Prolactin regulation by heparin-binding growth factors expressed in mouse pituitary cell lines

Prolactin (PRL) secretion is regulated by both inhibitory and stimulatory factors. Dopamine is the primary inhibitor, but multiple factors stimulate PRL gene expression and release. These can be divided into two categories: those that rapidly stimulate PRL release and those that induce the PRL gene followed by increased release. The pituitary intermediate lobe (IL) contains a PRL-releasing factor (PRF) that rapidly stimulates PRL release. From a mouse IL tumor, we established a non-melanotroph cell line, mIL5, which secretes a factor that stimulated PRL gene expression and release in vitro. This PRF activity did not rapidly stimulate PRL release and bound to heparin. Our objective was to examine the regulation of PRL by heparin-binding proteins and characterize the PRF activity produced by mIL5 cells. PRL gene expression and release was determined using GH3 cells, stably transfected with a PRL promoter/luciferase reporter (GH(3)/luc). After screening mIL5 cells by reverse transcriptase polymerase chain reaction, we found that they expressed two heparin-binding growth factors basic fibroblast growth factor (FGF-2) and heparin-binding epidermal growth factor (HB-EGF) which were considered strong candidates for PRL transcriptional regulatory activity. To determine whether the activity produced by mIL5 cells is attributed to FGF-2 or HB-EGF, three approaches were used: heparin-affinity chromatography, Western blotting, and immunoneutralization. The PRF activity in conditioned media eluted from heparin with 1 M NaCl whereas both FGF- 2 and HB-EGF eluted with >1 M NaCl. Neither growth factor was detectable in mIL5 cells by Western blotting. Antibodies directed against FGF-2 and HB-EGF, alone or together, did not abolish this activity from mIL5 cells. In conclusion, FGF-2 and HB-EGF are potent stimulators of PRL gene expression and release but do not account for most of the endogenous PRL gene activity in mIL5 cells. The distinct heparin-binding factor that stimulates PRL gene transcription remains to be identified.