Pit-1 Binding Sequences Permit Calcium Regulation of Human Prolactin Gene Expression

Abstract

This study examines the regulation of the human PRL (hPRL) gene promoter by intracellular calcium. Deletants of the 5'-flanking region of the hPRL gene and constructs consisting of the thymidine kinase promoter linked to the first or second proximal Pit-1 binding site were fused to the bacterial chloramphenicol acetyl transferase (CAT) reporter gene. With the complete 5-kilobase pair (kbp) hPRL promoter sequence the calcium channel agonist Bay K8644 induced a significant 2-fold increase in CAT reporter gene expression and the antagonist verapamil a 4.5-fold reduction, using GH3 cells cultured in physiological levels of calcium. The transcriptional response to calcium influx was similar with a series of 5'-deleted hPRL-CAT constructs including those that comprised the proximal (up to 740 bp) or distal (-1300- to -1700-bp) sequences alone. When treating cells cultured in low calcium conditions the induction with the hPRL promoter increased to 5-fold on the addition of exogenous calcium and Bay K8644. The pituitary-specific expression of the hPRL gene is conferred by the interaction of the pituitary-specific factor Pit-1 with several binding sites located in the 5'-flanking DNA, of which three are located in the proximal region. This suggested that Pit-1 binding sites may be involved in the calcium response.(ABSTRACT TRUNCATED AT 250 WORDS)

Binding of a 100‐kDa ubiquitous factor to the human prolactin promoter is required for its basal and hormone‐regulated activity

“…For this purpose, we used the dihydropyridine drug Bay K8644, which enhances Ca2+ influx through the voltage-sensitive Ca2+ channels [Ill. Fig. 4 shows that the activity of the hPRL promoter (p164CAT) was stimulated 2.1-fold by the Ca2+ ionophore Bay K8644, as reported previously [16]. This stimulation is promoter specific, since thymidine-kinase -promoter activity was not affected by the drug.…”

Expression of Pit-1 and related proteins in diverse human pituitary adenomas

“…These studies were approved by the Central Manchester Health Authority Clinical Research Ethical Committee. GH3 rat pituitary tumour cells were obtained from the European Collection of Animal Cell Cultures, PHLS, and grown as previously described (Hoggard et al 1991) in Ham's F-10 medium supplemented with 10% horse serum and 5% fetal calf serum.…”

“…It is involved in the tissue-specific expression of the GH and prolactin genes in somatotrophs and lactotrophs respectively, and is probably also involved in the expression of the thyrotrophin (TSH)-ß gene in thyrotrophic cells (Rosenfeld, 1991). Pit-1 is centrally involved in the hormonal regulation of the rat prolactin gene (Fox et al 1990;Iverson et al 1990; Yan & Bancroft, 1991) and the human prolactin gene (Hoggard et al 1991;Peers et al 1991). This regulation may involve more than one isoform of Pit-1, as an alternative splice variant has different transcriptional effects for the GH and prolactin genes (Konzak & Moore, 1992;Morris et al 1992;Theill et al 1992).…”

EGF stimulates Pit-1 independent transcription of the human prolactin pituitary promoter in human breast cancer SK-BR-3 cells through its proximal AP-1 response element

“…TRH, EGF, thyroid hormones, glucocorticoids, estrogens and by second messengers Ca2+ and cAMP (Peers et al, 1990Hoggard et al, 1991;Nalda et al, 1997;Pernasetti et al, 1997;Van De Weerdt et al, 2000;reviewed in Muller et al, 1998). It displays many biological activities triggered through binding to its specific transmembrane receptor (PRLR).…”