Mutually Dependent Elements in the Neurotensin/Neuromedin N Gene Promoter Integrate Multiple Environmental Stimuli in PC12 Cells: a Thesis

This thesis examines the structure and regulated expression of the gene encoding the neuroendocrine peptides neurotensin and neuromedin N (NT/N gene). Previous studies have shown that expression of NT/N mRNA and NT peptide in PC12 cells are strictly dependent on simultaneous exposure to combinations of nerve growth factor (NGF), glucocorticoids, activators of adenylate cyclase, and lithium ion. My objective was to characterize the cis-regulatory DNA sequences involved in regulated expression of this gene. The initial focus of this study was an analysis of the structure, tissue-specific expression, and exon evolution of the rat NT/N gene. Nucleotide sequence comparisons between the rat gene and the canine and bovine cDNA sequences indicated that the predicted structure of a 170 amino acid precursor protein is highly conserved. Furthermore, the close similarity between the two cDNAs suggested that identical precursor proteins are expressed in neural and endocrine tissues. RNA analysis revealed that the gene is transcribed to yield two distinct mRNAs, 1.0 kb and 1.5 kb in size. The two mRNA species differ only in the size of their 3\u27 untranslated regions. Interestingly, the smaller mRNA is predominant in the gastrointestinal tract, while both mRNAs are equally abundant in all neural tissues examined, except the cerebellum, where no expression was observed. Transient transfection assays were used to delineate the rat NT/N gene cis-regulatory DNA sequences. Progressive deletion of the NT/N 5\u27 flanking region revealed that sequences between -216 and +56 of the NT/N gene are sufficient to confer the full spectrum of responses of the endogenous gene to either of two reporter genes. A detailed mutational analysis of the NT/N control region indicated that it is composed of an array of inducible cis-regulatory elements, including an AP-1 site, two cAMP-responsive elements (CREs), and a glucocorticoid-responsive element (GRE). Specific mutations to the AP-1 site and either CRE suggested that these elements are functionally interdependent. I propose that this array of cis-regulatory sequences in the NT/N transcriptional control region serves to integrate multiple environmental stimuli into a unified transcriptional response. To further examine the role of the AP-1 site and CREs in the NT/N promoter, reporter genes containing either a single or multiple AP-1 or CRE sites were expressed in PC12 cells and protein kinase A-deficient PC12 cells treated with forskolin, NGF, and lithium, either individually, or in combination. The results indicated that lithium and NGF markedly activate promoters containing multiple AP-1 sites, but not a single site, and that these effects were additive. Both agents potentiated forskolin-induced activation of promoters containing a single or multiple CREs, but had no effect, individually. Also, in contrast to the activation of multiple AP-1 sites by lithium and NGF, activation of the NT/N promoter and promoters containing CREs is absolutely dependent on protein kinase A activity. These results suggested that promoters containing multiple AP-1 sites, or a single AP-1 site in the context of nearby active CREs, are selectively activated by lithium and NGF in PC12 cells. Based on the results of this thesis I have proposed a model to account for the complex transcriptional regulation of the NT/N gene in PC12 cells. I have also addressed the relevance of these findings to the mechanisms of phenotypic plasticity of embryonic neural crest cells, NGF-induced neuronal differentiation, and the pharmacological actions of lithium

beta-Actin messenger RNA localization and protein synthesis augment cell motility

In chicken embryo fibroblasts (CEFs), beta-actin mRNA localizes near an actin-rich region of cytoplasm specialized for motility, the lamellipodia. This localization is mediated by isoform-specific 3\u27-untranslated sequences (zipcodes) and can be inhibited by antizipcode oligodeoxynucleotides (ODNs) (Kislauskis, E.H., X.-C. Zhu, and R.H. Singer. 1994. J. Cell Biol. 127: 441-451). This inhibition of beta-actin mRNA localization resulted in the disruption of fibroblast polarity and, presumably, cell motility. To investigate the role of beta-actin mRNA in motility, we correlated time-lapse images of moving CEFs with the distribution of beta-actin mRNA in these cells. CEFs with localized beta-actin mRNA moved significantly further over the same time period than did CEFs with nonlocalized mRNA. Antizipcode ODN treatment reduced this cell translocation while control ODN treatments showed no effect. The temporal relationship of beta-actin mRNA localization to cell translocation was investigated using serum addition to serum-deprived cultures. beta-actin mRNA was not localized in serum-deprived cells but became localized within minutes after serum addition (Latham, V.M., E.H. Kislauskis, R.H. Singer, and A.F. Ross. 1994. J. Cell Biol. 126:1211-1219). Cell translocation increased over the next 90 min, and actin synthesis likewise increased. Puromycin reduced this cell translocation and blocked this induction in cytosolic actin content. The serum induction of cell movement was also inhibited by antizipcode ODNs. These observations support the hypothesis that beta-actin mRNA localization and consequent protein synthesis augment cell motility

Isoform-specific 3\u27-untranslated sequences sort alpha-cardiac and beta-cytoplasmic actin messenger RNAs to different cytoplasmic compartments

