The role of cis-acting destabilizing RNA sequences in the determination of endocrine gene expression has been investigated using a novel paradigm, in which the differential regulation of two alternatively polyadenylated RNA transcripts may be observed both in vivo and in vitro. In the rat anterior pituitary gland in vivo, we have shown that, after the termination of an estrogen stimulus, a 1.7-kilobase (kb) vasoactive intestinal peptide (VIP) RNA containing an extensive 3'-untranslated region (UTR), is preferentially down-regulated with respect to a 1.0 kb VIP transcript that is uniquely abundant in this tissue. Differential regulation of the anterior pituitary VIP transcripts can be modeled in an explant culture system in which we defined both transcriptional and posttranscriptional phases of VIP gene regulation in vitro, and showed that selective down-regulation of the 1.7-kb transcript is posttranscriptional. Inhibitors of transcription and translation have also allowed us to show in vitro that differential regulation of VIP transcripts occurs through an active process that appears to involve the synthesis of a labile, destabilizing factor. In order to confirm the role of RNA destabilization as the primary mechanism of differential posttranscriptional regulation, we have also performed cell-free stability assays in which explant extracts were incubated with 32P-labeled run-off transcripts corresponding to the two alternatively polyadenylated VIP RNAs. The resultant estimates of RNA half-life showed significantly lower values for the synthetic VIP transcript containing the 3'-UTR. Our findings demonstrate the presence of functional destabilizing sequences in the 3'-UTR of the rat VIP RNA which appear to act in the physiological control of VIP gene expression.

It is well established that oestrogens can stimulate prolactin (PRL) secretion as well as the expression of the vasoactive intestinal peptide (VIP) gene whose product is also a potent PRL secretagogue. Previous evidence has supported both an autocrine and a paracrine role for pituitary VIP in PRL release in vitro; however, the cellular origin of VIP in pituitary tissue still remains poorly defined. In these studies, we have demonstrated by in situ hybridisation that VIP RNA is detected in the anterior pituitaries of chronically hyperoestrogenised rats, but not in those of untreated animals. Using a double-probe labelling procedure, VIP RNA has been shown to be present in a subpopulation of PRL-producing cells, while colocalisation of VIP and GH RNA was not observed. VIP gene expression in the rat anterior pituitary gland was characterised by the presence of two alternatively polyadenylated transcripts, 1.7 kb and 1.0 kb in size. We have generated a probe specific for the 1.7 kb transcript and double-labelling studies also showed definitive colocalisation with PRL mRNA. Our results demonstrating the presence of VIP RNA in PRL-producing cells thus suggest that VIP may play an autocrine role in PRL hypersecretion under conditions of oestrogen-induced hyperplasia.

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