led us to investigate the role of NT in the enterohepatic circulation of BA. In fasted, anesthetized rats with common bile ducts cannulated for bile collection, intravenous NT infusion (10 pmol. kg(-1). min(-1)) enhanced BA output relative to control over 3 h in animals administered donor bile into the duodenum (30 microl/min). This suggested that the effect of NT was on the return of BA from the intestine to the liver, which is rate determining in the normal process. In rats prepared as described above and administered [(3)H]taurocholate ([(3)H]TC; 5 mM, 1 ml) duodenally, NT infusion (3-10 pmol x kg(-1) x min(-1)) increased the [(3)H]TC recovery rate in bile approximately twofold, whereas sulfated CCK-8 (12-50 pmol x kg(-1) x min(-1)) had no effect. To investigate the roles of endogenous NT and CCK, we administered [(3)H]TC into the rat duodenum or lower jejunum and tested the effect of the NT antagonist SR-48692 (2 nmol x kg(-1) x min(-1)) or CCK-A antagonist lorglumide (100 nmol x kg(-1) x min(-1)). SR-48692 reduced the [(3)H]TC recovery rate by congruent with 50% and congruent with 24% in the duodenum and jejunum, respectively, whereas lorglumide had no effect. These results suggest that NT or a similar peptide is an endogenous regulator of enterohepatic BA cycling, which acts by enhancing BA uptake in the intestine

Sequences required for induction of neurotensin receptor gene expression during neuronal differentiation of N1E-115 neuroblastoma cells

The promoter region of the mouse high affinity neurotensin receptor (Ntr-1) gene was characterized, and sequences required for expression in neuroblastoma cell lines that express high affinity NT-binding sites were characterized. Me(2)SO-induced neuronal differentiation of N1E-115 neuroblastoma cells increased both the expression of the endogenous Ntr-1 gene and reporter genes driven by NTR-1 promoter sequences by 3-4-fold. Deletion analysis revealed that an 83-base pair promoter region containing the transcriptional start site is required for Me(2)SO activation. Detailed mutational analysis of this region revealed that a CACCC box and the central region of a large GC-rich palindrome are the crucial cis-regulatory elements required for Me(2)SO induction. The CACCC box is bound by at least one factor that is induced upon Me(2)SO treatment of N1E-115 cells. The Me(2)SO effect was found to be both selective and cell type-restricted. Basal expression in the neuroblastoma cell lines required a distinct set of sequences, including an Sp1-like sequence, and a sequence resembling an NGFI-A-binding site; however, a more distal 5\u27 sequence was found to repress basal activity in N1E-115 cells. These results provide evidence that Ntr-1 gene regulation involves both positive and negative regulatory elements located in the 5\u27-flanking region and that Ntr-1 gene activation involves the coordinate activation or induction of several factors, including a CACCC box binding complex

Synergistic activation of neurotensin/neuromedin N gene expression by c-Jun and glucocorticoids: novel effects of Fos family proteins

The cis-regulatory region of the neurotensin/neuromedin N (NT/N) gene integrates diverse environmental signals in the neuroendocrine PC12 cell line, resulting in remarkable synergistic regulation. An AP-1 site appears to play a pivotal role in cooperative NT/N gene activation, as mutations in this site decrease responses to all inducer combinations by at least an order of magnitude. Here we report that c-Jun acts synergistically with glucocorticoids to activate the NT/N promoter, and that Fos family proteins have novel regulatory effects on this interaction. Cotransfection of individual pCMV-AP-1 expression plasmids revealed that c-Jun most potently activates the NT/N promoter and that costimulation with dexamethasone results in a further 6- to 12-fold increase in expression. Unlike its general inhibitory effects on glucocorticoid regulation in other systems, c-Fos potentiated activation by glucocorticoids when coexpressed with c-Jun, and Fos B had a similar, but more limited, positive effect. In contrast, Fra-1 reversed the direction of glucocorticoid regulation, and Fra-2 abolished synergism. AP-1, cAMP response element, and glucocorticoid response element motifs are required for full cooperative activation by either c-Jun or c-Jun/c-Fos and glucocorticoids. These results indicate that NT/N promoter activation involves synergistic interactions between specific AP-1 complexes and ligand-activated glucocorticoid receptor, and similar mechanisms may regulate NT/N gene expression in central neurons

The rat gene encoding neurotensin and neuromedin N. Structure, tissue-specific expression, and evolution of exon sequences

Recombinant DNA clones encoding the neurotensin/neuromedin N precursor protein have been isolated from both bovine hypothalamus cDNA and rat genomic libraries using a heterologous canine cDNA probe. Nucleotide sequence analysis of these clones and comparison with the previously determined canine sequence has revealed that 76% of the amino acid residues are conserved in all three species. The protein precursor sequences predicted from bovine hypothalamus and canine intestine cDNA clones vary at only 9 of 170 amino acid residues suggesting that within a species identical precursors are synthesized in both the central nervous system and intestine. The rat gene spans approximately 10.2 kilobases (kb) and is divided into four exons by three introns. The neurotensin and neuromedin N coding domains are tandemly positioned on exon 4. RNA blot analysis has revealed that the rat gene is transcribed to yield two distinct mRNAs, 1.0 and 1.5 kb in size, in all gastrointestinal and all neural tissues examined except the cerebellum. There is a striking variation in the relative levels of these two mRNAs between brain and intestine. The smaller 1.0-kb mRNA greatly predominates in intestine while both mRNA species are nearly equally abundant in hypothalamus, brain stem, and cortex. Sequence comparisons and RNA blot analysis indicate that these two mRNAs result from the differential utilization of two consensus poly(A) addition signals and differ in the extent of their 3\u27 untranslated regions. The relative combined levels of the mRNAs in various brain and intestine regions correspond roughly with the relative levels of immunologically detectable neurotensin except in the cerebral cortex where mRNA levels are 6 times higher than anticipated

Higher level organization of individual gene transcription and RNA splicing

Visualization of fibronectin and neurotensin messenger RNAs within mammalian interphase nuclei was achieved by fluorescence hybridization with genomic, complementary DNA, and intron-specific probes. Unspliced transcripts accumulated in one or two sites per nucleus. Fibronectin RNA frequently accumulated in elongated tracks that overlapped and extended well beyond the site of transcription. Splicing appears to occur directly within this RNA track, as evidenced by an unambiguous spatial separation of intron-containing and spliced transcripts. Excised introns for neurotensin RNA appear free to diffuse. The transcription and processing site of the fibronectin gene localized to the nuclear interior and was associated with larger transcript domains in over 88 percent of the cells. These results support a view of nuclear function closely integrated with structure