neuromedin-n and Pheochromocytoma

Nerve growth factor (NGF) cooperates with glucocorticoids, activators of adenylate cyclase, and lithium to induce the expression of teh gene encoding the neuropeptides neurotensin and neuromedin N (NT/N gene) in PC 12 pheochromocytoma cells. High level expression requires simultaneous treatment with three or all four inducers. To examine the mechanism underlying this complex synergism, we have examined the effects of protein kinase inhibitors and other agents which influence intracellular signal transduction on NT/N gene expression. Two structurally similar bacterial alkaloids, staurosporine and K-252a, inhibit several protein kinases in vitro, including protein kinase C and cyclic nucleotide-dependent kinases. K-252a has been reported to specifically inhibit the effects of NGF on PC12 pheochromocytoma cells. Surprisingly, staurosporine in combination with other inducers markedly potentiated NT/N gene expression. In contrast, K-252a had no effect on NT/N gene expression when added simultaneously with other inducers. Expression of the NT/N gene was also potentiated by the phorbol ester 12-O-tetradecanoylphorbol-13-acetate, which directly activates protein kinase C, and by bradykinin, which stimulates phosphatidylinositol turnover in PC12 cells, and these effects were not blocked by staurosporine. Staurosporine was generally more effective in stimulating NT/N gene expression when used in inducer combinations that did not include NGF. These results, taken together with recent evidence that staurosporine is also able to induce neurite outgrowth from PC12 cells, suggest that the effects of staurosporine and NGF may converge, in part, on a common intracellular target.

Topics: Adrenal Gland Neoplasms; Alkaloids; Bradykinin; Gene Expression Regulation; Humans; Nerve Growth Factors; Neurotensin; Peptide Fragments; Pheochromocytoma; Protein Kinase C; RNA; Signal Transduction; Staurosporine; Tetradecanoylphorbol Acetate; Tumor Cells, Cultured

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' 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.

Topics: Adrenal Gland Neoplasms; Amino Acid Sequence; Animals; Base Sequence; Chromosome Mapping; Fibroblasts; Gene Expression; Genes, Regulator; Molecular Sequence Data; Neurotensin; Peptide Fragments; Phenotype; Pheochromocytoma; Pituitary Gland; Promoter Regions, Genetic; Rats; Tumor Cells, Cultured

Lithium perturbs intracellular signal transduction pathways used by neurotransmitters, suggesting that changes in receptor signalling may underlie its actions in the treatment of manic depressive illness. Little attention, however, has been directed toward possible additional actions at the level of specific gene expression, particularly of genes encoding neurotransmitters or neuromodulators. In PC12 pheochromocytoma cells, lithium dramatically potentiates increases in intracellular levels of the neuropeptide neurotensin and the mRNA encoding it, caused by combinations of nerve growth factor, dexamethasone, and the adenylate cyclase activator, forskolin. This result demonstrates that lithium can profoundly influence the expression of a specific neuropeptide gene in a previously unanticipated manner and suggests that changes in gene expression might be involved in its therapeutic activity.

Topics: Adrenal Gland Neoplasms; Animals; Cell Line; Chlorides; Colforsin; Dexamethasone; Genes; Kinetics; Lithium; Lithium Chloride; Nerve Growth Factors; Neurotensin; Peptide Fragments; Pheochromocytoma; Transcription, Genetic