The possible role of cyclic AMP (CAMP) in the regulation of the vasopressin (VP) gene was tested in two cellular expression systems: one cell line with endogenous VP expression and the other which was transiently with a VP promoter-luciferase fusion gene. 8,Bromo-CAMP stimulated the VP mRNA content about 4-fold in the human VP-expressing small cell lung carcinoma cell line GLC-8. The luciferase activity in P19 embryonal carcinoma cells which were transiently transfected with -174 to +44 of the 5'-flanking region of the human VP gene linked to the firefly luciferase gene, was stimulated about 2-fold by the CAMP analogue. The results indicate that CAMP plays a role in the upregulation of the VP gene and hence point to several putative nucleotide motives in the promoter functionally conferring this response.
The role of glucocorticoids and second messenger systems in the regulation of the vasopressin (VP) gene was studied in the human small cell lung carcinoma cell line GLC-8. Small cell lung carcinoma GLC-8 cells express VP mRNA and contain both glucocorticoid and mineralocorticoid receptors. Treatment with the synthetic glucocorticoid dexamethasone when added alone at 10(-8) M had no effect on the VP mRNA level and decreased the level by 30% at 10(-6) M. However, the effect of dexamethasone changed to positive when cells were simultaneously treated with cAMP-enhancing agents. VP mRNA levels, which were elevated by 1.5- to 2-fold by the cAMP-enhancing agents alone, increased a further 1.5- to 3-fold by dexamethasone. Thus, the combined effect of dexamethasone and cAMP stimulation was a 3- to 7.5-fold increase in VP mRNA levels. Long term treatment with the phorbol ester 12-O-tetradecanoyl-phorbol-13-acetate (TPA) reduced the VP mRNA level by 75%. The TPA-suppressed VP mRNA levels could be up-regulated about 6-fold by simultaneous treatment with 8-bromo-cAMP. Dexamethasone did not alter the TPA-suppressed VP mRNA levels. These results indicate that both cAMP and protein kinase-C pathways as well as glucocorticoid receptors are involved in the regulation of VP mRNA levels and that these factors interact. This leads to a negative or positive response of VP gene expression to glucocorticoids in a state-dependent manner. The interactions may be of significance in a physiological context and relate to the different regulation of VP-expressing systems in the brain.
Oxytocin (OT) plays a role in reproduction at the level of the pituitary and mammary glands and uterus. This OT is synthesized in the hypothalamo-neurohypophyseal system (HNS). A number of observations have suggested that estrogens regulate the production of OT in the HNS. In this study the effect of 17beta-estradiol on the activity of the OT gene promoter was examined as well as the effect of 17beta-estradiol in vivo on OT messenger ribonucleic acid (mRNA) and peptide revels in the rat HNS. Vasopressin (VP) and its mRNA were also determined in the in vivo studies. The direct transcriptional stimulation of OT gene expression by 17beta-estradiol was studied in two different heterologous expression systems. When a plasmid having nucleotides -363 to +16 of the rat OT gene fused to the firefly luciferase reporter gene was co-transfected with an estrogen receptor expression vector in P19 embryonal carcinoma cells, luciferase activity was stimulated 80-fold by 17beta-estradiol. In estrogen receptor containing MCF-7 cells transfected with a plasmid having nucleotides -188 to +16 of the rat OT gene fused to the chloramphenicol acetyl transferase gene, 17beta-estradiol induced the expression of the chloramphenicol acetyl transferase gene through the cloned promoter element. After in vivo treatment of ovariectomized rats with 17beta-estradiol, levels of OT mRNA and VP mRNA were measured in microdissected supraoptic and paraventricular nuclei as well as VP and OT levels in these nuclei and the pituitary gland. As compared to non-treated ovariectomized rats there was no difference in contents of OT mRNA and VP mRNA in these hypothalamic nuclei and in levels of the peptides in paraventricular nuclei and the pituitary gland. A 30% reduction of the OT content of the supraoptic nuclei only was found, while the VP content did not change. To explain the results immunocytochemical analyses of the hypothalamus were performed, showing that the estrogen receptor was absent in the magnocellular neurons of the supraoptic and paraventricular nuclei. The results demonstrate that the 5'flanking region of the OT gene confers estrogen-sensitivity to transcription of the OT gene. This potential to respond to estrogens is not used in the OT-producing neurons of supraoptic and paraventricular nuclei probably due to the absence of the estrogen receptor.
The presence of 3 different neuropeptide mRNAs with a strict cell-specific expression in vivo was investigated in 13 tumor cell lines from neuroendocrine and in 23 tumor cell lines from non-neuroendocrine origin. Northern blots showed no expression of mRNA for vasopressin (VP) in the 36 tested cell lines. Very low oxytocin (OT) mRNA hybridization signals were detected in the rat pituitary tumor cell line GH4C2 and the rat pancreas tumor cell line RIN5. Both the rat pituitary tumor cell line AtT-20 and the human myeloid leukemia cell line K562, contained proopiomelanocortin (POMC) mRNA. The low incidence of VP, OT and POMC gene expression in the tested tumor cell lines was not influenced by treatments inducing differentiation. In contrast, the cholecystokinin (CCK) gene which is widely present in nervous and endocrine systems was abundantly expressed in the human primitive neuroepithelioma cell line SK-N-MC and its clonal derivative SK-N-MC-IX-C. The results indicate that the expression of neuropeptide genes is very rare in tumor cell lines. The lack of expression in undifferentiated cells agrees with the appearance of expression after day 13 of the embryogenesis when maturation of neurons begins.
The cholinergic human neuroepithelioma cell line SK-N-MCIXC expressed mRNAs for the neuropeptides cholecystokinin (CCK), neuropeptide Y, gastrin-releasing peptide (GRP) and enkephalin. The CCK transcript of about 800 nt was present at very high levels and CCK-like peptides immunoreactive to a C-terminal CCK octapeptide antiserum were present in the cell line and its medium. This clonal neuronal cell line provides a unique model system to identify cis-and trans-acting factors responsible for neuron-specific expression and regulation of the CCK gene. Furthermore, the pluripotent properties of the undifferentiated cell line may open studies on neuronal differentiation at the level of co-expression of neuropeptides and transmitters.
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