PURIFICATION OF HEPARIN-BINDING EPIDERMAL GROWTH FACTOR-LIKE GROWTH-FACTOR FROM PIG UTERINE LUMINAL FLUSHINGS, AND ITS PRODUCTION BY ENDOMETRIAL TISSUES

Pig uterine luminal flushings contain at least four heparin-binding growth factors (HBGF) that stimulate fibroblast [H-3]thymidine incorporation. One of these factors, which appeared to be a relatively minor HBGF, was eluted from heparin affinity columns by 1.0 M NaCl and was found to compete with I-125-epidermal growth factor (EGF) for binding to an endometrial carcinoma cell line. This EGF receptor (EGF-R)-binding property was abolished by an antiserum to heparin-binding EGF-like growth factor (HB-EGF) that specifically blocks binding of HB-EGF to the EGF-R Reverse-phase HPLC resulted in the purification of two EGF-R-binding activities correlated with 13 500 and 17 000 M(r) proteins that reacted with an antiserum raised against residues 9-26 of human IIB-EGF. Uterine extracts also contained an EGF-R-binding factor that was eluted from heparin by 1.0 M NaCl and was antagonized by NB-EGF antiserum. Endometrial mRNA subjected to reverse transcriptase-polymerase chain reaction (RT-PCR) and nested PCR through the use of HB-EGF-specific primers yielded fragments of the predicted size. Cloning of the nested PCR product revealed a 380-bp porcine HB-EGF cDNA sequence that was 78-85% homologous to primate or rodent HB-EGF. HB-EGF was immunohistochemically localized primarily to the luminal epithelium in both pregnant and nonpregnant animals.