Plasma membrane-bound
carboxypeptidase-D (CPD) cleaves C-terminal
arginine from extracellular substrates. In the cell,
arginine is converted to
nitric oxide (NO). We have reported that up-regulation of CPD
mRNA/
protein levels by 17β-estradiol and
prolactin (PRL) in
breast cancer cells, and by
testosterone in
prostate cancer cells, increased NO production and cell survival. The CPD promoter contains a consensus γ-
interferon-activated sequence (GAS) and 3 putative
androgen response elements (ARE.1, ARE.2, ARE.3) that could potentially bind PRL-activated
transcription factor Stat5 (
signal transducer and activator of transcription 5) and the liganded
androgen receptor (AR), respectively. This study showed that
synthetic androgen R1881 and PRL elevated CPD
mRNA/
protein levels in human MCF-7 and T47D
breast cancer cells in a time-/dose-dependent manner. PRL/
R1881-elevated CPD expression was blocked by
actinomycin-D, and a CPD promoter construct containing these GAS and AREs was stimulated by PRL or
R1881, indicating transcriptional regulation by both
hormones.
Luciferase reporter assays showed that GAS and the adjacent ARE.1 only were active. Mutation of GAS in the ΔGAS-CPD construct (ARE.1 intact) abolished CPD promoter activity in response to PRL and, surprisingly, to
R1881 as well. ΔGAS-CPD promoter activity was restored by PRL+R1881 in combination, and enhanced by ectopic Stat5, but abolished by Stat5 gene knockdown.
Chromatin immunoprecipitation analysis confirmed binding of activated Stat5 and liganded AR to GAS and ARE.1, respectively. Activated Stat5 also induced binding of unliganded AR to ARE.1, and liganded AR induced binding of unactivated Stat5 to GAS. In summary, PRL and
R1881, acting through Stat5 and AR, act cooperatively to stimulate CPD gene transcription in
breast cancer cells.