Pituitary adenylate cyclase-activating polypeptide (PACAP), a neuropeptide with 
trophic and cytoprotective effects, has been shown to affect cell survival, 
proliferation, and also differentiation of various cell types. The high PACAP 
level in the milk and its changes during lactation suggest a possible effect of 
PACAP on the differentiation of mammary epithelial cells. Mammary cell 
differentiation is regulated by hormones, growth factors, cytokines/chemokines, 
and angiogenic proteins. In this study, differentiation was hormonally induced 
by lactogenic hormones in confluent cultures of HC11 mouse mammary epithelial 
cells. We investigated the effect of PACAP on mammary cell differentiation as 
well as release of cytokines, chemokines, and growth factors. Differentiation 
was assessed by expression analysis of the milk protein β-casein. 
Differentiation significantly decreased the secretion of interferon gammainduced 
protein (IP)-10, "regulated upon activation normal T cell expressed and 
presumably secreted" (RANTES), insulin-like growth factor-binding protein 
(IGFBP)-3 and the epidermal growth factor receptor (EGFR) ligands, such as 
epidermal growth factor (EGF) and amphiregulin (AREG). The changes in the levels 
of IP-10 and RANTES may be relevant for the alterations in homing of T cells and 
B cells at different stages of mammary gland development, while the changes of 
the EGFR ligands may facilitate the switch from proliferative to lactating 
stage. PACAP did not modulate the expression of β-casein or the activity of 
hormone-induced pathways as determined by the analysis of phosphorylation of 
Akt, STAT5, and p38 MAPK. However, PACAP decreased the release of EGF and AREG 
from non-differentiated cells. This may influence the extracellular 
signal-related transactivation of EGFR in the non-differentiated mammary 
epithelium and is considered to have an impact on the modulation of oncogenic 
EGFR signaling in breast cancer.