The influence of oestrogen on PBF expression, secretion and invasion in MCF7 cells

RJ Watkins; ML Read; VE Smith; G Lewy; MC Eggo; LS Loubiere; E Vasilopoulou; K Boelaert; JA Franklyn; CJ McCabe

Pituitary tumor transforming gene binding factor (PBF) is a relatively uncharacterized gene implicated in endocrine neoplasia. Given the presence of putative oestrogen response elements (ERE) in its promoter, we assessed PBF regulation by oestrogen. PBF mRNA expression was induced maximally at 48 h by 20 nM diethylstilbestrol in ERalpha-positive MCF-7 cells (2.1±0.1-fold, P<0.001, N=6). PBF protein expression levels were also significantly up-regulated by 10 nM diethylstilbestrol (2.5±0.4-fold, P<0.05, N=4) and 20 nM diethylstilbestrol (2.3±0.5-fold, P<0.05, N=4). Close analysis of the PBF promoter showed that the region -399 to -291 relative to the transcriptional start site contains variable repeats of an 18 bp sequence housing a putative ERE half-site (gcccctcGGTCAcgcctc). Sequencing the PBF promoter from 90 DNA samples from normal subjects revealed that individuals may be homozygous or heterozygous for between 1 and 6 repeats of the ERE, with overall allele frequencies of 1.1% (1 ERE), 77.8% (3 EREs), 3.9% (4 EREs), 14.4% (5 EREs) and 2.8% (6 EREs). Pulse chase experiments revealed that approximately 20% of endogenous PBF is secreted. Secretion of transiently over-expressed wild type and mutated PBF in MCF-7 cells was enhanced by mutations lacking putative endocytosis motifs, and abrogated by mutations targeting predicted glycosylation sites. We next assessed whether PBF might play a role in invasion. Oestrogen treatment (2.3±0.2 fold, P<0.001, N=9) as well as PBF over expression (5.2±1.9 fold, P<0.01, N=6) significantly increased the invasiveness of MCF-7 cells through matrigel. Given that PBF is a potent transforming gene, we propose that oestrogen treatment in post-menopausal women may up-regulate PBF expression, leading to PBF secretion and increased cell invasion. Further, the number of ERE half sites in the PBF promoter may significantly alter the response to oestrogen treatment in individual subjects

Endocrine Abstracts

P170