We demonstrate that in differentiating myoblasts, the mRNAs encoding two actin isoforms, beta-cytoplasmic, and alpha-cardiac, can occupy different cytoplasmic compartments within the same cytoplasm. beta-actin mRNA is localized to the leading lamellae and alpha-actin mRNA is associated with a perinuclear compartment. This was revealed by co-hybridizing, in situ, fluorochrome-conjugated oligonucleotide probes specific for each isoform. To address the mechanism of isoform-specific mRNA localization, molecular chimeras were constructed by insertion of actin sequences between the Lac Z coding region and SV-40 3\u27UTR in a reporter plasmid. These constructs were transiently expressed in a mixed culture of embryonic fibroblasts, myoblasts and myotubes, beta-galactosidase activity within transfectants was revealed by a brief incubation with its substrate (X-gal). Since the blue-insoluble reaction product co-localized with the specific mRNAs expressed from each construct, it was used as a bioassay for mRNA localization. Transfectants were scored as either perinuclear, peripheral or nonlocalized with respect to the distribution of the blue product. The percentage of transfectants within those categories was quantitated as a function of the various constructs. This analysis revealed that for each actin mRNA its 3\u27UTR is necessary and sufficient to direct reporter transcripts to its appropriate compartment; beta-actin peripheral and alpha-actin perinuclear. In contrast, sequences from the 5\u27UTR through the coding region of either actin gene did not localize the blue product. Therefore, 3\u27UTR sequences play a key role in modulating the distribution of actin mRNAs in muscle cells. We propose that the mechanism of mRNA localization facilitates actin isoform sorting in the cytoplasm

Beta-actin mRNA localization is regulated by signal transduction mechanisms

Beta-actin mRNA is localized in the leading lamellae of chicken embryo fibroblasts (CEFs) (Lawrence, J., and R. Singer. 1986. Cell. 45:407-415), close to where actin polymerization in the lamellipodia drives cellular motility. During serum starvation beta-actin mRNA becomes diffuse and non-localized. Addition of FCS induces a rapid (within 2-5 min) redistribution of beta-actin mRNA into the leading lamellae. A similar redistribution was seen with PDGF, a fibroblast chemotactic factor. PDGF-induced beta-actin mRNA redistribution was inhibited by the tyrosine kinase inhibitor herbimycin, indicating that this process requires intact tyrosine kinase activity, similar to actin filament polymerization and chemotaxis. Lysophosphatidic acid, which has been shown to rapidly induce actin stress fiber formation (Ridley, A., and A. Hall. 1992. Cell. 790:389-399), also increases peripheral beta-actin mRNA localization within minutes. This suggests that actin polymerization and mRNA localization may be regulated by similar signaling pathways. Additionally, activators or inhibitors of kinase A or C can also delocalize steady-state beta-actin mRNA in cells grown in serum, and can inhibit the serum induction of peripherally localized beta-actin mRNA in serum-starved CEFs. These data show that physiologically relevant extracellular factors operating through a signal transduction pathway can regulate spatial sites of actin protein synthesis, which may in turn affect cellular polarity and motility

Colony-stimulating factor-1 (CSF-1) rescues osteoblast attachment, survival and sorting of beta-actin mRNA in the toothless (tl-osteopetrotic) mutation in the rat

We have shown that in the osteopetrotic rat mutation toothless (tl) osteoblasts are absent from older bone surfaces in mutants and that mutant osteoblasts in vivo lack the prominent stress fiber bundles polarized along bone surfaces in osteoblasts from normal littermates. Our recent data demonstrate that in normal osteoblasts in vitro beta- and gamma-actin mRNAs have different, characteristic intracellular distributions and that tl (mutant) osteoblasts fail to differentially sort these mRNAs. Because bone resorption and formation are highly interdependent and injections of CSF-1, a growth factor, increase bone resorption and growth in tl rats, we examined the effects of CSF-1 treatment on osteoblast survival and ultrastructure in vivo and ability to sort actin mRNAs in vitro. Neonatal CSF-1 treatment of mutants restores osteoblasts on older bone surfaces, normalizes the intracellular distribution of stress fibers in osteoblasts in vivo and promotes normal sorting of beta-actin mRNA in mutant osteoblasts in vitro without normalizing gamma-actin distribution. These data suggest the beta- and gamma-actin mRNAs in osteoblasts are sorted by different mechanisms and that the differential sorting of beta-actin mRNA is related to the characteristic polarization of stress fibers in osteoblasts and their survival on bone surfaces. This experimental system can be used to explore the relationships and regulation of these aspects of cell and tissue biology

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

Sequences responsible for intracellular localization of �-actin messenger RNA also affect cell phenotype

Abstract. We have characterized the structure and function of RNA sequences that direct B-cytoplasmic actin mRNA to the cell periphery were mapped to two segments of 3'-untranslated region by expression of LacZ/B-actin chimeric mRNAs in chicken embryo fibroblasts (CEFs). A 54-nt segment, the "RNA zipcode; and a homologous but less active 43-nt segment each localized B-galactosidase activity to the leading lamellae. This zipcode contains the full activity, and mutations or deletions within it reduce, but do not eliminate, its activity, indicating that several motifs contribute to the activity. Two of these motifs, when multimerized, can regenerate almost full activity. These sequences are highly conserved in evolution, since the human/3-actin zipcode, positioned identically in the YUTR localizes equally well in chicken cells. Complementary phosphorothioate oligonucleotides against the zipcode delocalized endogenous/3-actin mRNA, whereas those complementary to the region just outside the zipcode, or sense oligonucleotides, did not. Actin mRNA or protein levels were unaffected by the antisense treatments, but a dramatic change in lamellipodia structure, and actin stress fiber organization was observed using the same antizipeode oligonucleotides which delocalized the mRNA. Hence, discrete 3'UTR sequences direct B-actin isoform synthesis to the leading lamellae and affect cell morphology, presumably through the actin cytoskeleton. TIN is a highly abundant structural constituent of all eukaryotic cells integral to a variety of cellular functions. As a major constituent of the cytoskeleton or myofilaments, it is essential for the maintenance of cel